extraction des protéines de canola par des solutions aqueuses

EXTRACTION DES PROTÉINES DE CANOLA PAR DES SOLUTIONS AQUEUSES ÉLECTRO-ACTIVÉES,

OPTIMISATION DES CONDITIONS D’EXTRACTION ET ÉTUDE DE LEURS

PROPRIÉTÉS TECHNO-FONCTIONNELLES

Thèse

Alina Gerzhova

Doctorat en sciences et technologie des aliments

Philosophiae Doctor (Ph. D.)

Québec, Canada

© Alina Gerzhova, 2016

EXTRACTION DES PROTÉINES DE CANOLA PAR DES SOLUTIONS AQUEUSES ÉLECTRO-ACTIVÉES,

OPTIMISATION DES CONDITIONS D’EXTRACTION ET ÉTUDE DE LEURS

PROPRIÉTÉS TECHNO-FONCTIONNELLES

Thèse

Alina Gerzhova

Sous la direction de :

Mohammed Aider, directeur de recherche Martin Mondor, codirecteur de recherche

iii

Résumé

Le tourteau de canola représente une importante source de protéines de haute valeur

nutritionnelle et pourrait faire l’objet de multiples utilisations dans l’alimentation humaine.

Toutefois, l’extraction conventionnelle de ces protéines, généralement réalisée en milieu

fortement alcalin, altère leurs qualités et fonctionnalités. De plus, l'engouement pour les

technologies vertes, justifié par la pollution générée par l’industrie agroalimentaire et la

consommation accrue de l’énergie par celle-ci, incite les industriels à innover en utilisant des

méthodes d'extraction alternatives respectueuses des principes du développement durable.

Dans ce contexte, l’électro-activation, une technologie novatrice et prometteuse pour

l’industrie alimentaire, a fait l’objet d’une étude détaillée sur son utilisation pour produire

des solutions d’extraction ayant un pouvoir extractif élevé. Elle consiste à appliquer un

champ électrique pour activer des solutions aqueuses, légèrement salines, pour les utiliser

dans la réalisation de différentes réactions chimiques, y compris l’extraction de protéines.

Ce projet vise à démontrer la preuve du concept de l’efficacité de l’électro-activation

à être utilisée comme technologie d’extraction sans utilisation d’acides et de bases et à étudier

son potentiel pour l’extraction des protéines à partir de tourteau de canola.

Le premier objectif consistait à étudier l'effet du pH, de la concentration du tourteau

de canola et de sel sur le taux d'extraction des protéines par la méthode d’extraction

conventionnelle. Il en est ressorti que seul le pH a un effet significatif sur le taux d’extraction.

Cette étude a servi de témoin pour la comparaison avec l’extraction par la technologie de

l’électro-activation en solution.

Le deuxième objectif visait à étudier les propriétés alcalines des solutions électro-

activées (SEA) en fonction du type de configuration du réacteur d’électro-activation,

l'intensité du courant électrique, la concentration du sel ajouté et le temps d’électro-activation

(de traitement). Les résultats obtenus ont montré que toutes les solutions électro-activées

après 10 minutes ont toutes été caractérisées par un pH très alcalin (pH > 10), mais leur

alcalinité titrable a varié de manière significative en fonction du temps d’électro-activation

et de l'intensité du courant appliqué (les facteurs les plus importants), ainsi que de la

concentration du sel et du type de configuration. Cette étude a montré qu'en modifiant les

paramètres d’électro-activation, il était possible de moduler les propriétés alcalines des

iv

solutions aqueuses électro-activées pour une éventuelle utilisation comme conditions

optimales pour l'extraction des protéines à partir de tourteau de canola.

Le troisième objectif consistait à évaluer et comprendre l’effet de l’électro-activation

sur le taux d'extraction des protéines du tourteau de canola et leurs propriétés physico-

chimiques. Dans cette optique, les solutions électro-activées (SEA) optimisées et une solution

de NaOH (témoin) ont été utilisées. Les résultats obtenus ont montré que le taux d'extraction

était plus élevé avec l’utilisation des SEA. De plus, les résultats obtenus par l’analyse SDS-

PAGE indiquaient que les protéines extraites par les SEA ont été nettement moins dénaturées

comparées à celles extraites avec du NaOH. L’analyse par FTIR a révélé la présence de pics

plus étroits, témoignant de la structure native secondaires des protéines extraites par électro-

activation.

Le quatrième objectif visait l’étude des propriétés fonctionnelles des protéines de

canola extraites par électro-activation et leur comparaison avec les propriétés de protéines

extraites par la méthode conventionnelle avec du NaOH. Les résultats obtenus ont montré

des améliorations dans les propriétés de surface telles que l'émulsification et la taille des

particules pour les protéines extraites par des SEA. En outre, les protéines extraites par

électro-activation étaient plus efficaces pour abaisser la tension de surface liée à leur taux de

flexibilité et taux d'adsorption plus élevé.

Le cinquième objectif a fait l’objet d’une étude dans laquelle les protéines extraites

par électro-activation sous forme de concentrés et d'isolats ont été ajoutées à une formulation

de biscuits sans gluten afin d'étudier leur comportement dans une réelle matrice alimentaire.

La formulation de biscuits sans gluten est à base d’une combinaison de farine de sarrasin

biologique cultivé au Québec et de farine de riz. Les résultats obtenus ont permis de

démontrer la pertinence d’ajouter des protéines de canola dans ce genre de produit. En effet,

par rapport à une farine sans gluten seule, l'ajout des protéines de canola a nettement amélioré

la texture des biscuits, la valeur nutritionnelle et l’acceptabilité générale du produit, ce qui a

était soutenu par une évaluation sensorielle.

Finalement et compte tenu des résultats obtenus à chacune des étapes ce projet de

doctorat, il est possible de conclure que les solutions aqueuses électro-activées (SEA) sont

des agents d'extraction puissants qui offrent des perspectives prometteuses pour l'extraction

de protéines de canola et pourraient être appliquées à d'autres tourteaux oléagineux. En plus,

v

leur utilisation comme substitut d’agents chimiques d’extraction en fait la preuve de

l’adéquation totale avec les concepts du développement durable.

vi

Abstract

Canola meal is an alternative source of valuable proteins which can be valorized for

human consumption. However, conventional extraction which implies the use of chemical

alkali and acids has a serious impact on their quality and functionality. In addition, today’s

trend towards “eco-friendly” technologies urges scientists to look for alternative methods of

protein extraction.

A novel technology of electro-activation (EA) has been reported to produce solutions

with high extraction potential by subjecting dilute salt solutions to the energy of external

electric field. Therefore the aim of the work was to study the efficiency of electro-activated

solutions for protein extraction from canola meal as a “green” emerging approach.

The first objective was to find the optimal conditions for higher extractability from

canola meal by studying the effects of pH, meal to solvent ratio, and salt concentration. It

was found that pH of the extracting medium had the most significant effect on protein

extractability. This study served as a reference template for further comparison with

extraction by electro-activated solutions (EAS).

Second objective aimed to study the properties of electro-activated solutions

depending on the type of configuration, current intensity, salt concentration, and the time of

treatment. All the solutions after 10 min treatment were characterized by highly alkaline pH

(>10) gradually attaining pH ~12, yet their titratable alkalinity differed significantly

depending on the time and the current intensity as the most important factors as well as on

the salt concentration and cell configuration. However, most importantly, it was shown that

by changing the parameters of electro-activation, optimal conditions for proteins extraction

can be modulated.

Third objective focused on protein extraction from canola meal. Electro-activated

solutions optimized in the second objective were compared to chemical base in terms of

protein extractability, and their effect on the physico-chemical properties and secondary

structure. The results showed comparably higher extractability rates for electro-activated

solutions as well as less damaged proteins as was reflected in the FTIR spectra and SDS-

PAGE analyses.

The fourth objective was to study the proteins` functionality, which showed certain

improvements in surface related properties such as emulsion activity index and droplet size

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for proteins extracted by electro-activated solutions. Also they were more efficient in

lowering the surface tension which pointed at their higher flexibility and adsorption rate.

Finally, for the fifth objective the extracted proteins in the form of concentrates and

isolates were added to gluten-free biscuit formulation in order to investigate their behavior

in a real food matrix. In comparison with gluten free flour blend consisting of the mixture of

rice and buckwheat flours, the addition of canola proteins markedly improved the texture of

biscuits, their nutritional value, and general acceptance which was supported by sensorial

test. Considering the results obtained at each of the stages it is possible to conclude that

electro-activated solutions are powerful extracting agents providing promising perspectives

for the extraction of proteins from canola and other oilseeds. Moreover their utilization will

allow to eliminate the use of chemical bases for the extraction step which well corresponds

to the aims stated by the concept of sustainable development.

viii

Table of contents

Résumé .............................................................................................................................................. iii Abstract ............................................................................................................................................. vi Table of contents ............................................................................................................................. viii List of tables ..................................................................................................................................... xii List of figures .................................................................................................................................. xiv

List of abbreviations ....................................................................................................................... xvii Acknowledgments ........................................................................................................................... xix

Préface ............................................................................................................................................... xx

Introduction ......................................................................................................................................... 1

1. CHAPTER 1: Literature review .................................................................................................. 4

Proteins ............................................................................................................................... 4

1.1.1. General information .................................................................................................... 4

1.1.2. Role of proteins in nutrition and their nutritive value ................................................ 5

1.1.3. Functional properties .................................................................................................. 5

1.1.4. Sources of proteins .................................................................................................... 18

Canola ................................................................................................................................ 20

1.2.1. Historical remarks ..................................................................................................... 20

1.2.2. Production, economical interest ............................................................................... 23

1.2.3. Meal processing ........................................................................................................ 24

1.2.4. Composition, nutritive and biological value of canola meal ..................................... 25

1.2.5. Proteins of canola ...................................................................................................... 31

1.2.6. Methods of protein extraction .................................................................................. 33

1.2.7. Effect of processing conditions on the quality of proteins ....................................... 36

1.2.8. Possible application ................................................................................................... 39

Electro-activation as an emerging technology .................................................................. 41

1.3.1. History and development .......................................................................................... 41

1.3.2. Principles ................................................................................................................... 42

1.3.3. Phenomenon of EAS .................................................................................................. 47

1.3.4. Reactors design ......................................................................................................... 49

1.3.5. Application of EAS ..................................................................................................... 53

2. CHAPTER 2: Problematic, research hypothesis and objectives ............................................... 58

2.1. Problematic ....................................................................................................................... 58

Research hypothesis.......................................................................................................... 58

Main objective ................................................................................................................... 58

Specific objectives ............................................................................................................. 59

3. CHAPTER 3: Total dry matter and protein extraction from canola meal as affected by pH and salt addition and the use of Zeta-potential/turbidity to optimize the extraction conditions .............. 60

Contextual transition ......................................................................................................... 60

ix

Résumé .............................................................................................................................. 61

Abstract ............................................................................................................................. 62

Introduction ...................................................................................................................... 63

Materials and methods ..................................................................................................... 65

3.6.1. Materials ................................................................................................................... 65

3.6.2. Proximate analysis ..................................................................................................... 65

3.6.3. Extraction .................................................................................................................. 66

3.6.4. Protein precipitation ................................................................................................. 66

3.6.5. Total dry matter and protein extractability .............................................................. 66

3.6.6. Turbidity and Zeta Potential (ζ) measurements ........................................................ 67

3.6.7. SDS-Gel electrophoresis ............................................................................................ 67

3.6.8. Experimental design and statistical analysis ............................................................. 68

Results and discussion ....................................................................................................... 69

3.7.1. Extractability .............................................................................................................. 69

3.7.2. Protein and ash composition of extracts .................................................................. 77

3.7.3. Precipitability based on Zeta Potential and Turbidity ............................................... 78

3.7.4. Gel Electrophoresis ................................................................................................... 81

Conclusion ......................................................................................................................... 83

Acknowledgments ............................................................................................................. 84

4. CHAPTER 4: Monitoring of pH and alkalinity changes in the electro-activated aqueous solutions generated in the cationic compartment. The effect of salt concentration, current intensity, time and cell configuration ................................................................................................................ 85

Contextual transition ......................................................................................................... 85

Résumé .............................................................................................................................. 86

Abstract ............................................................................................................................. 87

Introduction ...................................................................................................................... 88

Materials and methods ..................................................................................................... 91

4.5.1. Chemicals .................................................................................................................. 91

4.5.2. Ion-exchange membranes ......................................................................................... 91

4.5.3. Reactor design. Configurations of electro-activation cells ....................................... 91

4.5.4. Protocol of electro-activation ................................................................................... 92

4.5.5. Analysis methods ...................................................................................................... 92

4.5.6. Statistical analysis ...................................................................................................... 92

Results and discussion ....................................................................................................... 93

4.6.1. Configuration 1 .......................................................................................................... 93

4.6.2. Configuration 2 .......................................................................................................... 97

4.6.3. Configurations 3 and 4 ............................................................................................ 101

4.6.4. The effect of type of configuration ......................................................................... 102

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Conclusion ....................................................................................................................... 106

Acknowledgments ........................................................................................................... 107

5. CHAPTER 5: A comparative study between the electro-activation technique and conventional extraction method on the extractability, composition and physicochemical properties of canola protein concentrates and isolates ..................................................................................................... 108

Contextual transition ....................................................................................................... 108

Résumé ............................................................................................................................ 109

Abstract ........................................................................................................................... 110

Introduction .................................................................................................................... 111

Materials and methods ................................................................................................... 113

5.5.1. Chemicals ................................................................................................................ 113

5.5.2. Ion-exchange membranes ....................................................................................... 113

5.5.3. Configurations of the electro-activation reactor .................................................... 114

5.5.4. Experimental ........................................................................................................... 114

5.5.5. Chemical analysis .................................................................................................... 115

5.5.6. Total dry matter and protein extractability ............................................................ 116

5.5.7. SDS PAGE ................................................................................................................. 116

5.5.8. FTIR .......................................................................................................................... 117

5.5.9. Statistical analysis .................................................................................................... 117

Results and discussion ..................................................................................................... 117

5.6.1. Changes in pH .......................................................................................................... 118

5.6.2. Extractability ............................................................................................................ 122

5.6.3. Composition ............................................................................................................ 125

5.6.4. SDS PAGE ................................................................................................................. 128

5.6.5. FTIR .......................................................................................................................... 132

Conclusion ....................................................................................................................... 137


6. CHAPTER 6: Study of the functional properties of canola protein concentrates and isolates extracted by electro-activated solutions .......................................................................................... 139


Résumé ............................................................................................................................ 140

Abstract ........................................................................................................................... 141

Introduction .................................................................................................................... 142

Materials and Methods ................................................................................................... 144

6.5.1. Raw materials and extraction methods .................................................................. 144

6.5.2. Nitrogen solubility index ......................................................................................... 144

6.5.3. Water absorption capacity (WAC) ........................................................................... 145

6.5.4. Fat absorption capacity (FAC) ................................................................................. 145

6.5.5. Surface characteristics ............................................................................................ 145

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6.5.6. Surface active properties ........................................................................................ 147



6.6.1. Nitrogen solubility index ......................................................................................... 149

6.6.2. Water absorption capacity ...................................................................................... 150

6.6.3. Fat absorption capacity ........................................................................................... 152

6.6.4. Surface characteristics ............................................................................................ 153

6.6.5. Surface active properties ........................................................................................ 157

Conclusion ....................................................................................................................... 165


7. CHAPTER 7: Incorporation of canola proteins extracted by electro-activated solutions in the gluten free biscuit formulation of rice-buckwheat flour blend: Assessment of quality characteristics and textural properties of the product. ............................................................................................ 167


Résumé ............................................................................................................................ 168

Abstract ........................................................................................................................... 169

Introduction .................................................................................................................... 170

Materials and methods ................................................................................................... 172

7.5.1. Materials ................................................................................................................. 172

7.5.2. Proximate analysis ................................................................................................... 173

7.5.3. Cookie preparation .................................................................................................. 173

7.5.4. Analytical tests ........................................................................................................ 174



7.6.1. Proximate analysis ................................................................................................... 175

7.6.2. Dimensions and cookie spread ratio ....................................................................... 176

7.6.3. Dough characteristics .............................................................................................. 179

7.6.4. Cookie characteristics ............................................................................................. 181

7.6.5. Surface colour ......................................................................................................... 183

7.6.6. Moisture content and water activity ...................................................................... 185

7.6.7. Microstructure analysis ........................................................................................... 187

7.6.8. Preliminary sensory evaluation ............................................................................... 189

Conclusion ....................................................................................................................... 191


8. CHAPTER 8: Conclusion and perspectives ............................................................................ 194

General conclusion .......................................................................................................... 194

Perspectives .................................................................................................................... 196

References ....................................................................................................................................... 198

xii

List of tables

Table 1.1: Indispensable amino acid requirements (FAO/WHO/UNU, 2007). ..................... 4

Table 1.2: Various protein functionalities and their application in food. Adapted from Fennema (1996). ..................................................................................................................... 6

Table 1.3: Worldwide production of major oilseeds and their meals in MMT and their protein contents. Adapted from Ramachandran et al. (2007); USDA (2013). ..................... 20

Table 1.4: Proximate composition of canola meal. Adapted from Newkirk et al. (2003). .. 25

Table 1.5: Amino acid composition of canola. Adapted from Newkirk et al. (2003), Downey (1990). .................................................................................................................... 26

Table 1.6 : Carbohydrate content of canola meal. Adapted from Newkirk (2011). ............. 27

Table 1.7: Mineral composition of canola and soybean meal. Adapted from Bell et al. (1999); Downey (1990); Newkirk (2011). ........................................................................... 28

Table 1.8: Extraction methods and conditions used for canola and rapeseed. ..................... 37

Table 3.1: Proximate chemical composition of canola oil cake on a dry weight basis. ....... 69

Table 4.1: Variables and their coded values. ........................................................................ 97

Table 4.2: Comparison of pH and alkalinity between two configurations for different current intensities within 0.01 M NaCl concentration. ....................................................... 103

Table 4.3: Comparison of pH and alkalinity between two configurations for different current intensities within 0.1 M NaCl concentration. ......................................................... 104

Table 4.4 : Comparison of pH and alkalinity between two configurations for different current intensities within 1 M NaCl concentration. ............................................................ 105

Table 5.1: Proximate chemical composition of canola oil cake on a dry weight basis. ..... 118

Table 5.2: Comparison of pH and alkalinity between two configurations for I = 0.3 A and within different NaCl concentrations. ................................................................................ 119

Table 5.3: Comparative characteristics of protein concentrates. ........................................ 125

Table 5.4: Comparative characteristics of protein isolates. ................................................ 127

Table 6.1: Absorption capacities of electro-activated protein isolate (EAPI), conventional protein isolate (CPI), electro-activated protein concentrate (CPC), and conventional protein concentrate (CPC)............................................................................................................... 151

Table 6.2 : Surface pressure at air-protein solution interface. ............................................ 154

Table 6.3 : Interfacial tension between oil and protein solution. ....................................... 155

Table 6.4: Foaming capacity of protein isolates and concentrates. .................................... 159

Table 7.1: Control biscuit formulation. .............................................................................. 173

Table 7.2: Proximate analysis of flours and proteins. ........................................................ 176

Table 7.3: Physical parameters of cookies made from flour blend of rice and dark (toasted) buckwheat and rice and green (non-toasted) buckwheat. ................................................... 177

Table 7.4: Changes in surface colour of biscuits prepared from the blend of rice and dark buckwheat with the addition of canola proteins. ................................................................ 183

xiii

Table 7.5: Changes in surface colour of biscuits prepared from the blend of rice and green buckwheat with the addition of canola proteins. ................................................................ 184

xiv

List of figures

Figure 1.1: Major destabilization processes in emulsions. Adapted from Tadros (2013). ... 12

Figure 1.2 : Triangle of U showing the relationship of major Brassica species. Adapted from Ahuja et al. (2010). ...................................................................................................... 21

Figure 1.3: Decrease in glucosinolates and erucic acid levels in rapeseed/canola. Adapted from Daun et al. (2011). ....................................................................................................... 22

Figure 1.4: Number of varieties of canola in Canada. Adapted from Daun et al. (2011). ... 22

Figure 1.5: Processing of canola by prepress solvent extraction. Adapted from Daun (2004). .............................................................................................................................................. 24

Figure 1.6: Dissociation of 12S globulin under external conditions. Adapted from Mieth et al. (1983) ............................................................................................................................... 32

Figure 1.7: Basic electrolytic cell. Adapted from Zeng and Zhang (2010). ......................... 44

Figure 1.8 : Electrolytic cell showing the passage of electrons and some products of electrode reactions. Adapted from Chaplin (2000). ............................................................. 46

Figure 1.9: Flow type electrochemical modules. Adapted from Leonov et al. (1999). ........ 50

Figure 1.10: A schematic representation of a three-call electro-activation reactor. Adapted from (Liato et al., 2015a). ..................................................................................................... 51

Figure 1.11: Example of a CEM with fixed SO3 groups. Adapted from (Bazinet et al., 1998a). .................................................................................................................................. 53

Figure 1.12 : Electro-activation unit for protein extraction form sunflower oil cake, where 1-tank with oilcake; 2 - cathodic chamber; 3 - membrane; 4 – anodic chamber; 5 – tank with anolyte; 6 – peristaltic pump; 7 – anode; 8 – cathode; 9 – filter; 10 – oilcake; 11 – extracting solution. Adapted from Plutakhin et al. (2005). .................................................. 56

Figure 3.1: Influence of the solution pH on the total dry matter extractability from canola meal. ..................................................................................................................................... 70

Figure 3.2: Influence of the solution pH on the protein extractability from canola meal. ... 70

Figure 3.3: The effect of salt concentration on total dry matter (a) and protein extractability (b) at pH 10. .......................................................................................................................... 73



Figure 3.6: Protein and ash content of extracts obtained with 10% canola meal concentration. ....................................................................................................................... 78

Figure 3.7: The influence of the solution pH on the Zeta potential of the canola protein. ... 79

Figure 3.8: The influence of the solution pH on the turbidity of canola proteins solution. . 80

Figure 3.9: SDS-PAGE of canola proteins A) without NaCl addition and B) with an addition of 1 M NaCl. ........................................................................................................... 82

Figure 4.1: Configuration 1 of the electro-activation reactor used for the generation of the electrolysed water. ................................................................................................................ 93

xv

Figure 4.2: The effect of current intensity and salt concentration over time on the pH monitoring of electro-activated solutions, configuration 1. ................................................. 94

Figure 4.3 : The effects of salt concentration, current intensity and time on the alkalinity: a) time hold constant at maximum value (60 min); b) current intensity hold constant at maximum value (0.2 A); c) concentration hold constant at maximum value (1 M). ........... 96

Figure 4.4: Configuration 2 of the electro-activation reactor used for the generation of the electrolysed water. ................................................................................................................ 97

Figure 4.5: The effect of current intensity and salt concentration over time on the pH monitoring of electro-activated solutions, configuration 2. ................................................. 99

Figure 4.6: The effects of salt concentration, current intensity and time on the alkalinity: a) time hold constant at maximum value (60 min); b) current intensity hold constant at maximum value (0.2 A); c) concentration hold constant at maximum value (1 M). ......... 101

Figure 4.7: Configuration 3 (a) and 4 (b) of the electro-activation reactor used for the generation of the electrolysed water. .................................................................................. 102

Figure 5.1 : Configurations of the reactor used for EA corresponding to: a) Configuration 1 (C1); b) Configuration 2 (C2). ............................................................................................ 114

Figure 5.2: Changes in pH during EA for I = 0.3 A: a) C1; b) C2. .................................... 119

Figure 5.3: Changes in pH during extraction by EAS: a) C1, 0.01 M NaCl; b) C1, 0.1 M NaCl; c) C1, 1 M NaCl; d) C2, 0.01 M NaCl; e) C2, 0.1 M NaCl; f) C2, 1 M NaCl. ....... 120

Figure 5.4: Total dry matter extractability for I = 0.3 A: a) conventional extraction; b) extraction with EAS in C1; c) extraction with EAS in C2. ................................................ 123

Figure 5.5: Protein extractability for I = 0.3 A: a) conventional extraction; b) extraction with EAS in C1; c) extraction with EAS in C2. ................................................................. 124

Figure 5.6 : Molecular profiles as analyzed by SDS PAGE: a) EAPC extracted with 0.01 M electro-activated NaCl solution; b) EAPC extracted with 1 M electro-activated NaCl solution; c) CPC extracted with 0.01M and 1 M NaCl solutions. ...................................... 129

Figure 5.7 : Molecular profiles as analyzed by SDS PAGE: a) EAPI extracted with 0.01 M electro-activated NaCl solution; b) EAPI extracted with 1 M electro-activated NaCl solution; c) CPI extracted with 0.01 M NaCl solution; d) CPI extracted with 1 M NaCl solution. .............................................................................................................................. 132

Figure 5.8: Deconvulved FTIR spectra: a) Protein concentrates extracted with 0.01 M NaCl; b) Protein concentrates extracted with 1 M NaCl. ................................................... 133

Figure 5.9 : Deconvulved FTIR spectra: a) Protein isolates extracted with 0.01 M NaCl; b) Protein isolates extracted with 1 M NaCl. .......................................................................... 137

Figure 6.1: The solubility behavior of canola proteins as a function of pH, where EAPI is electro-activated protein isolate, CPI is conventional protein isolate, EAPC is electro-activated protein concentrate and CPC is conventional protein concentrate. ..................... 150

Figure 6.2: Surface hydrophobicity of protein isolates and concentrates as a function of pH. ............................................................................................................................................ 157

Figure 6.3: Foam stability as a function of time: A) FS of proteins isolates, B) FS of protein concentrates. ....................................................................................................................... 160

xvi

Figure 6.4: Emulsifying properties of canola protein isolates and concentrates: A) Emulsion activity index; B) Droplet size. ........................................................................................... 162

Figure 6.5: Emulsion stability index of the proteins. ......................................................... 163

Figure 6.6: Creaming stability of canola protein-stabilized emulsions. ............................. 165

Figure 7.1: Cookies prepared with rice and dark buckwheat flours (1), rice and green buckwheat flours (2) with the incorporation of electro-activated canola protein concentrate and isolate: (A) Control; (B) 3% EAPC; (C) 6% EAPC; (D) 9% EAPC; (E) 3% EAPI; (F) 6% EAPI; (G) 9% EAPI. .................................................................................................... 178

Figure 7.2: Dough characteristics: (A) dough prepared with grey buckwheat; (B) dough prepared with green buckwheat. ......................................................................................... 180

Figure 7.3 : Cookie characteristics: (A) cookies prepared with grey buckwheat; (B) cookies prepared with green buckwheat. ......................................................................................... 182

Figure 7.4 : Moisture of baked biscuits. ............................................................................. 186

Figure 7.5: Water activity (aw) levels for baked biscuits. ................................................... 186

Figure 7.6: Scanning electron microscopy at 1000x magnification of: (A) rice flour; (B) grey buckwheat flour; (C) green buckwheat flour; (D) EAPC; (E) EAPI at 300x magnification; and (F) EAPI at 1000x magnification. ....................................................... 187

Figure 7.7 : Scanning electron microscopy of the cross section of selected biscuits at 1000x magnification: (A) 3% EAPC; (B) 6% EAPC; (C) 9% EAPC; (D) 3% EAPI; (E) 6% EAPI; (F) 9% EAPI; (G) control. .................................................................................................. 189

Figure 7.8 : Preliminary sensory characteristics of canola protein enriched cookies......... 191

xvii

List of abbreviations

A Alkalinity Å Angstrom a* CIE color space co-ordinate: degree of greenness/redness ACE Angiotensin-i converting enzyme ADF Acid detergent fiber AEM Anion-exchange membrane ANC Acid neutralization capacity ANOVA Analysis of the variance ANS Anilino-1-naphthalenesulfonic acid AOAC Association of analytical communities b* CIE color space co-ordinate: degree of blueness/yellowness C1 Configuration 1 C2 Configuration 2 CEM Cation-exchange membrane CM Canola meal CMC Carboxymethyl cellulose CPC Conventional protein concentrate CPC_10 Conventional protein concentrate extracted at pH 10 CPC_12 Conventional protein concentrate extracted at pH 12 CPI Conventional protein isolates CS Creaming stability DIR Direct alkaline extraction EA Electro-activation EAI Emulsion activity index EAPC Electro activated protein concentrate EAPC_C1_60 Electro-activated protein concentrate, treated in the configuration 1 for 60 min EAPC_C2_60 Electro-activated protein concentrate, treated in the configuration 2 for 60 min EAPC_C1_10 Electro-activated protein concentrate, treated in the configuration 1 for 10 min EAPC_C2_10 Electro-activated protein concentrate, treated in the configuration 2 for 10 min EAPI Electro activated protein isolate EAS Electro-activated solutions ESI Emulsion stability index FAC Fat absorption capacity FC Foaming capacity FID Flame ionization detector FS Foaming stability FTIR Fourier transform infrared spectroscopy

xviii

GC Gas chromatography GF Gluten free HE Height of the emulsion HS The height of the serum layer I Electric current IEM Ion exchange membrane kDa Kilo Dalton L* CIE color space co-ordinate: degree of lightness LAL Lysinoalanine LDL Low-density lipoprotein LDV Laser Doppler velocimetry MMT Million metric tones MW Molecular weight NDF Neutral detergent fiber NSI Nitrogen solubility index NSPS Nonstarch polysaccharides NTU Nephelometric turbidity units PDCCAS Protein digestibility corrected amino acid score PER Protein efficiency ratio pI Isoelectric point PMM Protein micellar mass RFI Relative fluorescence intensity SD Standard deviation SDS PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis SEM Scanning electron microscopy SHMP Sodium hexametaposphate TPA Texture profile analysis TRIS Tris(hydroxymethyl)aminomethane UV Ultraviolet WAC Water absorption capacity

xix

Acknowledgments

It has been incredible 3 years of experience, meetings, challenges and achievements. And it

would not be possible without people to whom I want to express my sincere gratitude.

Hereby, I want to thank my professor Mohammed Aider for the opportunity he offered me

as well as for his faith in me and encouragements especially in the beginning of my thesis. I

also want to thank for his guidance, support and the ability to turn me back to the topic

without deviating from the subject and dissipating my attention on things less relevant. I also

want to thank my co-director Martin Mondor for reading and correcting my articles as well

as for his precise recommendations and pieces of advice. I kindly appreciate the participation

of Dr. Marzouk Benali and would like to acknowledge the members of the committee Dr.

Charles Lavigne and Dr. Wassef Ben Ounis for reading and evaluating my thesis.

I cannot forget the precious contribution of Diane Gagnon, who helped a lot thorough all

these years of my thesis spent in the lab. Thank you for your kindness, sympathy and

responsiveness, not to mention the candies in your office. I want to thank Pascal Lavoie and

Pierre Côté for the technical support in the lab, and Ahmed Gomaa for his help with

understanding FTIR.

I would like to express my sincere gratitude to the friends I met here from all over the world.

Truly they are real specialists passionate about what they do. So many times when I needed

a piece of advice they were there for me. Thank you also for sharing our best moments here.

Valerie Carnovale, Shyam Suwal, Sergei Mikhaylin, Attara Hell, Alex Kastyuchik, Stanislav

Stefanovski, Mayank Pathak, Mathieu Persico, Nassim Naderi, and many others. I have never

seen such a bright multinational company. You become our family here.

I am who I am thanks to my dear mom and my sister. There are no words capable of

describing my gratitude and my feelings to what you mean to me. While writing this part I

tended to write “we” or “us” because all of this would be impossible without my dearest

husband, my best friend and advisor Cheslav Liato. Thank you for your love, support, and

inspiration. Next to you I forget that I am so far from my homeland.

Finally, I thank you, my dear reader for appreciating my work.

xx

Préface

Cette thèse de doctorat est composée de huit chapitres et les résultats sont présentés

sous forme d’articles scientifiques soumis ou publiés dans des revues internationales avec

comité de lecture.

Le premier chapitre est une revue de littérature détaillée dans laquelle trois éléments

principaux ont été traités : le tourteau de canola avec le potentiel qu’il présente en tant que

source de protéines de haute valeur nutritionnelle et technologique, la technologie d’électro-

activation comme alternative à l’élimination d’agents chimiques utilisés pour la valorisation

du tourteau de canola et finalement le potentiel offert par les protéines de canola pour la

production de produits sans gluten.

Le deuxième chapitre est consacré à énoncer l’hypothèse de recherche qui découlait

du premier chapitre, de l’objectif général du présent projet de doctorat et des objectifs

spécifiques qui serviront à atteindre l’objectif principal et à confirmer l’hypothèse de

recherche.

Le troisième chapitre est un article scientifique qui a été rédigé suite à la réalisation

du premier objectif spécifique et qui consistait à étudier l'effet du pH, de la concentration du

tourteau de canola et de sel sur le taux d'extraction des protéines par la méthode d’extraction

conventionnelle. Cette étude a servi de témoin pour la comparaison avec l’extraction par la

technologie de l’électro-activation en solution. Cet article est publié dans Food Chemistry.

Alina Gerzhova, Martin Mondor, Marzouk Benali, Mohammed Aider. (2015). Study of total

dry matter and protein extraction from canola meal as affected by the pH, salt addition and

use of Zeta-potential/turbidimetry analysis to optimize the extraction conditions. Food

Chemistry, 15, 243-252.

Le quatrième chapitre est un article scientifique consacré à la réalisation du

deuxième objectif spécifique qui visait à étudier les propriétés alcalines des solutions électro-

activées (SEA) en fonction du type de configuration du réacteur d’électro-activation,

l'intensité du courant électrique, la concentration du sel ajouté et le temps d’électro-activation

(de traitement). Cet article est soumis à Engineering in Food and Agriculture.

Alina Gerzhova, Martin Mondor, Marzouk Benali, Mohammed Aider. (2015). Study of a

self-generation process of alkaline aqueous solutions by using the electro-activation

technology. Food Bioscience, Soumis.

xxi

Le cinquième chapitre est un article scientifique qui a traité du troisième objectif

spécifique de cette thèse de doctorat et consistait à évaluer et comprendre l’effet de l’électro-

activation sur le taux d'extraction des protéines du tourteau de canola et leurs propriétés

physico-chimiques. Cet article est publié dans Food Bioscience.

Alina Gerzhova, Martin Mondor, Marzouk Benali, Mohammed Aider. (2015). Study of the

functional properties of canola protein concentrates and isolates extracted by electro-

activated solutions as non-invasive extraction method. Food Bioscience 12, 128-138.

Le sixième chapitre est un article scientifique qui a traité du quatrième objectif

spécifique de ce projet. Il visait l’étude des propriétés fonctionnelles des protéines de canola

extraites par électro-activation et leur comparaison avec les propriétés de protéines extraites

par la méthode conventionnelle avec du NaOH. Cet article est publié dans Food Bioscience.

Alina Gerzhova, Martin Mondor, Marzouk Benali, Mohammed Aider. (2015). A

comparative study between the electro-activation technique and conventional extraction

method on the extractability, composition and physicochemical properties of canola protein

concentrates and isolates. Food Bioscience 11, 56-71.

Le septième chapitre est également réalisé sous forme d’un article scientifique qui a

traité du cinquième objectif spécifique de cette thèse de doctorat. Il a fait l’objet d’une étude

dans laquelle les protéines extraites par électro-activation sous forme de concentrés et

d'isolats ont été ajoutées à une formulation de biscuits sans gluten afin d'étudier leur

comportement dans une réelle matrice alimentaire. Cet article est publié dans International

Journal of Food Science and Technology.

Alina Gerzhova, Martin Mondor, Marzouk Benali, Mohammed Aider. (2015). Incorporation

of canola proteins extracted by electro-activated solutions in gluten free biscuit formulation

of rice-buckwheat flour blend: Assessment of quality characteristics and textural properties

of the product. International Journal of Food Science and Technology, Accepted.

Le huitième chapitre est une conclusion générale et des recommandations

spécifiques pour une meilleure valorisation des résultats de cette thèse de doctorat. En effet

et compte tenu des résultats obtenus à chacune des étapes de ce projet de doctorat, il est

possible de conclure que l’hypothèse de recherche énoncée au début de ce projet a été vérifiée

et validée par la réalisation de cinq objectifs spécifiques.

xxii

Madame Alina Gerzhova, candidate au doctorat en Sciences et technologie des

aliments, est l'auteure principale des cinq articles scientifiques présentés dans cette thèse de

doctorat. Elle a été responsable de la conception, la planification et de la réalisation des

expériences ainsi que de la rédaction et de la correction des articles. Dr Mohammed Aider,

directeur de recherche de la candidate et responsable du projet, a activement participé à toutes

les étapes de réalisations de ce projet de doctorat. Dr Martin Mondor, codirecteur de

recherche de la candidate, a activement pris part à toutes les étapes de réalisation de cette

thèse de doctorat. Dr Marzouk Benali, membre actif et participant à ce projet, a activement

participé à la réalisation de toutes les étapes du projet.

1

Introduction

Nutrition has always played an important role in human`s life with its main aim being to

provide with all the essential ingredients required for healthy existence. Unfortunately, the

problem of malnutrition and hunger is still a serious worldwide concern. According to The

United Nations Food and Agriculture Organization (FAO) about 795 million out of 7.3

billion people in the world were suffering from chronic undernourishment in 2012-2014

(FAO, 2014). It is an oppressive statistic, in spite of the significant decrease in the number

of undernourished people in comparison with the period 1990–1992, and it is aggravated by

the constant increase of the world population. According to United Nations Statistical Office,

the world population has grown from 2.5 billion in 1950 to 7.2 billion people in the mid-2013

and is predicted to increase by almost one billion people within the next twelve years reaching

10.9 billion by 2100. This however is not accompanied with the corresponding increase in

food sources, proteins in particular. Malnutrition implies first of all the protein energetic

deficiency which is a lack of proteins and calories in the diet. Proteins are indispensable for

the normal growth and development and their inadequate intake may lead to failure to thrive

in children and serious health disorders in adults.

That is why there is an increased interest worldwide towards non-conventional protein

sources the most cheap and abundant represented by plant proteins. In spite of that fact the

utilization of plant proteins in human consumption is rather limited. Most of them are used

as forage for animals to obtain conventional sources of proteins such as meat, eggs, and milk

which is substantially inefficient. Due to the animal metabolism 6 kg of plant proteins are

needed in order to produce 1 kg of animal proteins, thus 85 % of possible protein and energy

sources are wasted and turned into polluting emissions coming from animals` metabolism

(Arntfield, 2004; Wu et al., 2014). Furthermore, a hundred times more water is needed to

produce 1 kg of animal proteins in comparison with plant proteins

(http://www.canolacouncil.org, 2015a). The land used to produce animal feed could be

reduced tenfold if it was aimed for human consumption only. Regarding this and an increase

in population a more efficient utilization of plant proteins is needed. Indeed, the situation will

become critical when the protein production from animal sources reaches its maximum

capacity (Agriculture and Agri-Food Canada, 2014). This urges the food industry to search

and develop alternative sources.

2

Oilseeds such as soybean, sunflower, canola and especially the press cake left after oil

extraction are interesting protein sources which can be valorized (Rodrigues et al., 2012).

Among them canola is the leading crop in Canada and the second largest oilseed crop in the

world, left behind by soybean only (Dessureault, 2012; http://www.canolacouncil.org,

2015b). The seed of canola consists of 40% oil approximately and 17-26% protein, whereas

canola meal (a by-product of canola oil extraction) has up to 50% protein on a dry basis

(Aider and Barbana, 2011). Until relatively recently canola (rapeseed) press-cake has been

regarded and utilized mostly in animal nutrition due to the presence of anti-nutritive factors;

however the emergence of low glucosinolates and low erucic acid cultivar opened new

possibilities for its utilization as a food grade protein supplement. Its balanced amino-acid

composition and adequate digestibility can compete with such a widely consumed plant

based protein source as soybean (Tan et al., 2011a). The most promising utilization of canola

proteins is in the form of isolated protein powder, characterized by increased shelf life, higher

microbial stability, lower weight and improved portability (Arntfield, 2004).

The idea of utilization of canola proteins in food industry is supported by their interesting

functional properties including foaming, gelling, water holding, and emulsification properties

(Aachary and Thiyam, 2011; Aluko and McIntosh, 2001; Khattab and Arntfield, 2009). As

an ingredient it can potentially replace whey proteins, casein, and egg yolk in such widely

consumed products as mayonnaise (Morris, 1992), emulsion-type meat products (Cumby et

al., 2008; Mansour et al., 1996; Thompson et al., 1982; Yoshie-Stark et al., 2006), bread

(Kodagoda et al., 1973a; Shahidi, 1990), and pasta (Alireza Sadeghi and Bhagya, 2008).

However, the successful utilization of canola proteins is challenged by the choice of

extracting technology.

Conventional extraction with the use of sodium hydroxide and subsequent precipitation with

hydrochloric acid have a detrimental effect on quality and functionality of proteins

(Rodrigues et al., 2012). In addition it creates a large amount of effluents which need to be

further processed. The use of chemical acids and bases cause environmental, safety and

health hazards and stimulate researchers to develop alternative “green” methods (Shi et al.,

2012). One of the promising techniques for protein extraction implies the energy of electric

current in the form of electro-activated aqueous solutions. The conversion of the electric

energy into chemical creates a necessary conditions such as pH and alkalinity which can be

3

utilized for proteins extraction (Aider et al., 2012b). No chemical bases are therefore required

which make the EA an environmentally friendly technology.

The aim of the project was to investigate the use of electro-activated solutions for protein

extraction from canola meal as well as its effect on structural and functional characteristics.

This will expand the current knowledge on the utilization of EAS and possibly become a

novel extracting method allowing to valorize canola oil cake for its application in the food

sector.

4

1. CHAPTER 1: Literature review

Proteins

1.1.1. General information

The word protein is defined as “any of a group of complex organic compounds, consisting

essentially of combinations of amino acids in peptide linkages that contain carbon, hydrogen,

oxygen, nitrogen, and usually, sulfur” (Morris, 1992). All of them regardless of their origin

are formed of twenty amino-acids. It is their order and conformation that provide such a great

variety resulting in more than 1010 different species of naturally occurred proteins (Walstra,

2002).

Some amino-acids were found to be more important than others. In the early studies in rats

performed by Rose (1947) the lack of certain amino acids resulted in poor growth, loss in

weight and eventually death, whereas other did not have that impact. It was found that these

amino acids could not be synthesized by organism and were called essential. Requirements

for protein consumption are basically the need in essential amino-acids which must be

satisfied. Daily requirements for adults are presented in the Table 1.1.

Table 1.1: Indispensable amino acid requirements (FAO/WHO/UNU, 2007).

Amino acid Indispensable amino-acid requirements mg/kg per day mg/g protein

Histidine 10 15 Isoleucine 20 30 Leucine 39 59 Lysine 30 45 Methionine+cysteine 15 22 Methionine 10 16 Cysteine 4 6 Phenylalanine+Tyrosine 25 38 Threonine 15 23 Trpytophan 4 6 Valine 26 39

Total indispensable amino acids 184 277

5

1.1.2. Role of proteins in nutrition and their nutritive value

Among other foodstuffs supplying energy to a human body proteins were characterized as

“unquestionably the most important of all known substances in the organic kingdom” (Lewis,

1948). Derived from a Greek word “protein” means “first” or “primary” due to its important

role in sustaining life since it contains nitrogen, essential for the structure of the cell, a basic

component of protoplasm (Li-Chan, 2004). It is difficult to overestimate the role of proteins

in human nutrition. Dietary proteins as a source of amino-acids are used by the human body

to synthesize its proper proteins. Apart from being the builders of new tissues and cells they

play specific role in the maintenance of body functions, in controlling and regulating body

processes (metabolism, osmotic regulation), catalyzing reactions, contracting muscles,

transporting and storing nutrients, protecting against oxidative stress, infection, and bleeding

etc. (Fennema, 1996; Wu et al., 2014).

The lack of proteins in a diet may lead to serious health problems in adults. In children, who

are more susceptible to protein malnutrition, protein deficiency causes impaired growth and

results in decreased immune function and increased susceptibility to infectious disease (Wu

et al., 2014).

1.1.3. Functional properties

Apart from nutritional aspect proteins are widely used in food technology performing specific

functions. Food preferences are normally based on the sensory characteristics such as texture

and mouthfeel, flavour, colour, and appearance. Sensory characteristics of food are the result

of multiple interactions between various functional ingredients and proteins have a great

influence on the sensorial perception. For a protein to be useful as an ingredient in certain

type of foods e.g. cakes, biscuits, sauces, etc. it must possess multiple functionalities or

functional properties. Kinsella and Melachouris (1976b) describes functional properties as

“physicochemical property which affects the processing and behavior of protein in food

systems”. Functional properties of proteins used in various types of foods are shown in Table

1.2.

Functional properties greatly vary depending on the type of protein, methods of extraction

and precipitation, drying, concentration, or modification (enzymatic, alkaline, or acid

hydrolysis, chemical), as well as environmental conditions, notably temperature, pH, and

6

ionic strength. In addition food proteins are normally composed of several proteins each

having their proper functionalities (solubility, isoelectric point, susceptibility to denaturation,

etc.) and thus a specific protein preparation will not always reflect the properties of the total

protein but rather of one of the components (Kinsella and Melachouris, 1976b).

Table 1.2: Various protein functionalities and their application in food. Adapted from Fennema (1996). Functionality Mechanism Food

Solubility Hydrophilicity Beverages

Viscosity Water binding, hydrodynamic size

and shape

Soups, gravies, and salad

dressings, deserts

Water binding Hydrogen bonding, ionic hydration Meat sausages, cakes, and

breads

Gelation Water entrapment and

immobilization, network formation

Meats, gels, cakes,

bakeries, cheese

Cohesion

adhesion

Hydrophobic, ionic, and hydrogen

bonding

Meats, sausages, pasta,

baked goods

Elasticity Hydrogen bonding, disulfide

crosslinking

Meats, bakery

Emulsification Adsorption and film forming at

interfaces

Sausages, bologna, soup,

cakes, dressings

Foaming Interfacial adsorption and film

formation

Whipped toppings, ice

cream, cakes, desserts

Fat and flavor

binding

Hydrophobic bonding, entrapment Bakery products,

doughnuts

Physical and chemical properties are involved in the formation of protein functionality

including size; shape; amino acid composition and sequence; net charge and distribution of

charges; hydrophobicity/hydrophilicity; secondary, tertiary and quaternary structures;

molecular flexibility / rigidity; and finally ability to interact with other components. A

challenging task is the prediction of functional properties through the analyses of molecular

properties. Until now it has not been completely succeeded despite all the available

information mostly because the behavior in model system differs from the one in real foods.

7

Nevertheless, considerable progress has been achieved towards the understanding of the

structure-function relationships. It is not an easy task to correlate the physical and chemical

properties with a specific functionality. However, in general terms functional properties can

be regarded as a set of three molecular aspects depending on the interactions (Fennema, 1996;

Gauthier, 2012):

• Hydration properties – based on protein-water interactions: solubility,

dispersibility, wettability, swelling, thickening, water absorption and water-holding

capacity

• Surface related properties – based on protein-interface interaction: emulsification,

foaming, flavor binding, pigment binding

• Hydrodynamic/rheological properties – based on protein-protein interactions:

elasticity, viscosity, cohesiveness, chewiness, adhesion, stickiness, gelation, dough

formation, texturization.

1.1.3.1. Hydration properties

The majority of foods contain water and thus interactions of macromolecules such as proteins

with water are of great importance. Most of the proteins functional properties e.g. solubility,

water absorption and water holding ability, viscosity, emulsification, foaming, gelation

depend on the water-protein interactions.

1.1.3.1.1. Water absorption and water holding capacity

The ability to absorb or bind and hold water is very important in several food preparations

especially in low and intermediate moisture foods such as baked and comminuted meat

products. In the absence of sufficient water they do not dissolve but rather imbibe water. This

characteristic plays a major role in the texture and consistence and confers body, thickening

and viscosity to such products. As an example the ability to bind and retain meat juices in

beef patties and frankfurter type meat emulsions enhances mouthfeel and flavor, in bakery

products it prolongs the shelf life (Kinsella, 1979a). When substituting one protein for

another in formulations the information on the water absorption and holding is crucial since

without any adjustments there is a risk to obtain a product with non-acceptable organoleptic

properties, either too dry or too moist.

8

Methods of analysis

The capacity to absorb or bind water is measured by subjecting the dried protein powder to

water vapor of a known relative humidity when it reaches its equilibrium. It is governed by

the amount of polar and non-polar groups exposed at the surface and related to the amino-

acid composition of the protein as well as its conformation. The ability to retain water against

gravitational flow known as water holding capacity (sometimes also referred in the literature

as water absorption capacity) is however even more important. It is analyzed by subjecting

the protein to the excess of water and centrifuging the protein dispersion (Fennema, 1996).

Factors affecting water absorption

It is dependent on the environmental factors such as pH, ionic strength, type of salts, and

temperature. By changing the pH of the medium the net charge of the protein molecule

changes too which impacts protein-water interactions. At isoelectric point (pI) they are

minimal increasing above and below pI. Water hydration properties reach their maximum at

pH 9-10 in most proteins (Fennema, 1996). An increase in temperature normally decreases

the water bonding capacity due to the decrease in hydrogen bonding. However a denatured

protein might have higher water absorption capacity due to the protein unfolding and

liberating previously buried groups unlike protein solubility (Cheftel et al., 1985). Low salt

concentrations (<0.2M) enhance protein hydration, however at higher concentrations it

usually decreases due to the increased water-salt interactions.

1.1.3.1.2. Solubility

Proteins solubility is one of the most important parameters as it influences other properties

such as foaming, emulsifying, gelling and thickening. Good solubility gives wider

possibilities of the potential applications of proteins and is one of the most practical indexes

of the extent of denaturation (Kinsella and Melachouris, 1976b). Good protein solubility

initially is required in some functional properties such as emulsification or foam formation

in order to allow fast migration to the oil/water or air/water interfaces. On the contrary for

some properties such as water absorption high solubility is not beneficial. Thus, it has been

shown that water absorption capacity can be improved by preliminary denaturation (Cheftel

et al., 1985).

9

Methods of analysis

Most of the time nitrogen solubility index (NSI) is used as a practical and quick test. Proteins

are complex structures with great variety of forms and conformations. They possess both

hydrophobic and hydrophilic areas, which determine their behavior in solution. Therefore,

the solubility of a protein depends on the properties of the groups that are situated at the

surface level (Rodrigues et al., 2012). Hydrophobic patches are involved in protein-protein

interactions which are accompanied by decreased solubility, whereas hydrophilic ones are

responsible for protein-water interactions and therefore improve solubility. According to the

solubility in different solvents, proteins are classified into: albumins that are soluble in water;

globulins, soluble in diluted salt solutions; glutelins, soluble only in acid or alkaline solutions;

and prolamins that are soluble in 70% ethanol. Two latter groups are highly hydrophobic

(Fennema, 1996).

Factors affecting the solubility

Solubility depends on many factors such as pH of the solutions, ionic strength, temperature

or the presence of organic solvents.

pH: The pH of the medium is one of the factors having the highest impact on solubility. It is

closely related to the isoelectric point of each protein where they carry a net charge equal to

zero. Below the isoelectric point (pI) proteins are charged positively whereas above the pI

the net charge is negative. Under such conditions solubility is provided by electrostatic

repulsions and by the hydration of the charged residues. For most proteins minimum

solubility is observed at isoelectric point where they show low interaction with water so that

the solubility curve gains the U-shaped form when plotted against pH. The solubility

measurements in a wide pH range give a good indication of a potential or limitation for a

protein as a functional ingredient (Kinsella and Melachouris, 1976b).

Temperature: When proteins are heated to the temperatures higher than 50°C solubility

decreases as links implied in protein stabilization are disrupted and protein unfold which

liberates the hydrophobic residues buried inside the molecule in its native state. Unfolding

alters the protein-solvents interactions making protein-protein interactions more favorable

and resulting in protein aggregation (Cheftel et al., 1985).

10

Ionic strength: Ionic strength of the solution greatly impacts the protein solubility. At low

salt concentrations the solubility is increased due to the interactions between ions of salt and

protein charges thus decreasing electrostatic interactions between charged groups of a protein

molecule. This is known as “salting-in” effect. On the contrary, high salt concentrations

decrease the solubility due to the competition for the water molecules between salt and

proteins and lead to protein precipitation known as “salting-out” (Arakawa and Timasheff,

1985).

1.1.3.2. Surface related properties

Many food products are composed of two immiscible layers, a hydrophilic and a hydrophobic

one (e.g. water and oil or water and air) and can be classified as emulsion-type products or

foam-type products. These food systems are rather unstable because of a great surface tension

between two phases and need a surface active agent capable of bringing two phases together.

Proteins are amphiphilic substances by nature, which means they contain both hydrophilic

and hydrophobic patches and therefore are capable of lowering the surface tension between

two immiscible phases performing emulsion or foaming properties. In spite of the fact that

all proteins are ampiphilic by their nature their surface activity varies greatly depending on

the ratio of hydrophobic and hydrophilic patches. In addition protein conformation which

includes the flexibility of the polypeptide chain, adaptability to the environmental changes,

disposition of the hydrophobic and hydrophilic residues is also of great importance

(Fennema, 1996). There are three main features that determine a good surfactant: (1) ability

to rapidly migrate to the interface; (2) ability to rapidly unfold and adsorb at the interfaces;

(3) ability to form a strong and cohesive film that can withstand the thermal or mechanic

stress (Dickinson, 1998). The speed of migration depends on the size of a molecule, small

and compact proteins will migrate faster. The distribution of hydrophilic and hydrophobic

amino acids on a protein surface influences the rapidity of protein adsorption at the interfaces.

Hydrophobic proteins migrate to the interface and adsorb more readily (Nakai, 1983). During

adsorption protein molecule unfold and orient its hydrophobic parts towards air or oil phase

and hydrophilic towards water. Structural rigidity or flexibility of a folded protein thus has a

great impact. Compact globular proteins stabilized by covalent bonds will take time to unfold

whereas proteins with random coil type of structural organization will unfold and adsorb

11

much more readily. It may also be affected by extrinsic factors, such as the ionic strength,

pH and temperature. Upon unfolding new liberated protein areas also might affect the

functionality at the interfaces (Damodaran, 1989). Viscoelastic film that is formed after

influences the stability of the system. It is favoured by some degree of surface denaturation,

which amplifies protein-protein interaction and enhances cohesive forces between the

proteins in the film (Kinsella, 1981).

Interfacial properties can be evaluated by measuring the interfacial tension. Surface

hydrophobicity was also successfully employed in predicting the proteins functionality at the

interfaces (Damodaran, 1989).

Although the principles of foam and emulsion formation are similar, the requirements for a

surfactant differ and thus the protein that performs well at air-water interfaces may not act

the same at oil-water interfaces.

1.1.3.2.1. Emulsion properties

Emulsions are two-phase systems consisting of water and oil. Their ratio determines the type

of emulsion to be formed either oil in water or water in oil. When the dispersed phase is oil

and the continuous phase is water the oil in water emulsion is formed and vice versa.

Examples of oil in water emulsion would be mayonnaise or vinaigrette whereas butter

represents a water in oil emulsion (Gauthier, 2012). To form an emulsion an adequate amount

of energy is needed to be applied which is generally achieved by using mixers, homogenizers,

or other appliance in order to disperse one phase in another. For two immiscible phases such

state is thermodynamically unfavorable due to the high free energy of the interface between

the two phases. As all the systems tend to equilibrium reached when the contact area between

two immiscible phases is minimal, various destabilization processes arise leading to phase

separation. Various forces are involved in phase separation and may take place

simultaneously making it more difficult to distinguish them.

Destabilization processes in emulsions

The most common destabilization processes are shown in the Figure 1.1. Creaming and

sedimentations are caused by external forces usually gravitational or centrifugal. Due to the

differences in the density, droplets with lesser density will move to the top, or if their density

is higher will move to the bottom. Flocculation results from droplet aggregation due to van

12

der Waals forces when there is no sufficient repulsion to keep them apart. Although being

immiscible two liquids normally have mutual solubility up to a certain degree. In emulsion

with polydisperse distribution smaller droplets disappear with time as they diffuse to the bulk

and become deposited on the larger droplets. This phenomenon is known as Ostwald

ripening. Coalescence takes place when droplets approach and fusion together in order to

reduce the surface area and is related to the strength of the film formed around the droplets.

When it is not strong enough it becomes thin and finally disrupts leading to the formation of

larger droplets. Finally phase inversion may take place with time or if conditions were

changed transforming oil in water emulsion into water in oil emulsion and vice versa (Tadros,

2013).

Figure 1.1: Major destabilization processes in emulsions. Adapted from Tadros (2013).

Emulsifiers can slow down the phase separation. According to Kinsella (1979a) the

stabilization of emulsified droplets is achieved by formation of a charged layer around the

oil droplets creating mutual repulsions and/or by the formation of film around the droplets

by solutes such as proteins.

Methods of analysis

There is no standardized test for analyzing the emulsification properties (McWatters and

Cherry, 1981). Three different methods such as emulsifying capacity, emulsion stability,

emulsifying activity, and droplet size distribution are most commonly used for the analysis

of the emulsifying properties of proteins (Kinsella and Melachouris, 1976b).

13

Emulsifying capacity is determined as the amount of oil that can be emulsified by a protein.

It is measured by conductivity or viscosity measurements under constant addition of oil until

the phase inversion occurs. The collapse of emulsion is accompanied by a sudden drop in

viscosity or conductivity, or an increase in resistance (Kinsella and Melachouris, 1976b).

Emulsion activity is measured as the available contact area between two phases by estimating

the turbidity of a diluted emulsion (Karaca et al., 2011a). This method was proposed by

Pearce and Kinsella (1978) based on the Mie theory of light scattering, which states that the

interfacial area of an emulsion is twice its turbidity (Cameron et al., 1991).

Emulsion stability can be evaluated by several methods. Most of them subject the emulsion

to various external perturbations such as heating, centrifugation, etc. and note the amount of

oil released after centrifugation (Kinsella and Melachouris, 1976b). Another approach is to

measure the changes in droplet size (Tan et al., 2014) or turbidity (Zhu et al., 2010) with the

time. The degree of creaming has been monitored by using the creaming index (Karaca et al.,

2011b). Emulsions are stored in the glass cylinder for a certain period of time until the

creamed layer became visible on the top of the emulsion. Creaming index was measured as

the relation of total height of the emulsion (HE) and the height of the serum layer (HS). New

generation of instruments such as turbiscan can directly provide with the information about

the degree of creaming, sedimentation, flocculation or coalescence (Álvarez Cerimedo et al.,

2010).

Droplet size is an important characteristic of an emulsion and the most important factor to

predict the coalescence. Smaller droplets are less prone to coalescence because of the smaller

film area between them which lowers the probability of its rupture. Smaller droplets also

decrease the rate of creaming (Fennema, 1996).

Factors affecting emulsion properties

Emulsifying properties can be influenced by several factors including intrinsic (pH, ionic

strength, presence of other substances, type of protein, etc.) and extrinsic factors (type of

appliance used to prepare the emulsion, the energy input, rate of shear, etc.) (Fennema, 1996).

Protein solubility and hydrophobicity: Protein solubility facilitates emulsion formation,

however high solubility (100%) is not required as normally high soluble proteins as well as

low-soluble proteins do not perform well as emulsifiers. Hence 25-80% solubility range is

suitable in most cases, however the minimum solubility required for good emulsification

14

varies depending on the protein (Fennema, 1996). Emulsifying properties are also correlated

with the surface hydrophobicity which can be used to predict emulsifying properties of some

proteins (Damodaran, 1989). Partial denaturation may improve emulsification as it facilitates

protein unfolding due to increased molecular flexibility and surface hydrophobicity

(Fennema, 1996).

pH and ionic strength: Proteins are normally low-soluble at their isoelectric point which

hampers the emulsion formation. Most of proteins have a compact structure at their pI which

also impedes the unfolding and adsorption, yet it can stabilize the film already created and

enhance emulsion stability (Cheftel et al., 1985; Fennema, 1996). On the other hand, the

interfacial films formed by proteins are relatively thin and electrically charged and therefore

are particularly sensitive to pH and ionic strength of the medium. At pH proximate to

isoelectric point and when the ionic strength exceeds a particular level they tend to flocculate

as there is not enough electrostatic repulsion between the droplets to overcome the various

attractive interactions (McClements, 2004). Experimental data on different proteins are rather

contradicting showing that one proteins have the optimal emulsifying properties at their pI

whereas other performs better at pH far from isoelectric point (Cheftel et al., 1985).

Protein concentration: Protein concentration affects differently the emulsion properties.

Emulsion capacity and efficiency in general will decrease above a critical concentration

whereas emulsion stability will increase (Kinsella, 1979a; Kinsella and Melachouris, 1976b).

Type of appliance: As mentioned before small droplets give more stable emulsions. The size

of droplets is influenced by the energy input and the type of appliance used to prepare an

emulsion. High pressure homogenizers are the most effective in producing fine droplets and

thus resulting in a more stable emulsion. It is followed by sonication (using ultrasound) and

mechanical stirrers such as Ultraturrax (Walstra, 1993).

1.1.3.2.2. Foaming properties

Foams are also two-phase systems composed of gas bubbles (air) dispersed in liquid (water)

and requiring a surface active agent such as protein. Many food products are foams by their

nature e.g. cakes, bread, meringue, whipped cream, mousses, marshmallow, ice cream, etc.

Their smooth texture and mouthfeel are due to these dispersed micro bubbles (Fennema,

1996). In order to create foam it is necessary to provide the energy to the system same as for

15

creating emulsions. It can be done by whipping, bubbling, or shaking a protein solution.

Foams differ from emulsion by their much bigger contact area as the dispersed phase (gas)

occupies a much bigger volume than in emulsion. Due to that fact the majority of foams are

instable (Cheftel et al., 1985).

Destabilization processes in foams

There are three major destabilization processes notably draining, gas diffusion, and rupture

of liquid lamella. Draining occurs due to the gravitational effect as the liquid is heavier than

the gas it tends to drain towards the bottom with the time. Difference in pressure might also

cause draining. If the pressure in gas bubbles is high they tend to press on one another making

the liquid drain. Evaporation that takes place during storage might also be the cause of

draining. When the foam surface becomes dry, the films around the bubbles lose their

elasticity, which makes them weaker and favors their approach. Gas diffusion from small to

large bubbles takes place due to the dissolution of gas in the liquid phase. Finally, protective

films tend to become thin and fragile resulting in the collapse of the foam (Cheftel et al.,

1985; Gauthier, 2012).

Similar to the emulsion formation when air is purged through the liquid the air bubbles tend

to coalesce to minimize surface exposure. This, however may be prevented by the presence

of an appropriate surfactant capable of forming an interfacial film barrier (Kinsella, 1981).

Formation of foams is similar to emulsion formation. First proteins diffuse to the air-water

interface to reduce the interfacial tension, after that they unfold and orient their polar parts

towards water and non-polar towards air and finally they form a continuous film which

protects air bubbles and stabilizes the foam (Kinsella and Melachouris, 1976b).

Analysis of foaming properties

Foaming properties are analyzed by several methods. Foaming capacity, foam expansion or

overrun is the ability to form foams and refers to the maximum volume increase of protein

dispersion, achieved by the incorporation of air by whipping, stirring, or aeration. Foaming

power measures the increase in volume, when the gas is purged through the protein solution.

Basically all of them are based on the measurement of the initial volume of a protein solution

and the final volume of the foam followed by stirring or gas introduction. Foam stability is

16

the ability of a formed foam to retain its volume which is measured as a volume decrease

with the time (Kinsella and Melachouris, 1976b).

Factors affecting foaming properties

A number of factors influence foaming properties such as type of protein and its composition,

its solubility, hydrophobicity, concentration, method of preparation, external factors such as

pH, temperature, the presence of salts, sugars, lipids, and finally, the method of measurement

(Kinsella and Melachouris, 1976b).

Internal factors or molecular properties

The stability of foams is governed by three principal factors: low interfacial tension, high

viscosity of the liquid phase and strong, cohesive, and elastic films (Cheftel et al., 1985).

Protein-based foams first of all depend on the intrinsic molecular properties of the protein

such as conformation, flexibility, molecular size, shape, amino acid sequence, surface

polarity and hydrophobicity, charge, etc. (Kinsella and Melachouris, 1976b). However, foam

formation and foam stabilization require somewhat different properties, hence some of the

properties desired for foam formation, do not provide the stability whereas molecular

characteristics which impart foam stability may slow down the foam formation. Thus,

foaming capacity mostly depends on the rate of adsorption, hydrophobicity and flexibility of

the molecule, whereas the stability is influenced by the rheological properties of the film

formed around the bubble (Fennema, 1996). To perform well in foams surfactant should be

soluble in the liquid phase and capable of rapid migration and orientation to form an

interfacial film around newly developed gas bubbles. Same as in emulsions flexible proteins

are better foaming agents. On the other hand they are poorly structured (low in secondary

and tertiary structures) which results in coarse rapidly-formed and less stable foams

(Kinsella, 1981). Foam stability on the other hand requires strong and viscoelastic film,

impermeable for air which can be provided by globular proteins with high molecular weight

and more rigid structure partially resistant to unfolding (Gauthier, 2012). Globular proteins

due to their rigid structure will unfold and spread much slower so that it will take more time

for them to build foam however it will be more stable. Surface viscosity is an important factor

influencing the stability. High surface viscosity and high film yield result in strong and

viscoelastic foams that are resistant to stress as they can expand and compress if necessary.

17

On the other hand high viscosity is not desirable during the initial foam formation as it slows

down the process of migration and adsorption (Kinsella, 1981).

External factors

pH: Foamability and foam stability is high at the isoelectric point for proteins which are

soluble at their pI due to the lack of repulsive forces which favors the formation of protein-

protein interactions and of a viscous film at the interface. In addition at pI more proteins are

adsorbed at the interface because of the lack of repulsions between the interface and the

adsorbing molecules. For proteins that are poorly soluble at their pI only those that are soluble

will participate in foam formation. Insoluble fraction, however, can also be adsorbed and

contribute to foam stabilization. Under such conditions the volume will be low but the

stability will be high. At pH other than isoelectric point the foaming capacity of proteins is

good but the stability is not (Fennema, 1996).

Salts: For some proteins foamability and foam stability increases with an increase in salt

concentration due to the neutralization of charges by ions of salt. However, it depends on the

type of the salt present and the solubility characteristics of the protein in the presence of that

salt. For some proteins it can have the opposite effect. When the salting-in effect is implied

it decreases the surface denaturation (unfolding) and as a result foaming properties. Sodium

chloride also can reduce surface viscosity and rigidity of protein films (Kinsella, 1981).

Sugars: The addition of sugars in general improves the foam stability as it increases the bulk

phase viscosity which decreases the drainage. On the other hand it decreases the foaming

ability.

Lipids: The presence of even traces of lipids (0.1%) significantly impairs the foaming

properties. Polar and surface active lipids adsorb themselves at the interfaces obstructing the

protein adsorption. The lack of viscoelastic properties for such foams result in rapid collapse

during whipping (Cheftel et al., 1985; Fennema, 1996).

Protein concentration: An increase in protein concentration results in the formation of stiff

foams made of small bubbles and high viscosity. Higher viscosity promotes the formation of

firm films at the interfaces which improves foam stability (Cheftel et al., 1985).

Temperature: Limited heat treatment without thermal coagulation provokes partial

unfolding and facilitates foam formation. The stability of such foams however is reduced due

to decreased viscosity. It also depends on the type of a protein and its structural flexibility.

18

The greater the rigidity of native protein, the greater the improvement caused by heating

(Kinsella, 1981).

The method of foaming: In order to form a foam the time and intensity of whipping should

be chosen in such a way that they allow sufficient unfolding and adsorption of proteins. Too

fast agitation may result in protein denaturation, aggregation and precipitation due to

increased shear rate and thus decrease foaming properties (Fennema, 1996).

1.1.3.3. Other properties

1.1.3.3.1. Oil binding capacity

The ability of protein to bind fat is a very important characteristic in certain foods such as

meat replacers, cakes, cookies as it is associated with the flavor retention and improved

mouthfeel (Kinsella and Melachouris, 1976b). It is based on protein-lipid interactions and is

measured similarly to water absorption capacity by the addition of liquid oil to the protein

powder, mixing and holding for a specific amount of time, centrifuging and determining the

amount of absorbed oil. Such method accounts for the amount of oil physically entrapped by

the protein. It is influenced by the type of protein, its solubility, hydrophobicity, and protein

concentration. Proteins having higher solubility exhibited lower fat absorption. Increase in

protein concentration increased the oil binding by proteins as observed by Hutton and

Campbell (1981).

1.1.4. Sources of proteins

Conventional sources of proteins are still represented by animal sources such as meat, egg,

and fish. However, the interest towards alternative protein sources has grown dramatically

over past years. The world’s situation regarding malnutrition in developing countries, high

cost of proteins from animal sources, health concerns such as intolerances to animal proteins

or conscious refuse from consuming animal proteins has led to a substantial search for

proteins from alternative sources which could fully replace those from animal ones.

Alternative sources have been thoroughly studied in recent years with proteins derived from

plants being the most cheap and abundant.

19

1.1.4.1. Cereals

Wheat, rice, barley, oats, sorgo, corn are the principal cereal crops. Protein content of cereals

is rather low in both quantity and quality. The highest amount of proteins is found in wheat

(12-14% on a dry basis), followed by millet and sorghum (10.5%), maize (9%) and rice

having the lowest amount of proteins (5-7%) (USDA, 2012). Beside that cereals are deficient

in some essential amino-acids, particularly in lysine. Almost all of the cereal proteins have it

in insufficient quantity. Apart from lysine other amino acids such as tryptophan, threonine,

or methionine appear to be limiting depending on the type of cereal. Another limiting factor

of cereal as a major source of proteins is their high content of carbohydrates which leads to

low protein-to-calories ratio (Bressani and Elias, 1968).

1.1.4.2. Legume seeds

Legumes represent another group of protein-rich material. The most commonly cultivated

legumes are pea, chick-pea, broad bean, lentils and the common bean. Their protein content

may reach up to 30% on a dry basis and their overall protein quality is accepted to be higher

than those of cereal proteins. Contrary to the cereal proteins legumes are a good source of

lysine, however, they lack sulfur-containing amino-acids and tryptophan (Bressani and Elias,

1968). On the other hand the digestibility of legume proteins is lower due to the presence of

poorly digestive carbohydrates which cause certain inconveniences such as flatulence. These

problems are related to the fact than the human digestive system lacks the enzymes

responsible for the hydrolysis of these sugars which leads to their anaerobic fermentation in

the large intestine with gas release (Rodrigues et al., 2012).

1.1.4.3. Oilseeds

Oilseeds are plants used in the industry for oil production due to their high oil content.

Soybean, rapeseed, flaxseed, cotton, peanut and sunflower are the most widely produced

oilseeds. Apart from high oil content they are characterized by the elevated percentage of

proteins and balanced content of carbohydrates. Protein content of the oil cake left after oil

extraction depending on the seed ranges between 35% and 60% on a dry basis (Moure et al.,

2006). Nutritive value of oilseeds proteins is generally higher in comparison with legume

and cereal proteins. Soybean is limited in sulfur-containing amino-acids but at the same time

is considered to be a good source of lysine. Peanut, sesame and cottonseed are limited in

20

lysine although they have high content of methionine. Soybean is the most studied and the

most widely spread oilseed culture used in both human and animal diet. However, the

proteins of canola are also being extensively studied and their quality is not inferior to the

soybean ones (Bressani and Elias, 1968). Rapeseed or canola is widely used in oil production.

Worldwide production of major oilseeds as well as the protein content of their meals is shown

in the Table 1.3.

Table 1.3: Worldwide production of major oilseeds and their meals in MMT and their protein contents. Adapted from Ramachandran et al. (2007); USDA (2013).

Soybean Canola Cottonseed Sunflower

Oilseed production, MMT 281.66 66.49 44.39 41.76

Oil cake production, MMT 188.06 36.92 15.66 16.30

Oil cake protein content, % 47.5 39.9 40.3 34.1

Canola

1.2.1. Historical remarks

Canola and rapeseed are the part of the most widespread family of plants cultivated by human

– the Brassicaceae or Cruciferae. Cabbage, cauliflower, turnip, Brussel sprouts, and radish

all belong to this family. Although being the part of one family they are greatly differed by

seed size, seed color, and chemical composition. Rapeseed is tolerant to the low temperatures

and thus can be grown in regions which are not suitable for soybean and sunflower.

One of the earliest cultivated species of rapeseed is suggested to date back as much as 3000

years ago in India, later being introduced to China and Japan (Shahidi, 1990). The wide

spread of cultivars is believed to be due to the birds migration who were attracted by small

size seeds rich nutrients. Unlike other oilseeds rapeseed comes from several species. Figure

1.2 shows the three basic ones: B.nigra, B.oleracea, and B.rapa which by natural

hybridization centuries ago gave rise to the other species, B. carinata, B. juncea, and B. napus

(Daun et al., 2011).

21

Figure 1.2 : Triangle of U showing the relationship of major Brassica species. Adapted from Ahuja et al. (2010).

In Canada rapeseed appeared just before the World War II and by the end of the war

significant amounts were produced in Western Canada. It quickly adapted to the Canadian

prairies and soon became the major oilseed crop, producing seeds not only for domestic use

but also for export. In 1970 about 70 to 90% of the edible oils were produced in Canada by

harvesting around 1 million tons of rapeseed. Yet there was a concern about the composition

of fatty acids and erucic acid in particular. Some works from Europe demonstrated that the

high intake of erucic acid could lead to heart lesions. Apart from erucic acid first cultivars

were high in glucosinolates which prevented their use as high protein supplement for animal

rations. After various breeding programs and due to the introduction of gas chromatography

which allowed to separate fatty acids and to study the genetics behind the fatty acid

composition of rapeseed the new cultivar low in erucic acid and glucosinolates was

introduced in 1974 by Dr. Baldur Stefansson at the University of Manitoba. The development

of low glucosinolates and erucic acid rapeseed crops is shown in the Figure 1.3.

22

F

Figure 1.3: Decrease in glucosinolates and erucic acid levels in rapeseed/canola. Adapted from Daun et al. (2011).

Figure 1.4: Number of varieties of canola in Canada. Adapted from Daun et al. (2011).

The further transformation from low erucic rapeseed to canola as it is known today took

several decades of constant improving and releasing of new varieties. The major tasks apart

from reducing anitutritive factors were to improve yield of crops by producing herbicide

resisting cultivars. For 30 years the amount of varieties has increased by around a hundred

times since 1980 (Figure 1.4). Today canola oil is recognized as GRAS (generally

recognized as safe) and as one of the healthiest oils due to its lowest composition of saturated

fatty acids (Daun et al., 2011). Therefore canola is the world's only "Made in Canada" crop

23

and its greatest agricultural success story. Name canola comes from the Canadian Canola

Association and is a contraction of ‘Canadian oil, low acid’ (Rodrigues et al., 2012).

To be named canola, an oilseed plant must meet this internationally regulated standard "Seeds

of the genus Brassica (Brassica napus, Brassica rapa or Brassica juncea) from which the oil

shall contain less than 2% erucic acid in its fatty acid profile and the solid component shall

contain less than 30 micromoles of any one or any mixture of 3-butenyl glucosinolate, 4-

pentenyl glucosinolate, 2-hydroxy-3 butenyl glucosinolate, and 2-hydroxy- 4-pentenyl

glucosinolate per gram of air-dry, oil-free solid" (http://www.canolacouncil.org, 2015b). The

term rapeseed does not have any regulatory basis and in general is used in relation to oilseed

rape cultivars with 45% or more erucic acid in oil and seed meals that are either high or low

in glucosinolates and mainly used for non-edible purposes such as lubricants and hydraulic

fluids (Raymer, 2002). However, this designation is not used worldwide and there are

rapeseed cultivars that have low erucic acid and glucosinolates (Arntfield Susan, 2011).

1.2.2. Production, economical interest

Today canola is Canada`s most valuable crop, bringing $19.3 billion to the Canadian

economy each year, providing more than 249 000 Canadian jobs and $12.5 billion in wages.

From 14 crushing and refining plants about 10 million tons of canola seed can be processed

giving about 3 million tons of canola oil and 4 million tons of canola meal annually. Around

90% of totally produced canola is exported as seeds, oil or meal to 55 markets around the

world including United States (65% of oil exports, 90% meal export), China, Japan, and

Mexico (http://www.canolacouncil.org, 2015a). Worldwide canola/rapeseed is the second-

largest oilseed grown crop after soybeans (Arntfield Susan, 2011; Daun et al., 2011).

The use of canola is not restricted to the oil production; the press cake left after oil extraction

is a good source of protein for poultry, fish and cattle and potentially a source for edible

protein for human. It also can be used as a food ingredient due to its functionalities as

texturized protein, emulsion or foaming agent, as a basis for edible protein films, etc. It also

found application in the biodiesel production. The possible technical application includes

fiber production for textile, asphalt emulsions, packaging films, detergents, etc. (Thiyam-

Hollaender et al., 2013; Wanasundara, 2011).

24

1.2.3. Meal processing

The processing of seeds which eventually results in canola meal or oil cake – a basis for the

protein extraction starts with flaking (to facilitate the oil removal) and cooking in order to

inactivate the enzyme myrosinase which catalyzes the hydrolysis of glucosinolates. It is

performed in stack cookers with temperatures reaching as high as 100-120 °C (Thiyam-

Hollaender et al., 2013). After that the flakes are transported to the screw press, also known

as an expeller allowing to extract about 70% of the oil. The remaining oil is extracted with

the help of solvents, normally hexane, using counter-current extraction process. Thereby

obtained “white flakes” contain less than 1% of oil (Daun, 2004). After oil extraction the

meal is desolvenized and toasted, where the residual solids are removed by steam heating the

meal at 130 °C, followed by drying and cooling. The general scheme of canola oil extraction

is shown in the Figure 1.5.

Figure 1.5: Processing of canola by prepress solvent extraction. Adapted from Daun (2004).

25

1.2.4. Composition, nutritive and biological value of canola meal

1.2.4.1. Nutritive and biological value of canola meal

Canola meal or oil cake (presscake) is what is left after oil extraction and constitutes 60 % of

the seed. In Canada a blend of Brassica napus, B. rapa, and B. juncea are used for canola oil

production by prepress solvent extraction. The nutrient composition of canola meal is

affected by environmental conditions during growth and harvest conditions as well as

processing of the seed and meal (Newkirk, 2011).

Table 1.4: Proximate composition of canola meal. Adapted from Newkirk et al. (2003).

Component Average

Crude protein (Nx6.25: %) 36 Oil (%) 3.5 Linoleic acid (%) 0.6 Ash 6.1 Crude fiber (%) 12 Tannins (%) 1.5 Sinapine (%) 1.0 Phytic acid (%) 3.3 Glucosinolates (µmol/g) 7.2

Proteins

Protein is the most abundant fraction in canola meal, depending on variety it can reach up to

40% on a dry basis. The quality of a protein can be characterized by the protein efficiency

ratio (PER) which is a gain in body mass related to protein intake. According to Friedman

(1996) the PER below 1.5 describes a protein of low or poor quality; between 1.5 and 2.0, an

intermediate quality; and above 2.0, good to high-quality. For canola proteins the PER has

been estimated as 2.64, which is higher than PER of soybean meal equal to 2.19 (Tan et al.,

2011a). Canola has been reported to be a source of well-balanced amino acids (Table 1.5),

which makes it attractive for the utilization as feed but also as a protein source for human

diet. Similarly to other oilseeds canola is limited in lysine, however rich in cystine and

methionine (http://www.canolacouncil.org, 2015b). It is also rich in sulfur-containing amino

acids such as glutathione and homocysteine, which are effective regulators in antioxidant

defense processes (Fleddermann et al., 2013). Another way to characterize the quality of

26

proteins is by analyzing the digestibility of amino acids by PDCCAS (Protein Digestibility

Corrected Amino Acid Score). The investigations showed that canola meal meets the

requirements for essential and non-essential amino acids in adults and 10-12 years old

children according to the PDCCAS. In the work of Klockeman et al. (1997) the PDCCAS of

canola amino acids were >1.00 for 10-12-year-olds and adults. Another work showed the

PDCCAS for rapeseed proteins equal to 0.83-0.93 which is comparable to animal sources

such as egg (1.00), milk (1.00), and fish (1.00) (Fleddermann et al., 2013). Nitrogen

digestibility of canola (rapeseed) proteins is equal to 93.3% - 97.3% which is comparable to

animal proteins notably egg (98%), casein (95%), and collagen (95%) and higher than plant

proteins of wheat (91%) and beans (78%) (Fleddermann et al., 2013).

Table 1.5: Amino acid composition of canola. Adapted from Newkirk et al. (2003), Downey (1990).

Amino-acid Canola/rapeseed* Canola/rapeseed** Soybean** Alanine 1.57 4.36 4.2 Arginine 2.08 5.78 7.2 Aspartate+asparagine 2.61 7.25 11.7 Cystine 0.86 2.39 1.6 Glutamate+glutamine 6.53 18.14 18.7 Glycine 1.77 4.92 4.2 Histidine 1.12 3.11 2.6 Isoleucine 1.56 4.33 4.5 Leucine 2.54 7.06 7.8 Lysine 2.00 5.56 6.4 Methionine 0.74 2.06 1.3 Methionine+cystine 1.60 4.44 ND Phenylalanine 1.38 3.83 5.0 Proline 2.15 5.97 5.1 Serine 1.44 4.00 5.1 Threonine 1.58 4.39 4.0 Tryptophan 0.48 1.33 1.3 Tyrosin 1.16 3.22 3.2

*Amounts shown are in grams per hundred grams of canola meal with 36% of protein ** Amounts shown are in grams per hundred grams of protein

In addition, a first cross over study in humans showed that the bioavailability of canola

proteins was similar to soybean proteins and that they can be used as effectively in human

nutrition (Fleddermann et al., 2013).

27

Carbohydrates

Carbohydrates compose a relatively small part of canola seed. The carbohydrate`s content is

rather complicated and is mostly represented by pectins (50%), cellulose (24.1%), and

pentosans (25.9%) (Table 1.6) (Bell, 1984). Total dietary fiber was estimated to be around

27.3-30.1% (Slominski et al., 1994). Comparatively to soybean canola meal has higher

amount of fiber from the hull which unlike soybean is not eliminated and represents about

30% of meal weight. It is, however, poorly digestible and its removal would significantly

enhance the digestibility and metabolizing energy (Bell, 1993). Cellulose constitutes the

major part of hull, followed by hemicellulose and lignin. Canola has been reported to contain

8-10% sucrose, 2-3% oligosaccharides, 20-22% nonstarch polysaccharides (NSP), and 5-8%

lignin and polyphenols. Starch is presented only in trace levels. Among low molecular weight

carbohydrates soluble sugars constitute about 48% and include D-glucose, D-fructose, D-

galactose, sucrose melibiose, raffinose, manninotriose and stachyose as reserve

carbohydrates, most of them however will be washed out during processing due to their

solubility. These compounds are known as the principal cause of flatulence. (Bell, 1984).

Table 1.6 : Carbohydrate content of canola meal. Adapted from Newkirk (2011).

Component Average Starch (%) 5.1 Sugars (%) 6.7

Sucrose (%) 6.2 Fructose + glucose (%) 0.5

Cellulose (%) 4.5 Oligosaccharides (%) 2.2 NSPs (%) 15.7 Soluble NSPs (%) 1.4 Insoluble NSPs (%) 14.4 Crude fiber (%) 11.7 ADF (%) 16.8 Acid detergent lignin (%) 5.1 NDF (%) 20.7 Total dietary fiber (%) 32.3

NSPS, nonstarch polysaccharides

ADF, acid detergent fiber

NDF, neutral detergent fiber

28

Vitamins and minerals

Mineral content of canola meal greatly depends on the location and the soil where it was

grown reflecting the uptake of soil minerals. Overall canola meal is a good source of essential

minerals and vitamins such as choline, biotin, folic acid, niacin, riboflavin, and thiamine

(Table 1.7) (Newkirk, 2011).

Table 1.7: Mineral composition of canola and soybean meal. Adapted from Bell et al. (1999); Downey (1990); Newkirk (2011). Mineral Average

Canola meal Average Soybean meal

Calcium (%) 0.64 0.3

Phosphorus (%) 1.12 0.63

Magnesium (%) 0.56 0.27

Copper (mg/kg) 6.2 23

Zinc (mg/kg) 68.2 43

Iron (mg/kg) 188 119

Manganese (mg/kg) 55 29

Biotin (mg/kg) 0.96 0.3

Choline (mg/kg) 6500 2614

Folic acid (mg/kg) 2.3 0.7

Niacin (mg/kg) 156 28

Pantothenic acid (mg/kg) 9.3 16.3

Pyridoxine (mg/kg) 7 6

Riboflavin (mg/kg) 5.7 2.9

Thiamine (mg/kg) 5.1 5.6

Vitamin E (mg/kg) 13 3

1.2.4.2. Bioactive properties

Canola proteins are also one of the promising sources of bioactive compounds. Thus, the

isolates itself and their hydrolysates were analyzed for Angiotensin-I Converting Enzyme

(ACE) inhibitory activity, antioxidant properties, bile acid-binding capacity, and anti-

thrombotic activity (Aider and Barbana, 2011).

1.2.4.2.1. ACE inhibitory activity

ACE is known to participate in the blood pressure regulation by converting the inactive

peptide Angiotensin I into Angiotensin II which is the peptide associated with the

29

development of various cardiovascular diseases. The inhibition of its formation was

successfully used for the treatment of hypertension and related organ damages. However, the

use of synthetic ACE-inhibitory drugs has been reported to have the side effects (Aachary

and Thiyam, 2011). The natural inhibitory action of peptides derived from canola

hydrolysates was first studied by Marczak et al. (2003), and later by Yoshie-Stark et al.

(2006) and Wu and Muir (2008). Both studies showed strong inhibitory potential, however

further studies in vivo were required.

1.2.4.2.2. Antioxidant capacity

It is known that the oxidation processes in the body give rise to the production of free radicals

which can damage cell structure and be precursors of serious disorders such as oxidative

stress-originated diseases (e.g., cardiovascular and neurodegenerative diseases), and cancer

(Apak et al., 2013). The results of Xue et al. (2009) and Cumby et al. (2008) proved that

rapeseed protein hydrolysates could be utilized as a source of bioactive peptides with

antioxidant properties. The antioxidant capacity was reported to be dependent upon peptide

concentration and composition. Also other minor substances such as phenolics which

possibly were co-extracted also could have contributed to the free radicals scavenging

activity of canola proteins.

1.2.4.2.3. Bile acid binding capacity

It has been demonstrated that the intake of dietary plant protein has hypocholesterolemic

effect; this is to say it reduces the LDL (low-density lipoprotein) cholesterol in the blood

vessels, responsible for various heart diseases and atherosclerosis (Aachary and Thiyam,

2011). The hypocholesterolemic effects of plant proteins might be attributed to its bile acid

binding capacity. As the body uses cholesterol to produce bile-acids, their binding promotes

further cholesterol decomposition to produce more bile acids to replace those that have been

lost (Pandolf and Clydesdale, 1992). The study of Yoshie-Stark et al. (2006) and Yoshie-

Stark et al. (2008) showed that canola proteins were able to bind the bile acids. They also

noted that precipitated protein isolate had higher bile acid binding capacity in comparison

with ultrafiltered which could be due to the higher concentration of fiber.

In addition, antidiabetic (Mariotti et al., 2008a), antithrombotic (Zhang et al., 2008),

anticancer (Xue et al., 2010), and antiviral (Yust et al., 2004) properties have been noted for

canola/rapeseed proteins.

30

1.2.4.3. Antinutritive factors

1.2.4.3.1. Glucosinolates

Canola is distinguished by low content of glucosinolates (10-12 µmol/g and lower) in

comparison with rapeseed cultivars (120–150 µmol/g) which is one of the major

improvements achieved by breeders (Bell, 1993). Total glucosinolate content of Canadian

canola meal was reduced to about 7.2 µmol/g (Daun et al., 2011; Newkirk et al., 2003). There

are also cultivars with the content in glucosinolates as low as 1.66 and 0.53 µmol/g (Bell et

al., 1991). Glucosinolates on their own are non-toxic, the problem arises during their

hydrolysis into nitriles, isothicyonates, and thiocyanates catalyzed by the enzyme myrosinase

(Bell, 1993). High glucosinolates rapeseed cultivars lead to reduced feed intake, enlarged

thyroid, reduced plasma thyroid hormone levels, and may as well affect organ liver and

kidney (Aachary and Thiyam, 2011; Bell, 1984). The glucosonolate`s content of today`s

canola meal was claimed to be of little consequence (Newkirk, 2011). A two-solvent oil

extraction method was developed which allowed to reduce the amount of glucosinolates in

meals to trace levels (Naczk et al., 1985). On the other hand both intact glucosinolates and

their metabolites have the ability to give various types of biological effects (Bernhoft, 2010).

Reduced risk of cancer due to their consumption has been reported (Song and Thornalley,

2007; Verhoeven et al., 1996).

1.2.4.3.2. Phytic acid

In canola as well as in many other plants and oilseeds phosphorous is stored mainly in the

form of phytic acid which is considered an antinutritive factor reducing the bioavailability of

proteins as well as the absorption of minerals, especially Zn. It is known to react with basic

proteins by the formation of insoluble protein-phytate complexes and to inhibit the action of

the digestive enzymes, such as pepsin, trypsin and α-amylase (Rodrigues et al., 2012).

However, the inhibition of α-amylase decreased the glycemic index of the food which is

beneficial for diabetics as it may aid them to control blood glucose (Harland and Morris,

1995). Also it binds di-and trivalent metals, such as calcium, magnesium, zinc, and iron, to

form poorly soluble compounds that are not readily absorbed from the intestine (Liener,

1994). Out of 1.22% of total P in canola meal 0.53 % is phytate-bound. In comparison with

soybean, canola however, provides twice as much non-phytate phosphorus (Bell, 1993). On

31

the other hand, phytic acid was reported to have a positive effect and was claimed to act as

an antioxidant and an anticancer agent (Harland and Morris, 1995; Lott et al., 2000).

1.2.4.3.3. Phenolic compounds

Phenolic compounds include simple phenols, phenolic acids, coumarins, flavonoids,

stilbenes, hydrolysable and condensed tannins, lignans, and lignins (Naczk and Shahidi,

2004). Phenolic acids and condensed tannins are the predominant types of phenolics in

canola/rapeseed. The effect of tannins on palatability and protein digestibility in canola meal

has been reported to have less negative effects as compared to other plants (Daun et al., 2011).

Phenolic acids are present in the form of free, esterified and insoluble-bound acids and are

derivatives of benzoic and cinnamic acids (Naczk et al., 1998b). They have traditionally been

associated with a bitter flavor, dark colour and astringency of canola meal. In addition they

can form complexes with proteins and essential amino acids making them less bioavailable.

Sinapine is the most abundant phenolic acid ester in canola meal and is considered to be a

major contributor to the unpleasant flavor (Naczk et al., 1998a). However, it has been stated

that at the levels found in canola meal it did not affect feed intake or growth rate (Daun et

al., 2011). Also during the conventional processing of canola meal to obtain protein

concentrates and isolates, the content of phenolics was reduced by 60-83% (Naczk and

Shahidi, 2004).

1.2.5. Proteins of canola

1.2.5.1. Structure

Rapeseed/canola is known to have a complicated protein composition with wide distribution

of isoelectric points and molecular weights (Gillberg and Törnell, 1976). Quinn and Jones

(1976) reported around 30 different protein species in rapeseed meal with isoelectric points

situated mostly in neutral region. By solubility in various solvents canola protein consists of

approximately 70% of salt soluble globulins, up to 20% of alcohol soluble prolamins, and

10% to 15% water-soluble albumins (Tan et al., 2011a). They are also classified by the

sedimentation coefficient in Svedberg units (S) which shows the speed of sedimentation of a

macromolecule in a centrifugal field. Canola proteins are mainly composed of high-

molecular weight and low-molecular weight units determining their nutritive and functional

32

properties. According to the corresponding sedimentation coefficient canola consists of two

main classes of storage proteins: 12S cruciferin, which is a salt-soluble globulin and a 2S

napin, which is a water-soluble albumin (Karaca et al., 2011a). They account for 60% and

20% respectively of the total protein in a seed (Wu and Muir, 2008). They differ not only by

their molecular mass but also by the types of molecular interactions involved in structure

stabilization which determines their functionalities.

1.2.5.1.1. Globulin 12S cruciferin

Cruciferin is an oligomeric protein with high molecular mass (300 - 310 kDa) and several

subunits. The quarternary structure of a protein obtained by x-ray scaterring represents a

trigonal antiprism consisting of 6 ordered subunits composed of two polypeptide chains (α =

30 kDa and β = 20 kDa) stabilized by intramolecular disulfide bridges and noncovalent

interactions. It has been stated that the quarternary structure and subunit was similar to other

11/12-S seed proteins revealing certain degree of homology between these proteins (Prakash

and Narasinga Rao, 1984; Prakash and Rao, 1986; Schwenke, 1994). Similar to other oilseed

proteins of this group it can dissociate under the influence of external factors. The scheme of

dissociation is shown in the Figure 1.6.

Figure 1.6: Dissociation of 12S globulin under external conditions. Adapted from Mieth et al. (1983)

The dissociation to the 7S fraction which is presented as a half 12S fraction indicates the

oligomerisation of canola globulin. The 2-3S units may also split under the effect of

detergents such as sodium dodecylsulphate and reducing agents such as mercaptoethanol to

smaller units of 18000 and 31000 g/mol molar masses (Mieth et al., 1983). Cruciferin is

classified as a “neutral protein” with the isoelectric point equal to 7.25 ± 0.10 (Schwenke et

al., 1981). However, upon dissociation numerous subunits were found to have isoelectric

points in a wide range of pH from 4.75 to 9.15 (MacKenzie, 1975). The type of a cultivar

may also have an impact on the ratio of acidic to basic amino-acids influencing the isoelectric

point as well as the secondary structure. Secondary structure was mostly characterized by

33

circular dichroism. According to the findings it is low in α-helix (11%) but is relatively high

in β-pleated structure (50%) (Tan et al., 2011a).

1.2.5.1.2. Albumin 2S napin

A small molecular weight protein fraction named napin is a basic protein with the isoelectric

point situated in the pH range of 9-10 and molar mass between 12 and 17.7 kDa. It consists

of two polypeptide chains of 4.5 kDa and 10 kDa linked by inter- and intra-molecular

disulphide bonds which ensures the maintenance of the conformation at high and low pH

values as well as at different electrolyte concentrations (Mieth et al., 1983). For napin α-

helical structure is predominant (40% to 46%) while β-sheet conformation accounts for only

12%. The high degree of amidation of the amino acids is responsible for the strongly basic

character of the low-molecular rapeseed protein (Schwenke, 1994). It is a hydrophilic protein

by nature which was shown by measurements through fluorescent probes (Jyothi et al., 2007).

The disulfide links are the major stabilizing bonds (Wanasundara, 2011).

1.2.6. Methods of protein extraction

1.2.6.1. Protein solubilization

Method of extraction influences first of all the composition and the structure profile of the

obtained product which is in close relationship with functional properties. Protein extraction

from canola meal same as from other plants involves two consecutive processes such as

solubilization in the aqueous medium followed by separation of solubilized proteins from the

residual meal. Solubilization is conventionally performed in alkaline medium adjusted with

sodium hydroxide to pH 9-12. The time of extraction varies from 30 min to 2 h in most cases

with constant stirring or shaking. This method allowed obtaining up to 80% of proteins from

laboratory prepared meal (low temperatures treatment) (Gillberg and Törnell, 1976; Tzeng

et al., 1990b), whereas industrially obtained meals normally gave lower rates due to the fact

that an important part of proteins was denatured during oil extraction and desolvenization.

After that the proteins are centrifuged in order to separate the supernatant with solubilized

proteins from the meal residues and either oven dried or lyophilized. The speed and the time

of centrifugation also vary. Such extraction has been named direct alkaline extraction (DIR).

Although being rather effective in terms of protein yield, this method has its drawbacks.

Harsh alkaline medium have negative impact on the quality of extracted proteins leading to

34

their denaturation, reduced digestibility, and damage to some amino acids (lysine and

cysteine). It was reported that proteins prepared by this procedure have poor solubility thus

being ill-suited for using as food ingredients (Tan et al., 2011b). Moreover, it leads to salt

formation during the pH adjustment which is not desirable (Sari et al., 2013). Salts are also

extensively used in order to increase the protein extractability due to salting-in effect. In most

cases NaCl is the salt of choice, using different molarities from 0.2 to 2M. They gave lower

total extractabilities and needed an additional step such as dialysis to remove salts. The use

of polyphosphates notably sodium hexametaposphate (SHMP) as an effective complexing

agents in protein isolation has been reported (Tzeng et al., 1988b). However the protein

extractability was largely dependent on the type of meal preparation and was reduced for

industrially prepared meal (Thiyam-Hollaender et al., 2013). Another method is known as

Osborne scheme based on the consecutive extraction of proteins in water, salt solution,

ethanol, and alkaline solution. Thus, albumins, globulins, prolamins and glutenins are

extracted one after another (Osborne, 1912). This method is particularly useful in case where

specific protein fractions are needed. On the other hand, it is not economical to scale up at

the industrial level due to long processing time, high water consumption and the need for

dialysis to separate salts used for globulin extraction. Extraction by the use of enzymes has

also been utilized as an alternative to alkaline extraction as it is held in milder conditions and

thus has less negative impact on the environment (Sari et al., 2013).

1.2.6.2. Protein separation

1.2.6.2.1. Isoelectric precipitation

As the aqueous extract together with proteins contain other substances, it is subsequently

subjected to other treatments in order to remove the impurities. After extraction and

centrifugation the supernatant is subsequently treated with acid (most often HCl) in order to

precipitate the solubilized proteins at their isoelectric point or subjected to ultrafiltration and

diafiltration. The majority of works report 2 isoelectric points, one situated in pH acid region

(pH 3-5) and the other in neutral region (pH 6-8) (Ghodsvali et al., 2005; Pedroche et al.,

2004; Quinn and Jones, 1976; Tan et al., 2011b). However, commercial canola meals might

have only one point of minimum solubility situated in acidic region (between pH 4-5) as a

result of heat treatment during meal processing (Naczk et al., 1985). Isoelectric precipitation

35

is widely used in the industry as it allows to obtain 96-99% of protein in the precipitate, is a

rapid and effective technique that might be easily adapted to a large scale, does not require

sophisticated equipment and is relatively inexpensive (Zaman et al., 1999). However, the use

of acids brings about the formation of salts which further should be separated. The following

washing step results in the formation of substantial amounts of chemical effluents. Also it

may result in protein denaturation impairing its further utilization as a food ingredient and

decreasing its nutritive value. After centrifugation and washing to remove salts the

precipitates are dried or lyophilized prior to utilization.

1.2.6.2.2. Ultrafiltration and diafiltration

Ultrafiltration can be used instead of isoelectric precipitation. It serves to separate protein

molecules from low molecular substances which were co-extracted and is performed under

pressure ranging from 1-15 bar using membranes with cut-off values of 2 - 300 kDa (Lewis,

1996). Diafiltration is an extension of ultrafiltration performed by the addition of water and

further filtration so as to wash out the low-molecular weight substances which might still be

found after ultrafiltration. It allows to obtain proteins of higher solubility and better

functional properties. In addition, it allows the reducing of nutritive factors which have

smaller molecular weight comparing to proteins (Rodrigues et al., 2012).

Despite the aforementioned advantages it found few applications in the industry first of all

due to the economics of the process, cost of membranes and their maintenance (Brennan et

al., 2006). Secondly, as opposed to the separation of dairy proteins where ultrafiltration is

extensively used, plant proteins require preliminary extraction as the starting meal is a solid

material and proteins need to be pulled out first. In general the extraction is performed at high

pH values where vegetable proteins have their maximum solubility and where their

hydrolysis might occur. The ultrafiltration in such conditions may therefore lead to the loss

of nitrogen (Lewis, 1996). In addition, most of the proteins in the extract are close to their

solubility limits so the following concentration might enable them to precipitate leading to

membranes fouling. This phenomenon appearing on the surface of the membrane results in

the flux decline with the time as feed components accumulate in the membrane pores as well

as on the membrane surface. In addition it significantly reduces the working life of the

membrane. Membrane fouling is a very important factor limiting the use of membranes in

the separation of food proteins. In some cases, a flux decline may be so important that it

36

makes membrane processes unpractical for protein isolation. Problem of membrane fouling

has been reported by Sayed Razavi et al. (1996), Bazinet (2005), Alibhai et al. (2006). A

substantial review on membrane fouling was presented by Fane and Fell (1987) and Marshall

et al. (1993).

1.2.7. Effect of processing conditions on the quality of proteins

The quality of a product which implies nutritive value of canola meal and extracted proteins

as well as functional properties strongly depends on the processing conditions starting from

milling, oil extraction, desolvenization and protein isolation. Each step can bring about

changes in quality some of which are irreversible. These changes are accompanied by the

structural transformations due to the changes in conformation and interactions with other

seed compounds and may lead to protein denaturation (Mieth et al., 1983).

1.2.7.1. Effect of meal processing

Being an efficient process for oil extraction it is detrimental for the quality of the meal due

to high temperatures and the use of solvents. High temperatures have by far the most drastic

effect on the quality of the meal. While processing the meal is subjected several times to the

action of high temperatures. During milling the temperature can increase due to the shear

stress, causing protein denaturation. Two methods of canola meal preparation, a conventional

screw-pressed and a cold-milled techniques were compared by Manamperi et al. (2012).

Results showed that milling temperature have significant effect on protein quality, yield and

functionality. Meal desolvenization is the most critical step for the protein quality where the

product is exposed to temperatures over 100°C for a long term (1 to 2h) inevitably leading to

their damage (Thiyam-Hollaender et al., 2013). The results on protein extractability of

industrially toasted meal showed that it had a significantly higher amount of non-extractable

proteins in the residues in comparison with the other meal samples not subjected to heat,

irrespective of protein extraction method (Tan et al., 2011b). In the work of Samadi et al.

(2013) the seeds were heat treated and the changes in secondary structure were monitored

showing important changes in the content of α-helix and β-sheet indicating protein

denaturation. A significant decrease in the availability and the digestibility of amino-acids

due to heating process has also been reported. A 7-11% reduction in lysine content has been

found in toasted meal in comparison with non-toasted presumably due to the Maillard

37

reactions which occur during toasting which is the final step of desolvenisation (Newkirk et

al., 2003).

Table 1.8: Extraction methods and conditions used for canola and rapeseed.

Source and type of defatting

Extraction solution

pH Time Meal to solvent ratio

Maximum Protein,%

Precipitation technique

Reference

Laboratory defatted rapeseed

H2O adjusted with NaOH, CaCl2 (aq)

1-12 30 min 1:20 >80 HCl, pH 2.5-6 (Gillberg and Törnell, 1976)

Laboratory defatted Canola

5% NaOH 9.5-12 30 min 1:18 69.5 HCl, pH=3.5, 7.5, ultrafiltration

(Ghodsvali et al., 2005)

Laboratory defatted Ethiopian mustard

H2O adjusted with NaOH

10-12 60 min, twice

10% 79.2 0.5 N HCl pH=3.5;5 CaCl2

(Pedroche et al., 2004)

Canola laboratory defatted

0.1M NaOH - 20 min 1:10 No data 0.1M HCl, pH=4

(Aluko and McIntosh, 2001)

Commercial/ laboratory defatted canola

H2O adjusted with NaOH

10.5-12.5 30 min-2h

1:18 >80 HCl, pH=3.5, Ultrafiltration, diafiltration combined

(Tzeng et al., 1990b)

Commercial/ laboratory defatted rapeseed

0-3% SHMP* or NaOH (aq)

1-12 30 min 1:6-1:21 >80 HCl, pH=3.5, CaCl2, ultrafiltration, diafiltration

(Tzeng et al., 1988b; Tzeng et al., 1988a)

Soybean, Rapeseed microalgue

Enzyme assisted Depending on enzyme

3h-24h 1:25 80, 15-30

- (Sari et al., 2013)

Commercial/ laboratory defatted canola meal

Osborne scheme/ 0.1NaOH

- 1h each solvent

1:10 No data vacuum-filtered, dialyzed, 1M HCl, pH 4.0

(Tan et al., 2011b)

Laboratory defatted canola

Osborne scheme - 4h, 4h, 1h

1:5 No data vacuum-filtered, dialyzed

(Manamperi et al., 2012)

laboratory defatted rapeseed

10%NaCl, 0.01M sodium pyrophosphate

pH 7 90 min 3 times

1.3:35, 1:50

67 dialysis (Bhatty et al., 1968)

Commercial canola meal

0-1M NaCl or CaCl2,

0.1 to 0.4% NaOH

2-12 60 min 5% 92.9 Acetic acid, pH 3.5

(Klockeman et al., 1997)

Commercial canola meal

0.01-0.1M NaC1/NaH2PO4

5.5-6.5 60 min No data No data ultrafiltration (Ismond and Welsh, 1992)

*SHMP – sodium hexametaphosphate

38

Important changes have also been observed in the studied functional properties of canola

proteins as shown by Khattab and Arntfield (2009). Boiled and roasted meal had significantly

lower nitrogen solubility, emulsion and foaming capacities comparing to raw meal. Reduced

nitrogen solubility of protein isolates obtained from toasted meal as compared to non-toasted

meal was reported by Naczk et al. (1985).

The alternative technologies include aqueous extraction and the use of enzymes which

significantly increases the price of processing and is hardly competitive with the conventional

technology in economic sense (Embong and Jelen, 1977; Thiyam-Hollaender et al., 2013).

For the production of canola meal and oil the major point is the economic side of the question

that is why although alternative techniques to conventional processing exist the finished

product is not compatible in terms of price. Recently the use of fluidized-bed desolvenizing

system introduced by Dr. Weigel Anlagenbau GmbH has been utilized for gentle solvent

removal under reduced temperatures (Adem et al., 2014; Thiyam-Hollaender et al., 2013).

Meals were significantly lighter in colour and had higher protein dispersibility index.

Implementation of a new technology will open new perspectives for the industrial production

of high quality meal.

1.2.7.2. Effect of protein production

The meal subjected to high temperatures on the stage of oil extraction has already partially

damaged proteins and thus a high quality product cannot be obtained even if mild conditions

are to be used for protein extraction.

The harsh alkaline treatment extensively used for protein isolation in order to maximize the

yield could additionally damage the amino-acids such as a cystine, lysine, arginine and

possibly serine. During extraction step the formation of amino acid derivatives such as

lysinoalanine (LAL) which drastically decreases the nutritive value as well as digestibility

has been noted (De Groot and Slump, 1969). Also it has been reported to result in amino

acids racemization making them unavailable for a human body (Maga, 1984). The effect of

pH, temperature and time of treatment with regard to amino acid degradability and LAL

formation was analyzed by De Groot and Slump (1969) in various samples and by Deng et

al. (1990) in rapeseed meal and isolates particularly. The combined effects of pH,

temperature and long treatment resulted in the most significant changes in amino-acids.

However, the samples which were not subjected to temperatures and prolonged treatment,

39

contained only low levels of LAL. Less than 500 µg/g of LAL which is similar to that found

in commercially produced casein and soybean protein isolates was detected in rapeseed

proteins extracted even at pH 12. Klockeman et al. (1997) also did not detect any LAL in

canola protein isolates extracted with 0.4% NaOH. Therefore, alkali extracted protein isolates

were claimed to not represent a significant health hazard due to the LAL content (Deng et

al., 1990). On the other hand extraction at high pH is accompanied by the oxidation of

phenolic compounds which gives bitter taste and dark colour to the isolates. The dark colour

is also a concern when it comes to food utilization. Many researches were seeking the ways

to avoid the darkening or to decolorize the product. Hydrogen peroxide and sodium sulphite

were mostly used as decolorizing agents (El-Kadiri et al., 2013; Keshavarz et al., 1977; Tzeng

et al., 1990b).

Functional and physico-chemical properties were largely affected by the type and pH of

extracting medium (Mwasaru et al., 1999a; Mwasaru et al., 1999b). Higher pH of the

extracting medium allows to extract more protein but of reduced quality. The reduced

solubility and precipitability are the indication of protein alteration. Pedroche et al. (2004)

compared the functional properties of meal to those extracted at pH 10 and pH 12. In general,

the functional properties except water binding and relative foam stability impaired with the

increase in pH of extraction explained by the protein denaturation. Only isolates obtained at

pH 10 had functional properties comparable to those of the defatted meal.

1.2.8. Possible application

Proteins as already mentioned have been used as food ingredients due to their functional

properties which is their ability to confer specific characteristic to the food, either sensorial

(texture and mouthfeel) or technological (foaming, emulsifying, gelling, etc.). The area of

possible protein application is based on their functional properties but is not limited to them.

The knowledge of functional properties of isolated protein is not a guaranteed knowledge of

its behavior in food systems where numerous factors might apply. Thus, an interaction with

other components as well as processing conditions have a great influence and are able to

either enhance or impair the performance of a protein in a food matrix (Arntfield, 2004).

Hence the behavior in model systems might differ from their behavior in real products

(Luyten et al., 2004).

40

The addition of protein isolates to food formulations has two aims, first – increase the

nutritive value, and second – improve the texture. There are few works devoted to the

feasibility of canola proteins utilization in various food matrixes. One of them is comminuted

meat products, where the gelling capacity of canola proteins was utilized. The addition of

protein isolates improved cooking yield, reduced the shrinkage and gave more tender meat

patties (Thompson et al., 1982). Several authors showed improved water holding capacity

and improved cooking yield in meat products (Cumby et al., 2008; Mansour et al., 1996) as

well as improved taste and aroma (Yoshie-Stark et al., 2006).

Another possible application is baked goods or extruded products. The studies on the

inclusion of canola proteins in baked goods are scarce and mostly refer to the improvement

of nutritive value. Incorporation of canola proteins in bread has been attempted up to 18-20%

of flour with further increase resulting in denser bread and decreased sensorial properties

(Kodagoda et al., 1973a; Shahidi, 1990). Bread systems might be challenging as the

distinctive texture, loaf volume as well as sensorial characteristics of bread are attributed to

the gluten of wheat. However, in other baked systems such as cookies which do not rely on

gluten visco-elastic properties the incorporation of other proteins may add to the textural

properties, especially in those systems which do not contain wheat (gluten free products).

Such products mostly based on starchy flours have low nutritional value as well as poor

texture. The utilization of proteins thus can perform two roles. So far the possibility of

improving texture in baked gluten-free products by canola proteins has not been regarded.

A recent trend of functional food, such as protein enriched products show that in future they

will play an important role in our lives. More and more enriched products are coming to the

market. With the continuous supply of food grade oil there will be a constant supply of

protein from oilseeds. The range of products where oilseed proteins can be incorporated is

wide, however their utilization in human nutrition should be corroborated by the relevant

studies to receive the commercial recognition as in case of soybeans (Arntfield, 2004).

Canola meal is a highly potential source of edible proteins and a potential ingredient

possessing interesting functional properties which can be implemented in the food

production. However its performance is greatly perturbed during processing such as

conventional extraction. Alternative technologies have been proposed, however all of them

are not competitive with conventional extraction in terms of price. That is one of the reasons

41

why the production of protein from canola has not become commercially available for

purchase. Hence, a method that will combine the effectiveness and the cost of a conventional

technique and at the same time will give the product of a higher quality might solve this

problem

Electro-activation as an emerging technology

In recent years there has been a significant increase in the development of new extraction and

separation techniques with the emphasis on using “green technologies”. The majority of

conventional technologies imply the utilization of organic and inorganic chemicals such as

solvents, acids, bases, etc. in order to extract the targeted components from plant materials

some of which drastically affects the quality of a product. Strict regulations on the

employment of such techniques due to the environmental, safety and health concerns and an

increased awareness of consumers about the use of chemical agents urge the food industry to

search for alternative methods. It is amplified by the demand for a higher quality product that

traditional processing cannot satisfy. “Green” methods on the contrary are environmentally

friendly and cause less air pollution and industrial waste (Shi et al., 2012).

Also an increase in scientific interest in using the conventional sources of energy such as

electric power for non-conventional purposes has been noted (Aider et al., 2012b). Thus, an

application of a direct electric current in separation processes could be an alternative method

to the existing conventional technologies. The electric energy transformed into the chemical

energy allows to decrease the use of chemical acids and alkali providing environmentally

friendly “green” technology for improved food processing application.

1.3.1. History and development

The use of electric current in technology has been known since 1802 when a Russian scientist

Petrov observed that the liberation of gases near the electrodes was accompanied by the

acidification of water near the anode and alkalization near the cathode. Thirty years later

Faraday stated the world-known electrolysis laws. This gave a rise to a new branch of science

known as electrochemistry (Tomilov, 2002). Electro-activation (EA) as a technology itself

accounts for more than a 100 years, when its unique physico-chemical properties were

observed by Russian scientists while working with drilling mud (Prilutskii and Bakhir, 1999).

42

In 1972 a Russian engineer Bakhir noted that electro-activated solutions (EAS) prepared

from low concentrated salt solutions had properties different from chemically alkalized or

acidified solutions. However, he also observed that these properties were not constant and

tended to decrease with the time finally becoming equal to their chemical counterparts. Due

to these observations such solutions were called electro-activated (Tomilov, 2002). Since that

time EAS were utilized in many fields mostly as a powerful disinfecting agent (Lelianov et

al., 1991; Prilutskii et al., 1996; Sato et al., 1989; Sergunina, 1968). However, the lack of

theoretical and experimental studies hindered the development of EA as a technology and

prevented its broader application (Gnatko et al., 2011). In recent years the number of studies

devoted to Electro-activation technology increased dramatically showing its high potential

for the utilization in food and other industries (Aït-Aissa and Aïder, 2015; Aït Aissa and

Aïder, 2013b; Huang et al., 2008; Kastyuchik, 2015; Koffi et al., 2014; Liato, 2015; Liato et

al., 2015c; Thorn et al., 2012).

1.3.2. Principles

Electro-chemical activation occurs as a result of physico-chemical processes during which

the water and the dissolved particles find themselves in the nonequilibrium thermodynamic

state characterized by abnormal physico-chemical properties (Plutakhin et al., 2013). It takes

place in the near-electrode layer after subjecting water or brine solutions to the action of

external electric field (Aider et al., 2012b; Plutakhin et al., 2013; Tomilov, 2002). Water

molecules activated by the electric field fall into a metastable state resulting in higher activity

and modified physico-chemical properties such as pH, oxido-reduction potential, electro

conductivity, etc. With the time, however, the solutions are prone to relaxation, finally

coming into equilibrium (Aider et al., 2012b). The non-equilibrium processes are the

quintessence of electro-activation. When in most electro-chemical processes the equilibrium

state is sought, for EA the parameters are selected in such a way allowing to move as far as

possible from the equilibrium conditions of the flow of electro-chemical reactions (Tomilov,

2002). As a technology EA is based on the well-known phenomenon of electrolysis (Aider

et al., 2012b; Plutakhin et al., 2013; Tomilov, 2002).

43

1.3.2.1. Water electrolysis

Water electrolysis is its decomposition with the liberation of hydrogen and oxygen gases as

a result of the passage of electric current (Chaplin, 2000). The decomposition of water is

performed in the electrolytic cell, a basic example of which is shown in the Figure 1.7

consisting of two electrodes immersed in the aqueous solution known as electrolyte. The

electrodes are connected to the positive and negative charges of electric source and are termed

anode and cathode respectively. When water or a brine solution is subjected to the direct

electric current via two immersed electrodes, positively and negatively charged ions start to

migrate through the solutions towards oppositely charged electrodes to transfer the charge

(Bazinet, 2005). At the electrode`s surface a number of reactions occur leading to the

liberation of hydrogen and oxygen gases. Electric energy is transformed into the chemical

energy which gives rise to the reactions that cannot be performed under normal conditions

(Plutakhin et al., 2013). This also leads to the formation of new substances and to the

structural and compositional changes in water structure (Petrushanko and Lobyshev, 2001).

1.3.2.2. Charge transfer

The set of reactions which takes place as a result of applied current is linked to the transport

of electric charge (Bard and Faulkner, 2000). For electrolysis the electric charge is carried

out by two different species - electrons in the electrode and migrating ions in the electrolyte.

Due to the presence of two phases (electrode-electrolyte) and the fact that the charge is

transported by different carriers the change of charge carrier takes place at the interface. On

one side charge carriers of one type transfer the charge to the carriers of the other type. This

is possible due to the chemical reactions which imply both charge carriers, known as electro

chemical or electrode reactions. On the anode, as a result of chemical reaction electrons are

generated and pass from the solution to the electrode. On the cathode electrons are transferred

from the electric circuit to the electrolyte. The withdrawal of electrons on the anode is

equivalent to the oxidation of an element whereas the addition of electrons equals to the

reduction process (Bagotsky, 2006).

44

Figure 1.7: Basic electrolytic cell. Adapted from Zeng and Zhang (2010).

1.3.2.3. Electrode reactions

Due to the presence of different cationic and anionic species as well as molecules a number

of different processes can take place simultaneously at the electrodes. The type of reactions

depends on the electrode material, ionic species in the solution, and other factors. Depending

on the material some electrodes can participate in the reactions (Bagotsky, 2006), however it

is not the subject of the current study as for electro-activation non-consumable (inert)

electrodes are utilised. They serve primarily as a stock and a sink of electrons and permit the

electron transfer without participating themselves in the reactions (Brett and Brett, 1993).

The principal reactions which take place during water electrolysis are water oxidation on the

anode with O2 liberation (Eq. 1.1; 1.2) and water reduction on the cathode with H2 release

(Eq. 1.3; 1.4). It is also accompanied by pH changes near anode and cathode. As a result an

acidic solution is generated in the near anode layer and an alkaline solution is produced in

the near cathode layer. In order to obtain two solutions with different properties such as acidic

45

anolyte and alkaline catholyte the electrolytic cell can be separated with the porous

diaphragm or an ion-exchange membrane (Aider et al., 2012b).

Anode oxidation:

2Н2О (l) − 4ē→ 4Н+ (aq) + О2 (g) ↑ (Eq. 1.1)

4OH–(aq) → O2↑ (g) + 2H2O (l) + 4ē (Eq. 1.2)

Cathode reduction:

2Н2О (l) + 2ē→ Н2↑ (g) + 2ОН −(aq) (Eq. 1.3)

2H+ (aq) + 2ē → H2↑ (g) (Eq. 1.4)

where (l), (g) and (aq) are liquid, gas or aqueous solution.

Provided that only hydrogen and oxygen molecules as well as charged species participate in

electrode reactions twice as much hydrogen is liberated compared to oxygen gas (Aider et

al., 2012b). The near electrode layer is the area of high intensity where the voltage can reach

up to hundred thousand volts, yet it is rather thin. This layer is also known as double diffusive

layer (Tomilov, 2002).

As the water itself poorly conducts the electric current, to facilitate the charge transfer and

to decrease the system resistance salts are usually added (Chaplin, 2000). Most often salts

like NaCl, KCl, Na2SO4, and NaHCO3 are used. In the solution they dissociate and exist as

ionic species e.g. Na+, K+, Cl-,SO42-, HCO3

-, etc., carrying positive and negative charges

(Bagotsky, 2006). When different anionic and cationic species as well as not dissociated

molecules are present in the solution several different reactions might occur at the same time

due to the competition between ions which move towards the electrodes (Anshitz et al.,

2008). This yields new solutes and compounds which can further react between each other

(Figure 1.8). The electrode reactions are numerous and rather complex. Some of the

reactions products are unstable and quickly undergo further transformations (Chaplin, 2000).

46

Figure 1.8 : Electrolytic cell showing the passage of electrons and some products of electrode reactions. Adapted from Chaplin (2000).

1.3.2.3.1. Cathode reactions

The sequence of the redox reactions in the cathodic compartment depends on the activity of

metals. All metals can be divided in three groups: (1) Active metals (from Li to Al), (2)

medium active (Al to H), and (3) low active (metals after H). This classification is known as

the reactivity series and was made according to the magnitudes of the electrode potentials. In

this series metals are placed from the most active (having the lowest electrode potential) to

the least active ones (having the highest electrode potential). With an increase of the standard

electrode potentials the reducing capacity of metals decreases. Thus all the metals placed

before hydrogen (first and second groups) have more negative potential and can expel the

hydrogen from the diluted mineral acids (Vapirov et al., 2000). Metals of the first group

hence are not prone to reduction. On the cathode instead the hydrogen is reduced according

to Eq. 1.3. Cations of the second group are reduced together with the hydrogen ions (Eq. 1.3

and Eq.1.5). Finally, the cations of low active metals reduce themselves on the cathode (Eq.

1.5).

Men+ + nē → Me0 (Eq. 1.5)

If cations of different ions are present in the solution, then first metal to be reduced will be

the one with the highest potential followed by the metals with lower potentials (Anshitz et

al., 2008). When NaCl salt are used cathode reactions also yield sodium hydroxides as well

47

as a number of highly active reducers and metastable aquacomplexes of hydroxyl anion (ОН-

), hydroperoxide anion (НО2-), molecular oxygen ion-radical (O2

-), oxygen anion (O2-),

peroxide anion (О22-) and metastable aquacomplexes of hydrogen superoxide (НO2), free

hydrogen radical (Н•), free peroxide radical (НO2•) (Belovolova et al., 2006; Gnatko et al.,

2011). Catholyte thus saturated with the reducers gains the enhanced adsorbing capacity

(Leonov et al., 1999).

1.3.2.3.2. Anode reactions

At the anode oxidation of the acid residuals takes place. Those that do not contain oxygen

oxidize themselves e.g. Eq. 1.6. Those that contain oxygen are not oxidized at the anode,

instead the oxygen from the water is liberated (Eq. 1.1).

2Cl− - 2ē → Cl2↑ (Eq. 1.6)

Simultaneously a number of active oxidizing compounds are generated presented by a

mixture of peroxide compounds such as НO•– (hydroxyl radical); НО2− (peroxide anion); О2

(singlet molecular oxygen); О2− (superoxide anion); O3 (ozone); O• (atomic oxygen) and

chlorine-oxygen compounds notably HClO (hypochlorous acid); ClO− (hypochlorite ion);

ClO• (hypochlorite radical); and ClO2 (chlorine dioxide). Some of them can further react

with each other neutralizing themselves, e.g. reaction of hypochlorous acid with hydrogen

peroxide (Eq. 1.7) or reaction of hydrogen peroxide with ozone (Eq.1.8) (Bakhir, 2012b;

Gnatko et al., 2011).

HClO + H2O2 →O2↑+ H2O + HCl (Eq. 1.7)

H2O2+ O3→ 2O2↑+ H2O (Eq. 1.8)

1.3.3. Phenomenon of EAS

It is difficult to highlight a single reason of the electro-activation phenomenon. Instead there

is a number of factors explaining the high reactivity of EAS. As was already mentioned it

can be done only when the system is in thermodynamically unstable state which is achieved

by the use of external electric field. The electric field supplies the energy required for the

electro-chemical reactions to launch. This energy is the highest near the electrodes surface,

where they contact with the electrolyte (Plutakhin et al., 2013). As a result of treatment

48

anolyte and catholyte acquire such properties that cannot be modulated in the solutions not

subjected to electro-chemical effect (Sprinchan et al., 2011b).

The activated state of the solutions is not perpetual. With the time the anomalous properties

vanish and the thermodynamic equilibrium is established with the pH and oxido-reduction

potential inherent to typical solutions known as relaxation (Sprinchan et al., 2011b). The

phenomenon of relaxation demonstrates the presence of metastable particles and the fact of

the existence of activated state. Thus EAS exist only during the period of relaxation

(Prilutskii and Bakhir, 1999).

One of the suggested explanations is the formation of various products during water

electrolysis. Some of them are rather stable and can exist in the medium for a certain amount

of time, other immediately enter the subsequent reactions (Gnatko et al., 2011). Several

groups of reactive substances can be discerned. First group represents stable reaction

products such as bases and acids. They explain the alkaline properties of the catholyte and

acid properties of the anolyte. Second group is highly active metastable compounds such as

molecular ions НО2-, О2

-, ОН- as well as free radicals. And third refers to the long-lived quasi-

stable structures formed in the near electrode surface area which could be presented as free

structural complexes, hydrated ion shells, molecules, or radicals (Sprinchan et al., 2011b).

The factors of the second group enhance the redox properties of the catholyte such as an

electron donor. Finally the third group of factors is responsible for the catalytic activity of

the catholyte. Specifically the products of the second and third groups cause the modification

of the activation energy barriers between the components of the system. Their generation is

possibly only under the condition of electro-chemical treatment (Sprinchan et al., 2011b).

Next possible explanation is connected to the water state and the modification of its structural

and energetic characteristics (Gnatko et al., 2011; Petrushanko and Lobyshev, 2001). When

EA is performed under the condition of the low mineral concentration the water electrolysis

at the electrodes surface is performed under high voltages which cause the structural

modification of water molecules. As a result of hydrogen bonds rupture the water structure

gains electron donor and electron acceptor properties near cathode and anode. Such water

easily forms aqua complexes (hydrated ionic complexes) which enhance the reaction and

catalytic activity of EAS. In the solutions with high mineral salt concentrations the voltage

is low and water structure is less prone to changes, thus the properties of such solutions

49

mainly depend on the products of electrolysis reaction. (Prilutskii and Bakhir, 1999). The

resonance effect of water clusters resulting from co-vibrating dipoles of water molecules as

well as charged species is also one of the factors used to explain the non-contact EA. Such

dipole systems are unstable and rapidly collapse in static, whereas in dynamic state provided

by electric field it becomes possible (Aider et al., 2012b; Shironosov, 2000). Finally the

formation and the presence of gaseous microbubbles also favors the anomalous properties of

solutions in activated state (Gnatko et al., 2011).

The evidence of the existence of activated state was shown by Pastukhov and Morozov

(2000) who studied the EAS by means of Raman spectroscopy. According to the study the

vibrational spectra of electrochemically treated solutions and their chemical analogues were

different in the spectral region between 700 and 2700 cm-1. A broad peak was obtained for

EAS in this region which was not present for their chemical counterparts. The intensity of

the peak also decreased with the time indicating the relaxation. The spectra of EAS were

similar to those of concentrated alkali and acids in spite of the fact that they had low

mineralization. This led to the assumption that EAS might have properties similar to those of

concentrated acids and bases (Aider et al., 2012b; Plutakhin et al., 2013).

1.3.4. Reactors design

As already mentioned the process of electro-activation takes place in the near electrode layer,

where the resistance can reach up to several thousand volts per cm. This layer is

comparatively thin (around 0.1 µm), thus a design and a construction of a reactor with optimal

characteristics was not an easy task (Tomilov, 2002).

1.3.4.1. Flow-type reactor

First attempts to design a reactor for the production of electro-activated solutions were made

in 1974 – 1975. Twenty years later the reactor has been released. It was a flow type reactor

made of several parallel placed flow electrochemical modules (Figure 1.9) with external

tubular and internal rod-like electrodes. A tubular ceramic ultrafiltration diaphragm was

placed between them. Such configuration allows to treat the volumes of water equally

saturating them with metastable active particles and provides unipolar water treatment when

it passes through anodic and cathodic chambers (Leonov et al., 1999). The volumetric flow

rates usually range between 0.5–1.9 l/min (Aider et al., 2012b). As a result two solutions are

50

obtained notably anolyte and catholyte with physico-chemical properties determined by the

current intensity, mineral composition and the volume of treated solution. Since that time a

number of technologically improved reactors appeared not only for technological purposes

but also for domestic use producing electro-activated solutions with disinfecting, sterilizing,

stabilizing, conservation, extracting, and healing properties (Bakhir, 2012a).

Figure 1.9: Flow type electrochemical modules. Adapted from Leonov et al. (1999).

Many other types of reactors have been used worldwide. In 1999, only in Japan over 30

different types of reactors were functioning. More on different types of reactors can be found

in Aider et al. (2012b); Tanaka et al. (1999).

1.3.4.2. Stationary type of reactors

Together with flow type reactors the stationary types are also used. In this case the solutions

are treated in batches of 1-15 l and the time of EA varies between 3 and 115 min. The voltages

of 9–120 V are used for electro-activation with the current intensity being set from 0.7 to 20

A (Aider et al., 2012b).

Outlet C

Outlet A

Anode

Diaphragm

Outlet A

Outlet C

Inlet A Inlet C

Direction ofwater flows

Anode

Outlet A

Diaphragm

Outlet C

Cathode

Cathode

51

For large scale production flow- type reactors are more practical, whereas in the lab mostly

a stationary type of cell is used for electro-activation (Aider et al., 2012b). A three-

compartment reactor consisting of the anodic, cathodic and central chambers was used in the

works of Aït Aissa and Aïder (2013b), Koffi et al. (2014), and Liato et al. (2015b) (Figure

1.10). Anodic and cathodic compartments separated by the ion-exchange membranes allow

to produce solutions with oxidizing (acid anolyte) and reducing (alkaline catholyte)

properties. By changing the parameters such as type of configuration, current intensity, time

of treatment, types of salts and their concentrations the solutions with desired properties can

be obtained. Central compartment has also been utilised for the analysis of a non-contact

EAS (electro-activated solution) (Liato et al., 2015b).

Figure 1.10: A schematic representation of a three-call electro-activation reactor. Adapted from (Liato et al., 2015a).

1.3.4.3. Requirements for reactors and its functional parts

In order to be efficient reactors must meet the following requirements. First, a large active

electrode area is needed per unit volume. Second is a uniform distribution of the electric

potential on the electrode surface. Finally, rather high current density, good mass transport

towards the electrodes, and low power consumption are required (Gnatko et al., 2011).

Certain requirements exist for the electrodes and membranes as they are the functional parts

of the reactor.

52

1.3.4.3.1. Electrodes

A very important characteristic is the chemical stability of the electrodes (Aider et al., 2012b).

They must not participate in the chemical reactions themselves being chemically inert as well

as insoluble in the products of electrolysis Thus typical materials used for electrodes are

platinum or titanium coated with porous layers of a metal oxide catalyst (e.g. RuO2, TiO2,

SnO2, IrO2) due to their high stability, selectivity, electro-chemical reactivity, corrosion

resistance and operating life (Thorn et al., 2012; Trasatti, 1987; Trasatti, 2000). Chemically

inert graphite electrodes also have been utilised, however they have some disadvantages such

as their porosity and friability. Gas bubbles can accumulate in the pores thus reducing the

active working surface of electrodes (Plutakhin et al., 2013).

1.3.4.3.2. Membranes

Membranes are used in order to separate the reaction products and increase the effectiveness

of the process. There are several types of membranes used in electro-activation, however the

most utilised are ion-exchange membranes such as cation exchange membranes (CEM) and

anion exchange membranes (AEM) as they allow to separate the ionic species according to

their charge. Both of them are monopolar, this is to say that they allow only one type of ions

to pass. CEM is permeable for cations and rejects anions, for AEM it is the opposite. They

are normally made of a macromolecular material known as skeleton with fixed ionisable

groups which can be ion-exchange resins (Bazinet et al., 1998a). The skeleton can be made

completely from the ion-exchange material, in this case membranes are called homogenous

or the filler can be added in order to increase the mechanical strength, such membranes are

called heterogeneous (Shaposhnik, 1999). Typical groups fixed on the CEM are sulfonic

(SO3-), carboxylic (COO-), arsenic (AsO3

2-), or phosphoric (PO32-) groups and alkyl

ammonium groups (NR3+, NHR2

+, NH2R+) for AEM (Bazinet, 2005). When the membrane

is placed in the solution it is neutralized by the ions of the opposite charge known as counter

ions or compensator ions. These ions are used to carry the electric current in the membrane.

For CEM counter ions are positive, whereas for the AEM they are negative (Figure 1.11)

(Bazinet et al., 2010). Ions move inside the membrane through its pores and canals and

include the processes of sorption on the surface of the membrane, transport, and desorption

(Yaroslavtsev and Nikonenko, 2009). The selectivity of membranes depends on the pore

sizes which can vary from 10 to 100 Å. Higher pore size is less selective and allow more ions

53

with the same charge to pass than do membranes with small pores. Typically pore size of 10

- 20 Å are used (Bazinet et al., 1998a).

Figure 1.11: Example of a CEM with fixed SO3 groups. Adapted from (Bazinet et al., 1998a).

1.3.5. Application of EAS

Unique properties of EAS open doors to their wide application in many fields including water

preparation, chemical industry, agriculture, livestock breeding, medical and food industries

(Golokhvast et al., 2011). As it was aforementioned anolyte and catholyte possess largely

differing properties determined by their composition. The presence of strong oxidizers in the

anolyte makes it a powerful cleaning and disinfecting agent having highly pronounced

biocide properties. The catholyte on the other hand rich in reducers gains considerable

absorption and catalytic activity as well as detergent capacity (Prilutskii and Bakhir, 1999).

Hence, their utilisation is dictated by the stated goals.

1.3.5.1. Cleaning and disinfection

The utilisation of EAS as disinfectant has several advantages. They were shown to be more

environmentally friendly than traditional cleaners. Furthermore they are not harmful for

human health e.g. not corrosive to skin, mucous membrane, or organic material as compared

to chemical acids and bases (Huang et al., 2008). No changes in body weight nor any

abnormal effects were found in mice after digesting electro-activated solutions (Morita et al.,

2011).

Antimicrobial properties of the anolyte also known as strongly acidic electrolyzed water,

electrolyzed strong acid aqueous solution, superoxidized water, electrolyzed oxidizing water,

etc. have been utilized by many authors as an alternative to chlorine and hydrogen peroxide

54

(Gnatko et al., 2011; Huang et al., 2008). It showed the high antimicrobial effect against

numerous pathogens, some of them are: Pseudomonas aeruginosa, Staphylococcus aureus,

S. epidermidis, E. coli O157:H7, Salmonella Enteritidis, Salmonella Typhimurium, Bacillus

cereus, Listeria monocytogenes, Mycobacterium tuberculosis, Campylobacter jejuni, etc.

(Drees et al., 2003; Kim et al., 2000; Sato et al., 1989; Vorobjeva et al., 2004). It also showed

the inhibitory effect on fungal species such as: Alternaria spp., Bortrytis spp., Cladosporium

spp., Fusarium spp., Aspergillus spp., and yeasts: Saccharomyces cerevisiae (Guillou and El

Murr, 2002), Kloeckera and Candida spp (Lustrato et al., 2006), and many other. Various

mechanisms were proposed, however the main is the high oxidation potential of electro-

activated water which leads to damage of cell membranes. There are also indications of

antiviral capacity against blood borne pathogenic viruses such as hepatitis B virus (HBV),

hepatitis C virus (HCV), and human immunodeficiency virus (HIV) (Kitano et al., 2003;

Sakurai et al., 2003; Selkon et al., 1999) by destruction of viral surface proteins, nucleic acids

and RNA (Huang et al., 2008). Thus, electro oxidized water have been used for disinfection

of processing equipment (Abe, 1999; Ayebah and Hung, 2005; Walker et al., 2005), washing

freshly cut fruits and vegetables (Al-Haq et al., 2001; Gil et al., 2015; Izumi, 1999; Park et

al., 2001), disinfecting poultry, meat, and seafood (Ozer and Demirci, 2006; Park et al.,

2002; Yang et al., 1999). A substantial review on the application of electro oxidized water as

a disinfectant is presented by Huang et al. (2008). Electro-activated solutions have also been

used in combination with other treatments such as sterilisation of canned vegetables in order

to decrease the sterilisation temperatures (Genois, 2014; Liato et al., 2015c).

1.3.5.2. Other applications

Other than disinfection EA was used for the stabilisation of the maple sap soft drink by its

acidification directly in the anodic compartment of a 3-cell reactor (Koffi et al., 2014). EAS

were used in dough instead of tap water for making bread and macaroni (Prilutskii and

Bakhir, 1999). Their activity on yeast activation for bread baking resulted in lower

fermentation time and higher bread volume (Nabok and Plutakhin, 2009). Other examples

include the catalysis of starch hydrolysis (Leonov et al., 1999), the germination of barley

(Plutakhin et al., 2014), thermal coagulation of beetroot pectin (Shazzo et al., 2005), acid

mine drainage neutralization (Kastyuchik, 2015), etc.

55

The utilisation of catholyte is based on its catalytic and reducing properties. Catalytic

properties were utilised for synthesis of lactulose from lactose in a three cell stationary

reactor (Aider and Gimenez-Vidal, 2012; Aït Aissa and Aïder, 2013b). In the other study the

possibilities of simultaneous separation of protein concentrate and lactulose synthesis were

analyzed by EA of whey in a flow type reactor (Sprinchan et al., 2011b).

As an extracting agent EAS were used for hop extraction (Khrapenkov et al., 2004), pectin

production from apple and beet cake, extraction of propolis and ginseng (Prilutskii and

Bakhir, 1999).

1.3.5.3. Protein production

Extraction of proteins by the use of electro-activated solutions is another perspective

application which can be used for the valorisation of proteins from vegetable sources and by-

products or for improving existing technologies on the protein preparation from conventional

sources. For protein extraction and precipitation necessary alkaline and acid conditions are

created in the cathodic and anodic chambers.

Electro acidification of soy protein extract using the technology of electro dialysis was

performed by Bazinet et al. (1998b); Bazinet et al. (1997). Protein extract was treated in the

electrochemical cell separated from electrodes by the ion exchange membranes to precipitate

the proteins. The obtained isolates were higher in protein and lower in ash content, and

showed equal or improved functional properties in comparison with commercial samples. At

the same time the quality of isolates were improved (less denatured) regarding the solubility

profiles. The method was improved later in the work of Mondor et al. (2004) by adding the

ultrafiltration membrane which helped to obtain more pure isolates but also increased the

cost of extraction. Although the mentioned technology used for acidification is

fundamentally different from electro-activation as in this case protein precipitation was based

only on the pH of the medium and not on the metastable state of the solution, it gives the

indication of the possibility of performing the EA technique for protein extraction.

Electro-coagulation of whey performed in electrolyser with gradual increase and decrease in

pH in order to separate whey proteins was realized and patented by Bologa et al. (1996). The

thermal coagulation of proteins from Lucerne (alfalfa) leaf juice using EA technology was

performed by Plutakhin et al. (2004). Later, protein isolates were produced from sunflower

oilcake in a flow type reactor by the same author - Plutakhin et al. (2005). The schematic

56

representation of extracting unit is shown in the Figure 1.12. The sunflower meal was added

with water (1:10) in a tank (1) equipped with capronic filter. Tank (1) was connected to the

peristaltic pump (6) which pumped the solution through cathodic chamber until required pH

was reached. A 5-time passage was needed to reach pH 11. In the anodic chamber at the same

time the NaCl solution with the concentration of 50 mg/l was circulated. After extraction the

protein solution was pumped through the anolyte chamber until the pH reached 4

(corresponding to the isoelectric point). In order to prevent the thermal coagulation of

proteins the parameter of current intensity and the speed of circulation were chosen in such

a way that the temperature did not exceed 40 °C. As a result 34% of protein was extracted

using electro-activation compared to 39% obtained using alkaline extraction with NaOH. The

losses of protein were summoned by the local overheating due to the contact with the surface

of electrodes which could be solved by modification of the unit construction (increasing the

surface of electrodes) and by increasing the solvent to meal ratio (Plutakhin et al., 2005).

Figure 1.12 : Electro-activation unit for protein extraction form sunflower oil cake, where 1-tank with oilcake; 2 - cathodic chamber; 3 - membrane; 4 – anodic chamber; 5 – tank with anolyte; 6 – peristaltic pump; 7 – anode; 8 – cathode; 9 – filter; 10 – oilcake; 11 – extracting solution. Adapted from Plutakhin et al. (2005).

Other works on protein extraction include a series of patents differing by technical

characteristics and conditions of extraction (Koschevoi et al., 1997; Koshchaev et al., 2005;

Petenko et al., 2003; Plutakhin et al., 2006a, b). Regarding all the aforementioned information

the advantages of using EAS over traditional approach become evident. First of all, they

allow to reduce the use of chemical bases and acids and potentially produce protein products

of higher quality. The conditions, required for protein extraction and precipitation are

generated using the electric field by the passage of electric current through the salt solution.

57

Secondly, the amount of preparative steps is also reduced. In comparison with conventional

extraction which includes preparation of NaOH and acid separately, EA needs only the

preparation of salt solution. Third, the conditions of extraction can be monitored by

controlling the parameters of electro-activation. However, there is a lack of studies on the

relationship between the parameters of the EA process and properties of electro-activated

solutions (Aider et al., 2012b). Also the effects of EAS on the quality of obtained proteins

such as structural characteristics and functional properties have not been studied.

58

2. CHAPTER 2: Problematic, research hypothesis and objectives

2.1. Problematic

The literature review showed increasing demand for alternative protein sources to satisfy the

needs of continuously growing world`s population. Proteins from renewable plant sources

are considered as the most promising. These sources are being properly studied in order to

show that they are suitable for human consumption and the idea of their valorization is close

to its realization. New protein sources require appropriate extraction techniques based on the

knowledge of the physico-chemical properties, spatial arrangement, process requirements, as

well as techno-functional properties of these proteins. While conventional processing

significantly alters the quality and nutritional value of the extracted proteins, new processing

techniques are needed. In addition, regarding the environmental situation ``green`` methods

(eco-friendly) are welcomed and encouraged worldwide. Human start taking after the

nature`s best phenomena, which leads us to eco-friendly techniques, one of which is electro-

activated solutions. With their help the utilization of chemical acids and alkali can be

significantly reduced or even completely eliminated which will as well be positively reflected

on the quality of extracted proteins. Such proteins can be further incorporated into different

food matrices conferring required properties. But before implementing any novel

methodology or technology, a proper research should be conducted to confirm that such

technique is beneficial compared to conventional processing.

Research hypothesis

The use of electro-activated aqueous solutions, as alkali replacement, will allow to extract

proteins from canola press-cake with their further utilisation as a product ingredient.

Main objective

The main objective of this doctoral thesis is to study the electro-activated solutions in terms

of their generation, protein extraction capacity and their impact on proteins physico-chemical

and techno-functional properties.

59

Specific objectives

In order to verify the stated hypothesis and to achieve the main objective, the following

specific objectives were highlighted:

1. To study and to find the optimal conditions for protein extraction from canola meal

by performing conventional alkaline technique as a comparative basis for the electro-

activation method.

2. To investigate the process of generating electro-activated aqueous solutions

depending on the influence of different operating conditions in order to obtain

solutions with alkaline properties as a replacement of chemical alkali.

3. To perform the extraction of canola proteins by using electro-activated aqueous

solutions

4. To study and analyse the impact of electro-activation on the physico-chemical and

functional properties of canola proteins.

5. To incorporate the proteins extracted by the electro-activation technology in a gluten

free food matrix (biscuits) so as to verify their behaviour in real food systems.

60

3. CHAPTER 3: Total dry matter and protein extraction from canola

meal as affected by pH and salt addition and the use of Zeta-

potential/turbidity to optimize the extraction conditions

Contextual transition

Nowadays there is a need in alternative protein sources with high nutritive value and adequate

functional properties. One of the possible sources is canola, a leading oilseed crop in Canada.

Although there are works devoted to the extraction of canola proteins the amount of cultivars,

different pre-treatments techniques and tests make it difficult to choose the optimal extracting

conditions and give sometimes contradictory information. In our case canola meal as a by-

product of oil production was directly obtained from the industry and was used for all the

analysis. The aim was to screen the number of factors affecting the extractability and to see

which protein profiles are extracted. It was also of interest to verify whether conditions

optimal for protein extraction also give the highest dry matter extractability.

This chapter is presented as an article entitled: “Total dry matter and protein extraction from

canola meal as affected by pH and salt addition and the use of Zeta-potential/turbidity to

optimize the extraction conditions”.

The authors are: Alina Gerzhova (Ph. D. candidate: planning and realization of the

experiments, results analysis and manuscript writing), Martin Mondor (Thesis co-director:

scientific supervision, article correction and revision), Marzouk Benali (Scientific

collaborator of the project: correction and revision of the manuscript) and Mohammed Aïder

(Thesis director: scientific supervision, article correction and revision).

61

Résumé

La matière sèche et les protéines ont été extraites à partir de tourteau de canola sous

différentes conditions. Nous avons observé que le pH du milieu d'extraction, la concentration

du tourteau de canola et la concentration du sel ont des effets différents sur l’extractibilité de

la matière sèche totale et des protéines, avec le pH ayant l’effet le plus significatif. Le plus

fort taux d'extractibilité de la matière sèche (42.8 ± 1.18%) a été obtenu avec 5% de tourteau

de canola à pH 12 sans addition de sel, tandis que l'extractibilité maximale des protéines

(58.12 ± 1.47%) a été obtenue avec 15% de tourteau sous les mêmes conditions. La plus

faible quantité de matière sèche extraite (26.63 ± 0.67%) a été obtenue avec 5% de tourteau

de canola à pH 10 sans addition de sel, alors que la plus faible quantité des protéines a été

obtenue avec 10% de tourteau à pH 10 et 0.01 M NaCl. Les analyses de turbidité et du

potentiel zeta ont démontré que le rendement maximal de protéine est atteint à pH 4 – 5;

intervalle de pH correspondant au point isoélectrique de la majeure partie des protéines de

canola. Les analyses réalisées par SDS-PAGE ont démontré que l'addition du sel augmente

la solubilité des protéines de canola, particulièrement la fraction de la cruciférine, alors que

le sel pourrait également réduire l'extractibilité de la napine.

62

Abstract

Total dry matter and proteins were extracted from canola meal (CM) under different

conditions. It was observed that pH of the extraction medium, CM concentration, and salt

concentration influenced the extractability of total dry matter and proteins, with the pH of the

extracting medium having the most significant effect. The maximum extractability of total

dry matter (42.8 ± 1.18%) was obtained with 5% CM at pH 12 without salt addition whereas

the maximum total protein extractability (58.12 ± 1.47%) was obtained with 15% CM under

the same conditions. The lowest amount of dry matter (26.63 ± 0.67%) was obtained with

5% CM at pH 10 without salt addition and the lowest protein amount was observed with 10%

CM at pH 10 in 0.01 M NaCl. Turbidity and ζ-potential measurements indicated that the

optimum pH for obtaining the maximum yield of protein was within the range of pH 4 and

pH 5. In our investigation, Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-

PAGE) showed that although, addition of salt increased the solubility of canola proteins

specifically the cruciferin fraction, it reduced the extractability of napin.

63

Introduction

Canola is Canada's one of the most important and valuable crop. Canada is the largest

canola producer and exporter in the world. It is not only a principal source of vegetable oil

but also the second most important oilseed crop after soybean (Pan et al., 2011). It is used for

producing edible oils with a high content of monounsaturated fatty acids and low levels of

saturated fatty acids. Canola meal is a by-product of oil extraction with a high protein content

and dietary fiber. Depending on the type of meal it can contain up to 50% protein (dry basis)

and 20% carbohydrate (Aider and Barbana, 2011; Yoshie-Stark et al., 2008). Canola protein

content and amino-acid composition are similar to soybean and it can be used not only as

forage for livestock, but in the food industry owing to its interesting functional properties

(Aider and Barbana, 2011; Aluko and McIntosh, 2001; Fleddermann et al., 2013). Canola

protein has also been attributed with bioactive properties (Aider and Barbana, 2011; Khattab

et al., 2010; Wu and Muir, 2008; Yoshie-Stark et al., 2006; Yoshie-Stark et al., 2008).

However, the complex composition of canola proteins (Gillberg and Törnell, 1976;

Kodagoda et al., 1973b; Tzeng et al., 1990b; Wu and Muir, 2008) makes the standardization

of extraction conditions a challenging task. Here it is important to note that a high alkaline

medium (pH values ≥10) significantly increases the protein extractability. In addition, it has

been also stated that the extraction of phytic acid (an anti-nutritional factor) remains low at

high alkaline conditions (Ghodsvali et al., 2005; Tzeng et al., 1990b; Tzeng et al., 1988a).

The effect of extraction conditions on the composition, amino-acids and functional

properties of the extracted protein isolates have been previously investigated (Aluko and

McIntosh, 2001; Khattab and Arntfield, 2009; Pedroche et al., 2004; Tzeng et al., 1988b;

Tzeng et al., 1988a; Xu and Diosady, 1994; Yoshie-Stark et al., 2006). There also have been

attempts to reduce the amount of anti-nutritional factors (glucosinolates, polyphenols and

phytic acid) present in the meal as they might negatively impact the organoleptic and

nutritional properties of the final product (Diosday et al., 1984; El-Kadiri et al., 2013;

Ghodsvali et al., 2005). The influence of the solvent used for defatting the meal on the protein

yield has also been investigated (Tzeng et al., 1990b). The polypeptide profile and the

composition of canola proteins has been studied by Aluko and McIntosh (2001); Klockeman

et al. (1997); Mimouni et al. (1990); Wu and Muir (2008). The two most abundant protein

fractions present in canola meal are the 12S globulin known as cruciferin and 2S albumin

64

known as napin. Since canola proteins mainly consist of water soluble albumins and salt

soluble globulins, the most common extraction technique employed are alkaline extraction

using aqueous NaOH solution and extraction with salt solutions such as CaCl2, sodium

hexametaphosphate (SHMP) and NaCl (Tan et al., 2011a; Tzeng et al., 1988b; Tzeng et al.,

1988a). A method known as protein micellar mass (PMM) has also been reportedly used,

where the meal is dissolved in a buffer containing 0.1 M NaCl/0.1 M NaH2PO4 (Ismond and

Welsh, 1992). An extraction method based on solubility of canola proteins in different

solvents (water, salt solution, acid or alkali and aqueous alcohol) known as Osborne scheme

has also been performed (Manamperi et al., 2012; Tan et al., 2011b). Nevertheless, despite

the numerous methods developed for the extraction of canola protein the conventional

extraction method, commonly known as direct alkaline extraction (DIR) that uses alkaline

medium, remains the most effective and utilised method. In addition, it is also a reference

method when newly developed techniques and processes are to be compared for their

efficiently and efficacy (He et al., 2013).

Alkaline extraction is traditionally followed by isoelectric precipitation. Canola

proteins have two isoelectric points, one in the range of pH 3-5 and the other between pH 6-

8 (Ghodsvali et al., 2005; Pedroche et al., 2004; Quinn and Jones, 1976; Tan et al., 2011b).

Isoelectric point is related to the composition of the side chains of proteins, and because the

type of protein can vary based on the type of canola meal investigated, contradictory

information regarding isoelectric points has been found in the literature. Ultrafiltration and

diafiltration can also be used following alkaline extraction for separating the extracted

proteins from the solvent (Tzeng et al., 1990b). Most of the studies done concerning

isoelectric point (pI) determination, use either isoelectric focusing or titration of the

supernatant with HCl with subsequent determination of nitrogen in precipitate in relation to

the total nitrogen extracted and plotting the values vs pH (Ghodsvali et al., 2005; Pedroche

et al., 2004; Tan et al., 2011b).

The quality of literature available highlights the importance of this topic; however,

most studies mainly focus on the characterization of the obtained protein isolates, and not on

the extractability of proteins and dry matter. Furthermore, apart from optimizing the

parameters for protein extraction, extraction of carbohydrates is also of particular interest as

it may have beneficial impact on the functional properties (Shao et al., 2013). This study

65

therefore, has been done to optimize parameters for protein extractability and total

extractability. Additionally it also compares the conditions optimal for protein and total

extractability. Specifically, the first objective was to determine the optimal conditions (pH,

ionic strength and solvent to meal ratio) for canola extraction and ensure maximum

extractability, to also understand the influence of these parameters on the total and protein

extractability, using the conventional technique. The second objective was to analyze and

understand the dynamics of precipitation of canola proteins at their isoelectric point by

studying the charge on the surface of particles (zeta potential) in suspension, in combination

with turbidity. This would provide useful information concerning the optimal conditions for

protein precipitation. The chosen method is not only efficient but also it is faster compared

to other conventional methods reported in the literature.

Materials and methods

3.6.1. Materials

Defatted canola meal was kindly provided by Bunge ETGO, Becancour, Québec,

Canada. Sodium hydroxide (NaOH) was purchased from VWR International LLC (West

Chester, PA, USA). Concentrated hydrochloric acid (HCl) was purchased from Fisher

Scientific (Mississauga, ON, Canada) and sodium chloride (NaCl) was purchased from

Caledon Laboratories LTD (Georgetown, ON, Canada).

3.6.2. Proximate analysis

Proximate analysis of the industrial canola meal was performed according to AOAC

International (2012). Moisture and total dry matter were determined by drying the weighed

sample in a Fisher Isotemp Vacuum Oven (Fisher Scientific, Montreal, QC, Canada)

according to method 925.09. Ash content was determined according to method 923.03 by

combusting a weighed portion of 5 g in the muffle oven until constant weight and the ash

content was expressed as the ratio between the weight sample after combustion and its dry

weight before combustion. Crude protein was determined following the Dumas method

992.23 with LECO Truspec FP-428 (Leco Corp., St. Joseph, MI, USA) using a conversion

factor of 6.25. Residual oil in the defatted meal was determined by Soxhlet extraction

66

according to method 925.85 using petroleum ether as extraction solvent. Carbohydrates were

determined by difference (Karaca et al., 2011a).

3.6.3. Extraction

Industrially defatted canola meal was weighed and dispersed in distilled water to give

final concentrations of 5%, 10% and 15% (w/w). For the experiments aiming to determine

the influence of the ionic strength on the extraction, canola meal was dissolved in NaCl

solution of the following concentrations: 0.01; 0.1 and 1 M. For the other values, the

extractability has been interpolated assuming the linear behavior. Extractions were carried

out at room temperature, for 1 h, under constant agitation using a magnetic stirrer. The pH of

the solutions was adjusted by the addition of 0.1–2 M NaOH solution dropwise so as to reach

the pH values of 10, 11 and 12 and maintained constant by the addition of aqueous NaOH as

required.

The slurry was subsequently centrifuged at 10 000 g during 30 min at room temperature so

as to separate insoluble material using Sorvall centrifuge RC-5C (Thermo Fisher Scientific,

Waltham, Massachusetts, USA). The supernatant was collected and the residues were freeze-

dried. All the extractions were performed in triplicate and the mean values were determined.

3.6.4. Protein precipitation

For the determination of turbidity and zeta potential the extract was prepared using

the following concentrations: 5% (w/w) of canola meal, dispersed in water with subsequent

pH adjustment with NaOH until a value of 12 is reached. These conditions were chosen as

those giving high percent of extractable nitrogen and total dry matter. Total time of extraction

was 1 hour. The slurry was centrifuged as described before. The supernatant was collected

and the isoelectric precipitation was performed by continuous addition of HCl with different

molarities (1 M-0.01 M) depending on the pH (higher concentrations were needed while

operating with the extremums) so as to reach the pH values from 12 to 2 with decrement of

1 pH unit and subsequently left for 2 h to allow aggregation.

3.6.5. Total dry matter and protein extractability

In order to determine the total extractability the aliquote of the supernatant in each

case was weighed and dried in the oven until constant weight. The extractability was

67

calculated as a ratio of dry matter in supernatant over total dry matter content. In order to

exclude the minerals added during the extraction the ash was determined in each case and

subtracted from the total extractable matter. Nitrogen content of the supernatant was

measured using Dumas method with LECO Truspec FP-428 (Leco Corp.). The aliquote of

the supernatant was weighed and oven dried overnight at 60°C before being analyzed. The

extractability was expressed as a function of nitrogen content in supernatant in relation to

nitrogen content in canola meal on a dry basis.

3.6.6. Turbidity and Zeta Potential (ζ) measurements

Turbidity was measured in nephelometric turbidity units (NTU) using The Hach

Model 2100AN Laboratory Turbidimeter (HACH LANGE GmbH Willstätterstr. 11, D-

40549 Düsseldorf, Germany). Samples adjusted to pH from 12 to 8 were used without

dilution whereas sample adjusted to pH values from 7 to 2 were analyzed using the dilution

factor of 2. All measurements were taken in triplicate and average values were recorded.

The zeta potential was measured as described by Narong and James (2006) using Zetasizer

2000 (Malvern Instruments Ltd., UK). This instrument measures the electrophoretic mobility

and the zeta potential on the particles in the solution using Laser Doppler velocimetry (LDV).

Samples adjusted to values from pH 12 to pH 2 were diluted to a concentration of

approximately 0.01% (w/w) using distilled water prior to the analysis, left for stabilization

for 2 h and subsequently analyzed. The signal recorded by the equipment was converted to

zeta potential according to Henry`s equation:

= ( ) (Eq. 3.1)

Where - Zeta potential, - electrophoretic mobility, - viscosity of the solution, -

dielectric constant, ( ) – Henry`s function that measures the ratio between the radius of

the particle and the thickness of the electric double layer. For the aqueous media and

moderate electrolyte concentration ( ) is 1.5, used in the Smoluchowski approximation.

The experiments were done at room temperature (23°C).

3.6.7. SDS-Gel electrophoresis

To assess the impact of tested conditions on the polypeptide profile of extracted

proteins the Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) was

68

performed following the method of Aluko and McIntosh (2001). Based on the results of

proteins yield at various conditions 4 samples were chosen for the test such as canola meal

and canola proteins extracted at pH 10, 11 and 12 with and without the addition of 1 M NaCl.

Specifically, 1% protein solutions were prepared and left overnight, followed by dilution with

deionized water so that the amount of proteins loaded onto the gel was approximately 20 µg.

Subsequently, diluted samples were dissolved in Tris-HCl buffer solution containing SDS

for non-reduced conditions. For reduced conditions samples were dissolved in Tris-HCl

buffer with addition of 5% (v/v) β-mercaptoethanol. Afterwards, the samples were boiled for

5 min, vortexed and 10 µl of each sample were loaded onto 4–20% gradient gels (#456-1093,

Bio-Rad, Canada). Precision plus Protein Kaleidoscope Standards of 10-250 kDa (#161-

0375, BioRad, Canada) were used as molecular weight standards. After staining and

destaining the gels were scanned using Chemi Doc XRS 170-807 (Bio-Rad, Hercules, CA,

United States of America).

3.6.8. Experimental design and statistical analysis

A full factorial experimental design was used to study the effect of different

experimental conditions on total extractability of the dry matter from canola meal obtained

after oil extraction from canola seed harvested in the Becancour region, Quebec, Canada.

The same conditions were studied regarding their effect on differential protein extractability.

The obtained data were used to optimize the experimental conditions. Zeta potential and

turbidity measurements were used to obtain the most favorable conditions for high protein

recovery from the total dry matter extracted from the meal. Each treatment was carried out

in a triplicate and mean values ± SD were calculated and used for the comparisons between

the different treatments at 95% confidence level with a protected LSD test. Detailed analysis

of the variance (ANOVA) was performed by using SAS software (Version 9.1, SAS

Institute, Cary, NC, USA). A decomposition of the ANOVA was also used to validate the

significance of each individual independent variable. Minitab software (Version 16, Minitab

Inc, State College, PA, USA) was used for the optimization of the extraction conditions.

69

Results and discussion

Proximate chemical composition of canola meal is presented in the Table 3.1. The results

are in accordance with those reported in the literature before (Pedroche et al., 2004; Tan et

al., 2011b).

Table 3.1: Proximate chemical composition of canola oil cake on a dry weight basis.

Moisture (%)

Protein (N*6.25,%)

Ash (%)

Fat (%)

Carbohydrate (%, by difference)

Total phosphorus (%)

10.51 ± 0.01 43.36 ± 0.71 7.19 ± 0.01 3.58 ± 0.07 35.36 2.21 ± 0.48

3.7.1. Extractability

In order to determine the influence of different parameters the total and protein

extractability were measured as a function of pH, canola meal concentration (solvent to meal

ratio) and salt concentration (ionic strength). In the next sections the impact of each of these

parameters on the extractability will be discussed.

3.7.1.1. Effect of pH

In the first experiment the effect of pH on the total dry matter and protein

extractability was studied. It has been previously shown that pH has a major influence on the

extractability of canola proteins (Ghodsvali et al., 2005; Klockeman et al., 1997; Nioi et al.,

2012). More alkaline medium allows more protein to be solubilized and this effect is more

important at low CM concentration. Similar tendency can be noticed regarding total dry

matter extractability. However, the gain in total extractability with an increase of the

extraction pH is less important than the one observed for protein extractability as shown in

Figures 3.1 and 3.2.

70

* CM – canola meal

Figure 3.1: Influence of the solution pH on the total dry matter extractability from canola meal.

* CM – canola meal

Figure 3.2: Influence of the solution pH on the protein extractability from canola meal.

0

10

20

30

40

50

60

10 11 12

Ext

ract

abili

ty,%

pH

CM 5%

CM 10%

CM 15%

0

10

20

30

40

50

60

10 11 12

Ext

ract

abili

ty, %

pH

CM 5%

CM 10%

CM 15%

71

There is a significant increase in protein extractability, by as much as 2.5 times, when the pH

is increased from 10 to 12 and by as much as 1.7 times for total extractability. Earlier studies

however reported higher results (Gillberg and Törnell, 1976; Quinn and Jones, 1976; Tzeng

et al., 1988a). In such conditions they managed to extract more than 85% of the proteins. In

our case this range of pH allowed to extract from 22 to 58% of canola proteins and from 26

to 43% of total dry solids which is supported by more recent works (Ghodsvali et al., 2005;

Pedroche et al., 2004).

On the other hand, extraction at pH > 10 has a negative effect on proteins

functionality. Some authors reported poorer functional properties of the proteins obtained at

harsh alkaline conditions such as pH 12 as a result of protein denaturation (Jyothirmayi et

al., 2006; Pedroche et al., 2004). These authors reported that only protein isolates obtained

at pH 10 had functional properties comparable to those of the defatted meal. In this context

the extraction at pH=10 would result in proteins with better functionality but would at the

same time result in the lowest yield.

3.7.1.2. Effect of canola meal concentration

Extractable protein increased from 22.4% to 28.5% and the total dry matter increased

from 26.6% to 29% as the concentration of CM increased from 5% to 15% for pH 10. For

pH 11 and 12 the corresponding increases were 29.5% to 39.8%, 30% to 33.1%, 53.9% to

58.2 and 42.8% to 42%, respectively (Figures 3.1 and 3.2). Most authors used 5%

concentration of CM or 1:18 solvent to meal ratio (Ghodsvali et al., 2005; Klockeman et al.,

1997; Tzeng et al., 1990b; Tzeng et al., 1988a) or 10% (Aluko and McIntosh, 2001; Pedroche

et al., 2004). As it might be noticed CM concentration influences the total extractability

mainly at lower pH values (pH 10) whereas at pH 11 this effect is reduced and at pH 12 there

is even a slight decrease in total extractability. Protein extractability increases with an

increase in CM concentration at all tested pH values. As the reduced use of solvents is more

desirable the 15% concentration seems more attractive. On the other hand extraction at such

a high concentration is complicated by its high viscosity. So a concentration of 10% would

be optimal.

3.7.1.3. Effect of salt concentration

It is generally recognized that salt in low concentrations enhances the protein

solubility due to “salting-in” effect which is explained by the interactions between ions in the

72

solution and the protein charges. These interactions weaken the protein-protein interactions

and increase the protein solubility. At higher salt concentration, on the contrary, the salting-

out effect takes place resulting in lower solubility due to the binding of the water molecules

by the ions of salt.

There is limited information about the effect of salt on the extractability of canola

proteins. Most of the studies analyzed the influence of ionic strength on their precipitability.

Thus Tzeng et al. (1990b) reported high protein yield due to “salting-in” effect that allowed

them to obtain 80% yield. In their study the 0.15 M CaCl2 was added during the precipitation

step in order to obtain the soluble protein. Ghodsvali et al. (2005) used the addition of 15%

CaCl2 also during the precipitation step. In the study of Aluko and McIntosh (2001) 0.02 M

and 0.15 M CaCl2 was added after the pH adjustment to 6. A lower protein yield in calcium

precipitated proteins in comparison with acid precipitated proteins has been reported. Thus,

the 6-16% reduction of protein yield was observed with salt-coagulated proteins compared

to those of acid-coagulated proteins (Soetrisno and Holmes, 1992). The authors explained

that fact due to higher pH used for salt-coagulated proteins (pH 6 compared to pH 4 for acid-

coagulated proteins).

Regarding the extractability of the proteins from other than canola sources, it was

reported that the addition of salt (0.1-0.5 M) broadened the pH range of the minimum

solubility and shifted it to the lower values (Eromosele et al., 2008; Jyothirmayi et al., 2006).

The salt addition has significantly increased the protein extractability at neutral pH region

and even at the range of minimum solubility close to the isoelectric point as shown in the

work of Oomah et al. (1994) for flaxseed meal. In the study of Lazos (1992) the protein

extractability from pumpkin seeds was significantly increased (from 4.5-8.7% to 12-55% )

at their minimum solubility with an increase in sodium chloride concentration. However, the

decrease in protein solubility was noticed in the range of pH 8-11. The work of Jyothirmayi

et al. (2006) showed higher nitrogen extractability at isoelectric point when sodium chloride

was added (60% compared to 26.8%). On the other hand the authors note that at pH 12 the

amount of nitrogen extracted was insensitive to the ionic strength. This might be explained

by the denaturation of globulin proteins at harsh alkaline medium, by various conformations

of protein molecules at different pH values, and by their interactions with other compounds

present in the meal (phytates, sugars, fibres). However, an increase in solubility usually is

73

associated with a decrease in the recovery yield of the precipitate (Gillberg and Törnell,

1976). The authors reported that only 25% of the dissolved proteins were recovered. As it

can be seen in Figures 3.3 to 3.5, our results revealed that the addition of salt influenced in

a different manner the total and protein extractability. In order to exclude the influence of

pH, the data will be compared within constant pH values at pH 10, 11 and 12, respectively.

3.7.1.3.1. Extraction at pH 10

Figure 3.3: The effect of salt concentration on total dry matter (a) and protein extractability (b) at pH 10.

a

b

74

Figure 3.3 shows the extrapolated results of the influence of NaCl concentration at

pH 10. Globally, the addition of salt had a positive effect on the extractability at all CM

concentrations. For protein extractability 5% CM concentration and 1 M NaCl will be the

most favorable conditions. In comparison with salt-free extraction the 0.01 M concentration

of NaCl didn`t influence the extractability at CM concentration of 5% (22.38 to 22.39% for

protein extractability and 26.63 to 26.90% for total extractability) and even a minor decrease

was observed in our case for 10% and 15% CM concentration (25.62 to 22.12; 28.03 to 27.36

and 28.53 to 25.74; 29.08 to 27.94 for protein and total extractability and CM concentration

of 10% and 15% respectively.), (data not shown). The 0.1 M concentration had some positive

effect (32.51 ± 0.31 % of total dry matter and 24.89 ± 0.91 % of proteins with 5% CM

concentration) whereas with 1 M NaCl at pH 10 and the concentration of CM 5% there was

a double increase in protein extractability and almost double increase in total extractability

22.39 ± 2.52 % to 40.87 ± 3.48 % and 26.63 ± 0.77 % to 39.88 ± 0.48 % respectively).

Therefore, the maximum yield was observed at the maximum concentration of salt e.g. 1 M

and at the concentration of canola meal of 5%. The optimal conditions at pH 10 will be 0.5

– 1 M NaCl and 5 – 6% CM concentration.


The extrapolated results for pH 11 are shown in Figure 3.4. As it can be seen the

conditions the most favorable for total extractability are not optimal for protein solubility.

Thus, the optimal conditions for total extractability are similar to those ones obtained at pH

10 e.g. 0.7 – 1 M NaCl concentration and 5 – 7% CM concentration where the increase in

protein extractability is not significant. For proteins the optimum is in the range of 8 – 15%

of CM concentration and NaCl 0.3 – 0.9 M. However, in comparison with the extraction at

pH 10 the increase observed with the addition of salt was lower and a 10% increase was

revealed in this case with the maximum yield for total dry matter at 5% CM concentration

and 1 M NaCl (from 29.98 ± 1.22% to 39.37 ± 3.13 %). Concerning the extractability of

proteins a 12% increase was observed in the whole range of CM concentration whereas

within one concentration the increase was only 4%. Maximal values were obtained at 0.1 M

NaCl and 15% CM concentration (40.57 ± 0.35 %), although with no salt added and under

75

the same conditions the results were (39.79 ± 3.54%) showing no statistically significant

difference (data not shown).


a

b

76


The effect of salt addition at pH 12 is shown in Figure 3.5. The pattern observed for

both extractabilities is also different. For the total extractability the tendency was similar to

those observed at pH 10 and 11 with the optimum in the range of maximal salt concentration

(0.6 – 1 M) and minimal CM concentration (5%). As for the protein extractability the

decrease is observed with an increase in salt concentration being minimal within the range of

0.4 – 1 M NaCl concentration and 5 – 10% CM concentration. In this case the maximal yield

is obtained with no salt added and at 15% CM concentration (58.12 ± 1.18%) (data not

shown). This value decreases to 42.99 ± 0.24% with an increase in salt concentration for 15%

CM concentration and from 53.91 ± 5.18% to 34.91 ± 0.93% and 54% to 39% for 5% and

10% CM concentration respectively (data not shown). In spite of the decrease the percentage

of extracted matter in general exceeds the levels extracted at lower pH values. However this

effect is due to the influence of pH rather than the influence of salt addition.

Our results demonstrated that the salt addition had a positive impact on extractability

only at pH 10 whereas at pH 11 this effect was minor and at pH 12 a negative effect was

observed. This is consistent with the data in the literature (Oomah et al., 1994). However, in

the work of Klockeman et al. (1997) an increase in protein extractability with an increase in

salt concentration at pH 11 has been noted. The NaCl concentration of 1 M does have an

effect on the extractability at lower pH values whereas lower concentrations such as 0.1 and

0.01 M have less pronounced influence. In spite of the positive effect of NaCl addition the

maximum protein extractability was observed in salt-free extraction at pH 12. Therefore, salt

extraction would be useful when a highly alkaline medium is not desired as in case when

functional properties need to be preserved.

77


3.7.2. Protein and ash composition of extracts

Protein content of extracts obtained at pH 10 without NaCl ranged between 27-32% with ash

being ~12% on a dry basis. When 1 M NaCl was used the percentage of protein was lower

(8-16% on a dry basis) due to the high content of salt added during extraction (60-70% of

a

b

78

ash) (Figure 3.6). At pH 11 and 12 the protein content was 32-37% and 39-45% respectively,

whereas ash composed 13-15% and 14-19% of the total dry solids. For 1M NaCl

concentration extract obtained at pH 11 had 8-19% protein and 55-75% ash and extract

obtained at pH 12 had 8-21% of protein and 56-66% of ash on a dry basis. The utilization of

1 M NaCl for protein extraction requires further desalting in order to purify the extracted

fractions.

Figure 3.6: Protein and ash content of extracts obtained with 10% canola meal concentration.

3.7.3. Precipitability based on Zeta Potential and Turbidity

The extract obtained during the first step represents a colloid system. This means that

solubilized particles are dispersed in the solution. Various repulsive and attractive forces are

involved and interactions between particles influence the stability of the system. The

character of these interactions dictated by the surface charge of the particles predetermines

the behavior of the system. The stability of such a system depends first of all on the pH of

the medium. It is a well-known fact that proteins are able to change their surface charge

depending on the pH of the medium. As the proteins have acid and basic groups their charge

depends on the pH and ionic strength of the medium (Benítez et al., 2007).

In order to obtain a desired protein yield the understanding of the precipitation

behavior and electro-kinetic characteristics is needed. One of the readily measurable electro-

kinetic characteristics is zeta potential which is an expression of the charge found on the

0

10

20

30

40

50

60

70

80

90

pH

10

pH

11

pH

12

pH

10

pH

11

pH

12

pH

10

pH

11

pH

12

pH

10

pH

11

pH

12

no NaCl 0.01 M NaCl 0.1 M NaCl 1 M NaCl

Pro

tein

an

d a

sh c

on

ten

t o

n a

dry

ba

sis,

%

Ash

Proteine

79

surface of the suspended particle. When the net charge of the particles is the same, the

repulsive forces are predominant which prevents their aggregation so that they remain in

suspension. On the other hand, when the net charge becomes neutral, the repulsive forces

become less important which results in the aggregation and precipitation of suspended

particles. It is known as isoelectric point precipitation.

Our study was not aimed to determine the effect of ionic strength on the zeta potential

so the measurements were taken under the same extraction conditions (5% CM concentration

and pH 12). The effect of pH on the zeta potential is shown in Figure 3.7. For canola meal

as it might be seen, the proteins carry net negative charge in the alkaline medium with

maximum value of net charge at pH 10. Further decrease in pH values causes a decrease in

net charge leading to the isoelectric point. However, at more alkaline medium there is an

increase in zeta potential which is probably due to the nature of proteins. As a matter of fact

one of canola`s main proteins is napin which is characterized by strong alkalinity due to a

high level of amidation of its amino acids (Aider and Barbana, 2011).

Figure 3.7: The influence of the solution pH on the Zeta potential of the canola protein.

The minimum net charge is observed at pH values around 4.3 which indicates that the

isoelectric point is in the range of pH 4-5. This is in agreement with previous works where

the highest precipitability has been reported at pH values between 4.5 and 5.5 (Ghodsvali et

al., 2005; Tan et al., 2011b). On the other hand, Quinn and Jones (1976) reported minimum

solubility for canola proteins at pH values 3.7-4.0 and 7.7-8.0 and Pedroche et al. (2004)

-70

-60

-50

-40

-30

-20

-10

0

10

20

30

2 3 4 5 6 7 8 9 10 11 12

Ze

ta p

ote

nti

al,

mV

pH

80

indicated two isoelectric points at pH 3.5 and 5. This might be due to different types of meal

used for the experiments with different profiles composition.

Another way of evaluating the maximum precipitability is by measuring the turbidity

of the suspension. Turbidity shows the amount of colloidal material present in the suspension.

The increase in turbidity is a reflection of an increase in flocculation rate. Therefore, the

maximum turbidity is supposed to be in the range of the minimal solubility of the proteins.

The turbidity has been measured over a range of pH values from 12 to 2. As it can be seen in

Figure 3.8 the maximum turbidity is in the range of pH 4-6 with its peak at pH 5. Initially

with no pH adjustment at pH 12 the turbidity of the slurry is close to zero. In the beginning

there are almost no changes in turbidity over the range of pH 12 to pH 9. The alkaline medium

corresponds to maximum solubility of the proteins; the repulsive forces are strong enough

which prevents the aggregation of the particles. When the pH becomes lower than 9 a rapid

increase in turbidity is observed which continues until reaching a pH of 5. Further decrease

in pH results in decrease of turbidity.

Figure 3.8: The influence of the solution pH on the turbidity of canola proteins solution.

0

2000

4000

6000

8000

10000

12000

2 3 4 5 6 7 8 9 10 11 12

Tu

rbid

ity

, N

TU

pH

81

The correlation coefficient between the zeta potential and the turbidity is -0.8 with

p=0.003 indicating strong linear relationship which can be noticed from Figure 3.7 and

Figure 3.8. The maximum turbidity is found at pH values between 4 and 5, similarly the

minimum net charge is also found at pH values between 4 and 6. This means that close to the

isoelectric point there is no energy barrier preventing flocculation so the turbidity of

suspension at this point is the highest. Slightly higher values of the turbidity might correspond

to complex protein composition of canola proteins. The range of pH 4 – 6 is the zone where

the proteins are the least stable. Even minor fluctuations in pH result in changes on the surface

of the particle.

3.7.4. Gel Electrophoresis

The results of SDS PAGE are presented in Figure 3.9A and 3.9B. Reducing and non-

reducing conditions were followed by 2 tests with and without NaCl addition. The aim was

to verify whether or not the salt addition influences the protein profiles extracted under pH

10, 11 and 12 and to which extent. As it can be seen, pH 10 and 11 give more prominent

protein profiles in comparison with pH 12 which is due to the protein denaturation in highly

alkaline medium leading to poor protein solubility.

It is known that canola`s main protein fractions are napin and cruciferin which differ

by their molecular weights, sedimentation coefficients, and functionalities. Napin is a 2S

albumin, whereas cruciferin is a 12S globulin. They also differ by their isoelectric points

which complicate their production (Manamperi et al., 2012).

The influence of salt addition can be regarded at pH 10 and 11 showing more

pronounced bands in Figure 3.9A for the molecular weights of 45-50 kDa and 27-30 kDa.

The former band disappeared under reducing conditions indicating the presence of disulfide

bonds whereas the latter became more intense due to formation of new polypeptides implying

other than disulfide linking. Fraction of 18 kDa becomes more distinct under reducing

conditions unlike the study of Aluko and McIntosh (2001) in which this fraction disappeared.

These bands can be assigned to cruciferin fraction which according to the literature is

presented by following molecular weight profiles: 50 kDa, 29.5 kDa and a minor band of 44

kDa (Wu and Muir, 2008).

82

Figure 3.9: SDS-PAGE of canola proteins A) without NaCl addition and B) with an addition of 1 M NaCl.

83

Napin is a low molecular weight basic protein that consists of two polypeptide chains

linked together by disulfide bonds. It usually constitutes from 15 to 45% of total canola

proteins with the molecular weight ranging from 12.5-14.5 kDa. Napin’s fraction of 15 kDa

and a minor band of 27.5 kDa (a dimer of napin) being water soluble is clearer in the Figure

3.9A. Under reducing conditions the bands fade due to the cleavage of disulfide links which

are main stabilizing bonds for napin. New bands of 9 and 13 kDa appeared under reducing

conditions due to the formation of new polypeptides. They are more intense in the first case

where no salt was added, suggesting that these bands are the part of water soluble proteins

linked by disulfide bonds. This is in accordance to the works of Nioi et al. (2012) and Wu

and Muir (2008) where napin and its dimer appeared with their molecular weights of 14 kDa

and 27.5 kDa from purified fraction and dissociated into small polypeptide chains under

reducing conditions highlighting the presence of disulfide links.

On the whole, salt addition contributed to higher solubility of canola proteins

specifically cruciferin fraction although it reduced napin extraction. Therefore, the extraction

conditions should be chosen with regards to the aims of the work as it will have a direct

impact on the functional properties of obtained product.

Conclusion

The impact of extraction conditions on total and protein extractability was studied.

Among all the parameters tested, the changes in pH had the strongest influence on both

extractabilities. Maximum extractability was obtained with pH 12. Extraction with NaCl

significantly increased the extractability only at pH 10. Taking into account the information

that functional properties might be affected in case of highly alkaline medium, the

combination of low pH (i.e. pH 10) with addition of NaCl would be a better alternative to

obtain a high protein yield. However, this could also imply to remove the added salt prior to

use of the ingredient for food formulation. The study of zeta potential and turbidity revealed

a strong correlation between both parameters and might be further used in the determination

of isoelectric points when different extraction procedures are tested since both methods are

fast in comparison with titration and protein dosage on the supernatant or precipitate. The

SDS-PAGE analysis showed that salt addition contributes to higher solubility of canola

proteins specifically cruciferin fraction although it reduces napin extraction. Therefore, the

84

extraction conditions should be chosen with regards to the aims of the work as it will have a

direct impact on the functional properties of obtained product.

Acknowledgments

This work was financially supported by the innovation in food support program that

was funded by contracts through the "Growing Forward" Program that occurred between the

Ministère de l'Agriculture, des Pêcheries et de l'Alimentation du Québec (Ministry of

Agriculture, Fisheries and Food of Quebec) and "Agriculture and Agri-Food Canada".

85

4. CHAPTER 4: Monitoring of pH and alkalinity changes in the electro-

activated aqueous solutions generated in the cationic compartment.

The effect of salt concentration, current intensity, time and cell

configuration


The previous section showed that pH and salt concentration have the major impact on protein

extractability from industrially defatted canola meal. In the following chapter such conditions

were modulated by electro-activated solutions generated in the cathodic compartment of a 2-

and a 3-cell reactor. The study of factors such as type of configuration, time of treatment,

current intensity, and salt concentration on the pH and alkalinity of solutions is presented

hereafter.

This chapter is submitted as an article entitled: “Monitoring of pH and alkalinity changes in

the electro-activated aqueous solutions generated in the cationic compartment. The effect of

salt concentration, current intensity, time and cell configuration”.





(Thesis director: scientific supervision, article correction and revision)

86

Résumé

Les propriétés des solutions aqueuses électro-activées à l’interface cathode/solution

(catholyte), notamment le pH et l'alcalinité, ont été étudiées. Un plan factoriel complet a été

appliqué pour examiner les effets de l'intensité du courant, de la durée du traitement et de la

concentration du sel sur le pH et l'alcalinité de solutions électro-activées traitées selon 4 types

de configuration du réacteur d’électro-activation. Les configurations de réacteur à deux et

trois compartiments, séparés par une membrane échangeuse d'anions (AEM) ou une

membrane échangeuse de cations (CEM) ou par les deux, ont été testés. L'intensité du courant

électrique et le temps du traitement avaient les effets les plus significatives pour toutes les

configurations testées. D'autre part, la concentration du sel a un effet significatif seulement

lorsque les configurations 2 et 4 ont été appliquées en raison de la disposition des membranes

dans le réacteur. Les valeurs maximales de pH et d'alcalinité ont été obtenues avec I = 0.2 A,

t = 60 min à toutes les concentrations de NaCl pour les configurations 1 et 3. Les mêmes

valeurs pour les configurations 2 et 4 ont été obtenues uniquement avec la concentration

maximale de NaCl de 1 M. Malgré le pH fortement alcalin (pH ~ 12), l’alcalinité de la

solution était comparable à celle du NaOH dilué (~ 0.03 M).

87

Abstract

The properties of catholyte (pH and alkalinity) were investigated in the current study. A full-

factorial design was applied in order to examine the effects of current intensity, time of

treatment and salt concentration on the pH and alkalinity of electro-activated solutions treated

in 4 types of configurations. Two and three-cell reactor configurations were tested with

compartments separated by either anion exchange membrane (AEM) or cation exchange

membrane (CEM) or by both of them. Current intensity and time were found to be highly

significant for all tested configurations. On the other hand, the salt concentration had a

significant effect only when configurations 2 and 4 were applied due to the membrane

disposition in the reactor. Maximal values of pH and alkalinity (25.46 ± 0.31mmol/l) were

obtained with I = 0.2A, t = 60 min with all NaCl concentrations for the configurations 1 and

3. Whereas the same values for the configurations 2 and 4 were achieved only with maximal

NaCl concentration of 1M. Despite highly alkaline medium (pH~12) the strength of the

solution was weaker than chemical alkali solutions and was comparable to dilute NaOH

(~0.03M).

88

Introduction

Not long ago water was regarded as a biochemically passive substance. Its major role

was to act as a solvent where the transformations of active substances took place. Only the

interactions of organic and inorganic substances with water were studied while water

structure and changes of its properties were out of interest. However, about 50 years ago a

theory of a structured water was developed and the possibility of changing its properties

without any chemicals was recognized (Prilutskii and Bakhir, 1999). During the analysis, the

water subjected to magnetism, sound, illumination, electric field, heating or freezing showed

new properties. These properties had an impact on the kinetics of chemical reactions taking

place in the water, on its biological, solubilizing, and cleaning activity. Such water treated by

one of the listed methods was called “activated”, which comprises the sum of effects or new

properties appeared during technological treatments and without elemental chemical changes

(Aider et al., 2012b; Prilutskii and Bakhir, 1999).

Thus, the water treated under the influence of external electric field was called the

“electro-activated” (EA) water. The presence of an electric field supplies an external energy

making possible non-conventional chemical reactions due to the high reactivity of the

solutions (Aider et al., 2012b). Electro activated water found numerous utilization in

environmental and food industry (Huang et al., 2008) and currently is widely used mostly as

a disinfectant. Its bactericidal effect on many pathogenic bacteria was reported in the

literature (Kiura et al., 2002; Rahman et al., 2010). In addition, improved extractive and

catalytic function were also stated (Aider et al., 2012b; Aït Aissa and Aïder, 2013a).

Electroactivation as a technology of producing activated water with specific

properties is based on a well-known process of electrolysis with its main principles studied

in the early 19th century (Aider et al., 2012b). The simplest scheme comprises two electrodes

anode and cathode immersed in the aqueus solutions and a current (power) supply. A

potential difference between two electrodes enables the reaction to occur (Bazinet, 2005).

The changes in water activity are closely related to the oxido-reduction electrode reactions.

The main reactions which take place in the electrolyzing cell are water oxidation on the anode

with O2 liberation (Eq. 4.1), water reduction on the cathode with H2 release (Eq. 4.2),

formation of the gaseous chlorine on the anode (Eq. 4.3), formation of very active oxidants

in the anodic compartment such as Cl2 O, ClO2, ClO−, HClO, Cl•, O2•, O3, HO2, OH• and

89

formation of overactive reducing agents such as ОН−, Н3−О2

−, Н2, НО2•, НО2

−, О2− in the

cathodic compartment (Tomilov, 2002).

2Н2О − 4е→ 4Н+ + О2 ↑ (Eq. 4.1)

2Н2О + 2е→ Н2↑ + 2ОН− (Eq. 4.2)

2Cl− − 2е→ Сl2↑ (Eq. 4.3)

Thus, under the application of external electric field the two types of solutions are

generated. An electrolyzed basic solution is produced on the cathode side possessing strong

reducing potential and electrolyzed acid solution posessing strong oxidating potential is

generated on the anode side (Hsu, 2005).

The main drawback of such a scheme is the fact that the products of reactions mix. In

order to separate them different types of membranes are used such as porous non-permeable

diaphragme or permselective anion or cation exchange membranes or both (Mani, 1991). The

membranes properties and their selectivity are well-explained in the literarure and can be

found elsewhere (Baker, 2012).

External electric field creates unique prerequisites for the reactions which under usual

conditions cannot be performed. As an example the simple reaction of NaCl hydrolysis under

normal condition will flow in the opposite direction but the application of external energy

allowed chlorine and NaOH production, which is currently used in the industry. A two-cell

reactor and a CEM are used for such a reaction. As a result chlorine ions are discharged with

gas release on the anode and Na+ ions migrate to the adjacent compartment where H+ ions

form gaseous H2 while Na+ ions together with OH- form alkali (Shaposhnik, 1999).

The differences between electroactivation and electrodialysis are not evident at first

glance. However, when water splitting by electricity is a physico-chemical transformation of

the medium composition leading to the appearance of H+ and OH- ions, acids, hydroxides,

peroxides, radicals and hypochlorites, the electroactivation assumes the acquisition of new

properties which cannot be explained by usual chemical reactions. Thus, the anolite and

catholite gain such properties namely pH and oxido-reduction potential which cannot be

modulated in usual chemical solutions (Leonov et al., 1999; Tomilov, 2002). In general,

while the electric field is applied the composition of the water near anode and cathode

changes and it is accompanied with appropriate reactions. The maximum intensity is reached

in the near electrode layer called double diffusive layer where actually the process of

90

activation takes place. As a result after excitation by an external electric field the water falls

into a metastable state characterized by abnormal physico-chemical characteristics due to the

electrons activity in the water (Aider et al., 2012b; Tomilov, 2002).

Properties of electro-activated solution (EAS) have been extensively studied

(Belovolova et al., 2006; Petrushanko and Lobyshev, 2001). The vibrational spectra of EAS

were studied by means of Raman Spectroscopy (Pastukhov and Morozov, 2000). Their

results showed a significant difference in Raman spectra between 700 and 2700 cm −1 of the

electro-activated water taken in the near-anode (anolyte) and near-cathode (catholyte)

regions and chemically acidified and alkalinized water. The results of Raman spectroscopy

showed the presence of H+ or OH− ions in the anolyte and catholyte respectively which are

known to be responsible for acidic and alkaline properties. Furthermore, concentrated acid

and alkaline solutions showed the bands similar to the Raman Spectra bands of anolyte and

catolyte. As a result it was assumed that properties of electroactivated solutions may be

similar to those of concentrated acids and alkali (Aider et al., 2012b). Leonov et al. (1999)

also stated that electrochemically activated solutions have properties similar to those of

conventional acids and alkalis. In order to verify the feasibility of such a suggestion the

properties of catholyte generated under chosen conditions were analyzed in the current study.

Knowing the main parameters that influence the process of electroactivation and

using the specific configuration the desired degree of EA can be achieved. However, the

studies on the correlation between constructional characteristics of the electrolyzers and

technological parameters of the process on one side, and functional properties of

electroactivated solutions on another side, are scarcely reported in scientific literature. The

empirical adjustment of parameters is used in most sources (Aider et al., 2012b).

The aim of this study was to investigate the parameters that influence the pH and the

strength of the generated solution namely alkalinity in the cathodic compartment by studying

the effect of cell configuration, salt concentration, current intensity and the time of treatment

and to compare the obtained solutions with conventional base such as NaOH.

91


4.5.1. Chemicals

Sodium chloride (NaCl) was purchased from Caledon Laboratories LTD

(Georgetown, ON, Canada). Sodium hydroxide (NaOH) was purchased from VWR

International LLC, West Chester, PA, USA. Concentrated hydrochloric acid (HCl) and

sodium sulphate (Na2SO4) was purchased from Scientific Fisher, Canada, CAS.

4.5.2. Ion-exchange membranes

Anion (AM-40) and cation (CM-40) exchange membranes were purchased from the

Publicly Traded Company Schekina-Azot (Shchekina, Russian Federation). Prior to

utilisation both membranes were washed with 96%-ethanol to remove the wax and prepared

according to manufacturer’s instructions before their use in the electro-activation reactor.

4.5.3. Reactor design. Configurations of electro-activation cells

Electro-activation reactor was made of transparent plexiglass columns with the

dimensions of L50xW50xH120 mm. Three- and two-compartment electro-activation

reactors were used resulting in 4 different configurations. For the first configuration

consisting of three chambers and shown in the Figure 4.1 AEM separated the anode and

central parts, whereas CEM was placed on the cathodic side, separating it from central

section. Second configuration had the same three compartments but this time the membranes

were swapped places (Figure 4.4). Third and fourth configurations had two compartments

separated by CEM and AEM, respectively (Figure 4.7).

The RuO2–IrO2–TiO2 electrodes 120x34x1 mm with working active area of 40 cm2 were

placed at the extremities of the cells and were connected to the positive side of a direct electric

current power supply for the anodic compartment and to the negative side for the cathodic

one. The values of the applied voltage and the intensity of the electric current were measured

using a current power supply (CSI12001X, Circuit Specialists, Inc, USA).

92

4.5.4. Protocol of electro-activation

This study primarily focused on analyzing the catholyte and its properties. A full

factorial design was used to test the effect of cell configuration, salt concentration, current

intensity and time on two parameters, namely, pH and alkalinity of the solutions. The EA

was carried out at three constant electric current intensities of 0.05, 0.1 and 0.2 A providing

the galvanostatic state with total time of treatment of 10, 30 and 60 min.

To ensure the current flow salt solutions were circulated in all compartments. The

cathodic compartment was filled with NaCl solution of following concentrations 0.01, 0.1

and 1 M. Anodic and central (where applicable) compartments were always filled with

Na2SO4 solution of constant concentration corresponding to 0.25 M to avoid the liberation

of toxic chlorine (Cl2) on the anode.

4.5.5. Analysis methods

During each treatment, samples of 5 ml were taken every 2 min within the first 10

min and then every 10 min. The pH of the samples was measured using a pH meter (Model

SR 601 C SympHony, VWR Scientific Products, USA) after which the samples were

returned back to the cell to minimize the impact of sampling.

To determine the alkalinity simple titration method was used (Clesceri, 2005) with some

modifications. The electroactivated solution was titrated with 0.1M HCl until neutral pH was

reached. The volume of acid used to neutralize the alkali was noted and used for the

calculation of alkalinity:

= ( )∗ ∗ , / , (Eq. 4.4)

Where is alkalinity, ( ) is molar concentration of acid used for titration, is volume

of tested solution taken for analysis, is volume of HCl used to neutralize the alkali.

4.5.6. Statistical analysis

The analysis of the variance (ANOVA) at 95% confidence level was performed using

MiniTab software to test the significance (p ≤ 0.05) of each independent input variable on

pH and alkalinity of the catholyte.

93


4.6.1. Configuration 1

Configuration 1 is shown in the Figure 4.1 together with main reactions which take

place during the electro-activation. This configuration is characterised by two main

processes: desalination in the central compartment and concentration in the compartments

found at the extremeties. The cathodic compartment is separated from the central one by

CEM. This configuration allows the transfer of cations from the central compartment and at

the same time prevents anions from leaving the cathodic compartment. Water electrolysis

takes place on the cathode with H2 release so over some period of time the OH- ions are

accumulated leading to a pH increase.

Figure 4.1: Configuration 1 of the electro-activation reactor used for the generation of the electrolysed water.

4.6.1.1. pH evolution

It is known that pH is a measure of the hydrogen ions expressed as a negative logarithm of

H+ concentration. In other words, it is a measure of the acidity or basicity of an aqueous

solution. Initial pH values varied from 5.6-5.9 depending on the distilled water used in the

laboratory. When the direct current was applied, the pH of the solution increased drastically

within the first few minutes and after 10 minutes of electroactivation it reached a plateau.

94

Figure 4.2: The effect of current intensity and salt concentration over time on the pH monitoring of electro-activated solutions, configuration 1.

a b c

d e f

95

The effect of salt concentration and current intensity on the pH of the cathodic solution, over

time, is shown in Figure 4.2 All figures have the same tendency. One can notice from

Figures 4.2a - 4.2c that for all three concentrations the higher the current intensity, the higher

the pH obtained after 60 minutes of operation. In addition, an alkaline pH (e.g. pH=10) for

I=0.2 A is reached within one minute of EA, within 2.5 min for I=0.1 A and within 5 min for

I=0.05 A. This is to say that with higher current intensity a pH increase was performed faster

in comparison with lower current intensities. As it can be seen from Figures 4.2d-4.2f there

is no significant difference between the curves indicating that the salt concentration does not

affect the pH.

4.6.1.2. Alkalinity

Alkalinity is a measure of the capacity to neutralize acids, also called acid

neutralization capacity (ANC). The major substances that will affect the water alkalinity are

hydroxide, carbonate, and bicarbonate. Alkalinity is often confused with the pH, assuming

solutions which are alkaline have pH higher than 7. However, to have high alkalinity a

solution should not always have high pH values. A solution containing carbonate alkalinity

will have pH around 8.3. In the current study, only hydroxide alkalinity is tested since the

other aforementioned components are not present in the solutions. Since the hydroxide ion

OH- is a strong base, high alkalinity will result in high pH (>10). The total alkalinity of a

solution is its ability to bind protons which also means an ability to resist changes in pH by

neutralizing acids. In other words the alkalinity measurements can be used for evaluating the

buffering capacity of the solutions (Warfvinge, 2011). Hence, it provides with useful

information concerning the strength of the EAS making possible their comparison with

conventional bases.

When analyzing the alkalinity of obtained solutions significant changes were found.

The Figure 4.3 shows the effect of tested parameters on the alkalinity of the catholyte. The

graph is depicted in coded values which are explained in Table 4.1. At the beginning the

titrated solution is low-sensitive to the changes in the concentrations of H+ ions (i.e. to the

addition of acid) and its pH is changing slowly. But close to the equivalent point even

marginal amounts of acid result in the drastic and quick changes in pH. Only the time and

the current intensity had a significant effect on the alkalinity (p<0.05). The effect of time and

concentration is supported by the first Faradey’s law which states that the mass of the

96

substance liberated on the electrode is directly proportional to the amount of electricity

flowing through the electrolyte and to the time (Damaskin et al., 2008). Regardless of the

NaCl concentration maximum alkalinity (26.84 ±1.24 mmol/l) was attained at maximum

current intensity (I=0.2 A) and within maximum time (t=60 min). Other values obtained after

60 min of treatment were 13.28 ± 0.48 and 6.65 ± 0.24 for current intensities of 0.1A and

0.05A (Figure 3c). The NaCl concentration did not have a significant influence on the

alkalinity of the solution (Figures 3a, 3b).

Figure 4.3 : The effects of salt concentration, current intensity and time on the alkalinity: a) time hold constant at maximum value (60 min); b) current intensity hold constant at maximum value (0.2 A); c) concentration hold constant at maximum value (1 M).

97

Table 4.1: Variables and their coded values.

Variable Coded values -1 0 1

NaCl concentration, M 0.01 0.1 1 Current intencity, A 0.05 0.1 0.2 Time, min 10 30 60

With an increase in current intensity the water electrolysis on the cathode becomes

more intensive as well as the formation of other active reducers, therefore more H+ and OH-

ions are generated. The former in turn are reduced on the electrode surface in the form of gas

and the latter are accumulated leading to a pH increase. Similarly more OH – ions are

accumulated with the time flow. Since the CEM separates two compartments hydroxyl ions

which tend to reach anode cannot escape the compartment being rejected by the membrane

according to Donnan exclusion and thus enhancing the alkaline strength of the solution. The

Na+ ions easily permeate through the membrane from the central compartment and together

with hydroxyls form strong base NaOH.

4.6.2. Configuration 2

Figure 4.4: Configuration 2 of the electro-activation reactor used for the generation of the electrolysed water.

Second configuration is shown in the Figure 4.4. It is similar to the previous one but

the membranes are swapped places. This modification leads to changes in the behavior of the

98

charged species. Contrary to the configuration 1, the middle compartment is a zone of

concentration whereas the two extremities compartments are zones of depletion. Anions now

can permeate through AEM while cations from the middle compartment are retained.

4.6.2.1. pH evolution

The evolution of the pH in the cathodic compartment for the configuration 2 is shown

in Figure 4.5. Similar to the previous configuration the pH increases as a function of time.

However, regarding the current intensity not all the curves follow the same tendency. The

difference is noticed at the lowest salt concentration. Thus, in low NaCl concentration the

current intensity has an impact only for 10 and 30 min of treatment while after it the curves

become almost identical (superimposed). For higher concentrations the curves are well-

separated and higher current intensities result in higher pH values as it was observed for the

configuration 1. Similarly, at constant low current intensity there is no difference between

solutions pH for different concentrations, however one can notice that for I=0.1A and 0.2A,

pH values are slightly lower at low NaCl concentration (0.01 M).

99

Figure 4.5: The effect of current intensity and salt concentration over time on the pH monitoring of electro-activated solutions, configuration 2.

a b c

d e f

100

4.6.2.2. Alkalinity

The alkalinity evolution is shown in Figure 4.6. Difference in configuration revealed

new parameter affecting the alkalinity of the solutions. While time and current intensity have

the same effect as in previous configuration (Figure 4.6c) a marked deviation is observed

concerning the effect of NaCl concentration (Figures 4.6a, 4.6b). The alkalinity changes

drastically within the range of NaCl concentration considered in this study. Thus, with

minimal quantity of salt present (0.01M) for 0.05A the alkalinity is 5.12 ± 0.25, increasing

to 6.90 ± 0.37 for 0.1M NaCl and 7.45 ± 0.78 for 1M NaCl. With 0.1A current intensity these

values are 8.47±0.04, 13.14 ± 0.06 and 14.25 ± 0.35. And finally for 0.2A the values of 11.55

± 0.07, 24.15 ± 0.49 and 28.15 ± 0.64 were obtained respectively for 0.01, 0.1 and 1M

concentrations of NaCl (Figure 4.6a). The increase is more important within the range of

salt concentrations of 0.01M and 0.1M, while between 0.1M and 1M it is less pronounced.

The effect of the type of configuration is more important with minimal salt

concentration and maximal current intensity in this compartment. Due to different

membranes disposition, the cathodic compartment becomes a zone of depletion. When

0.01M concentration is used there is a lack of current carriers which can be noticed by higher

voltage (data not shown). In addition, chlorine and hydroxyl ions are leaving the cathodic

compartment under the effect of external electric field. In order to compensate the lack of

ions in the compartment water dissociation on the membrane takes place so as to provide the

solution with new ions (Tanaka, 2010). Therefore newly generated H+ and OH- ions become

current carriers. In this case, the higher the water dissociation the lesser the alkalinity is.

Water dissociation leads to H+ and OH- ions generation, the latter by turn leave the

compartment and the former neutralize OH- ions formed during the water electrolysis. With

the time more ions are accumulated in the compartment and the voltage starts slowly to

decrease, less water is dissociated and the alkalinity increases.

When higher NaCl concentrations are used in the cathodic compartment the higher alkalinity

is observed as there are enough current carriers. Therefore, the concentration is as important

as time and current intensity for this configuration.

101

Figure 4.6: The effects of salt concentration, current intensity and time on the alkalinity: a) time hold constant at maximum value (60 min); b) current intensity hold constant at maximum value (0.2 A); c) concentration hold constant at maximum value (1 M).

4.6.3. Configurations 3 and 4

Third configuration is a two-compartment reactor composed of an anodic and a

cathodic chambers separated by a CEM (Figure 4.7a). Due to a closer electrodes disposition

than for the first two configurations, the voltage is lower (data not shown). There is less

distance to cover while transporting the electric charges between the electrodes. For the

cathodic compartment, this configuration is similar to the one of configuration 1. Basically,

the same reactions take place as it was presented for the first configuration. The only

difference is the absence of the middle compartment. However, contrary to the first

configuration there is a transfer of H+ ions from the anodic compartment together with Na+

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ions to the cathodic compartment. It is less favorable as hydrogen ions neutralize OH- thus

reducing the alkalinity. Overall, in terms of pH and alkalinity the results observed for that

configuration were similar to the one discussed for the first configuration (data not shown).

No decrease neither in pH nor alkalinity was observed despite the additional H+ ions

migration for this configuration presumably due to the range of chosen conditions. However,

it is possible that the difference will occur if the time of treatment and the current intensity

are increased.

Figure 4.7: Configuration 3 (a) and 4 (b) of the electro-activation reactor used for the generation of the electrolysed water.

The fourth configuration (Figure 4.7b) is also a two-compartment cell with an AEM

separating both electrode compartments. The study of pH and alkalinity showed the same

tendency as with configuration 2. However, the absence of the middle compartment resulted

in additional release of chlorine (Cl2) on the anode due to the migration of Cl- from the

cathodic compartment. Apart from that fact and a lesser distance between the electrodes no

difference was noticed. Similar to the second configuration all three parameters were

significant for the changes in alkalinity (not shown).

4.6.4. The effect of type of configuration

Due to the aforementioned information among 4 tested configurations only the first

two will be discussed. The major difference between configuration 1 and configuration 2 is

the impact of salt concentration on the alkalinity which is not significant in the first case but

have a pronounced impact in the second case. Table 4.2 shows the increase in alkalinity and

pH with an increase in time for the two configurations. As it can be noticed the pH changes

progressively with an increase in time and current intensity. Regarding pH no important

a b

103

difference between the two configurations has been observed. Concerning the alkalinity the

situation is quite different.

Table 4.2: Comparison of pH and alkalinity between two configurations for different current intensities within 0.01 M NaCl concentration.

C (NaCl) = 0.01 M, I = 0.050 A

Configuration 1 Configuration 2

τ (min) pH Sd ± Alkalinity Sd ± pH Sd ± Alkalinity Sd ±

10 10.56 0.02 0.92 0.11 10.75 0.07 1.00 0.06

30 11.02 0.13 3.20 0.06 11.00 0.02 2.73 0.10

60 11.34 0.05 6.90 0.14 11.42 0.06 5.12 0.25

C (NaCl) = 0.01 M, I= 0.1 A



10,00 10.88 0.03 2.34 0.05 10.91 0.01 2.15 0.13

30,00 11.24 0.06 6.50 0.57 11.23 0.02 4.75 0.21

60,00 11.54 0.02 12.85 0.78 11.39 0.04 8.47 0.04

C (NaCl) = 0.01 M, I=0.2 A



10 11.05 0.01 4.85 0.78 11.20 0.06 3.50 0.28

30 11.60 0.02 14.07 0.04 11.33 0.02 7.30 0.28

60 11.81 0.07 25.46 0.31 11.49 0.04 11.55 0.07

The data presented in the table show that for the first configuration within 0.01M

concentration of NaCl the maximum alkalinity of 25.46 ± 0.31 is reached within 60 minutes.

It increases gradually with an increase in time and current intensity. For the Configuration 2

an increase is also observed but it is not that abrupt. For the same time and current intensity

it was only 11.55 ± 0.07, more than half as much compared to the Configuration 1. Therefore

for the minimal NaCl concentration the alkalinity in configuration 1 reached its maximum

but in configuration 2 did not.

With an increase in the NaCl concentration the alkalinity of the first configuration was not

statistically different from the one observed at 0.01 M concentration, however the second

configuration showed significant difference and by its values the alkalinity was closer to the

first configuration (Table 4.3). Finally, at maximum concentration of 1M in the cell using

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the second type of configuration the maximum alkalinity was reached, whereas no significant

changes were observed in the cell with the first type of configuration (Table 4.4).

Table 4.3: Comparison of pH and alkalinity between two configurations for different current intensities within 0.1 M NaCl concentration.

C(NaCl) = 0.1M, I=0.05 A



10 10,68 0,01 0,93 0,04 10,83 0,04 1,27 0,01

30 11,22 0,00 3,28 0,03 11,29 0,05 3,25 0,07

60 11,56 0,04 6,62 0,17 11,56 0,04 6,90 0,37

C (NaCl) = 0.1 M, I= 0.1 A



10,00 11,03 0,08 2,03 0,10 11,08 0,01 2,16 0,20

30,00 11,48 0,01 7,08 0,03 11,55 0,02 6,77 0,24

60,00 11,76 0,01 13,80 0,08 11,81 0,01 13,14 0,06

C (NaCl) = 0.1 M, I=0.2 A



10 11,38 0,01 4,35 0,21 11,39 0,01 4,45 0,07

30 11,81 0,02 14,77 0,10 11,82 0,01 13,05 0,21

60 12,03 0,02 27,90 0,14 12,03 0,01 24,15 0,49

105

Table 4.4 : Comparison of pH and alkalinity between two configurations for different current intensities within 1 M NaCl concentration.

C (NaCl) = 1 M, I = 0.050 A



10 10,62 0,03 0,96 0,06 10,66 0,08 1,07 0,04

30 11,12 0,01 3,03 0,04 11,11 0,08 3,35 0,13

60 11,40 0,01 6,42 0,40 11,49 0,04 7,45 0,78

C (NaCl) = 1 M, I= 0.1 A



10,00 10,92 0,04 2,02 0,03 11,01 0,02 2,31 0,07

30,00 11,36 0,00 6,77 0,04 27,99 23,37 7,20 0,00

60,00 11,74 0,03 13,20 0,14 11,72 0,01 14,25 0,35

C (NaCl) = 1 M, I=0.2 A



10 11,24 0,01 4,39 0,01 11,28 0,03 4,77 0,18

30 11,74 0,02 13,32 0,11 11,80 0,02 14,40 0,03

60 12,00 0,01 27,15 0,35 11,96 0,00 28,15 0,64

Therefore, it seems that the second configuration is less effective in comparison with

the configuration 1 mostly due to the fact that targeted OH- ions leave the compartment

through the AEM. Configuration 1 prevents hydroxyl ions from leaving the compartment and

in addition allows the transfer of Na+ ions from the central compartment forming NaOH.

Solutions generated with the first configuration are independent of the salt concentration and

thus do not require high content of salt. For the second configuration the similar alkalinity

can be reached when 1M NaCl concentration is used.

The study of alkalinity of EAS carries precious information about the strength of the

obtained solutions. While no important variation was observed in terms of pH for the

solutions generated under different conditions significant differences were noted in terms of

alkalinity. This can be explained by activation energy of solutions and their metastable non-

equilibrium state which is the heart of electro-activation. Alkaline catholyte having the excess

of negative charges does not contain counter-ions and locally is electronegative even though

the system in total is electrically neutral (Belovolova et al., 2006). The activation energy of

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EAS comes from the effect of an electric field on dipole moments of water resulting in the

appearance of resonance microclasters (G, 2006; Plutakhin et al., 2013). This high resonance

energy of co-vibrating dipoles and charged ions in the near-electrode spaces explains the

abnormal properties of EAS. When compared to conventional base such as NaOH maximal

values of alkalinity obtained under tested conditions can give a solution equivalent to 0.03M

concentration of NaOH. If this concentration is to be increased, higher values of current

intensity as well as longer treatments are needed. On the other hand the product of EA is not

a concentrated solution but an activated one in a metastable state which significantly

increases their reactivity (Plutakhin et al., 2013). Such solutions need further investigations

in terms of their reactivity. Their behavior in various physico-chemical and biological

reactions is the subject of further analysis.

Conclusion

The effect of current intensity, time and salt concentration on the pH and alkalinity of aqueous

solutions obtained in the reactor of 4 different configurations has been investigated. Among

the four tested configurations a pairwise similarity was observed. Thus, configurations 1 and

3 were similar as were configurations 2 and 4. The time of treatment was the most important

parameter for the changes in pH of all EA solutions. High alkaline pH was achieved during

the first 10 minutes, yet further treatment did not result in marked distinction. Regarding the

current intensity, the salt concentration and the type of configuration the differences were

minor. The situation was quite different for the alkalinity. Although the pH of solutions in all

cases indicated highly alkaline medium, the alkalinity studies showed a significant difference

for different time and current intensity (for all 4 configurations) and salt concentration (for

configurations 2 and 4). Alkalinity was higher under higher current intensity and longer time

of treatment. For configurations 2 and 4 the alkalinity increased with an increase in salt

concentration which was explained by the membrane disposition. In general, the

configurations 1 and 3 seem more effective under the tested conditions as they keep the

required ions in the cell whereas other two configurations allow their transfer to the adjacent

compartment.

Despite highly alkaline pH which indicated the presence of OH- groups the generated

solutions according to maximally achieved alkalinity corresponded to a 0.03M NaOH

107

solution. Under the tested conditions the solutions comparable to strong NaOH were not

obtained. A substantial increase in current intensity and time of treatment is therefore

required in order to increase the alkalinity.

Particular interest is shown by the correlation between the pH and alkalinity. Solutions

obtained in configurations 1 and 2 did not show an important difference in terms of pH,

whereas in terms of alkalinity a substantial increase was observed. Further analyses on the

utilization of EAS are needed. Alkaline properties of the catholyte can be used in

technological processes where conventional bases are normally used. Potentially the

technology of EA can become a better alternative to the utilization of conventional bases in

many technological processes as it allows to refuse from chemical reagents and therefore is

a “green” and environmentally friendly technology.

Acknowledgments





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5. CHAPTER 5: A comparative study between the electro-activation

technique and conventional extraction method on the extractability,

composition and physicochemical properties of canola protein

concentrates and isolates


Based on the results obtained in previous two chapters the following section aimed to study

the electro-activated solutions for their capacity to extract proteins from canola meal and to

compare them to the traditional technique which uses the NaOH solutions. Also the effect of

such treatment was assisted by studying the physico-chemical properties of extracted

proteins.

This chapter is presented as an article entitled: “A comparative study between the electro-

activation technique and conventional extraction method on the extractability, composition

and physicochemical properties of canola protein concentrates and isolates”.






This article was published in the “Food Bioscience” (2015), Volume 11, 1, Pages 56-71

The results were presented at The World Congress of Food Science & Technology (IUFoST)

2014 in Montreal as a moderated poster, at The International Congress on Engineering and

Food (ICEF) 2015, and IFT conference 2015.

109

Résumé

La technologie d’électro-activation en solution a été utilisée avec succès pour l'extraction des

protéines à partir de tourteau de canola. Une solution alcaline a été générée dans le

compartiment cathodique du réacteur d’électro-activation sous l'influence d’un champ

électrique continu. Cette solution aux propriétés alcalines a été utilisée comme substitut du

NaOH vu ces propriétés d'extraction améliorées par rapport aux solutions chimiquement

alcalinisées. L'étude vise à vérifier l'efficacité de la solution électro-activée pour l'extraction

des protéines à partir de tourteau du canola en analysant son effet sur le taux d'extraction, la

composition et la structure secondaire des protéines extraites. Les paramètres testés

comprenaient la concentration de NaCl (0.01-1 M), la durée d'électro-activation (10-60 min)

et l'intensité du courant électrique (0.2 ; 0.3 A). L'électro-activation a été réalisée dans un

réacteur d’électro-activation à trois compartiments, séparés par des membranes échangeuses

d'ions, après quoi les solutions obtenues ont été utilisées pour l'extraction pendant une heure.

Le taux d’extractibilité maximale des protéines était de 34.32 ± 1.21% et a été obtenue avec

une solution électro-activée générée avec un courant électrique de 0.3 A ; et ce, quel que soit

le temps d'activation. L'extraction conventionnelle dans les mêmes conditions (pH 7-10) a

donné un taux d’extraction de 31.18 ± 1.89% de protéines. Les profils électrophorétiques des

concentrés des protéines et les isolats électro-activés analysés par SDS-PAGE ont été plus

clairs et visibles par rapport à ceux obtenus par la méthode d’extraction conventionnelle avec

du NaOH. L’étude réalisée par FTIR a révélé des différences importantes dans les structures

secondaires des protéines dépendamment des conditions de traitements utilisés ; notamment

le pH et la concentration du sel. Aussi, les analyses par FTIR ont clairement montré que les

protéines obtenues avec des solutions électro-activées étaient nettement moins dénaturées

que celles obtenues par la méthode conventionnelle d’extraction avec du NaOH.

110

Abstract

A novel technology of electro-activation was used for protein extraction from canola

meal. An alkaline solution was generated in the cathodic compartment under the influence of

electric field. It has been reported to have improved extractive properties when compared to

chemically alkalized solutions. The study aims to verify the efficiency of electro-activated

solutions for protein extraction from canola oil cake by analyzing the effect of extraction

method on the extractability rates, composition, and secondary structure of extracted

proteins.

The tested parameters included NaCl concentration (0.01-1M), duration of electro-

activation (10-60 min), and current intensity (0.2, 0.3 A). The electro-activation was

performed in a three-compartment cell separated by ion exchange membranes, after which

the obtained solutions were used for 1-hour extraction. Maximal protein extractability was

34.32 ± 1.21% obtained with the electro-activated solution generated under 0.3 A irrespective

of the activation time. The conventional extraction under the same conditions (pH 7-10)

yielded 31.18 ± 1.89% of proteins. Electrophoretic profiles of electro-activated protein

concentrates and isolates analyzed by SDS-PAGE were clearly more distinguishable

compared to those obtained by conventional method. FTIR study revealed considerable

difference in proteins’ secondary structures between different treatment conditions (pH and

salt concentration) as well as between conventional and electro-activated samples, showing

less denatured spectra for the latter.

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Introduction

The interest towards new protein sources has grown dramatically in the past few years.

Increasing malnutrition in developing countries, high cost of proteins from animal sources,

health concerns such as intolerance to animal proteins and a conscious choice of many to

refrain consuming animal proteins has led to a substantial search for alternative sources of

proteins which could replace conventional ones. Alternative sources have been thoroughly

studied in recent years with proteins derived from plants, bacteria and yeasts being the most

promising ones. Among them oilseeds are interesting options as the protein rich oil cake left

after oil extraction is a byproduct which can be valorized.

Canola has become an important agricultural crop in Canada and around the world. It

was developed in Canada primarily as a source of edible oil. Canola is also used for the

production of biodiesel and its byproduct, the protein rich canola oilcake also finds use as

forage for livestock. Other possible applications of canola meal proteins include adhesives,

plastics, and biocomposites (Gillberg and Törnell, 1976). The protein content of an oilcake

left after oil extraction accounts for 20-50% on a dry weight basis, similar to soybean which

is extensively used in food industry (Tan et al., 2011a). However, at present its utilization is

limited to the production of animal feed. Numerous studies done on canola proteins’

physicochemical, functional and bioactive properties indicate potential for it to be used in the

food industry (Aider and Barbana, 2011; Ghodsvali et al., 2005; Khattab and Arntfield, 2009;

Moure et al., 2006; Rodrigues et al., 2012; Wanasundara, 2011; Yoshie-Stark et al., 2006;

Yoshie-Stark et al., 2008). Currently the most common extraction technique is a direct

alkaline extraction method which comprises protein solubilization at a highly alkaline pH

≥10 with subsequent precipitation either at its isoelectric point or by the use of membrane

technologies. Processing in a highly alkaline medium allows to extract up to 60% (Ghodsvali

et al., 2005) or even up to 80% of total proteins (Gillberg and Törnell, 1976; Pedroche et al.,

2004) depending on the canola variety but at the same time causes undesirable modifications

such as protein denaturation, reduction of digestibility and loss of essential amino acids

(Rodrigues et al., 2012; Sari et al., 2013). Furthermore, the presence of antinutritive factors

is another limiting factor. However, the amount of antinutritive factors can be significantly

reduced by the use of ultrafiltration and diafiltration (Ali et al., 2011; Xu and Diosady, 2002).

112

In order to increase the protein yield and improve the qualitative characteristics of the

protein extracted from different vegetable sources, the effect of various conditions and

reagents such as salt (Eromosele et al., 2008; Karaca et al., 2011a), temperature (Gillberg and

Törnell, 1976), time of extraction, and meal to solvent ratio (Nioi et al., 2012) has been

studied. Alternative methods to direct alkaline extraction have also been investigated such

as protein micellar mass (Ismond and Welsh, 1992), the use of enzymes (Sari et al., 2013),

and Osborne scheme (Manamperi et al., 2012; Tan et al., 2011a, b).

One of the most promising methods for the protein extraction is the use of direct electric

current. Water subjected to an electric field known as electro activated solution (EAS) was

claimed to possess improved extracting, cleaning and disinfecting properties (Aider et al.,

2012b). The energy supplied by electricity transforms water to a metastable state,

characterized by abnormal physicochemical properties (Leonov et al., 1999; Plutakhin et al.,

2013). Electro-activation (EA) may be explained by the phenomenon of water electrolysis

and oxido-reduction reactions which take place on the electrodes under the influence of

electric field. This leads to drastic changes in physicochemical properties in the near

electrode layer, resulting in the formation of an acid solution in the anodic side and an

alkaline solution in the cathodic side. The products of oxido-reduction reactions responsible

for physicochemical activity of the solutions have been identified: 1) stable products of

electro-chemical reactions responsible for pH changes; 2) non-stable over reactive substances

such as ОН−, Н3−О2

−, Н2, НО2•, НО2

−, О2− ; 3) other products formed near the electrode

surface in the form of free structural complexes or hydrated shells of ions, molecules, radicals

and atoms (Sprinchan et al., 2011a). The simultaneous formation of aqua complexes which

can also increase the reactivity of the medium has also been documented (Leonov et al.,

1999). The abnormal properties of EAS were studied by analyzing the Raman spectra and

fluorescence and comparing them with that of chemically alkalized water (Belovolova et al.,

2006; Pastukhov and Morozov, 2000). The authors concluded that the metastable state of the

near-electrode solutions was the cause of EAS activity (Aider et al., 2012b). It was also

reported that the physicochemical properties of EAS could be manipulated by using various

configurations of the reactor, membranes, and the time of treatment (Liato et al., 2015b). The

EAS as mentioned before in addition to their acid and alkaline properties contain other

substances which increase their reactivity (Prilutskii and Bakhir, 1999; Tomilov, 2002).

113

These substances are very unstable, yet they might contribute to the extraction processes. In

the food industry electro-activation has been used for protein extraction from sunflower seed;

protein solution was pumped through the cathodic chamber to extract and subsequently

through the anodic chamber to precipitate the extracted proteins (Plutakhin et al., 2005).

Comparing the yield, the authors found that chemical extraction at (10.2%), was not

significantly higher than electro-chemical extraction (8.9%) showing the sufficiently high

efficiency of extraction of sunflower proteins by means of EA.

The aim of the current study was to use the catholyte from cathodic compartment for

protein extraction from canola oilcake and to compare them with conventional extraction in

terms of protein and total dry matter extractability, subunit composition and secondary

structure of extracted proteins, amount of total phosphorus (as an estimate of phytic acid

content), and the amount of free amino-acids. In order to find the optimal conditions the

effects of the type of configuration, salt concentration, time of treatment, and current intensity

were studied. Conventional extraction was put through similar conditions in order to compare

the efficiency of new technique.


5.5.1. Chemicals

Defatted canola meal was kindly provided by Bunge ETGO, Becancour, Québec,

Canada. Sodium hydroxide (NaOH) was purchased from VWR International LLC (West

Chester, PA, USA). Concentrated hydrochloric acid (HCl) and sodium sulphate (Na2SO4)

were purchased from Fisher Scientific (Mississauga, ON, Canada) and sodium chloride

(NaCl) was purchased from Caledon Laboratories LTD (Georgetown, ON, Canada).

5.5.2. Ion-exchange membranes

Anion exchange membrane (AEM) AM-40 and cation exchange membrane (CEM)

CM-40 were purchased from the Publicly Traded Company Schekina-Azot (Shchekina,

Russian Federation). Prior to utilisation both membranes were washed with 96%-ethanol to

remove the wax and prepared according to manufacturer’s instructions by soaking them in

the salt solutions before their use in the electro-activation reactor.

114

5.5.3. Configurations of the electro-activation reactor

Electro-activation reactor was made of transparent Plexiglas columns with the

dimensions of 50x50x120 mm (LxWxH). Two types of configurations of the three-

compartmental reactor were used (Figure 5.1). For the first configuration referred as C1

further in the text, the AEM separated the anode and central parts whereas CEM was placed

on the cathodic side separating it from central section. The second configuration (C2) had the

same three compartments but this time the membranes were swapped places. The RuO2–

IrO2–TiO2 electrodes 120x34x1 mm with working active area of 40 cm2 were placed at the

extremities of the cells and were connected to the positive side of a direct electric current

power supply for the anodic compartment and to the negative side for the cathodic one. The

values of the applied voltage and the intensity of the electric current were measured using a

current power supply (CSI12001X, Circuit Specialists, Inc. Mesa, AZ, USA).

Figure 5.1 : Configurations of the reactor used for EA corresponding to: a) Configuration 1 (C1); b) Configuration 2 (C2).

5.5.4. Experimental

5.5.4.1. Protocol of EA

The EA was carried out at two constant electric current intensities of 0.2 and 0.3 A with

total time of treatment of 10, 30, and 60 min. To ensure the current flow salt solutions were

circulated in all three compartments. The cathodic compartment was filled with NaCl

solution of the following concentrations 0.01, 0.1, and 1M. Anodic and central compartments

were always filled with Na2SO4 solution of 0.25M concentration.

a b

115

5.5.4.2. Extraction by EA

Industrially defatted canola meal was weighed and dispersed in EAS, 10% (w/w).

Extractions were carried out at room temperature for 1 hr, under constant agitation using a

magnetic stirrer. After that the slurry was centrifuged at 10,000 x g during 30 min at room

temperature so as to separate insoluble material using Eppendorf centrifuge 5804R

(Eppendorf AG, Hamburg, Germany). The supernatant was collected as electro activated

protein concentrate, further referred as EAPC. All the extractions were performed in triplicate

and the mean values were determined. To obtain electro activated protein isolate, further

referred as EAPI the pH of the supernatant was adjusted to pH 4.3. The precipitate was

collected by centrifugation (10,000 x g, 10 min with the help of Eppendorf centrifuge 5804R)

and washed three times with distilled water. The pellets were freeze-dried.

5.5.4.3. Conventional extraction

Industrially defatted canola meal was dispersed in aqueous NaCl solutions of the

following concentrations 0.01M, 0.1M, and 1M. The pH of the slurries was adjusted by

adding 2M NaOH solution dropwise so as to reach pH 7-10 and the extraction was held 1 hr

with agitation. After centrifugation the supernatant was collected as conventional protein

concentrate (CPC). The conventional protein isolates (CPI) were obtained by isoelectric

precipitation as was aforedescribed for EAPI.

5.5.5. Chemical analysis

Proximate analysis of the industrial canola meal was performed according to AOAC

International (2012). Moisture and total dry matter were determined by drying the weighed

sample in a Fisher Isotemp Vacuum Oven (Fisher Scientific, Montreal, QC, Canada)

according to method 925.09. Ash content was determined according to method 923.03 by

combusting a weighed portion of 5 g in the muffle oven until constant weight and the ash

content was expressed as the weight ratio between the sample after and before combustion.

Crude protein was determined following the Dumas method with LECO Truspec FP-428

(Leco Corp., St. Joseph, MI, USA) according to the method 992.23 using a conversion factor

of 6.25. Residual oil in the defatted meal was determined by Soxhlet extraction according to

method 925.85 using petroleum ether as extraction solvent. Carbohydrates were determined

by difference (Karaca et al., 2011a). Phytic acid being the principal storage form of

116

phosphorus in canola was estimated from the ash residues (Stack, 1996) as the total

phosphorus content and was analyzed according to Plaami and Kumpulainen (1991). The

amount of free amino-acids was analyzed by EZ-Faast (Phenomenex, Torrance, USA) using

GC-FID. All analyses were conducted in triplicate and mean values were calculated

5.5.6. Total dry matter and protein extractability

In order to determine the total dry matter extractability the aliquot of the supernatant in

each case was weighed and dried in the oven until constant weight. The extractability was

calculated as a ratio of dry matter in supernatant excluding the salt added during the

extraction over total dry matter content of the canola meal. Nitrogen content of the

supernatant was measured using Dumas method with LECO Truspec FP-428. The protein

extractability was expressed as a function of nitrogen content in the supernatant in relation

to nitrogen content in canola meal on a dry weight basis.

5.5.7. SDS PAGE

To assess the impact of tested conditions on the polypeptide profiles of extracted

proteins the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) was

performed following the method of Aluko and McIntosh (2001). Initially protein

concentrates were dialyzed at 4 °C using Spectra/Por 3 dialysis tubing with 3.5 kDa cutoff

against water refreshing several times. After that, concentrates were freeze-dried and 1%

protein solutions (w/w) were prepared and left overnight, followed by dilution with deionized

water so that the amount of proteins loaded onto the gel was approximately 20 µg. Similarly

1% solutions were prepared from protein isolates. Subsequently, diluted samples were

dissolved in Tris-HCl buffer solution containing SDS for non-reduced conditions. For

reduced conditions samples were dissolved in Tris-HCl buffer with the addition of 5% (v/v)

β-mercaptoethanol. Afterwards, the samples were boiled for 5 min, vortexed and 10 µl of

each sample were loaded onto 4–20% gradient gels (#456-1093, Bio-Rad, Canada). Precision

plus Protein Kaleidoscope Standards of 10-250 kDa (#161-0375, BioRad, Canada) were used

as molecular weight standards and the electrophoresis was run at 15 mA until the tracking

dye reached the bottom of the gel. After staining in 2.5% Coomassie Brilliant Blue R250 in

water–ethanol–acetic acid (4:5:1, v/v/v) and destaining with water–methanol–acetic acid

117

(10:4:1, v/v/v) the gels were scanned, using Chemi Doc XRS 170-807 (Bio-Rad Laboratories,

Inc, Hercules, CA, United States of America).

5.5.8. FTIR

FTIR was performed as described by Ellepola et al. (2005). The infrared spectra were

recorded at room temperature (22°C) using a Nicolet Magna IR 560 spectrometer (Thermo

Scientific, Madison, WI, USA) with continuous nitrogen supply. Canola protein dispersions

(5% w/w) were dissolved in 0.01 mol/l deuterated phosphate buffer (pD 7.4). All protein

dispersions were prepared in D2O instead of H2O since D2O has greater transparency in the

infrared region, 1600–1700 cm -1. To ensure complete H/D exchange, samples were prepared

the day before and left overnight at 4°C prior to infrared measurements. Samples were placed

between two CaF2 windows separated by 25 mm polyethylene terephthalate film spacer for

FTIR measurement. A total of 256 scans were averaged at 4 cm -1 resolution. To study the

amide I` region of the protein, Fourier self-deconvolutions were performed using the software

OMNIC 9.2.98. Band narrowing was achieved with a full width at half maximum of 22 cm−1

and with a resolution enhancement factor of 2.2 cm−1.


All tests were performed in triplicate, and the means ± standard deviations were used.

Data collected were subjected to analysis of variance at 5% confidence level. When

significant difference between treatments was found by ANOVA analysis, Tukey's multiple

range test was carried out for multiple comparison of the means using Minitab Software.


Proximate chemical composition of canola oil cake is shown in the Table 5.1. On the

whole these values are well correlated with those reported in the literature before. It had

43.36% of proteins which is in accordance with those reported by Pedroche et al. (2004) and

Tan et al. (2011b). When harvested, canola seeds contain 19.0% of protein and 54.2% of fat

(Yoshie-Stark et al., 2008). After defatting procedure (made industrially) in our case the oil

cake still had fat traces in the amount of 3.58%. Ash and moisture were also in the range of

values reported for canola oil cake.

118

Phytic acid is the major storage form of phosphorus in canola. Depending on the variety

it accounts for 3.0–6.0 g/100 g (Bell, 1993; Gilani et al., 2012; Tan et al., 2011a). In spite of

its known adverse effect such as binding minerals and proteins leading to mineral depletion

and deficiency it was also reported to have a beneficial effect. Phytic acid is an effective

chelator of iron and it prevents the formation of free radicals, thus working as an antioxidant.

In addition it seems to bind heavy metals such as cadmium and lead helping to prevent their

accumulation in the body (Lott et al., 2000).

Table 5.1: Proximate chemical composition of canola oil cake on a dry weight basis.

Moisture

(%)

Protein

(N*6.25,%)

Ash

(%)

Fat

(%)

Carbohydrate

(%, by difference) Total phosphorus (%)

10.51 ± 0.01 43.36 ± 0.71 7.19 ± 0.01 3.58 ± 0.07 35.36 2.21 ± 0.48

5.6.1. Changes in pH

5.6.1.1. pH increase during EA

During the EA pH in the cathodic chamber increases drastically within first 10 min

regardless of the reactor`s configuration (Figure 5.2) due to the oxido-reduction reactions on

the electrodes. After 10 min further increase is also observed, however it is not that abrupt.

In previous study the effect of current intensity, time of treatment, salt concentration and type

of configuration on the pH and the strength of the catholyte (alkalinity) was studied

(Gerzhova et al.). It was shown that time of treatment and current intensity had the most

important impact on the alkalinity of the catholyte for C1. For C2, in addition to the

aforementioned factors, salt concentration had a significant impact. The pH increase for the

C1 was mostly dependent on the time of treatment and to a lesser extent on the current

intensity. The effect of salt concentration was also present for the C2. Properties of chosen

solutions for I = 0.3 A are shown in the Table 5.2. Properties of the solutions obtained under

lower current intensities were shown previously (Gerzhova et al.). The most important

differences between two different configurations are observed at minimal NaCl concentration

of 0.01M.

119

time of EA, min0 10 20 30 40 50 60 70

pH

5

6

7

8

9

10

11

12

13

0.01M NaCl

0.1M NaCl

1M NaCl

time of EA, min0 10 20 30 40 50 60 70

pH

5

6

7

8

9

10

11

12

13

0.01M NaCl

0.1M NaCl

1M NaCl

Figure 5.2: Changes in pH during EA for I = 0.3 A: a) C1; b) C2.

Table 5.2: Comparison of pH and alkalinity between two configurations for I = 0.3 A and within different NaCl concentrations.


C(NaCl) τ(min) pH ± Sd Alkalinity ± Sd pH ± Sd Alkalinity ± Sd

0.01

10 11.32 ± 0.01 8.5 ± 0.99 11.19 ± 0.01 3.95 ± 0.35

30 11.78 ± 0.06 21.04 ± 0.08 11.45 ± 0.04 9.54 ± 0.03

60 12.09 ± 0.01 44.5 ± 0.99 11.65 ± 0.02 14.27 ± 0.04

0.1

10 11.38 ± 0.01 7.07 ± 0.04 11.57 ± 0.01 6.29 ± 0.21

30 11.81 ± 0.02 21.64 ± 0.03 11.95 ± 0.01 17.90 ± 0.28

60 12.03 ± 0.02 43.30 ± 0.57 12.13 ± 0.01 32.35 ± 0.35

1

10 11.44 ± 0.02 7.03 ± 0.01 11.62 ± 0.04 6.90 ± 0.06

30 11.94 ± 0.04 21.05 ± 0.07 12.09 ± 0.01 20.40 ± 0.28

60 12.25 ± 0.01 43.21 ± 0.28 12.33 ± 0.01 43.04 ± 0.06

5.6.1.2. pH decrease during extraction

Solutions chosen for the extraction all had alkaline pH. During the extraction a pH

decrease was observed which can be explained by the buffer capacity of the canola oilcake.

The pH decrease is shown in the Figure 5.3.

a b

120

Figure 5.3: Changes in pH during extraction by EAS: a) C1, 0.01 M NaCl; b) C1, 0.1 M NaCl; c) C1, 1 M NaCl; d) C2, 0.01 M NaCl; e) C2, 0.1 M NaCl; f) C2, 1 M NaCl.

121

Solutions obtained under I = 0.2 A had the same tendency as those obtained under I =

0.3 A but were significantly weaker, so not to overload the paper only the results obtained

for I=0.3 A were shown. No statistically significant difference was observed for the C1

between different salt concentrations, whereas for the C2 a considerable difference was noted

between 0.01M NaCl and the other two concentrations. Regarding 0.1 and 1M NaCl

concentrations for the C2 only the solution treated 60 min was significantly different from

the other ones.

Time of treatment had the major impact on the changes in pH during extraction. A 10-

min solution was the weakest, which was in agreement with alkalinity measurements (Table

5.2). Its pH decreased dramatically down to pH 6 once it was mixed with canola oilcake and

was kept within this value during the extraction. Such behaviour was observed for all treated

solutions regardless of their NaCl concentration or the type of configuration. For the 30-min

solution there was a difference between configurations. This solution was twice as strong as

the 10-min according to its alkalinity. The pH of the 30-min solution dropped down not that

abruptly and was maintained around pH 8 for C1. For C2 there was a significant difference

between all three concentrations. Finally, the solutions treated 60 min were the strongest for

the C1 which was also well correlated with their alkalinity. In C2 the strength of the solutions

increased with an increase in salt concentration. In this case it seems that the salt

concentration was the most important parameter as even after 60 min of treatment the pH of

the solution made with 0.01M NaCl decreased faster and to a lesser value in comparison with

solutions of higher salt content (Figures 5.3d, 5.3e, 5.3f). Thus, the strength of 60-min treated

solution in C2 was lower than the strength of solutions treated 30 min in the same

configuration but with higher salt content. Comparing two configurations it is possible to

conclude that for the C1 the higher the time of treatment and the current intensity the lesser

the pH decrease is, which means the stronger the solution is. For C2 only the solution with

1M NaCl solution could maintain the pH within the scope of C1. The ability to maintain the

pH is related to the strength of the solution, to its alkalinity or buffering capacity in other

words. Although the concentration of OH- is sufficient for producing the same value of pH

as in chemical analogues it is insufficient to maintain it within the constant value, its buffering

capacity is lower in comparison with strong alkali such as NaOH. In order to increase the

pH of extraction so as to simulate the conditions similar to the conventional extraction the

122

higher current intensity and the longer time of treatment are required. The high pH can

possibly be maintained when there is a constant generation of OH- ions. This can be reached

when performing the extraction directly in the cell. However, there is a risk of membrane

fouling or precipitating on the electrodes.

5.6.2. Extractability

According to the previous study the pH has the major effect on the extractability which

is in accordance with other publications (Ghodsvali et al., 2005; Klockeman et al., 1997; Nioi

et al., 2012). Canola proteins are characterized as very complicated due to the diversity of

their molecular weights and isoelectric points. Thus, Quinn and Jones (1976) reported over

30 protein species with two major proteins - cruciferin being a neutral protein of a high

molecular weight (300-310 kDa) and an isoelectric point around pH 7 and napin, a small

molecular weight protein (12.5-14.5 kDa) characterized by strong alkalinity with an

isoelectric point around pH 10-11 (Aider and Barbana, 2011; Karaca et al., 2011a). Minimal

solubility was reported to be around pH 4.3 and the majority of plant proteins have their

isoelectric points within slightly acid pH region (pH 4-5). With an increase in pH of

extracting medium the amount of extracted proteins increases dramatically especially in the

pH range of 10-12 due to the increase in proteins ionization.

5.6.2.1. Total dry matter extractability

Total dry matter extractability is shown in Figure 5.4. Both configurations: C1 and C2

(Figures 5.4b, 5.4c) did not show an important difference in comparison with conventional

extraction (Figure 5.4a). Extractability increases with an increase in NaCl concentration,

however the increase is not considerable. Between different times of EA the difference was

also not important which means that even with the solution obtained after 10 min of EA good

results can be obtained. The effect of current intensity was also present. Minimal amounts of

extracted dry matter under I=0.2 A was 19.67 ± 1.83% obtained in C1 with 10-min solution

of 0.01M NaCl which increased to 29.52 ± 1.30% for 60-min solution of 1M NaCl. Second

configuration showed slightly lower results, however the difference was not significant (not

shown). Higher current intensity (I=0.3 A) showed the same tendency and the results were

24.15 ± 0.51% to 32.03 ± 4.97% for C1 and 23.69 ± 0.18% to 30.54 ± 1.35% for C2 (Figures

5.4b, 5.4c).

123

Figure 5.4: Total dry matter extractability for I = 0.3 A: a) conventional extraction; b) extraction with EAS in C1; c) extraction with EAS in C2.

5.6.2.2. Protein extractability

The tested parameters impacted the protein extractability comparatively more than the

total dry matter with the current intensity being the most significant factor (p < 0.0001).

Maximum extractability under I = 0.2 A was 19.93 ± 1.39% obtained in C1, 1M NaCl

concentration and with the solution treated 60 min which was significantly lower in

comparison with conventional extraction. Other parameters with 0.2 A current intensity gave

even lower values (not shown). With I = 0.3 A results were comparable to those obtained

with conventional extraction (Figure 5.5). Overall, the tendency of the changes in

extractability as a function of other parameters (time of EA, salt concentration, type of

configuration) was the same for both tested current intensities, that is why only the higher

one (I = 0.3 A) will be discussed. The effect of time of EA was lower than expected.

According to the alkalinity studies the strength of the 10 min and 30 min solutions increased

by 3 times and for the 30 min and 60 min solutions - by 2 times. However, the difference in

the amount of proteins extracted by each of these solutions was not significant. Considering

the pH of the extraction medium as a key factor these results become logic. Apparently, the

protein extractability has low sensitivity to pH changes within the region of pH 7-10 and the

increase of the extractability is observed at pH values higher than 10. Thus, the pH of the

solution, electro-activated during 10 min was maintained around 7 during the extraction and

the values obtained in the first configuration are perfectly correlated with extraction held at

pH 7 adjusted by the addition of aqueous NaOH. For C1 the extractability was 26.29 ± 0.25%

for 0.01M NaCl, 31.18 ± 1.20% for 0.1M NaCl and 32.75 ± 2.59% for 1M NaCl.

Conventional extraction held at pH 7 gave 24.84 ± 0.93%, 28.41 ± 1.88% and 31.18 ± 1.89%

for the same NaCl concentrations. A 30 min solution maintained pH around 8 and allowed

pH

7 8 9 10

Ext

racta

bili

ty,

%

0

10

20

30

40

500.01 M

0.1 M

1 M

a time, min

10 30 60

Ext

rac

tab

ility

, %

0

10

20

30

40

50 0.01 M

0.1 M

1 M

b time, min

10 30 60

Ext

rac

tab

ility

, %

0

10

20

30

40

50 0.01 M

0.1 M

1 M

c

124

to extract 27.72 ± 0.92%, 30.82 ± 0.61% and 32.07 ± 2.42% whereas the results for the

conventional extraction held at pH 8 were 23.28 ± 0.90%, 25.30 ± 0.52% and 27.78 ± 0.27%

which is slightly lower. Finally, the 60-min solution with pH around 9-10 gave 33.82 ±

0.59%, 36.10 ± 1.24% and 38.06 ± 0.13% which was substantially higher in comparison with

conventional extraction at pH 10 (23.67 ± 0.19%, 27.98 ± 0.47% and 29.97 ± 1.69%).

Figure 5.5: Protein extractability for I = 0.3 A: a) conventional extraction; b) extraction with EAS in C1; c) extraction with EAS in C2.

An increase in protein extractability with an increase in salt concentration is due to the

”salting-in” effect for conventional extraction and for the solutions obtained in the C1. As

for C2, more pronounced increase is observed (Figure 5.5c). The positioning of the AEM

and CEM also significantly affected the protein extractability by the EAS. In C2, alkalinity

of the solution increased with an increase in salt concentration as described in previous work

(Gerzhova et al.). Briefly, an anion exchange membrane which was placed in the cathodic

chamber, separating it from the middle section allowed the migration of hydroxyl ions,

responsible for the alkalinity into the neighboring compartment. Cathodic chamber was the

zone of depletion (desalting) that led to water dissociation on the membrane in order to supply

the current carriers. A generation of H+ ions in the cathodic compartment limited the increase

in alkalinity by neutralizing OH- ions. This was especially pronounced when 0.01M NaCl

concentrations was used. With an increase in salt concentration the effect of water

dissociation decreased which is supported by an increased alkalinity and the higher amount

of extracted proteins (Figures 5.5). When 1M NaCl concentration was used the effect of ion

migration through the membrane was minor for such high salt concentration. This explains

the difference in the amount of extracted proteins between the two configurations.

pH

7 8 9 10

Ext

racta

bili

ty,

%

0

10

20

30

40 0.01 M

0.1 M

1 M

a time, min

10 30 60

Ext

rac

tab

ility

, %

0

10

20

30

40

0.01 M

0.1 M

1 M

b time, min

10 30 60

Ext

rac

tab

ility

, %

0

10

20

30

400.01 M

0.1 M

1 M

c

125

5.6.3. Composition

5.6.3.1. Composition of protein concentrates

The composition of protein concentrated is shown in Table 5.3. The proteins extracted

through conventional method (conventional protein concentrates) were designated with the

code CPC followed by the pH of extraction (e.g. CPC_10 corresponds to conventional protein

concentrate extracted at pH 10). The protein extracted with EAS (electro-activated protein

concentrates) were designated EAPC, followed by the type of configuration, and the time of

treatment (e.g. EAPC_C1_10 means conventional protein concentrate, obtained by extraction

with EAS, treated in the configuration 1 for 10 min).

Table 5.3: Comparative characteristics of protein concentrates.

Protein content, % Ash, % Total phosphorus, %

Free amino acids (total), %

C(NaCl), M 0.01 1 0.01 1 0.01 1 0.01 1

CPC_10 35.11 ±

1.29b

14.22 ±

0.62b

11.68 ±

1.68b

65.98 ±

1.10a

0.41 ±

0.02b

0.03 ±

0.02d

3.57 ±

0.32a

0.59 ±

0.23a

CPC_12 49.52 ±

1.17a

17.12 ±

0.81a

16.15 ±

0.52a

63.92 ±

0.84a

0.35 ±

0.05b

0.06 ±

0.01c

2.46 ±

0.42a

0.49 ±

0.16a

EAPC_1_10 29.88 ±

0.17c

12.75 ±

0.77b

12.40 ±

1.37b

69.01 ±

0.57a

0.74 ±

0.26b

0.15 ±

0.02a

4.51 ±

0.97a

0.76 ±

0.06a

EAPC_1_60 34.04 ±

2.53b

13.74 ±

1.41b

11.31 ±

1.75b

66.58 ±

3.45a

0.67 ±

0.33b

0.04 ±

0.02c,d

3.54 ±

0.4a

0.62 ±

0.05a

EAPC_2_10 27.95 ±

1.60c

12.78 ±

0.78b

11.36 ±

0.27b

63.69 ±

4.90a

1.33 ±

0.24a

0.09 ±

0.00b

4.90 ±

1.03a

0.77 ±

0.1a

EAPC_2_60 29.16 ±

0.76c

14.37 ±

0.40b

9.87 ±

0.42b

66.97 ±

1.05a

0.80 ±

0.19b

0.03 ±

0.00d

4.20 ±

0.55a

0.67 ±

0.16a

* Results represent the average of three determinations ± SD, values in the same column with different letters

are significantly different (p< 0.05)

The highest protein concentration was obtained by conventional extraction at pH 12.

First configuration after 60 min treatment yielded the amount of proteins statistically similar

to conventional one at pH 10. All the other treatments were not statistically different. All

protein concentrates extracted with 1M NaCl by either conventional method or by electro-

activation had significantly lower amounts of proteins on a dry weight basis. This can be

126

explained by the presence of the considerable amount of salt and thus an increased mineral

content. Only concentrates extracted by conventional extraction at pH 12 had statistically

higher values, whereas no difference was observed for EA concentrates and conventional

ones at pH 10. Ash content of the extracts treated with the addition of 1M NaCl ranged

between 63.69 ± 4.90% and 69.01 ± 0.57% in comparison with 9.87 ± 0.42% and 16.15 ±

0.52% for 0.01M NaCl. Although the amount of total phosphorus in the sample treated with

0.01M NaCl was statistically equal apart from the EAPC_C2_10, a slight decrease with an

increase in pH can be noticed. According to Gillberg and Törnell (1976) and Ghodsvali et al.

(2005) the extraction of phytic acid and nitrogen takes place concomitantly and nitrogen

extractability is influenced by the phytic acid. Ghodsvali et al. (2005) stated that maximum

interactions were at pH lower than pI, when anionic groups of phytic acid bind to cationic

groups of proteins as proteins are charged positively and the phytic acid is charged

negatively. In neutral region it reacts with metal ions and proteins to form a soluble ternary

phytic acid–cation–protein complex. At alkaline pH, both the protein and the phytic acid are

negatively charged and the protein–phytate interaction takes place only in the presence of

multivalent cations and through a salt linkage or an alkaline-earth ion bridge (Champagne et

al., 1985). At high pH (> 11) the interaction between protein and phytic acid decreases and

in the presence of sufficient amount of Ca or Mg the ternary complex will precipitate

(Champagne et al., 1985). In addition, it has been reported that at high pH, the insoluble

phytate exists in a fine colloidal suspension which can be removed by centrifugation (Tzeng

et al., 1990a). Lower amounts of total phosphorus (phytic acid) in extracts treated with 1M

NaCl solution can be explained by the presence of considerable amounts of salt and decreased

protein content. Working in high salt concentration also weakens the electrostatic interactions

of phytates with the proteins (Schwenke, 1994) which explains the lower amounts of phytic

acid in concentrates extracted with 1M NaCl solution in comparison with 0.01M. The

amount of free amino acids can be useful and practical indices for evaluating the protein

quality. No statistically significant difference was noticed within the same salt concentration.

However, smaller values were obtained in the solutions with 1M NaCl compared to 0.01M

which can be related to the total amounts of proteins as the latter had considerably higher

protein contents.

127

5.6.3.2. Composition of protein isolates

The protein, mineral and total phosphorus contents of protein isolates are shown in

Table 5.4. The conventional isolates were named similarly to concentrates: CPI followed by

the pH of extraction. Electro activated protein isolates were designated EAPI, followed by

the type of configuration and the time of treatment.

Table 5.4: Comparative characteristics of protein isolates.

Protein content, % Ash, %

Total phosphorus, %

C(NaCl), M 0.01 1 0.01 1 0.01 1

CPI_10 89.67 ±

0.90a

97.95 ±

1.60a

1.43 ±

0.06b

1.54 ±

0.09b

1.17 ±

0.38a

0.85 ±

0.11b

CPI_12 86.16 ±

0.54b

89.99 ±

1.88c

1.96 ±

0.04a

2.32 ±

0.47a

1.68 ±

0.41a

0.97 ±

0.22b

EAPI_1_10 90.87 ±

1.00a

94.51 ±

0.49a,b

0.84 ±

0.08c

1.19 ±

0.15b

1.58 ±

0.1a

1.72 ±

0.01a

EAPI_1_60 82.43 ±

0.47c

94.16 ±

0.46a,b

1.13 ±

0.4b,c

1.28 ±

0.02c

1.13 ±

0.23a

0.82 ±

0.1b

EAPI_2_10 91.62 ±

0.32a

90.08 ±

0.43b,c

0.30 ±

0.05d

1.11 ±

0.25b

1.26 ±

0.04a

1.04 ±

0.07a,b

EAPI_2_60 85.27 ±

0.04b

91.67 ±

0.37b,c

2.28 ±

0.17a

1.18 ±

0.01b

1.42 ±

0.29a

0.62 ±

0.03b

* Results represent the average of three determinations ± SD, values in the same column with different letters

are significantly different (p< 0.05)

The protein content of all isolates was rather high and the ash content was reduced

significantly in comparison with concentrates which means a higher purity of final product.

Interestingly, the higher protein content was observed in samples with milder treatment

conditions. Thus CPI_10, EAPI_C1_10 and EAPI_C2_10 had higher protein content

compared to other isolates. Regarding samples treated with 1M NaCl CPI_10, EAPI_C1_10

and EAPI_C1_60 had the highest protein content. In addition samples of 1M NaCl

concentration had significantly higher protein amount in comparison with those of 0.01 M

NaCl. High amounts of total phosphorus (phytic acid) can be explained by the higher purity

of the sample compared to protein extracts which contained other interfering substances such

as salts and carbohydrates. In addition higher salt concentrations help to reduce the

128

electrostatic interactions of phytates with proteins resulting in lesser amounts of phytic acid

found in the isolate extracted with 1M NaCl (Schwenke, 1994).

5.6.4. SDS PAGE

5.6.4.1. SDS PAGE of protein concentrates

Figure 5.6 shows polypeptide compositions of canola protein concentrates with their

major components analyzed by SDS-PAGE in the absence (non-reducing) and presence

(reducing) of β-mercaptoethanol.

Major bands in non-reducing conditions were identified between 37 and 50 kDa (as 45

kDa), two between 26 and 37 kDa (27 kDa and 32 kDa), and two between 15 and 20 kDa (as

16 kDa and 18 kDa). This was consistent with Tan et al. (2011b) who observed 5 main

fraction with the following molecular weights: 16, 18, 26, 30, 45, and 53 kDa. However, the

last subunit of 53 kDa was not present in our proteins. After comparing all three graphs it is

noteworthy that in spite of different extraction techniques the proteins have more or less

similar composition, apart from the CPC_12 that is distinguished by weaker marked bands

or their absence. However, it had a band of higher molecular weight around 250 kDa which

was not present in other fractions. Similar phenomenon was noticed by Tan et al. (2011b)

who used different solvents in order to extract separately albumin, globulin, glutelin and

prolamin fractions. The authors managed to extract higher molecular weight subunits in

glutelin fraction which were not observed in albumin and globulin fractions. Therefore, it is

possible that under higher pH, subunits of higher molecular weight could be extracted (Quinn

and Jones, 1976). The smeared pattern of CPC_12 is an indication of reduced protein

solubility as a result of protein denaturation due to harsh alkaline conditions. The similarity

in obtained profiles is due to the complexity of canola protein composition and the

simultaneous co-extraction of different protein profiles. It is known that canola proteins have

complicated composition. Its two major components 12S globulin (cruciferin, 300 kDa) and

2S albumin (napin, 14 kDa) considerably differ by their molecular weight, isoelectric point

and functionalities (Manamperi et al., 2012; Wu and Muir, 2008). Cruciferin being salt-

soluble however tend to be co-extracted together with albumin during the conventional

extraction as well as napin was found in extracts obtained with 1M NaCl concentration. That

is why minor difference was noticed between profiles extracted with 0.01M NaCl and 1M

129

NaCl. Thereby extracted fractions, in fact, are a mixture of two main proteins found in canola

such as cruciferin and napin.

Figure 5.6 : Molecular profiles as analyzed by SDS PAGE: a) EAPC extracted with 0.01 M electro-activated NaCl solution; b) EAPC extracted with 1 M electro-activated NaCl solution; c) CPC extracted with 0.01M and 1 M NaCl solutions.

There was a noticeable difference in SDS-PAGE profiles between the non-reduced and

reduced state. Under the reducing conditions two of the most distinctive profiles of 16 and

45 kDa disappeared and smaller fractions of 10, 12, and 22 appeared indicating that the

former fractions were linked by disulfide bonds and the latter are the result of other than S-S

interactions. Other bands of 18, 27 and 32 kDa were not affected by the presence of β-

mercaptoethanol and became more pronounced under reducing conditions which means that

other than S-S bonds are predominant. In reducing conditions new bands are more marked in

a b

c

130

0.01M NaCl concentrations. This was consistent with the work of Tan et al. (2011b) who

stated that globulin bands had less intensity compared to the polypeptide profiles of albumins

but on the whole polypeptide profiles of globulin and albumin fractions were very similar.

A comparison with conventional extracting technique revealed the difference in terms of 27,

32 and 44 kDa fractions which are similar in all EAPC and conventional ones extracted at

pH 10 (CPC_0.01_10 and CPC_1_10) but disappears at pH 12. Napin was reported to appear

on the gel in 14 kDa and 27.5 kDa (a dimer of napin) fractions according to Wu and Muir

(2008) who studied the purified samples of both major proteins from canola. In our case it

explains why the bands at 27 and 32 kDa are more pronounced with a lesser amount of salt.

The 14 kD fraction was not observed in our study however the band observed at 16 kDa is

presumably napin that upon the contact with β-mercaptoethanol give rise to two new bands

of 10 kDa and 6 kDa. It is also present in conventional concentrate extracted at pH 12 which

is explained by napin`s rigid structure low prone to structural changes at different pH

(Krzyzaniak et al., 1998). The band at 44 kDa is present in all EAPC and in CPC_10; however

it disappears in the extract obtained under higher pH (pH 12). This phenomenon is linked to

the protein structure as 12 S globulin is an oligomeric protein which dissociates into smaller

subunits under the influence of pH or ionic strength (Schwenke, 1994).

5.6.4.2. SDS PAGE of protein isolates

The non-reduced (without β-mercaptoethanol) and reduced (with β-mercaptoethanol)

SDS-PAGE patterns for protein isolates and canola meal displayed as CM are presented in

Figure 5.7. Similar to protein concentrates the protein isolates profiles look comparable,

however new bands of higher molecular weight revealed on the gel. These bands of 75 kDa,

100 and 110 kDa were not present in the concentrates and could result from protein

aggregation during the precipitation with HCl. These aggregates are linked together by

disulfide bonds which can be confirmed by their absence in reduced condition. In addition a

partial hydrolysis of high molecular weight proteins may take place with an increase in

extracting pH. Also the 12S subunits were reported to dissociate in the presence of 2-8M urea

solutions or at pH levels below 3.5 (Goding et al., 1970). As it can be noted in the Figure

5.7c and Figure 5.7d the band corresponding to 100 kDa disappeared at pH 12 and is lighter

at pH 11 in comparison with conventional extraction held at pH 10 and to those performed

with EAS. This was also observed by Jarpa-Parra et al. (2014) when lentil protein was

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subjected to different pH during extraction. In their case an alkaline pH created a progressive

dissociation of the 11S protein to lower molecular weight subunits (7S and 2S).

An evident difference between protein profiles of CPI and EAPI can be noticed which

increases with an increase in pH suggesting proteins denaturation (Figures 5.7a, b and 5.7c,

d). In the work of Mwasaru et al. (1999a) subunit composition of proteins isolates from

cowpea and pigeon pea was not affected by the extraction technique. In the study the authors

used micellar isolation and isoelectric protein methods and concluded that regardless of the

extraction technique and pH conditions extracted proteins exhibited similar electrical

mobility. In the other work where the influence of pH of extraction on the molecular profiles

was tested samples extracted at pH 8, 9, and 10 showed similar SDS-PAGE patterns. The

authors suggested that the protein compositions of the three extracts were similar and mainly

composed of globulin proteins (Jarpa-Parra et al., 2014). Consequently, it can be concluded

that protein composition is not influenced by the extraction technique in the range of pH 7-

10, however further pH increase provokes proteins denaturation which can be observed by

fading away of the bands in Figures 5.7c and 5.7d.

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*MW standard: molecular weight standard

**CM: canola meal

Figure 5.7 : Molecular profiles as analyzed by SDS PAGE: a) EAPI extracted with 0.01 M electro-activated NaCl solution; b) EAPI extracted with 1 M electro-activated NaCl solution; c) CPI extracted with 0.01 M NaCl solution; d) CPI extracted with 1 M NaCl solution.

5.6.5. FTIR

Although total protein concentration is an important parameter the understanding of

protein secondary structure is crucial for the understanding of its nutritive quality,

functionalities, availability and digestive behavior which determines its further utilization.

Even if the protein concentration is the same, the quality may differ due to the difference in

the secondary structures. For the proteins to maintain its biological and functional values it

should be folded in the same way as in the native state. The changes in environment influence

protein conformation and changes in pH and ionic strength are strong factors affecting it.

a b

c d

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5.6.5.1. Secondary structure of protein concentrates

Figure 5.8 shows the infrared spectrum of canola protein concentrates in the amide I`

region (1700-1600 cm-1).

Figure 5.8: Deconvulved FTIR spectra: a) Protein concentrates extracted with 0.01 M NaCl; b) Protein concentrates extracted with 1 M NaCl.

It depends on the secondary structure of the backbone and is hardly affected by the

nature of the side chain, thereby being the most commonly used region in monitoring the

changes in secondary structure. It is composed mainly of C=O stretching vibrations and is

extremely sensitive to changes in hydrogen bonding so that even subtle changes in the

secondary structure can be observed (Barth, 2007; Kong and Yu, 2007). Bands of β-pleated

sheets arise in easily distinctive peaks between approximately 1620 and 1640 cm-1 and in

some cases even below 1620 cm-1, showing more than one component and one weaker band

between 1670 and 1680 cm-1. Amide I` bands disposed between approximately 1650 and

1658 cm-1 are generally considered to be a characteristic of α-helical structures. The peaks

around 1616 ± 3 and 1689 ± 4 cm-1 are normally due to the formation of an intermolecular

hydrogen-bonded antiparallel β-sheet structure. Finally, the peak around 1645 cm-1 points at

a b

134

the presence of a disordered structure known as random coil (Dong et al., 1995; Surewicz et

al., 1993; Susi and Byler, 1988; Tang and Ma, 2009).

Five main bands were observed in the spectra after deconvultion, which, according to

the literature are β-structures predominantly (Figure 5.8). The main protein fractions in

canola proteins are cruciferin and napin as shown previously in the SDS PAGE profiles. The

secondary structure of cruciferin is performed mainly by β-sheet (50%) and low content of

α-helix (10%) conformations and was found similar to other 11S globulins (Schwenke et al.,

1983). Napin, on the other hand is characterized by a high content of α-helix structure (40-

46%) and a low content of β-sheet conformation (12%) (Schwenke, 1994). The quality of

protein can be judged by their peak intensities, a protein in the native state would have clear

peaks revealing α- and β-forms. A decrease in peak intensity, band shifting, an appearance

of new bands associated with aggregation or an increase in random coil structure would

indicate protein unfolding. When the protein is completely unfolded it loses its secondary

structure and together with it its value and functionalities. An evident decrease in peak

intensities can be observed in the Figure 5.8a from the top to the bottom as reflected by the

protein unfolding. The most pronounced secondary structure is carried by two top proteins

EAPC_C1_0.01_10 and EAPC_C2_0.01_10, their bands are identical, and therefore

secondary structures are comparable. The most prominent peak is the one at 1634 cm-1

attributed to β-sheet structure, followed by 1651 cm-1 associated with α-helix structure. The

presence of two peaks at 1618 cm-1 and 1688 cm-1 was claimed to be due to the formation of

an intermolecular hydrogen-bonded antiparallel β-sheet structure (Chehín et al., 1999; Dong

et al., 1995). Thus, protein aggregation judged by an increase in non-native intermolecular

β-sheet structures is the beginning of the loss of native protein which is a common reaction

to the application of thermal, physical or chemical stresses (Chehín et al., 1999; Chi et al.,

2003). The growth of aggregation bands is accompanied by the rise of another broad band

at 1645 cm-l, which can be assigned to unordered structure or random coil (Dong et al., 1995).

This was noticed for CPC_0.01_10 (Figure 5.8a), which still has some secondary structure;

however the peaks are significantly diminished and the appearance of a broad platform at

1645 cm-1 is observed. Finally, at pH 12 (CPC_0.01_12) more loss in the secondary structure

was observed, the protein was denatured with a broad spectrum within Amide I`, distributed

between 1600 and 1700 cm-1. Structural differences in Figure 5.8a are induced by the

135

different characteristics of the solvent which in one case is EAS and in the other case saline

solutions adjusted to desired pH. Clearly, the properties of a solvent play an important role.

The pH of a solution determines the charge on the protein molecule. Proteins are normally

stable against aggregation over narrow pH ranges and may aggregate rapidly in solutions

with pH outside these ranges. Thus, in the work of Jarpa-Parra et al. (2014) the pH change in

the range of 7-9 did not have an impact on the secondary structure of lentil proteins.

However, at extreme values protein unfolded exposing its buried sites and changing the

electrostatic interactions (Chehín et al., 1999). As the pH changes the number of charged

groups increases which by turn increases the charge repulsions. This is sufficient to overcome

the attractive forces (mostly hydrophobic and dispersive) resulting in protein destabilization

and partial unfolding (Chi et al., 2003). First it was thought that aggregation arose from

completely unfolded protein, however further research showed that aggregates were formed

from partly unfolded proteins (Chi et al., 2003). The protein`s native conformation is flexible

and does not exist as a single structure but as an ensemble of conformations and the

aggregation may take place even at physiological conditions (Chi et al., 2003). Aggregation

band and the presence of random coil both characterize protein unfolding, however, when

the presence of aggregates is an intermediate state and protein molecule can refold again as

it still has secondary structure, the random coil means the loss of secondary structure and its

properties. Therefore the unfolded state is normally devoid of any native structure and exists

as a structureless random coil (Salahuddin, 1984; Tanford, 1968). Figure 5.8b shows the

spectroscopic characteristics of protein concentrates in the presence of 1M NaCl. The bands

of the spectra are similar to those shown in Figure 5.8a, however all bands are shifted to the

higher values which indicates the weakening of H-bonding (Zhao et al., 2008). This was also

observed by Ma et al. (2001) who studied the effect of different salts on the conformation of

oat globulins. In addition, an appearance of a new band in EAPC_C1_1_10 which was not

present in Figure 5.8a was noted. This band centered at 1644 cm-1 as previously discussed

was assigned to a random coil conformation and the presence of this band indicates a higher

level of disorder in protein`s secondary structure. Similar to Figure 5.8a this band grows

from the top protein to the bottom one. The decrease in 1672, 1654, and 1635 cm-1 and an

increase in 1690, 1644 and 1618 cm-1 peak intensities suggest transition from ordered

conformation to random coils and aggregated strands. Salts are known to perturb protein

136

conformation by affecting both electrostatic and hydrophobic interactions via a modification

of water structure (Ma et al., 2001). Over 100 years ago Hofmeister discovered that adding

salts to egg white protein could alter its solubility (Kunz et al., 2004; Schwartz et al., 2010).

The degree of the solubility is governed by the type of ions, their concentration and their

ability to increase or decrease the hydrophobic interaction, resulting in “salting-in” or

“salting-out” effects. This phenomenon became known as ̀ `Hofmeister series`` or ̀ `lyotropic

series``. Salts with the ability of the ions to reduce the hydrophobic interaction may enhance

the unfolding tendency of proteins were called chaotropes or structure breakers and salts

which can increase the hydrophobic interactions were called ``kosmotropes``, or ``structure

makers`` (Schwartz et al., 2010). However such classification was argued (Zangi, 2009).

NaCl depending on its concentration can cause both ``salting-in`` and ``salting-out effects``,

at low concentrations working as a chaotropic salt but with an increase in molar concentration

it acts as a kosmotrope and stabilizes protein conformation. Thus, it could precipitate proteins

only in the presence of 3.5N and higher salt concentration (Kunz et al., 2004). In the current

work the salt concentration was chosen in order to increase the extractability of proteins

which was reflected on the protein spectra.

5.6.5.2. Secondary structure of protein isolates

The spectra of protein isolates were quite different in shape and peak intensities,

indicating that proteins were more stable in concentrates. Broader and flatter surface suggests

that protein`s native state was more affected in protein isolates in comparison with protein

concentrates. As opposed to concentrates all the isolates were subjected to isoelectric

precipitation which could explain the difference in their secondary structure. The net charge

on the protein due to the titration of all the ionizing groups led to intramolecular repulsion,

sufficient to overcome the attractive forces resulting in at least partial unfolding of the

protein. This is supported by the presence of the peak close to 1645 cm-1 corresponding to a

random coil which was not present in the concentrates (Figure 5.9a). The Figure 5.9b was

characterized by the higher level of disordered structure and the band shifting towards the

higher frequencies, similar to what was observed in the concentrates and explained by the

presence of NaCl. On the whole, the tendency was similar in protein isolates and

concentrates. With an increase in the severity of treatment the aggregation bands and those

related to random coil increased in intensities, while those related to the native structure

137

decreased. In spite of acidic precipitation step which had certain negative effect on both

conventional and EA isolates, the latter managed to retain more of the native structure.

..

Figure 5.9 : Deconvulved FTIR spectra: a) Protein isolates extracted with 0.01 M NaCl; b) Protein isolates extracted with 1 M NaCl.

Conclusion

EAS proved to be an effective and a better medium for protein extraction from canola

meal. In contrast to conventional techniques which use chemicals, EA is a “green

technology” as it utilizes electric current to generate solutions possessing desired properties.

Both quantitative and qualitative analyses were performed comparing the extraction

with EAS with the conventional method. Proteins extracted by EAS were of higher quality

as seen in the SDS PAGE and FTIR results, also, the protein extractability was higher when

compared to conventional extraction for same process parameters (pH and time). It was noted

that the protein yield could be increased by increasing the current intensity, time of treatment

and salt concentration. Another option is to perform the extraction inside the EA cell, which

a b

138

would ensure a constant generation of OH-. However, it would influence the quality of

extracted proteins, resulting in the conformational changes and possibly higher denaturation

rates. In addition, there is a risk of membrane fouling or precipitation on the electrodes.

The structure of proteins dictates its quality and functional properties which determines their

further industrial utilization and commercial significance. Therefore, next objective should

be to analyze and compare the functional properties of proteins obtained by EAS and

conventional extraction.

Acknowledgments





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6. CHAPTER 6: Study of the functional properties of canola protein

concentrates and isolates extracted by electro-activated solutions


Following the study of physico-chemical properties in the previous chapter which gave us

some indications of improved quality of proteins extracted by electro-activated solutions in

comparison with conventional method, the effect of electro-activated solutions on the

functional properties was monitored in the current chapter.

This chapter is presented as an article entitled: “Study of the functional properties of canola

protein concentrates and isolates extracted by electro-activated solutions”.






This article was accepted for the publication in the “Food Bioscience” (2015), Volume 12, 1, Pages 128-138

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Résumé

Cette étude a comparé les propriétés fonctionnelles des isolats et des concentrés de protéines

de canola produits par l’utilisation de solutions électro-activées par rapport à des protéines

extraites par la méthode alcaline conventionnelle. Pour les isolats, les résultats n’ont montré

aucune différence significative en termes de solubilité des protéines, le caractère hydrophobe

de la surface, l’absorption de l’eau et la capacité d'absorption du gras. Toutefois, pour les

concentrés de protéines, une plus grande capacité d'absorption du gras a été observée pour

les protéines extraites par des solutions électro-activées. En outre, certaines propriétés tensio-

actives ont également été améliorées, notamment l'indice d'activité d’émulsification qui était

plus élevé pour les protéines extraites par électro-activation. Également, la taille des

particules était plus petite pour les protéines extraites par la méthode d’électro-activation.

Dans tous les cas, les propriétés fonctionnelles ont été fortement dépendantes de pH du milieu

et le comportement des isolats et des concentrés était tout à fait différent dans la gamme de

pH proche du point isoélectrique, ainsi que dans les régions neutres et alcalines. Pour

l'ensemble des fonctionnalités, en général les résultats ont montré que les concentrés sont

plus efficaces à pH 4, tandis que les isolats donnent des meilleurs résultats à pH 7 et pH 9.

141

Abstract

This study compared the functional properties of canola protein isolates and

concentrates produced by electro-activated solutions and conventional alkaline extraction. In

the case of the isolates, the results showed no significant difference in terms of protein

solubility, surface hydrophobicity, water absorption and fat absorption capacity. However,

for the protein concentrates, higher fat absorption capacity was observed for the proteins

extracted by electro-activated solutions. Moreover, some surface active properties were also

enhanced, notably higher emulsion activity index and smaller droplet size were observed for

the proteins extracted by the electro-activation method. In all cases, the functional properties

were strongly dependent on the pH of the medium and the behavior of the isolates and

concentrates was quite different in the pH range close to isoelectric point, in neutral and

alkaline regions. For all the functionalities, generally the results showed that the concentrates

were more effective at pH 4, whereas isolates performed better at pH 7 and pH 9.

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Introduction

Proteins in the form of isolates or concentrates are important ingredients in many food

products, where they perform specific functions (Kinsella and Melachouris, 1976a). The

quality of a food protein is established by its nutritional and functional properties (Schwenke,

2001). Nutritional properties include nutritive value, which is the amount of essential amino

acids, their bioavailability, or in other words “properties affecting the body after passage of

food into the alimentary canal” (Akiva, 1981). Functional properties could be defined as

“physical and chemical properties which affect the behavior of proteins in food systems

during processing, storage, preparation and consumption” (Kinsella and Melachouris, 1976a)

or “properties influencing foods prior to entering the body” (Akiva, 1981). This term has

been widely used in close relationship to the industrial utilization of proteins in various food

components. The range of functional properties is very wide and shows whether the protein

can be incorporated in a food matrix so as to impart some specific property; e.g. organoleptic

(colour, flavor, mouthfeel), textural (viscosity, adhesion), rheological (gelation, dough

formation, elasticity), surface active (emulsification, foaming), etc. (Kinsella and

Melachouris, 1976a). These properties may be more important than nutritional properties

when the protein is applied not as a main component but as an ingredient in a complex food

system (Schwenke, 2001). Nutritive value is of little importance if the protein or the product

in which it is incorporated is not acceptable for eating due to inappropriate texture, mouthfeel,

appearance, or flavor (Kinsella and Melachouris, 1976a). Functional properties of a protein

are governed by the qualitative/quantitative content and the sequence of amino acids,

building long polypeptide chains of different conformations. Molecular size, shape, and net

surface charge are no less important. Yet not only the intrinsic protein properties but also

their interactions with other food ingredients will determine their further utilization. The

majority of food systems are multicomponent and their functionality is the result of numerous

interactions between the different components. Thus, emulsion includes the interplay

between proteins, water, and lipids, whereas foams are formed from water, protein and air.

In addition, the environmental conditions such as temperature, pH and ionic strength play a

significant role (Kinsella, 1981).

Among new and developing sources for food applications, proteins from oilseeds

deserve particular consideration. Although canola is mostly regarded as a source of healthy

143

oil, it has also been reported to be rich in balanced proteins, which could be found in the oil

cake left after oil extraction (Khattab and Arntfield, 2009; Ohlson and Anjou, 1979). Many

studies were devoted to various types of protein extraction and characterization and the

results were quite inspiring (Ghodsvali et al., 2005; Tzeng et al., 1988a). Its nutritive value

is similar to soybean proteins which are extensively used in food processing (Bell, 1993;

Delisle et al., 1984). Amino acid composition is balanced and highly suitable for use in

products for 10-12-year-olds and adults (Klockeman et al., 1997; Pedroche et al., 2004; Tan

et al., 2011a). Apart from having high biological value, canola proteins were found to possess

highly interesting functional properties which imparts their further utilization in the industry

so as to potentially substitute animal proteins (Aluko and McIntosh, 2001; Ghodsvali et al.,

2005).

The type of protein isolation technique directly influences its quality and composition

as well as functional properties by acting directly on their conformation and on the types of

proteins being extracted. A conventional direct alkaline extraction (DIR) which comprises

extraction at highly alkaline medium (pH >10) using diluted NaOH solutions or deionized

water with pH adjustment with precipitation at the isoelectric point has been reported to have

adverse effects on proteins. Harsh conditions used during extraction launch a set of

undesirable reactions such as protein denaturation, dissociation and amino acid racemization

(Moure et al., 2006; Pedroche et al., 2004). In addition, the use of chemicals generates large

amounts of pollutants which need further waste management and which negatively affect the

environment. Thus, a milder extraction method is required, which could solubilize proteins

without damaging their native conformation, maintain their activity and at the same time

would give high percentage of extracted proteins. In our previous work (Gerzhova et al.,

2015a), a novel technology that uses Electro-activated solutions (EAS) was used for protein

and total dry matter extraction from canola meal. This method uses an electric field to

produce alkaline solutions on the basis of water electrolysis and which have been claimed to

possess good extractive properties. To assess the quality of proteins extracted by EAS and

DIR, the analysis of secondary structure and physicochemical properties were performed and

revealed certain differences. Changes in protein secondary structure may cause changes in

the hydrophobicity or hydrophilicity, and structural stability of protein which are in close

144

connection with the functional properties such as water holding capacity, surface

hydrophobicity and emulsifying activity.

Considering that data on the functional properties would be useful in terms of

verifying the possibility of their incorporation in food matrix (Zhu et al., 2010). The aim of

this study was therefore to evaluate and compare the functional properties of protein isolates

and concentrates extracted by conventional technique and by means of alkaline electro-

activated solutions generated in an electro-activation reactor.

Materials and Methods

6.5.1. Raw materials and extraction methods

Protein isolates and concentrates were produced from defatted canola meal (kindly

provided by Bunge ETGO, Becancour, Québec, Canada). NaCl, HCl, NaOH were purchased

from Laboratoires MAT Inc (Montreal, Canada). Two extraction methods were used: the

conventional alkaline extraction at pH 10 in 0.01 M NaCl and the extraction by EAS

(reactor`s configuration I, 0.01 M NaCl solution in the cathodic compartment, current

intensity 0.3 A, electro-activation time 60 min) as described in our previous study (Gerzhova

et al., 2015a). For both methods, the extraction was conducted during 60 min. The protein

concentrates were obtained by freeze drying of the supernatant straight after extraction and

centrifugation at 10,000 x g, while the protein isolates were obtained by precipitation of the

proteins from the supernatant with 1 M HCl followed by freeze drying. Protein isolates and

concentrates extracted by EAS will be referred to as ``electro-activated protein isolate``

(EAPI) and ``electro-activated protein concentrate`` (EAPC) accordingly, and those

extracted by the conventional alkaline extraction as conventional protein isolate (CPI) and

conventional protein concentrate (CPC).

6.5.2. Nitrogen solubility index

Protein solubility was analyzed according to the method of Jarpa-Parra et al. (2014).

Concentrates and isolates were dissolved in distilled water to obtain a 0.5% protein

concentration and the pH of the suspension was adjusted to 2–10 by using 0.1 M HCl or

NaOH solutions. Afterwards they were stirred for 1 h and centrifuged during 10 min at 10,000

x g at 23°C with the help of Eppendorf centrifuge 5804R (Eppendorf AG, Hamburg,

145

Germany). After that, the supernatant was carefully decanted and the protein content was

analyzed by the bicinchoninic acid (BCA) protein assay (Fisher Scientific, Waltham, MA,

USA) with bovine serum albumin (BSA) as the protein standard. All the analyses were

performed in triplicate. Protein solubility was calculated as nitrogen solubility index (NSI)

as follows:

(%) = ∗ ( )

( )∗ (%) ∗ 100 (Eq. 6.1)

6.5.3. Water absorption capacity (WAC)

Water absorption capacity of the canola protein concentrates and isolates was

measured according to Ghodsvali et al. (2005). Sample of 1 g of each material was carefully

mixed with 10 ml deionized water in 15 ml centrifuge tubes with the help of a glass rode

during 30 s. This was done every 10 min, and after 30 min the tubes were centrifuged at 2,000

x g in the Eppendorf centrifuge 5804R for 15 min at ambient temperature (23°C). The

supernatant was decanted and the tube was inverted and drained for 15 min before being

weighed. The WAC was expressed as the amount of water absorbed per gram of sample.

6.5.4. Fat absorption capacity (FAC)

Fat absorption capacity was measured as described by Pedroche et al. (2004). Sample

of 0.5 g of each material was mixed with 6 ml of canola oil during 30 s with a glass rode

every 5 min and after 30 min, the tube was centrifuged in the Eppendorf centrifuge 5804R at

1,600 x g for 25 min at 23°C. Afterwards, the free oil was decanted and the FAC was

expressed as the amount of absorbed oil per gram of sample by weight difference.

6.5.5. Surface characteristics

Dispersions of 0.2% (w/w) protein content of isolates and concentrates were prepared

by solubilizing them in deionized water at room temperature (23°C) for 1 h at pH 4, 7, 9 and

left overnight.

6.5.5.1. Surface tension

Surface tension at the air-water interfaces was obtained by surface tension

measurements according to the Wilhelmy Plate method using KRÜSS 2570 Processor

146

Tensiometer (KRÜSS, Hamburg, Germany) at 20°C. The platinum plate and the sample

vessel were thoroughly cleaned at the end of each analysis and flamed with the help of a

Bunsen burner.

6.5.5.2. Interfacial tension

Interfacial tension between solutions and canola oil was determined by the Du Noüy

ring method using KRÜSS 2570 Processor Tensiometer (KRÜSS, Hamburg, Germany) at

20°C. A protein solution was first added to the vessel after which canola oil as a lighter phase

was carefully added and the measurements were taken every 2 min with a total of 10

measurements after layering of the oil. The interfacial tension between water (without

protein) and canola oil was also measured.

6.5.5.3. Surface hydrophobicity

Average surface hydrophobicity was determined using the fluorescent probe, 8-

anilino-1-naphthalenesulfonic acid (ANS) according to the method of Kato and Nakai (1980)

with some modifications as described by Karaca et al. (2011a). Protein dispersions of 0.01%

were solubilized in appropriate buffers at pH 4 (sodium phosphate citrate buffer), pH 7

(phosphate buffer), pH 9 (borate buffer) and diluted with the same buffer to obtain 0.002%,

0.004%, 0.006%, 0.008%, and 0.01 % (w/w) pure protein concentrations. All the solutions

were prepared in duplicate. After that, 10 µl of ANS (8.0 mM in 0.1 M phosphate buffer, pH

7) was added to one tube at each concentration containing a 2 ml sample and vortexed for 10

s, while 10 µl of a phosphate buffer was added to the other tube as a control which was also

vortexed. After keeping each sample in the dark for 15 min, the relative fluorescence intensity

(RFI) of each sample and control was measured with a Cary Eclipse Fluorescence

Spectrophotometer (Varian inc., Palo Alto, CA, USA) at excitation and emission

wavelengths (λex, λem) of 390 and 470 nm, respectively. The excitation and emission slit

widths were 5 nm. The net RFI at each protein concentration was determined by substracting

the corresponding blank and plotted versus protein concentration. Initial slope (S0) of the line

was calculated by linear regression analysis and used as an index of protein surface

hydrophobicity (H0).

147

6.5.6. Surface active properties

6.5.6.1. Foaming properties

Foaming properties were analyzed according to the method of Zhu et al. (2010).

Canola protein concentrates and isolates samples (0.2 g) were dispersed in 20 ml of deionized

H2O and the pH was adjusted to 4, 7, 9, by 0.01-1 N HCl or NaOH followed by 1 h stirring

with a magnetic stirrer at ambient temperature (23°C). The solutions were homogenized with

the help of Ultra Turrax T-25 (VWR International, RADNOR, PA, USA) at 22000 rpm

during 2 min. Foaming capacity (FC) was expressed as the volume difference before and

after whipping:

(%) = (Eq. 6.2)

Where V0 is the initial volume and Vt is a volume measure at different times after whipping.

Foaming stability (FS) was measured after 2, 5, 10, 20, 30, 40, 50, and 60 min and expressed

as:

(%) = (Eq. 6.3)

Where FC0 is a foaming capacity at 0 min that is to say right after whipping.

6.5.6.2. Emulsifying properties

Emulsifying properties were determined as Emulsion activity index (EAI) and

Emulsion stability index (ESI) by the turbidimetric technique of Pearce and Kinsella (1978).

First, aqueous protein solutions were prepared by dissolving protein samples to get 1% total

concentration in appropriate buffers as for the surface hydriophobicity analysis with pH 4, 7,

9. The suspension was then stirred overnight. Each preparation also contained 0.05% sodium

azide to prevent microbial growth. An oil-in-water emulsion was obtained by emulsifying 30

ml of protein solution with 10 ml of canola oil using UltraTurrax T-25 equipped with a S25

N-18G dispersing tool at 7500 rpm for 1 min, followed by stirring at 14500 rpm for another

minute. A 10µl sample was taken from the bottom immediately after the emulsion was

prepared and after 10 min of static storage and diluted with 0.1% SDS to give absorbance

between 0.1 and 0.6 at 500 nm. The absorbance was measured by HP 8453 UV visible

spectrophotometer (Agilent Technologies, Santa Clara, CA, USA) with SDS solution as a

148

blank using plastic cuvettes (1 cm path length). The EAI and ESI were then calculated as

follows:

= ∗( )∗ ∗ (Eq. 6.4)

= . (Eq. 6.5)

= (Eq. 6.6)

Where T is turbidity, DF - dilution factor, φ -volume of oil fraction, C- weight of protein per

unit volume of the aqueous phase (g/mL), 10 000 - the correction factor for square meters, l-

path length of the cuvette (1 cm), A0 is absorbance measured at 500 nm at 0 min, ∆A is

absorbance difference between 0 min and 10 min, (Fennema, 1996).

6.5.6.3. Creaming stability

Creaming stability was measured as described by Karaca et al. (2011a). The same

emulsion as for the EAI analysis was prepared, poured in glass tubes, kept at room

temperature, and was visually observed after 24 h. The height of the creaming layer was

measured and the creaming stability (CS) was expressed as follows:

(%) = ∗ 100 (Eq. 6.7)

Where Ht is the height of the turbid layer and He is the total height of the emulsion.

6.5.6.4. Droplet size

Average droplet size of emulsions was determined using the method described by Tan

et al. (2014). Emulsions were prepared as for the EAI analysis at three different pH values

(4, 7, and 9). The mean droplet size of the studied emulsions was analyzed by mean of

Mastersizer Nano 3000 (Malvern Instruments Ltd., Malvern, U.K) equipped with Hydro EV

dispersion unit. Refractive index and absorption were set at 1.467 and 0.001 respectively.

The obscuration in all measurements was kept at around 14%.


All the experiments were performed at least in duplicate and the data presented are

means ± standard deviation. Analysis of variance (ANOVA) at 95% confidence level was

performed with the Tukey`s test by using Minitab statistical program (Minitab Inc., State

College, PA, USA).

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6.6.1. Nitrogen solubility index

The solubility of canola protein isolates and concentrates is shown in Figure 6.1. For

all the samples, the lowest solubility with average values of 8.25 ± 0.90% for EAPI, 8.09 ±

0.85% for CPI, 22.08 ± 0.04% for EAPC and 20.88 ± 1.04% for CPC, respectively was

observed at the pH range between 4 and 5 which corresponds to pH range of isoelectric point

for canola proteins (Gerzhova et al., 2016; Tan et al., 2011a). For proteins, a U-shaped

relationship with the lowest point situated close to the isoelectric point has been noted

(Cheftel et al., 1985). Consistent to what was reported by other authors, the solubility

increases in both senses towards acid and alkaline pH and reaching its maximum at pH 10

with mean values of 56.73 ± 3.08%, 56.82 ± 3.57% for EAPI and CPI and 66.71 ± 4.15%

and 63.78 ± 3.37% for EAPC and CPC, respectively. These results are in good agreement

with the data reported previously in other works on canola or related to canola proteins (Aider

et al., 2012a; Mao and Hua, 2012; Pedroche et al., 2004; Zhu et al., 2010). The solubility of

concentrates was significantly higher within the whole tested range of pH, indicating more

hydrophilic character of the extracted proteins or less damaged structure. Isolates were

additionally subjected to the influence of acid in order to precipitate them, which could cause

the further unfolding of the molecules with the liberation of hydrophobic patches. As shown

in our previous study, the isolates and concentrates were quite different in their secondary

structures with the isolates being more unfolded as indicated by the presence of some peaks

which were not observed in the concentrates (Gerzhova et al., 2015a) . In the literature, it has

been stated that solubility can serve as an index of the extent of protein denaturation (Kinsella

and Melachouris, 1976a; Zhu et al., 2010). Moreover, proteins having low solubility over a

broad pH range are an indication of severe denaturation which have been shown to markedly

affect the functional properties of such proteins (Mwasaru et al., 1999a). Also part of soluble

proteins could have been lost during the precipitation step. In the present study, there was no

significant difference (p > 0.05) between the proteins extracted by electro-activated solutions

and conventional alkaline extraction. This can be explained by the fact that the pH of

extraction process in both methods was 10. Thus, it is possible to conclude that EAS did not

influence the solubility profiles within the selected range. However, it is important to mention

150

that protein isolate extracted by the electro-activation method had higher solubility in the pH

range from 5 to 9. This particularity makes it possible to enhance the use of canola proteins

in some applications where the solubility is a key point. This is justified by the fact that

among all the functionalities, solubility is probably one of the most important one, as it will

impact in almost all the other functional properties. High degree of solubility of the

ingredients predetermines its successful industrial utilization whereas poor solubility

significantly decreases the range of potential applications.

pH

0 2 4 6 8 10 12

NS

I, %

0

10

20

30

40

50

60

70

80

EAPI

CPI

EAPC

CPC

Figure 6.1: The solubility behavior of canola proteins as a function of pH, where EAPI is electro-activated protein isolate, CPI is conventional protein isolate, EAPC is electro-activated protein concentrate and CPC is conventional protein concentrate.

6.6.2. Water absorption capacity

The results on the water absorption capacity of the canola protein concentrates and

isolates produced by both methods (electro-activation vs conventional) are summarized in

the Table 6.1 and are in accordance with the other results already published on such or related

to canola proteins (Dev and Mukherjee, 1986; Pedroche et al., 2004; Thompson et al., 1982).

Statistical analysis of the obtained data did not show any significant difference (p > 0.05)

between the WAC of canola proteins extracted by EAS and DIR.

151

Table 6.1: Absorption capacities of electro-activated protein isolate (EAPI), conventional protein isolate (CPI), electro-activated protein concentrate (CPC), and conventional protein concentrate (CPC).

WAC FAC

EAPI 1.25 ± 0.05a 0.97 ± 0.01a

CPI 1.30 ± 0.13a 1.11 ± 0.04a

EAPC ND* 3.46 ± 0.16c

CPC ND* 3.02 ± 0.02b

*ND – not determined

** Results represent the average of three determinations ± SD, values in the same column with different

letters are significantly different (p< 0.05)

***WAC - Water absorption capacity

****FAC - Fat absorption capacity

Although some authors reported good WAC for canola or related to canola proteins

(Lin et al., 1974; Sosulski et al., 1976), high water absorption is not always a good indication,

since a compound with very high WAC may bind too much of water and as a result dehydrate

other components in the system (Xu and Diosady, 1994). No data on WAC of concentrates

has been obtained for both concentrates in the present study due to their high water solubility.

This was also previously reported in the works of Xu and Diosady (1994) and Manamperi et

al. (2012). In the case of Xu and Diosady (1994), highly soluble protein isolate with low

WAC was obtained by membrane processing from the water soluble protein fraction. It is the

insoluble proteins mainly that contribute to higher WAC as they tend to swell and imbibe

water, a phenomenon which was also observed by Zhu et al. (2010). If conventional

extraction is used, the WAC increases with an increase in pH of extraction. Some authors

reported inverse relationship with protein solubility (Mwasaru et al., 1999a). On the other

hand, the review of Hutton and Campbell (1981) on WAC showed that most of the studies

agree that there is no direct correlation between the ability to absorb water and the solubility

of proteins. Kinsella and Melachouris (1976a) studied the influence of pH on water

absorption capacity and stated that the pH had little effect on this parameter, which indicates

an absence of a direct correlation between water binding and protein solubility.

For practical considerations, solubility measurements do not provide with the

information on whether a protein will bind more or less water or contribute to the texture

profile of a product (Hermansson, 1979). Thus, WAC is used in such case. For example,

152

WAC is extremely important in a variety of meat products because it contributes to the

binding and retaining of meat juices, enhances mouthfeel and flavor of meat products. In

bakery products and cheeses, the WAC helps to prolong the product shelf life (Kinsella,

1979b). In other studies, protein has been reported to be primarily responsible for water

absorption. The ability of proteins to absorb water is related to their surface hydrophilicity as

the protein-water interactions starts at polar amino-acid sites on the protein molecule; i.e.

carbonyl, hydroxyl, amino, carboxyl, and sulfhydryl groups. However, the environment

which affects the protein conformation (secondary structure) is also very important. The

conformational transformation during unfolding may liberate the groups buried inside the

protein molecule thereby increasing or decreasing its hydrophilicity, depending on the nature

of those groups (Hutton and Campbell, 1981). Size, shape of the protein molecule as well as

lipids, carbohydrates and tannins associated with proteins also have an impact (Mao and Hua,

2012). It is noteworthy that the presence of carbohydrates may add to higher WAC due to

the presence of hydrophilic parts such as polar or charged side chains (Zhu et al., 2010).

6.6.3. Fat absorption capacity

Fat absorption capacity of the studied canola protein concentrates and isolates is

presented in the Table 6.1. Statistical analysis of the obtained results showed that

significantly higher values (p < 0.001) were obtained for canola protein concentrates

produced by both direct conventional alkaline and electro-activation methods than for the

isolates. At the same time, the results showed that there was no significant difference (p >

0.05) between FAC values of the isolates produced by the two methods. However, for the

concentrates, significantly (p < 0.001) higher values were obtained for protein produced by

the use of the electro-activation based extraction method.

Data obtained in this study on the FAC values of the canola protein concentrates and

isolates match up with the values obtained by Pedroche et al. (2004), Yoshie-Stark et al.

(2006), and Xu and Diosady (1994). The observed differences between the two extraction

methods in regards to the fat absorption capacity of the extracted materials can be explained

by the fact that FAC is regarded as the physical entrapment of oil and is highly affected by

protein content and protein conformation (Kinsella, 1979b). In this regard, in our previous

study we showed that the canola proteins extracted by EAS have significant conformational

153

differences in comparison with those extracted by the conventional method (Gerzhova et al.,

2015a) . The results on the capacity of canola proteins to bind fat materials are very important

for food applications. Indeed, protein-lipid interactions are involved in many other functional

properties. For example, lipoprotein complexes are functional components of egg yolk,

meats, milk, coffee whiteners, dough, and cake batters. This interaction enhances the

organoleptic acceptability of foods in which proteins are incorporated and impart better

flavor retention, texture and mouthfeel. However, in some types of foods such as doughnuts

or pancakes, high FAC may not be desired and the addition of soy flour is sometimes used

to prevent the excessive fat absorption during cooking (Hutton and Campbell, 1981).

6.6.4. Surface characteristics

Surface characteristics such as surface tension or interfacial tension can be used in

the evaluation of the surface activity of proteins. The force that acts at the interface between

two liquids or a solid and a liquid is called the interfacial tension or surface tension when it

acts between a gas and a liquid. Protein molecules being of amphiphilic nature and having

both hydrophobic and hydrophilic residues possess the ability to lower the tension between

two immiscible substances by migrating towards the interface and aligning there. As a result,

hydrophobic and hydrophilic residues enter aqueous and non-aqueous phases by unfolding

and stretching at the interface (Cheftel et al., 1985). The parameters such as the shape, size

of the molecule as well as its speed at which it can reach the interface are crucial. Main

surface characteristics of the studied proteins for both extraction methods are discussed

below.

6.6.4.1. Surface tension

The behavior of canola protein concentrates and isolates at the air-protein solution

interface is shown in the Table 6.2. Statistical analysis of the data showed that pH had minor

effect (p > 0.05) on the changes in the surface pressure. Nevertheless, proteins solubilized at

pH 7 and pH 9 had slightly lower values, i.e. better surface active properties in comparison

with pH 4, which can be explained by lower protein concentration as a result of lower

solubility at pH 4. Within the same pH, the results were as follows: no significant difference

(p> 0.05) between samples at pH4. At pH 7, EAPC and EAPI gave significantly lower values,

154

which means their surface characteristic were better compared to conventional ones. Finally,

at pH 9 only EAPI was significantly different.

Table 6.2 : Surface pressure at air-protein solution interface.

Surface Tension, mN/m

Sample pH 4 pH 7 pH 9

EAPI 46.97 ± 1.69a* 41.45 ± 0.56a** 42.57 ± 0.05a**

CPI 47.77 ± 1.23a* 45.43 ± 0.54bc* 45.50 ± 1.59b*

EAPC 49.37 ± 1.31a* 44.10 ± 0.2ab** 45.59 ± 0.46b**

CPC 48.67 ± 0.31a* 47.22 ± 0.01c* 47.95 ± 1.21b*

*Results represent the average of three determinations ± SD, values in the same column with different

letters are significantly different (p< 0.05)

6.6.4.2. Interfacial tension

A pH factor had significant effect (p < 0.001) on the interfacial tension at the oil-

protein solution interface as shown in Table 6.3. In comparison with pure water which gave

an interfacial tension value of 21.23 ± 0.12 mN/m, all of the produced canola protein samples

significantly reduced the interfacial tension. Proteins solubilized at pH 4 managed to lower

the tension by 50% and samples at pH 7 and pH 9 by 75%. It is important to mention that no

statistically significant difference (p > 0.05) between proteins extracted by EAS and by DIR

was found. It should also be noted that protein concentrates had slightly lower interfacial

tension values in comparison with isolates. Lower surface activity at pH 4 can be attributed

to the lower protein solubility, as aforementioned (Figure 6.1). High protein solubility is

necessary for rapid migration and adsorption at the oil-water interface (Karaca et al., 2011a).

In addition, poor solubility resulted in lower protein concentration which also contributed to

observed lower results. The percentage of proteins adsorbed at the interfaces was measured

by Liang and Tang (2013) and the results they reported showed that the least amount of

proteins was adsorbed at pH close to the pI, corresponding to the lowest solubility, a fact

which explained the higher interfacial tension and poorer functional properties.

155

Table 6.3 : Interfacial tension between oil and protein solution.

Interfacial tension, mN/m

Sample pH 4 pH 7 pH9

EAPI 10.65 ± 0.27abc* 5.52 ± 0.31a** 4.55 ± 0.50a**

CPI 11.82 ± 0.81ac* 5.95 ± 0.41a** 4.65 ± 0.43a***

EAPC 10.28 ± 0.39b* 5.24 ± 0.42a** 5.42 ± 0.19a**

CPC 11.84 ± 0.80c* 5.29 ± 0.36a** 5.83 ± 0.36a**

* Means followed by the same letter in the columns are not significantly different (P > 0.05). Values given are

means of duplicate determinations.

6.6.4.3. Surface hydrophobicity

Results on the surface hydrophobicity are shown in the Figure 6.2 and are in good

agreement with those of Wu and Muir (2008) who reported hydrophobic value of 950 for

canola protein isolate at pH 7. The analysis of the obtained data in this study showed no

significant difference (p > 0.05) between the EAPI and CPI, presumably due to the acidic

precipitation step in the protein isolation process which compensated the effects of extraction

treatment. However, a higher level of surface hydrophobicity was observed for CPC in

comparison with EAPC at pH 4. At pH 4, the S0 of the canola protein concentrates was

almost twice as much in comparison with the isolates. This result shows that at this pH, the

isolates and concentrates are in different conformations. As was shown in the previous study

(Gerzhova et al., 2015a), the FTIR spectra revealed considerable differences in the secondary

structures of isolates and concentrates, a factor which could significantly influence their

hydrophobicity. However, at pH 7 and pH 9, their surface hydrophobicity was smaller than

those of the isolates showing more hydrophilic character. Generally, the effect of the pH was

highly significant for all the samples (p < 0.001). A progressive decrease of the hydrophobic

character was observed with an increase in pH. The maximum hydrophobicity was obtained

at pH 4. This has been reported to be due to the different conformation of canola protein at

acidic conditions (Folawiyo and Apenten, 1996). An increase in hydrophobicity with a

decrease in pH has also been reported by Das and Kinsella (1989) and by Paulson and Tung

(1987) who observed a steep decrease in hydrophobicity from pH 3.5 to pH 6.5 and a gradual

decrease with further pH increase. In addition, hydrophobicity and solubility are closely

related. As aforementioned, solubility corresponds to the number of exposed hydrophilic

156

groups, whereas the hydrophobicity comes from the number of exposed hydrophobic groups.

The lowest solubility was reported to be around pH 4-5 for all the samples, therefore the

hydrophobicity was expected to be the highest at this pH interval. Although highly soluble

proteins usually perform better in most applications, for some functions a certain degree of

hydrophobicity is required.

Structure-function relationship approach has been used by chemists for years in order

to better understand and be able to predict certain functional properties by regarding protein

structure. Hydrophobicity is the one that is closely correlated with many functional

properties. However, the surface hydrophobicity rather than the average one should be

considered. In fact, the nonpolar amino acids which are buried inside the protein

molecule are unable to participate in any interactions and the surface hydrophobicity is

not correlated with the average one (Keshavarz and Nakai, 1979). The surface

hydrophobicity can give a good indication of protein solubility as well as surface properties

such as foaming and emulsifying. Kato and Nakai (1980) by measuring the surface

hydrophobicity managed to explain the surface properties of proteins by using analytical

protocols. Moreover, hydrophobicity has been reported to be negatively correlated with the

interfacial tension (Keshavarz and Nakai, 1979). The authors stated that the hydrophobic

proteins should be able to interact easily with oil and water molecules simultaneously.

However, the results presented in the current work show that this statement should be treated

with caution. In our case, the most hydrophobic proteins at pH 4 did not show the best surface

active properties. Lower protein solubility, and as a result lower protein concentration in the

solution, was insufficient to decrease the interfacial tension. Therefore, it is not the

hydrophobicity but a reasonable balance between the hydrophilic and lipophilic parts that

determines the good surface active properties and higher hydrophobicity will not necessarily

result in better functionalities (Nakai, 1983).

157

pH 4 pH 7 pH 9

Hydro

phobic

ity

0

1000

2000

3000

4000

5000

6000EAPI

CPI

EAPC

CPC

aa

b

b

a abb b a

ab b

* Bars with different letters are significantly different for a given pH.

Figure 6.2: Surface hydrophobicity of protein isolates and concentrates as a function of pH.

6.6.5. Surface active properties

Surface activity is the ability to lower the interfacial tension. The greater it is the faster

the molecule can migrate and absorb between the layers so as to decrease the tension. The

mobility of molecule is of great importance because the surface tension is affected by surface

concentration and increases with an increase of the latter (Kinsella and Melachouris, 1976a).

Most important from technological point of view surface properties are presented below.

6.6.5.1. Foaming properties

The results on foaming capacity (FC) of the concentrates and isolates produced by

both extraction methods are shown in the Table 6.4 and those of the foam stability (FS) are

shown in the Figure 6.3. For isolates, maximum foaming capacity was obtained with proteins

solubilized at pH 9. Slightly lower values were obtained at pH 7 and much lower values

obtained at pH 4. For the concentrates, the situation was the opposite with maximum values

obtained at pH 4, followed by pH 7 and, finally pH 9. As aforementioned, a lot of factors

influence the foaming capacity and the solubility known to be highly dependent on the pH of

the medium is one of them. The curves of pH versus the FC as well as the pH versus the

solubility were observed to be parallel (Mao and Hua, 2012). An increase in net charge on

the surface of the protein molecule weakens the hydrophobic interactions and favors the foam

158

formation by increasing the flexibility of the molecule and allowing it to spread at the

interface. Another important factor is the hydrophobicity. Townsend and Nakai (1983) who

studied the relationship between the hydrophobicity, solubility, and the foaming capacity

claimed that the optimum foaming capacity would be associated with those proteins whose

dispersibility is more than 40% and hydrophobicity more than 700 which is associated with

a good balance of hydrophilic and hydrophobic groups necessary for effective stabilization

of the air bubbles. Hydrophobicity was found maximum at pH 4 for both isolates and

concentrates where the solubility reached its minimum values (less than 10% for isolates and

around 20% for the concentrates). The results on FC are consistent with the aforementioned

statement for the isolates, whose foamability was the lowest at pH 4. However, the

concentrates showed quite a different tendency. In spite of the low solubility, the concentrates

showed the highest FC at this pH value. This can be related to the fact that their solubility at

pH was higher in comparison with the isolates, showing that 20% solubility in combination

with high hydrophobicity was sufficient to provide good foaming capacity. Similar

information was reported by Townsend and Nakai (1983) who obtained good results for

proteins with solubility around 20% and hydrophobicity above 500, a fact which led to the

conclusion that insoluble proteins might also contribute to the stabilization of air bubbles in

foams in which they can serve as a physical barrier to their coalescence. A decrease in the

foaming capacity at pH 7 and pH 9 for the concentrates is due to their decreased

hydrophobicity. In spite of their high solubility in this pH range, their hydrophobicity is lower

than 500, which suggests that hydrophobicity is a more important parameter for the FC than

solubility. This is in a good agreement with the statement that irrespective of the degree of

solubility, proteins with low hydrophobicity show poor foaming capacity (Townsend and

Nakai, 1983). The extraction method had significant effect (p < 0.001) on the foaming

properties of the canola proteins. The obtained results showed higher values in terms of FC

and FS for isolates extracted with the electro-activation based method in comparison with

those separated by the conventional alkaline extraction method for pH 4. Canola proteins and

napin in particular have been reported to possess good foaming properties (Aluko and

McIntosh, 2001; Nitecka et al., 1986; Nitecka and Schwenke, 1986). However, it is important

to consider that many other factors could have influenced resulting in higher values starting

from the type of meal used, extraction conditions and the method of analysis. Food foams

159

represent a medium where two immiscible substances such as gas (air) and liquid are mixed

with the help of a surface active agent (protein). They are usually measured as the volume

expansion upon the incorporation of air. To perform well in foams a protein should be soluble

in the liquid phase and be capable of rapid migration to the interface to form a film around

nascent gas bubbles (Kinsella and Melachouris, 1976a). The rate of migration of the

proteins is important, however the inherent surface activity and the ability of the protein

to unfold and reorient at the interface is crucial for foam formation. This is closely related

to the intrinsic molecular properties such as molecular size, shape, flexibility,

hydrophobicity, charge, etc. Thus, protein with higher surface hydrophobicity adsorbs more

readily at the interfaces. Flexible and loose molecules can unfold faster, whereas globular

proteins with rigid structure will take more time to adsorb and lower the interfacial tension

(Kinsella and Melachouris, 1976a).

Table 6.4: Foaming capacity of protein isolates and concentrates.

Foaming capacity, %

Sample pH 4 pH 7 pH 9

EAPI 28.92 ± 2.35a 60.46 ± 2.41a 73.64 ± 3.19a

CPI 19.62 ± 0.33b 57.83 ± 1.52a 66.91 ± 5.8a

EAPC 77.38 ± 5.23c 36.14 ± 3.99b 24.73 ± 3.53b

CPC 78.49 ± 8.63c 33.02 ± 2.76b 31.76 ± 3.33b

* Means followed by the same letter in the columns are not significantly different (P > 0.05). Values given are

means of duplicate determinations

The results on FS are shown in the Figure 6.3. The volume of the foam was reduced

gradually within 60 min of observation, showing that foams produced by canola proteins

were not stable. Overall stability of isolates was higher compared to the concentrates with

the exception of those solubilized at pH 4. FS also was higher at higher pH values. The results

are consistent with those obtained by Aluko and McIntosh (2001) who measured the FS after

30 min storage at room temperature for foams at pH 7 prepared from acid precipitated

isolates. Foaming stability is the ability to withstand gravitational and mechanical stress and

is critical if proteins are intended to be used as foaming agents. Not always properties needed

for a good foaming capacity are desirable for a good foaming stability. In general proteins

that possess good foaming properties exhibit poor foaming stability and vice versa. It is

160

noteworthy that foamability and stability are governed by two different sets of molecular

properties. While the foaming capacity depends on the rate of adsorption, hydrophobicity

and flexibility of the molecule, the stability mostly relies on the rheological properties of the

film formed around the bubble (Fennema, 1996). High surface viscosity and high film yield

are required for foam stabilization as they reflect strong cohesion between film forming

molecules. However, it is not desirable in the initial foam formation step (Kinsella, 1981).

time, min

0 10 20 30 40 50 60 70

FS

, %

0

20

40

60

80

100

120EAPI (pH4)

CPI (pH4)

EAPI (pH7)

CPI (pH7)

EAPI (pH9)

CPI (pH9)

time, min

0 10 20 30 40 50 60 70

FS

, %

0

20

40

60

80

100

120EAPC (pH4)

CPC (pH4)

EAPC (pH7)

CPC (pH7)

EAPC (pH9)

CPC (pH9)

Figure 6.3: Foam stability as a function of time: A) FS of proteins isolates, B) FS of protein concentrates.

6.6.5.2. Emulsifying Properties

6.6.5.2.1. Emulsion activity index and droplet size

Results on EAI are shown in the Figure 6.4A. For isolates, the EAI increased with an

increase in pH to attain its maximum at pH 9 with mean values of 22.10 ± 1.74 m²/g of

emulsifier and 19.01 ± 0.20 m²/g for EAPI and CPI, respectively, showing the highest contact

area between the two phases. Simultaneously, the data on the droplet size (Figure 6.4B) show

that the finest droplets were also obtained at the highest pH with average values of 22.37 ±

0.40 µm and 26.15 ± 2.32 µm for EAPI and CPI, respectively. The interfacial tension was

also the smallest at pH 9 showing better surface active properties of proteins at that pH (Table

6.3). The largest droplet size was obtained at pH close to the isoelectric point. Low solubility

at isoelectric point and the lack of repulsive forces results in low protein concentration which

is insufficient to cover the surface of all the droplets to act as a bridge between the oil droplets

leading to their flocculation (Wang et al., 2010). The formation of salt bridges at the

isoelectric point has been reported by Tornberg et al. (1997). The biggest droplet size around

A B

161

isoelectric point has also been reported by Tan et al. (2014) and Liang and Tang (2013) and

decreased when the pH deviated from it. Concentrates showed a different behaviour with an

increase in the emulsifying properties towards the lower pH value. The same tendency was

noticed when foaming properties were analyzed and explained by higher hydrophobicity at

pH 4 and higher solubility in comparison with isolates. Proteins soluble at their isoelectric

points should perform well because protein adsorption and viscoelasticity at an oil-water

interface is maximum near or at isoelectric pH (Kinsella and Melachouris, 1976a). Proteins

with a certain degree of solubility at isoelectric point have the highest EAI, whereas low-

soluble proteins show poor EAI (Fennema, 1996). On the other hand the emulsifying

properties of proteins tends to decrease when protein concentration is increased above a

critical concentration (Kinsella and Melachouris, 1976a). When the concentration of soy

proteins was relatively high an increased density made them less flexible to adsorb and unfold

at the limited oil droplet surface quick enough (Wang et al., 2010). Lower EAI of

concentrates at pH 7 and 9 could be explained due to the aforementioned phenomenon. In

general canola proteins have poor emulsifying properties and collapse easily (Aluko and

McIntosh, 2001). Low EAI could be due to high molecular weight of canola proteins and the

presence of disulfide bonds which reduce their flexibility and ability to unfold and adsorb at

the interfaces. Separated canola proteins` albumin and globulin fractions showed

comparatively better EAI (Tan et al., 2014). Low emulsion activity could also be due to

denaturation of the extracted proteins and a loss of functionality. No significant difference

was observed between protein isolates and concentrates extracted with EAS in comparison

with the proteins extracted by conventional method except for pH 9 where EAPI performed

significantly better than CPI which could be an indication of less damaged structure. This is

consistent with the previous study where the FTIR and SDS PAGE revealed that more native

structure was retained by proteins extracted by EAS (Gerzhova et al., 2015a). Emulsions of

fat and water are systems frequently encountered in food processing which, similar to foams,

are thermodynamically unstable and require a surfactant capable of stabilizing it.

Stabilization of emulsified droplets is performed by formation of a charged layer or a film

around the fat globules which provoke mutual repulsion. The kinetics of film formation is

greatly affected by protein composition and conformation; flexible molecules would unfold

easily, showing high surface activity. This lowers the interfacial tension and prevents droplet

162

coalescence (Kinsella, 1979b). Among the diversity of methods EAI estimates the contact

area between the two phases. According to Mie theory of light scattering, the turbidity of a

dilute suspension of spherical particles is related to its interfacial area which was used by

Pearce and Kinsella (1978) as the basis of the emulsifying activity index (Cameron et al.,

1991).

pH 4 pH 7 pH 9

EA

I

0

5

10

15

20

25EAPI

CPI

EAPC

CPC

aa

bb a

a

b b

a

b

c c

pH 4 pH 7 pH 9

Dro

ple

t siz

e,

µm

0

10

20

30

40

50

60

70EAPI

CPI

EAPC

CPC

a

b

cc

a aa

a

a

bab

bc

* Bars with different letters are significantly different (P < 0.05) for a given pH.

Figure 6.4: Emulsifying properties of canola protein isolates and concentrates: A) Emulsion activity index; B) Droplet size.

Data on the droplet size of emulsions reported in the literature on canola proteins

widely varies which can be explained mostly by varying emulsifying conditions. Droplet size

distribution is strongly dependent on the instrument used, intensity, and time and finer

droplets can be obtained when using vacuum homogenizers or sonication (Tornberg et al.,

1997). The variety of methods of analysis and the absence of a standardized technique make

it difficult to compare the results obtained in other studies. Same turbidimetric technique was

used in the works of Tan et al. (2014), Aluko and McIntosh (2001), and Karaca et al. (2011a).

Although higher results on EAI have been reported by Tan et al. (2014), the discrepancy in

the formula by which the EAI was calculated was found. The method of measuring the EAI

by turbidimetric technique was first introduced by Pearce and Kinsella (1978) and in the

original article the equation uses φ in the denominator instead of (1-φ). However, φ means

the volume fraction of the oil while (1-φ) represents the mass of protein in the unit volume

of emulsion and should be used instead (Fennema, 1996). The validity of such correction was

shown by Cameron et al. (1991). When recalculated the results obtained on protein isolates

at pH 4, 7, and 9 were similar to those reported by Tan et al. (2014).

A B

163

pH 4 pH 7 pH 9

ES

I

0

5

10

15

20EAPI

CPI

EAPC

CPC

ac

bcbc

c a

b

aa a

a a a


Figure 6.5: Emulsion stability index of the proteins.

6.6.5.2.2. Emulsion stability index

Data presented in the Figure 6.5 show that all the emulsions prepared with canola

proteins possessed low stability and underwent rapid coalescence within few minutes after

they were formed. It is an indication of the weak interfacial film formed around the oil

droplets. The thicker the film the lower is the coalescence rate. In addition, the coalescence

stability of droplets is closely related to the visco-elasticity and integrity of the interfacial

films (Liang and Tang, 2013). Low stability of emulsions can also be attributed to the rather

high droplet size. Smaller droplets are less susceptible to the coalescence due to smaller film

area between them and as a result a lower risk of its rupture (Fennema, 1996). For all the

emulsions, the average oil droplet size was greater than 10 µm, which was consistent with

the information reported by Wu and Muir (2008) (Figure 6.4B). The dependence of

coalescence stability on the droplet size has been also shown by Das and Kinsella (1989) who

found that the larger the droplet size, the higher the coalescence rate.

Generally, emulsions are thermodynamically unstable systems due to the high free

energy of the interface between the two phases. The equilibrium is reached when the contact

area between two immiscible substances is minimal. Having high surface area, the droplets

tend to reduce it and hence are subjected to various destabilization processes such as

creaming, flocculation, coalescence, and oiling off (Cameron et al., 1991). As the emulsion

activity can be estimated from the contact area between two phases, the stability of an

164

emulsion should be related to the constancy of the interfacial area. Stable emulsions would

possess the constant turbidity, providing that the interfacial area does not change with time

(Pearce and Kinsella, 1978). Changes in the average droplet size influence the turbidity

causing its decrease and hence can be interpreted as the coalescence. Coalescence is

accompanied by an increase in viscosity, increase in droplet size and decrease in surface area.

Among different destabilization processes that can occur in the emulsion, the changes in the

interfacial area point to the presence of coalescence and do not provide with the information

about creaming or flocculation as creaming itself does not cause a reduction in the interfacial

area of the whole emulsion and flocculation is reversed when the emulsion is diluted with

SDS solution (Pearce and Kinsella, 1978).

6.6.5.3. Creaming stability

Data on creaming stability is presented in Figure 6.6. No significant difference (p >

0.05) was found between the samples treated at different pH or different extraction

techniques, except for the CPI and CPC solubilized at pH 4 and pH 9. The visually observed

separation started almost right after the emulsification and two layers were clearly

distinguishable within one hour after the emulsion was prepared. Such rapid creaming could

be explained by the emulsifying efficiency of the device used to form the emulsion (Ultra

Turrax) which have been reported to give the most rapid creaming of the emulsion formed

when compared with other emulsifiers, notably valve homogenizers and sonication technique

(Tornberg et al., 1997). The rate of creaming depends on the average droplet size, with finer

droplets being more stable against creaming (Das and Kinsella, 1989; Fennema, 1996).

Creaming is one of the destabilization mechanisms characterized by the gravitational phase

separation; i.e. rising of the droplets that have lower density to the top which can be visually

observed. As a result, a turbid layer is formed at the top of emulsion with a transparent layer

at the bottom.

165

pH 4 pH 7 pH 9

Cre

am

ing s

tabili

ty

0

10

20

30

40

50EAPI

CPI

EAPC

CPC a

a

a

a

a

a a a

aa a a


Figure 6.6: Creaming stability of canola protein-stabilized emulsions.

Conclusion

The current study showed the effect of the isolation technique on the functional

properties of canola proteins. Two methods were compared; namely the isolation by electro-

activated solutions and conventional alkaline extraction and their effect on functional

properties such as total proteins solubility, water and fat absorption capacity, as well as their

performance at the interfaces has been studied. The results did not show significant difference

between the two methods in terms of protein solubility, surface hydrophobicity, WAC and

FAC, except for the concentrates separated by EAS that showed significantly higher fat

absorption capacity. In addition, proteins separated by EAS performed better at the interfaces,

EAPI and EAPC at pH 7 and 9 showed lower surface tension at air-protein solution interface

compared to CPI and CPC which points to their higher flexibility and higher adsorption rate.

Nevertheless their foaming properties were statistically equal. As for the emulsifying

properties, there was a significant difference between the EAPI and CPI at pH 9, where

isolate extracted by electro-activated solution showed higher emulsion activity index than the

conventional isolate. Also, isolates produced by electro-activated solutions created emulsions

with significantly smaller droplet size at pH 4 and pH 9 when compared to the conventional

process. However, the stability of the emulsions prepared from the electro-activated isolates

at pH 4 and pH 7 was slightly lower compared to the conventional ones at the same pH

166

values. Considerably different behavior between the protein isolates and concentrates at

different pH was noticed in spite of the fact that their surface activity as well as

hydrophobicity showed the same tendency. In general, protein concentrates performed better

at the isoelectric point in foams and emulsions, where isolates showed much poorer

properties. One of the reasons could be the loss of soluble proteins in the isolates at the

isoelectric point during acid precipitation procedure. On the other hand, isolates were

superior in terms of emulsifying activity index and foaming capacity at other pH which can

be explained by the hydrophobicity-hydrophilicity rate of proteins.

Acknowledgments





167

7. CHAPTER 7: Incorporation of canola proteins extracted by electro-

activated solutions in the gluten free biscuit formulation of rice-

buckwheat flour blend: Assessment of quality characteristics and

textural properties of the product.


In previous chapters the behaviour of extracted proteins was shown following the model

systems. However, the behaviour in model systems might differ from those encountered in

real foods. That is why in the current study the extracted proteins were incorporated into the

matrix of biscuits as one of the possible applications of canola proteins.

This chapter is presented as an article entitled: “Incorporation of canola proteins extracted by

electro-activated solutions in gluten free biscuit formulation of rice-buckwheat flour blend:

Assessment of quality characteristics and textural properties of the product”.






This article was accepted for the publication in the “International Journal of Food Science

and Technology” on 23.11.2015.

168

Résumé

Les protéines de canola extraites par des solutions électro-activées ont été incorporées dans

une formulation de biscuits sans gluten faits à base d’un mélange des farines de riz et de

sarrasin biologique. Les propriétés physiques et texturales ont été influencées de manière

significative par l’ajout des protéines de canola. Une augmentation du diamètre et d'épaisseur

des biscuits et une diminution des taux de propagation ont été notées pour tous les

échantillons supplémentés en comparaison avec le témoin fait de la même formulation mais

sans ajout de protéines de canola. La dureté des biscuits a diminué avec l'ajout de protéines.

Les modifications de dureté du biscuit (résistance à la rupture) ont été corrélées avec les

variations de dureté de la pâte. Les biscuits enrichis en protéines avaient une texture plus

légère et plus aérée comme observée par la microscopie électronique à balayage et avaient

moins d'amidon gélatinisé. Cette observation ainsi que l’épaisseur améliorée des biscuits sont

une indication de l'amélioration de la capacité de rétention de gaz et d'une structure plus

stable de la matrice. En outre, les biscuits ont été caractérisés par rapport à leur teneur en

humidité et l'activité de l'eau qui était plus faible. Enfin, le test sensoriel a montré des

améliorations significatives dans la texture et la sensation en bouche du produit expérimental.

Le goût, l'arôme et l'acceptabilité globale étaient également plus élevés par rapport au produit

témoin. En général, les résultats ont montré que les protéines de canola peuvent être utilisées

dans la formulation de biscuits sans gluten afin d'améliorer leurs caractéristiques texturales,

nutritionnelles et sensorielles.

169

Abstract

Canola proteins extracted by electro-activated solutions were incorporated into the gluten

free biscuits made from the blend of rice and buckwheat flours. The physical and textural

properties were significantly influenced by the addition of proteins. An increase in diameter

and thickness and a decrease in spread ratio were noted for all of the supplemented samples

in comparison with the control one. The hardness of biscuits decreased with the addition of

proteins as compared to the control. The changes in biscuit hardness or fracture strength were

in line with the changes in dough hardness. Protein enriched biscuits had lighter, more aerated

texture which was observed by scanning electron microscopy and had lesser amounts of

gelatinized starch. This and an increased thickness are an indication of improved gas holding

capacity and a more stabilized structure. Furthermore, they were characterized by lower

moisture and lower water activity. Finally, the sensorial test showed significant

improvements regarding textural characteristic and mouthfeel, the taste, aroma and overall

acceptability were also higher as compared to the control. Overall, the results showed that

canola proteins can be utilized in biscuit formulation in order to improve their textural,

nutritional, and sensorial characteristics as well as control the spread in cookies.

170

Introduction

A growing interest towards functional foods has encouraged researches to discover

new ingredients and new ways for their incorporation into the daily diet. In many aspects,

food now is regarded not only from nourishing point of view but also as a preventive or even

treating way of different diseases. Thus, gluten free (GF) products can be regarded as

functional food for those people who are gluten intolerant or suffer from celiac disease. As it

is known celiac disease is a lifelong disorder that arises in genetically predisposed people as

a reaction to gliadin fraction of wheat when consuming products containing gluten such as

bread, pasta or cookies. The list of cereals causing reaction also includes rye, barley and

possibly oats as they share a common taxonomy (Schober et al., 2003). The only known

treatment today is the lifelong adherence to a gluten free diet which created a separate market

niche. Although celiac disease according to different sources strikes around 1% of the

population (many cases are believed to be left undiagnosed), the demand for gluten free

products has increased drastically in recent years. Apart from celiacs (1%) and gluten

intolerants (6%), 22% of Canadians avoid eating gluten containing products due to various

reasons. In Canada, an annual growth rate of gluten free consumption of more than 26% has

been recorded from 2008 to 2012 and is expected to grow at least by 10% each year through

to 2018 (Agriculture and Agri-Food Canada, 2014). In spite of the growing popularity of GF

products and the choice offered in supermarkets, the quality of such products leaves much to

be desired. Mostly based on starchy flours like rice or corn/maize, they contain carbohydrates

but are poor in proteins and other essential nutrients such as vitamins and minerals as well as

fibers (Gallagher, 2008; Hager et al., 2012). In addition, the texture is also poor. The absence

of gluten, a structure making protein, results in a sandy mouthfeel and crumbly texture as

well as the lack of volume which poses a serious challenge for bread making industry

(Arendt, 2009). Biscuits on the contrary do not need developed gluten network and therefore

can serve a suitable matrix for incorporation of functional ingredients such as proteins to

gluten free food matrices. Moreover, they have relatively long shelf-life, good eating quality

and are also widely consumed (Yamsaengsung et al., 2012).

The research towards GF products aims to improve their textural and organoleptic

properties to give people who follow the GF diet better choices. In order to balance the

nutrients and increase the nutritive value, the addition of protein has been regarded by

171

different researchers. Biscuits were supplemented with milk proteins (Conforti and Lupano,

2004; Gallagher et al., 2005), safflower protein isolate (Ordorica-Falomir and Paredes-

López, 1991), soy protein isolate (Rababah et al., 2006) as well as other flours with high

protein content (Gambuś et al., 2009; Tyagi et al., 2007; Yamsaengsung et al., 2012). The

use of plant proteins is attractive due to several reasons and the cost is one of the most

important ones. Also, the search for alternative protein sources reveals new cultures which

can diversify the human diet. Among them, canola can rank with such a widely consumed

plant protein as soybean. A study in human showed that canola proteins can be considered to

be as efficient as soy proteins for a postprandial amino acid response (Fleddermann et al.,

2013). Being rich in highly digestible proteins as well as amino acids, it also possesses certain

important functional properties (Aider and Barbana, 2011; Tan et al., 2011a). In previous

works, canola proteins were isolated with the help of an emerging technology named electro-

activation and their physico-chemical, structural, and functional properties were investigated

(Gerzhova et al., 2015a, b). The results showed less damaged secondary structure and the

improvement in certain functional properties in comparison with proteins extracted with the

conventional alkaline extraction method (Aider et al., 2012b; Gerzhova et al.).

The use of composite flours in the production of GF cookies has been regarded.

Among them, rice and buckwheat were chosen as the base for the incorporation of canola

proteins. Numerous benefits of buckwheat flours have been noted such as lowering

cholesterol, blood lipid, blood sugar and reducing the occurrence of hyperlipidemia, obesity,

and diabetes (Cai et al., 2004). In addition it is characterized by high content of valuable

protein with a biological value equivalent to 93% of defatted milk and 81.5% of egg protein

as well as high content of minerals, organic acids, vitamins, resistant starch, total and soluble

dietary fibers, and polyphenols (Gambuś et al., 2009). Buckwheat flour has a strong flavor

due to the presence of phenolic compounds which can enhance the organoleptic properties

and improve the flavor (Sakač et al., 2015). The evaluation of buckwheat supplemented

cookies (30; 40; 50% of wheat flour) showed improved organoleptic properties in

comparison with control wheat cookie (Filipčev et al., 2011b). Rice, on the other hand, is the

common flour used in many GF formulations due to its bland taste, white colour, and highly

digestible carbohydrates (Rosell and Marco, 2008). The mixture of rice and buckwheat flours

has already been tested for the production of cookies in terms of sensorial properties (Sakač

172

et al., 2015; Torbica et al., 2012). A blend of rice and buckwheat flours (90/10; 80/20; 70/30)

showed good results giving significantly higher scores for almost all the tested sensorial

attributes when compared to wheat based samples (Torbica et al., 2012). However, some

works have claimed the decrease in sensorial characteristics of biscuits getting the lowest

score for taste, colour and texture characterizing them as crumbly and having a strong

aftertaste (Baljeet et al., 2010; Kaur et al., 2015; Yamsaengsung et al., 2012). Therefore, it

can be concluded that buckwheat does have a specific aroma which influence the

organoleptic properties. Sensorial tests conducted by different researches showed that

customer acceptance regarding buckwheat supplemented or buckwheat based cookies vary

greatly. However, its health benefits and the absence of gluten cannot be argued which

justifies the choice of buckwheat flour for its use in gluten free formulations.

Considering the aforementioned information, the main objective of this work was to

study the behavior of canola protein concentrates and isolates in the matrix of biscuit dough

and cooked biscuits by supplementing the rice-buckwheat flour blend with different

concentrations of canola proteins. The inclusion of canola proteins into the biscuit matrix

therein has two goals; notably to increase the nutritive value and to improve the textural

properties of the end product.


7.5.1. Materials

Rice flour was purchased from Erawan Marketing (Erawan Marketing Co., LTD,

Bangkok, Thailand). Organic dark grey buckwheat flour (T60) and green buckwheat flour

(T40) were purchased from Aliments Trigone (Aliments Trigone Inc., QC, Canada). Electro-

activation extracted canola protein isolate (EAPI) and concentrate (EAPC) were produced as

described in (Gerzhova et al., 2015a) by using the following parameters: reactor`s

configuration # 1, concentration of NaCl in the cathodic compartment 0.01 M, time of

electro-activation 60 min, and current intensity of 0.3 A. Margarine, sugar, honey, sodium

bicarbonate and salt were purchased from the local market.

173


Moisture (925.09), ash (923.03), fat (925.85), and protein content (992.23) were

determined according to the methods described in the 19th edition of AOAC International

(2012). Carbohydrates were determined by difference.

7.5.3. Cookie preparation

The control cookie was prepared according to Torbica et al. (2012) by mixing the rice

and the buckwheat flours at 80/20 ratio with slight modifications (Table 7.1). Carboxymethyl

cellulose (CMC) was not added to the biscuit dough in order to exclude its effect and to test

the effect of canola proteins on the texture. The incorporation of canola proteins was done by

substituting the part of rice flour by 3, 6 and 9%. First, dry ingredients were mixed together

and then combined with liquids. Honey was dissolved in water before adding it to the dry

mixture. The amount of water was decreased for samples containing 6 and 9% of EAPI or

EAPC to obtain the dough with good handling properties. Cookie dough was prepared in a

Kitchen Aid Professional mixer 550 Plus (St. Joseph, MI, USA) at speed # 2 for 10 min using

a K-beater. After that, the dough was sheeted to the thickness of 7 mm using a rolling pin

and two gauge stripes after which the cookies were cut to pieces of 60 mm in diameter, baked

for 12 min at 180 °C in a Whitefficiency Rational Self Cooking Center (Rational AG, Iglinger

Straße 62, Landsberg am Lech, Germany) and left to cool down for 2 h before sealing them

in zip lock bags for storage and further analyses which were performed 24 h after preparation.

Table 7.1: Control biscuit formulation.

Ingredient Amount, g % of the total Buckwheat flour 60 10.00 Rice flour 240 39.99 Sugar 75 12.50 Margarine 100 16.66 Salt 2.1 0.35 Sodium bicarbonate 3 0.50 Honey 15 2.50 Water 105 17.50 Total 600.1 100.00

174

7.5.4. Analytical tests

7.5.4.1. Texture profile analysis (TPA)

Dough test was performed with the help of TPA using TA-XT2 Texture Analyser

(Texture Technologies Corp., NY, USA) according to Wehrle et al. (1999). A 25 mm cylinder

aluminium probe and a 5 kg load cell were run at a test speed of 1 mm/s with 5 s recovery

between two strokes and a compression distance of 3 mm. TPA curves were analyzed for

hardness, springiness and cohesiveness.

7.5.4.2. Biscuit fracture strength

Biscuit fracture strength was determined using a TA-XT2 Texture Analyser (Texture

Technologies Corp., NY, USA) equipped with a 3-point Bending Rig and a 5 kg load cell. A

biscuit was placed on two base beams with 40 mm distance between them. The analyzer was

set at a return to start cycle, with a pre-test speed of 1.0 mm/s, test speed of 3.0 mm/s, and

post-test speed of 10.0 mm/s. The maximum force required to break the cookie and the travel

distance until the maximum force was reached were recorded using 9 biscuits.

7.5.4.3. Moisture content and Water activity (aw)

Moisture content in biscuits was analyzed according to the oven dry method (Sathe,

1999). Water activity was analyzed with the help of Aqualab 3TE water activity meter

(Decagon Devices, Inc., Pullman, WA, USA). A set of 3 biscuits taken from different parts

of the oven (front, medium, back) were ground in a Magic Bullet Single Shot blender prior

to the analysis in three replicates.

7.5.4.4. Colour

Top and bottom surface colour of biscuits were measured by using Minolta CR-300

Chroma Meter (Sensing Inc., Osaka, Japan) in triplicates 24 h after baking. The instrument

was calibrated using a standard light white reference tile. The results were expressed in terms

of CIE* values: L* (lightness), a* (redness to greenness - positive to negative values,

respectively), and b* (yellowness to blueness - positive to negative values, respectively)

values.

7.5.4.5. Diameter, thickness and spread ratio

The average of 6 biscuits was taken when measured the weight, diameter and

thickness of cookies. After cooling, the diameter of biscuits was measured using Vernier

caliper, after which they were rotated 90° and re-measured. The thickness of the biscuits was

175

measured by stacking them on top of each other and dividing by 6. An average of two

measurements was taken after re-stacking and re-measuring of the biscuits. Spread factor was

calculated by dividing the average value of diameter by the average value of thickness of

biscuits.

7.5.4.6. Scanning electron microscopy (SEM)

Microstructures of the flours and biscuits were analyzed with the help of Jeol, JSM-

6360LV Scanning Electron Microscope (Tokyo, Japan). Flour samples and biscuits were

prepared as described in Torbica et al. (2012) by mounting them on aluminum specimen stubs

with the help of double‐sided adhesive tape and were sputter-coated with gold. The analysis

of samples was performed at an accelerating voltage of 15 kV under high vacuum.

7.5.4.7. Preliminary sensory evaluation

Preliminary sensory evaluation of the cookies was conducted by a group of 8

volunteers from students of different nationalities both male and female who were habitual

cookie consumers. Cookie samples were served in a random order and evaluated according

to the acceptability of their texture, taste, aroma and overall appreciation using a 9-point

hedonic scale with 9 being “like extremely”, 8 – “like very much”, 7 – “like moderately”, 6

– “like slightly”, 5 – “neither like nor dislike”, 4 – “dislike slightly”, 3 – “dislike moderately”,

2 – “dislike very much”, and 1 – “dislike extremely”. Water was provided between each

sample.


Analysis of variance (ANOVA) was used in order to test the effect of protein addition

for the significance of differences. When it was detected, the Multiple Comparisons test

(Holm-Sidak method) (p<0.05) versus Control Group was performed using Sigma Plot

software (Systat Software, Inc., San Jose, CA, USA).



Proximate analysis of the flours and proteins on a dry basis is shown in Table 7.2.

Rice flour had significantly lower amounts of protein, ash, and fat in comparison with

buckwheat flours. Dark (toasted) buckwheat was characterized by higher amounts of protein,

ash and fat as compared to green (non-toasted) buckwheat. The data on the composition of

176

rice and buckwheat flours is consistent with what has been reported before (Altındağ et al.,

2015; Kaur et al., 2015).

Table 7.2: Proximate analysis of flours and proteins.

Sample Moisture,

(%)

Protein,

(N*6.25,%)

Ash,

(%)

Fat,

(%)

Carbohydrate,

(by difference, %)

Rice flour 10.75 ± 0.13 8.53 ± 0.21 0.37 ± 0.01 0.27 ± 0.02 80.08 ± 0.37

Buckwheat flour

(dark) 10.54 ± 0.07 11.32 ± 0.19 1.71 ± 0.00 2.15 ± 0.06 74.28 ± 0.32

Buckwheat flour

(green) 11.66 ± 0.33 9.56 ± 0.02 1.48 ± 0.01 1.81 ± 0.04 75.49 ± 0.4

EAPC 4.82 ± 0.05 34.04 ± 2.53 11.31 ± 1.75 2.60 ± 0.01 47.23 ± 4.34

EAPI 1.83 ± 0.04 82.43 ± 0.47 1.13 ± 0.4 8.02 ± 0.02 6.59 ± 0.93

7.6.2. Dimensions and cookie spread ratio

Physical parameters of cookies such as diameter, thickness and spread ratio are

summarized in Table 7.3. The diameter and the thickness of cookies increased with the

addition of either canola protein isolate or concentrate at all concentrations. The spread ratio

on the contrary was the highest for the control cookies in both green and grey buckwheat and

decreased with an addition of canola proteins. Significant difference (p < 0.001) was noted

for diameter, thickness and spread ratio for the samples containing 6% EAPC and 9% EAPC

versus control cookie. For green buckwheat cookies, the diameter and the thickness were

lower in comparison with the dark buckwheat cookies whereas the spread ratio was higher

for all of the samples.

177

Table 7.3: Physical parameters of cookies made from flour blend of rice and dark (toasted) buckwheat and rice and green (non-toasted) buckwheat.

Grey buckwheat Green buckwheat

Sample Diameter, mm

Thickness, mm

Spread ratio Diameter,

mm

Thickness,

mm

Spread

ratio

Control 6.10± 0.12d 0.88± 0.06c 6.98± 0.57a 5.95± 0.05c 0.83± 0.05e 7.20± 0.37a

3% EAPC 6.23± 0.08c 1.00± 0.07b 6.27± 0.39c 6.12± 0.06ab 0.92± 0.07d 6.67± 0.43c

6% EAPC 6.33± 0.07ab 1.19± 0.11a 5.37± 0.45e 6.26± 0.05a 1.13± 0.05b 5.56± 0.20e

9% EAPC 6.40± 0.06a 1.21± 0.06a 5.29± 0.20f 6.27± 0.05a 1.23± 0.05a 5.12± 0.16f

3% EAPI 6.20± 0.04c 0.96± 0.01bc 6.46± 0.05b 6.16± 0.03ab 0.87± 0.16e 7.18± 1.29a

6% EAPI 6.23± 0.1c 1.02± 0.02b 6.12± 0.22cd 6.18± 0.02ab 1.00± 0.01c 6.20± 0.08d

9% EAPI 6.27± 0.12ab 1.05± 0.04b 6.00± 0.09d 6.25± 0.01a 1.03± 0.04c 6.08± 0.23de

During the baking process, a set of transformations occur such as volume increase of

the product due to the gas production from leavening agents and water vaporization which

results in weight decrease (Chevallier et al., 2000a). Spread factor is a relation of biscuit

diameter to its thickness. It is considered to be a characteristic of the quality of the biscuit

and should be controlled. Depending on the type of cookies, high or low spread is desirable.

High spread ratio is expected in short dough biscuits which are high in both fat and sugar

content, whereas in semi-sweet formulations with lower amounts of sugar and fat, biscuits

may not flow or even shrink (Manley, 2011). As reported by Filipčev et al. (2011a), the well-

developed crumb is easier to achieve at low spread ratio. Control cookies were flat and soft

on the surface whereas those enriched with EAPC at 6% and 9% levels formed a nice crust

and resembled homemade cookies. The addition of EAPC at levels of 6% and 9% also

promoted the development of fine cracks on the surface (Figure 7.1) related to the

recrystallization of sucrose on the cookie surface during baking (Pareyt et al., 2008). On the

other hand, they were less friable and better maintained their integrity during storage. The

reduction of spread factor with an addition of canola proteins can be related to the

significantly increased thickness of biscuits. This increase was more pronounced with the

addition of protein concentrate. An increase in cookie thickness with the incorporation of

canola proteins indicates their ability to entrap and hold the CO2 leading to the expansion of

the cookies and thus acting as a structural element. Increase in thickness and reduction of

spread factor could also be related to the oil absorption capacity of canola proteins. As

reported by Sudha et al. (2007), the spread of the biscuits reduced significantly when fat

178

amount was lowered whereas a significant increase in the thickness was observed. In our

case, higher values for oil absorption capacity for canola protein concentrate were obtained

in comparison with protein isolate (3.02 ± 0.02 versus 0.97 ± 0.01) (Gerzhova et al., 2015b).

Figure 7.1: Cookies prepared with rice and dark buckwheat flours (1), rice and green buckwheat flours (2) with the incorporation of electro-activated canola protein concentrate and isolate: (A) Control; (B) 3% EAPC; (C) 6% EAPC; (D) 9% EAPC; (E) 3% EAPI; (F) 6% EAPI; (G) 9% EAPI.

The increase in cookies thickness and the reduction of the spread factor with

incorporation of safflower proteins has been reported by Ordorica-Falomir and Paredes-

López (1991). The addition of soy protein isolate also decreased the spread ratio of biscuits

(Rababah et al., 2006). In the other work the diameter of the biscuits increased with the

incorporation of sodium caseinate but decreased with the addition of whey proteins, whereas

both proteins decreased the thickness of the cookies (Gallagher et al., 2005). This information

shows that the behavior of the cookie during baking greatly depends on the type and

properties of its ingredients. Different proteins and different flours may have different impact

179

on the cookies dimensions. For more details, the effect of flour properties on cookie quality

has been elucidated in the work of Mancebo et al. (2015).

7.6.3. Dough characteristics

With the addition of canola proteins the handling characteristics of the dough

changed. As shown in the Figure 7.2, hardness of the dough significantly decreased with an

addition of 3% of canola protein concentrate and isolate comparatively to the control.

However, an increase in hardness was observed when the levels of protein concentrate was

increased. This could be related to the amount of water in the formulation as water in the

dough acts as a plasticizer (Faridi and Faubion, 1990). Water content was kept constant for

control cookie, 3% EAPI and 3% EAPC, however, the addition of 6% EAPC and 9% EAPC

as well as 6% EAPI and 9% EAPI required less water to obtain the dough of good

consistence. If the amounts of water were not adjusted the dough would become too liquid

and impossible to handle. The amount of water needed for the dough is related to the water

absorption capacity of its components. In bread making, rice flour based doughs require

very high hydration in comparison with wheat flour doughs to achieve an appropriate

consistency (Rosell and Marco, 2008). Rice flour is rich in starch, responsible for higher

water absorption as around 46% of flour’s total absorption is associated with the starch

(Hazelton et al., 2004; Mancebo et al., 2015). Protein concentrate on the contrary is highly

soluble and poorly binds water as was observed in our previous study (Gerzhova et al.,

2015a). The addition of canola protein isolate decreased the dough hardness, however further

increase in protein isolate concentration did not have any significant impact. This observation

is in good agreement with the literature, since a decrease in dough hardness with an increase

in protein concentration was noted by Gallagher et al. (2005) who studied the addition of

dairy protein powders. They remarked that dough became softer and more workable and at

the same time more mechanically resistant. Another study on the addition of some protein

isolates to the gluten free dough showed that the addition of soybean protein isolate caused a

decrease in the hardness, whereas the addition of pea protein increased this parameter (Marco

and Rosell, 2008). The role of gluten in the cookie system has also been studied and some

contradictory results have been reported. Even though the gluten network is not developed

in biscuits, its levels influence the dough hardness. Thus, in the work of Pareyt et al. (2008)

higher gluten levels decreased the dough hardness whereas in the work of Fustier et al. (2008)

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the opposite has been found which could be due to different formulations used and different

concentration of the components.

Figure 7.2: Dough characteristics: (A) dough prepared with grey buckwheat; (B) dough prepared with green buckwheat.

The springiness increased with the addition of 3% of canola proteins for both isolates

and concentrates, and decreased with an increase in the level of proteins added. Springiness

refers to the elastic properties of the dough and its ability to spring back, recover after

deformation. Usually the elastic properties are referred to the presence of gluten and the

development of strong gluten network is undesirable in cookies formulation. On the other

hand, a certain degree of springiness should be present as it makes the dough easier to handle.

Since our formulation is gluten free, it can be concluded that canola proteins confer certain

00,10,20,30,40,50,60,70,80,91

0

500

1000

1500

2000

2500

3000

Hardness Springiness CohesivenessA

00,10,20,30,40,50,60,70,80,91

0

500

1000

1500

2000

2500

3000

Hardness Springiness CohesivenessB

181

elasticity to the dough and works as a structural agent. Similar observation was made by

Schober et al. (2003) on the addition of the soybean flour rich in proteins to the gluten free

dough and biscuits. In our case the addition of canola proteins made dough softer and

springier, easier to handle. The decrease in springiness with further increase in protein

concentration same as for hardness could be explained by the lesser amount of water used to

prepare the dough.

Cohesiveness is a parameter that could explain the structural integrity of the product.

Dough experiences a number of stresses before being baked. Thus, dough having adequate

cohesiveness will be more tolerant of handling during sheeting and cutting. No significant

difference was found between the compared samples. Cohesiveness varied from 0.22 to 0.28

for both types of buckwheat flours. Cohesiveness values of 0.2 – 0.3 have been reported for

biscuits supplemented with whey protein concentrate, which however increased to 0.3 – 0.35

with the addition of 10 and 15% of sodium caseinate (Gallagher et al., 2005).

7.6.4. Cookie characteristics

The hardness of the cookies, expressed in terms of force required to break it, is

presented in Figure 7.3. The hardness of the cookies decreased with an addition of canola

protein concentrate at 3% level but increased with an addition of 6% and 9% EAPC for both

grey and green buckwheat. The addition of EAPI at all levels significantly (p < 0.001)

decreased the hardness of biscuits. Similar tendency was noted for the dough hardness, which

is in accordance with Sudha et al. (2007) who found a high positive correlation between the

dough and the cookie hardness. Biscuits prepared from the flours higher in protein have been

reported to have harder structure as a result of strong adherence between proteins and starch

(Altındağ et al., 2015). It has been previously reported that biscuits supplemented with 10

and 15% of sodium caseinate were significantly harder than the control, whereas the addition

of whey proteins resulted in lower hardness similar to the control (Gallagher et al., 2005).

The authors explained this observation by the difference in water bonding capacities of tested

proteins. In the works of Conforti and Lupano (2004) and (Tyagi et al., 2007) the addition of

proteins also resulted in decreased hardness. Another positive correlation has been revealed

between biscuit thickness and the peak force required to break the biscuits (snap test)

(Gallagher et al., 2003). The results obtained in the current work match with the

182

aforementioned statement as the hardness of cookies increased with an increase in

incorporation level of EAPC same as the thickness of cookies increased.

Figure 7.3 : Cookie characteristics: (A) cookies prepared with grey buckwheat; (B) cookies prepared with green buckwheat.

As shown in Figure 7.3 the distance was significantly higher (p < 0.001) for all the

samples containing canola proteins in comparison with the control and it increased with an

increase in protein level. The distance represents the bending characteristics of the cookie. A

biscuit which is more flexible will bend before it breaks resulting in a longer distance. EAPC

had more pronounced bending characteristics (flexibility) than EAPI. The snap characteristic

of biscuits has been stated to be largely influenced by their dimensions. Thus, an increase in

0

2

4

6

8

10

12

14

0200400600800

10001200140016001800

Dis

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e, m

m

For

ce, g

Force DistanceA

0

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4

6

8

10

12

14

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Force DistanceB

183

distance was observed with an increase in the thickness of the biscuit and this observation

was in good agreement with the literature (Wehrle et al., 1999).

7.6.5. Surface colour

Table 7.4: Changes in surface colour of biscuits prepared from the blend of rice and dark buckwheat with the addition of canola proteins.

Parameter L* a* b*

Surface upper bottom upper bottom upper bottom

Control 58.66 ± 0.32a 44.06 ±

0.33a

4.37 ± 0.24a 8.04 ±

0.29d

23.31 ± 0.94c 18.28 ± 0.94c

3% EAPC 58.09 ± 0.60a 46.59 ±

3.66a

3.10 ± 0.37c 8.74 ±

0.14c

26.46 ± 0.49ab 21.02 ± 1.76a

6% EAPC 58.63 ± 0.83a 44.49 ±

1.32a

2.53 ± 1.93d 9.05 ±

0.28b

27.97 ± 0.53a 21.27 ± 2.70a

9% EAPC 57.27 ± 0.25a 42.46 ±

0.47b

4.65 ± 0.21a 9.33 ±

0.33a

27.46 ± 0.51a 19.14 ±

0.98ab

3% EAPI 58.87 ± 2.82a 45.47 ±

0.36a

3.73 ± 0.58b 8.59 ±

0.00c

22.71 ± 1.28c 19.14 ±

0.03ab

6% EAPI 57.89 ± 1.58a 45.56 ±

0.10a

3.81 ± 0.23b 8.47 ±

0.30c

22.07 ± 1.50c 19.29 ±

0.27ab

9% EAPI 58.08 ± 0.14a 45.54 ±

0.55a

3.13 ± 0.29c 9.09 ±

0.02b

22.17 ± 0.37c 19.35 ±

0.07ab

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Table 7.5: Changes in surface colour of biscuits prepared from the blend of rice and green buckwheat with the addition of canola proteins.

Parameter L* a* b*

Surface upper bottom upper bottom upper bottom

control 59.31 ± 0.73a 45.49 ± 0.22a 2.88 ± 0.18b 7.98 ± 0.11d 32.75 ± 0.71ab 20.50 ± 1.41a

3% EAPC 56.98 ± 1.00b 41.38 ± 3.57c 2.50 ± 0.39b 9.21 ±

0.39ab

34.16 ± 1.05a 21.01 ± 2.26a

6% EAPC 58.04 ± 2.14ab 41.95 ±

2.66bc

2.51 ± 0.32b 9.46 ± 0.13a 32.16 ± 4.36ab 20.79 ± 2.77a

9% EAPC 57.17 ± 2.33b 38.86 ± 1.11d 3.49 ± 0.09a 9.53 ± 0.06a 31.23 ± 2.47ab 18.01 ±

1.37ab

3% EAPI 59.61 ± 0.14a 44.06 ±

2.96ab

2.26 ± 0.19c 8.80 ±

0.19bc

29.53 ± 3.78b 21.75 ± 2.44a

6% EAPI 58.82 ± 0.04a 44.19 ±

1.19ab

2.31 ± 0.13c 8.90 ± 0.11b 29.04 ± 1.09b 21.05 ± 1.01a

9% EAPI 57.93 ± 1.37ab 44.21 ± 3.55a 3.37 ± 0.15a 9.04 ± 0.86b 27.80 ± 0.58c 20.79 ± 2.62a

Colour properties of the cookies prepared with blends of rice and buckwheat flours

with a supplementation with canola proteins are summarized in Tables 7.4, 7.5. For the top

surface of cookies prepared with grey buckwheat flour, no significant difference for L* and

a* parameters was found. However b* values indicating the yellowness showed significant

difference for cookies added with EAPC at all levels versus control cookie (p < 0.001). The

yellowness of the cookies increased with an addition of canola protein concentrate whereas

isolate did not bring any changes. This could be related to the colour of the added proteins

and the high contents of soluble sugars in the canola protein concentrate. These sugars may

enhance the intensity of the Maillard reactions during the baking process. Moreover, canola

protein concentrate is yellowish whereas the isolate is closer to neutral white colour when

hydrated which can be due to the presence of other compounds including polyphenols in the

concentrate. For the bottom surface, significant difference (p < 0.001) was found within a*

(redness) values for all cookies apart from those supplemented with 3% EAPI and 6% EAPI,

whereas lightness and yellowness of all the samples were statistically similar. The redness of

the bottom surface of the biscuits containing canola protein concentrate was comparatively

higher than the control or those containing canola protein isolate. In comparison with the top

surface, the bottom surface had higher a*, b*, and L* values in all the samples. Similar

185

observation has been reported by Torbica et al. (2012) for a*. For cookies prepared from

green buckwheat no significant difference (p > 0.05) was found for the top surface. For the

bottom surface, the a* parameter was significantly different (p < 0.001) for cookies prepared

with 6% EAPC and 9% EAPC, respectively.

Colour development during baking comes not only from the colour of ingredients

used in formulation but also from the non-enzymatic browning known as Maillard reactions

which take place between reducing sugars and amino groups of specific amino acids. Thus,

higher protein content of enriched biscuits can contribute to the darker colour (Gallagher et

al., 2003). Other reactions which impart the development of specific colour during baking of

the final product are sugar caramelization and starch dextrinization (Chevallier et al., 2000a).

The lesser amounts of water in biscuits prepared with canola protein concentrate could also

enhance the caramelization of sugars giving the darker surface with reddish-brown hues

(Mancebo et al., 2015). The amount of available water (free water) and its distribution are

also important factors for Maillard browning and a strong positive correlation has been

established between L* values and water activity levels (Gallagher et al., 2003). The effect

of protein addition on the crust colour could have been more pronounced if the formulation

did not contain honey which is high in fructose which is a reducing sugar participating in

Maillard reactions.

7.6.6. Moisture content and water activity

Moisture content and water activity showed similar tendency (Figures 7.4 and 7.5)

decreasing with the addition of protein concentrate, and not showing significant difference

with the addition of canola protein isolate. Biscuits containing 9% EAPC had the lowest

moisture content (5.75 ± 0.26% and 6.04 ± 0.49% for grey and green buckwheat flour,

respectively) as well as the lowest water activity (0.48 ± 0.01% and 0.48 ± 0.04%) which is

a measure of food dryness and susceptibility of a product to spoilage (Filipčev et al., 2011b).

As mentioned before, canola protein concentrate is highly soluble and do not actively bind

water which is why the initial amount of water was reduced in the formulation for those

samples. This explains the reduction in moisture content with an increase in EAPC

concentration. Other works on the contrary reported increased moisture content with the

incorporation of protein products or other flours explained by their higher water retention

capacity (Torbica et al., 2012; Tyagi et al., 2007). Lower water activity in biscuits

186

supplemented with protein is due to the less available water in the system which has also

been observed by Gallagher et al. (2005) on the addition of whey protein concentrate and

sodium caseinate. The high positive correlation (0.928) between moisture and water activity

has been reported by Schober et al. (2003) which led to the conclusion that it is the amount

of water in the baked biscuit which had the strongest effect on the available water.

Figure 7.4 : Moisture of baked biscuits.

Figure 7.5: Water activity (aw) levels for baked biscuits.

0

2

4

6

8

10

Grey Buckwheat Green buckwheat

0

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0,2

0,3

0,4

0,5

0,6

0,7

0,8

Grey buckwheat Green buckwheat

187

7.6.7. Microstructure analysis

Microstructure of rice and buckwheat flours as well as canola proteins is shown in

Figure 7.6. The most abundant and visible ingredient in rice and buckwheat flours is starch.

Rice flour is composed of fine polygonal-shape starch granules which are less than 5 µm and

grouped in agglomerates. Both grey and green buckwheat flours are represented by round

shape starch granules densely packed in clusters. This is in a good agreement with Mariotti

et al. (2008b), Torbica et al. (2012), and (Hager et al., 2012). The images obtained for green

and grey buckwheat flours are very similar showing no difference on the microstructural

level. EAPC is composed of uneven fragments of different shapes and sizes whereas EAPI

is represented by rather large particles of irregular shape.

Figure 7.6: Scanning electron microscopy at 1000x magnification of: (A) rice flour; (B) grey buckwheat flour; (C) green buckwheat flour; (D) EAPC; (E) EAPI at 300x magnification; and (F) EAPI at 1000x magnification.

The micrographs of baked biscuits enriched with EAPC are presented in Figures

7.7A-C, those enriched with EAPI in Figures 7.7D-F and control sample is shown in Figure

7.7E. As the microstructures of green and dark buckwheat were identical only the biscuits

made with green buckwheat are presented here. The matrix of a baked cookie has been

characterized as a composite of fat, sugar, and proteins in which intact or partially damaged

starch granules are embedded. The sugar that melts during baking and become glassy after

cooling provides the structure cohesiveness and link fat, proteins and starch granules

188

(Chevallier et al., 2000a). The starch granules when heated in water undergo the swelling and

the disruption of molecules while losing its crystallinity which is known as gelatinization.

The further heating leads to the formation of a continuous phase of solubilized molecules and

a discontinuous phase of swollen, amorphous starch granules (Arendt et al., 2008). It has

been stated that in biscuit system starch granules are mostly left intact due to low moisture

content and the presence of sugars which prevents starch gelatinization (Chevallier et al.,

2000a; Chevallier et al., 2000b). In Figures 7.7A, D, and G more starch granules are

gelatinized in comparison with other micrographs which can be due to the higher water

content of these samples. In the Figures 7.7A, B, C, and F more intact granules can be

discerned. Also the amount of gelatinized starch is not the same in the center and on the top

surface. Chevallier et al. (2000a) compared the center of biscuits and the top surface and

found that in the biscuit center some starch granules were swollen and some granules have

partially lost their birefringence whereas the top surface due to water evaporation from the

surface during baking showed more intact starch granules. Filipčev et al. (2011a) compared

the microstructures of the dough and the cooked biscuits and found them very similar.

Torbica et al. (2012) also reported intact starch granules in cookies based on rice/buckwheat

formulation. The presence of carboxymethyl cellulose (CMC) due to its hydration capacity

could be the reason of lower water content in the biscuit system preventing starch

gelatinization.

189

Figure 7.7 : Scanning electron microscopy of the cross section of selected biscuits at 1000x magnification: (A) 3% EAPC; (B) 6% EAPC; (C) 9% EAPC; (D) 3% EAPI; (E) 6% EAPI; (F) 9% EAPI; (G) control.

The surface of the cross sections of biscuits supplemented with canola proteins looks

more uneven including some cavities and presumably revealing some structure capable of

holding air cells during baking resulting in thicker biscuits. The control sample as well as

those containing 3% EAPI and 3% EAPC has less pronounced cavities. Regarding the

volume increase during baking (Table 7.5), the control sample was the densest followed by

a 3% EAPI and 3% EAPC supplemented cookies which showed the minimal volume

increase.

7.6.8. Preliminary sensory evaluation

Even though the health aspect and nutritive value is of great importance the sensory

characteristics remain the most important ones in terms of customer acceptance and thus

cannot be neglected. The results of the sensorial test are presented in the Figure 7.8. Only

190

the cookies made with the green buckwheat were taken for the test as such having milder

taste and more appealing yellowish colour (based on the preliminary test). In general, all of

the samples were rated between 6 and 7 for their appearance with no statistical difference

between them (p > 0.05). Aroma and taste are sometimes difficult to distinguish for most

people and as was shown by the analysis the scores for these two characteristics were very

similar. As judged by the panelists the taste and aroma of supplemented cookies were

increased with the addition of protein isolate and 9% EAPI got the highest appreciation.

Protein concentrate was admitted to add a specific flavor and aftertaste which was not equally

judged by different panelists. However, the taste and the aroma are not critical and can be

easily corrected by the addition of spices. The most crucial difference was noted in terms of

texture which also had a great impact on the overall appreciation. The control cookie had the

lowest score (2.50 ± 0.76) and was characterized as hard and dry. All the samples got

significantly higher marks compared to control (p < 0.001). The highest scores for texture

was assigned to the cookie with 9% EAPC (8.13 ± 0.64), characterized as crunchy and

slightly moist and making a specific noise when bitten. These features made it pleasant to

bite and resulted in the highest acceptance among the panelists. Other samples, notably 3%

EAPC and 6% EAPC got 5.00 ± 1.60 and 7.25 ± 1.83, respectively. Cookies supplemented

with protein isolates were similar to EAPC supplemented cookies and were rated accordingly

(5.88 ± 1.25, 6.38 ± 0.92, and 7.75 ± 1.04, for 3% EAPI, 6% EAPI, and 9% EAPI

respectively). Regarding overall acceptability the sample with 9% EAPI scored the maximum

among all the others (7.00 ± 0.76), whereas the control cookie got the minimum (4.38 ± 1.06).

The results of the test showed improved sensorial characteristics of gluten free enriched

cookies. Not only the protein addition resulted in a healthy gluten-free snack but was also

positively evaluated by judges in terms of organoleptic properties.

191

Figure 7.8 : Preliminary sensory characteristics of canola protein enriched cookies.

Conclusion

Canola protein concentrates and isolates were successfully incorporated into the

gluten free biscuits made from a blend of rice and buckwheat flours at the ratio of 80/20 with

3, 6 and 9% protein as substituting levels of the rice flour. The addition of canola proteins

needed the recipe adjustment for water content. Less water was needed for cookies enriched

with either protein isolates or concentrates in order to achieve the dough which can be

192

handled. Analyses showed significant difference for quality parameters of dough and

biscuits. The thickness and the diameter of biscuits enriched with 6% EAPC and 9% EAPC

were significantly higher in comparison with the control which resulted in lower spread ratio.

The addition of canola proteins produced significantly thicker biscuits with more aerated

texture which is an indication of improved gas holding capacity of the dough. This was also

observed in the micrographs of protein supplemented cookies. The addition of canola

proteins significantly changed the textural properties of dough and biscuits making them less

hard but springier. The amount of water in the initial formulation had a great impact on

textural parameters as well as on the moisture and water activity. Higher amount of water led

to the softer and less springy dough with higher moisture content and water activity. As the

addition of proteins required less water, the hardness increased with further increase in

protein concentration. The results on the colour changes showed a significant increase in the

product yellowness with the addition of EAPC. Other than that, the addition of canola

proteins did not bring significant changes for the lightness or redness/greenness of the end

product. The changes in biscuit hardness were in line with the changes in dough hardness.

Indeed, hardness of the cookies, expressed in terms of force required to break it decreased

with addition of canola protein concentrate at 3% level but increased with an addition of 6%

and 9% EAPC for both grey and green buckwheat. The addition of EAPI at all levels

significantly decreased the hardness of biscuits making them softener and more pleasant for

tasting. From sensorial point of view, addition of canola proteins to cookies made from a

blend of rice and buckwheat flours makes it possible to produce gluten free product with

good acceptability. Indeed, sensorial test showed improved overall acceptability as well as

taste and aroma of protein supplemented cookies; however the most significant changes were

noted in terms of texture. Gluten free rice and buckwheat cookies in the absence of a structure

making agents were hard, dry, and brittle whereas canola proteins imparted crunchiness,

slight moistness and as a result a pleasant mouthfeel.

Finally, the current work showed that canola proteins can be added to the gluten free

biscuit formulation in order to improve its nutritive value and texture, and control the spread.

However, the optimization of the formulation is needed followed by the sensorial analysis in

order to produce biscuits with acceptable organoleptic quality. Further work should be aimed

193

for the optimization of the formulation which can significantly improve the organoleptic and

handling properties

Acknowledgments





194

8. CHAPTER 8: Conclusion and perspectives

General conclusion

Current work focused on two main objectives, one of which was to investigate the properties

of electro-activated solutions regarding protein extraction from an industrial by-product of

oil production which is canola meal. The number of benefits of canola protein has been

reported among which are balanced amino-acid content and functional properties which can

be exploited in the production of many conventional foods. As for EAS their disinfecting and

catalytic functions are being extensively studied and there are indications of their extractive

ability as well.

Although canola proteins have been previously studied the amount of cultivars and the

various techniques used for the extraction of proteins made it difficult to choose the best

suitable extraction parameters. Furthermore the data on the simultaneous extraction of dry

matter has not been reported. Which is why the first objective was directed at the selection

of optimal parameters for maximal protein extraction and the verification whether such

parameters will result in the highest dry matter extraction or not. Meal to solvent ratio, pH

and a salt concentration were tested in connection with it. The most significant parameter

was pH of the extracting solution which was in accordance with published results. Salt

concentration did not have a significant impact on protein extractability in spite of the fact

that the main fraction is represented by globulins. SDS PAGE showed numerous fractions

with molecular weights varying from 7 to 100 kD revealing complicated protein composition.

A new approach was chosen for the determination of isoelectric point which combined the

turbidity and zeta-potential measurements. As was shown maximum turbidity was correlated

with minimal ζ-potential corresponding to zero net charge on the surface of protein molecule.

Second objective was aimed to modulate the parameters optimal for protein extraction from

canola meal with regards to results obtained in the first objective. Although electro-activated

solutions are becoming more and more popular due to their unique properties literature

review failed to provide with information required for choosing such parameters.

Considering that fact 4 types of configurations, three salt concentrations, three different times

of treatment and three current intensities were analyzed for modulating the extraction

conditions. Their effects on the pH and alkalinity propagation have been studied. The results

195

showed a significant difference between these two parameters. Alkalinity was shown to be

the most important parameter describing the force of electro-activated solutions. It was

largely dependent on the time of treatment and on the current intensity. The ionic strength of

the solution was significant only for two configurations showing the importance of the ion-

exchange membrane disposition in electro-activation reactor. More specifically the choice of

the membrane can improve or impair the efficiency of alkalinity generation as was shown

with configuration 2 where the withdrawal of anions from cathodic compartment enabled

water dissociation on the membranes surface neutralizing the alkalinity. Also for two and

three compartment cells similar results were obtained, which allowed us to reduce the number

of configuration for the next objective.

In the third objective the catholyte generated in the three-cell reactor has been investigated

for its extraction capacity and its effect on protein composition, secondary structure and

functional properties. Same analyses were performed on the proteins obtained following the

conventional technique. In general the effect of extracting solutions was comparable,

however certain improvements of the new technique were observed. Thus, the SDS PAGE

test showed clearer and more distinguishable bands which signified that proteins were less

denatured. Furthermore the FTIR study also proved that statement by providing narrower

peaks associated with native structure for protein extracted with EAS. As in case of

conventional technique where the quality of proteins following the FTIR peaks and SDS

PAGE bands was reduced with an increase in pH of extraction, the quality of proteins

extracted with EAS decreased with an increase in time of treatment.

In terms of functionalities studied in the fourth objective, proteins extracted by EAS showed

improved surface active properties suggesting their utilization in related food systems. Also

protein isolates and concentrates showed different behaviour depending on the pH at which

they were studied. Concentrates were more soluble within all the tested pH range and

performed better at their isoelectric point. Isolates were comparatively less soluble due to the

additional acid treatment for protein precipitation and to the loss of soluble proteins during

precipitations step.

Last objective was to verify the behaviour of proteins in real foods systems where gluten free

biscuits were chosen as an example of application. Baked goods are mostly prepared from

wheat flour, which is not suitable for people sensitive to gluten. Growing demand for gluten-

196

free products has been attributed to the increasing awareness of customers, improved

diagnostics as well as progressive marketing. The biggest problem in gluten free products is

the lack of structure, associated with lower sensorial characteristics. Also most of them are

poor in nutriments. The addition of canola proteins exhibited two roles, increased the

nutritive value and enhanced the structure. As a result of protein addition the textural and

sensorial characteristics were significantly improved.

Overall the results answered the stated hypothesis and showed the potency of EAS as an

extracting agent. The study revealed some benefits of using EAS as alternatives to

conventional extracting method, however it poses some new questions and leaves room for

further investigations.

Perspectives

Although this PhD work endeavoured to cover as much as possible, some of the observations

were restricted by the limits of chosen parameters. The addition of more points and more

factors might supply with the important information which would substantially improve the

quality parameters. For further perspectives there are several directions. First is adjusting the

parameters of EA and testing their effect on same physico-chemical and functional

properties. For example, the extraction of proteins using EAS treated for lesser amounts of

time e.g. 10-45 min might extract the proteins of higher quality, although reduce the

extractability rate. Other extracting parameters might also allow to extract different fraction

of proteins. Since canola consists of a mixture of proteins with largely varying properties, the

possibility of their fractionation while extracting seems highly appealing.

Furthermore, the protein precipitation can be performed by using the EAS treated in anodic

compartment respectively. This will allow to use efficiently both solutions anolyte and

catholyte at the same time. Another possibility is to perform the protein extraction directly in

the cathodic compartment while being connected to the electric power. For this the constantly

generated products of electrodes reactions will immediately react with meal which should

influence the extraction process.

It will be of interest to try EAS on other oilseed proteins such as soybean, sunflower or

leguminous such as pea.

197

The gelling properties have not been investigated within the scope of the current research

project, however gelling systems are frequently encountered in food systems, and their study

will broaden the spectrum of possible industrial application.

The possibility of protein incorporation in other food systems (pasta, bread, simulated meat

products) should also be studied and possibly compared to the protein that already found a

wide application, such as soybean.

The bioavailability of proteins themselves and in food products should also be checked

primarily in order to see their degradation in digestive tract, which can be performed by

performing simulated digestion. Finally, clinical tests on tolerance and allergenicity will be

needed to provide the data on safety of such preparation so that it could reach the commercial

scale.

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