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RECHERCHE D'ALTERNATIVES AUX NITRATES ET NITRITES DANS LES PRODUITS CARNÉS
Mémoire
Anne Patricia Kouassi
Maîtrise en génie agroalimentaire - Génie agroalimentaire
Maître ès sciences (M.Sc.)
Québec, Canada
© Anne Patricia Kouassi, 2015
iii
Résumé
L'objectif principal de cette maîtrise était de développer des formulations d'épices
capables de remplacer les nitrites et nitrates dans les produits carnés tout en gardant la
même durée de conservation que ces derniers ainsi que leurs propriétés organoleptiques,
antibactériennes et antioxydantes. Un criblage qualitatif puis quantitatif des épices a été
réalisé et trois épices ont été sélectionnées, les clous de girofle, le cumin et la cannelle.
Puis, des poudres de fruits ont été testées pour approcher la couleur rosée que donnent
les échantillons contenant les nitrites. L'analyse sensorielle effectuée par la suite a fait
ressortir le caractère sucré de la poudre de fruit et des propriétés très intéressantes de
certaines formulations par rapport à l'ajout des nitrites. Enfin, une analyse technico-
économique a été réalisée et a montré une très faible augmentation du coût de
production des produits carnés en utilisant nos formulations d'épices et de poudre de
fruit.
v
Abstract
The aim of this study was to develop formulations that can replace nitrates and nitrites
in meat-based products while keeping the same shelf life as nitrites and also, their
organoleptic, antimicrobial and antioxydant properties. A qualitative and quantitative
screening of spices have been made and three spices were selected, cloves, cumin and
cinnamon. Different fruit powders were tested to get closer to the pinkish color which
nitrites give to meats. Then, an organoleptic tests were performed on a panel of tasters
and the results have helped identify the sweetness of the variety of fruit powder and
very interesting sensory properties of several formulations in relation to the addition of
nitrites. Finally, a techno-economic analysis was performed and showed a small
increase in cost of production of meat products using spices and fruit powder, due to a
slight increase in the cost of raw materials.
vii
Table des matières
Résumé ............................................................................................................................. iii
Abstract ............................................................................................................................. v
Table des matières .......................................................................................................... vii
Liste des tableaux ............................................................................................................. xi
Liste des figures ............................................................................................................. xiii
Liste des abréviations .................................................................................................... xvii
Remerciements ............................................................................................................... xix
Avant propos .................................................................................................................. xxi
Introduction ....................................................................................................................... 1
Chapitre 1: Revue de la littérature .................................................................................... 3
1.1. Green Alternatives to Nitrates and Nitrites in Meat-Based Products – A Review ........................................................................................................................... 4
1.1.1. Résumé ........................................................................................................ 5
1.1.2. Abstract ....................................................................................................... 6
1.1.3. Introduction ................................................................................................. 7
1.1.4. Definition of nitrates and nitrites ................................................................ 9
1.1.5. Use of nitrates and nitrites as additives in food products ......................... 13
1.1.6. Physicochemical properties of Nitrites and Nitrates ................................. 14
1.1.7. Regulation of nitrite and nitrate ................................................................ 17
1.1.8. Toxico-kinetics of nitrates and nitrites ..................................................... 18
1.1.9. Effect of nitrates and nitrites ..................................................................... 20
1.1.10. Alternative of nitrites and nitrates used for preserving meat products . 24
1.1.11. Advantages in using spices as an alternative of nitrates and nitrites .... 26
1.1.12. Different types of spices ........................................................................ 28
1.1.13. Grapes ................................................................................................... 34
1.1.14. Conclusion............................................................................................. 35
1.1.15. Acknowledgements ............................................................................... 35
1.1.16. References ............................................................................................. 36
1.2. Spice use in food: Properties and benefits –A review ...................................... 42
1.2.1. Résumé ...................................................................................................... 43
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1.2.2. Abstract ..................................................................................................... 44
1.2.3. Introduction ............................................................................................... 45
1.2.4. General description of spices .................................................................... 46
1.2.5. Chemical properties of spices ................................................................... 49
1.2.6. Main applications of spices ....................................................................... 54
1.2.7. Advantages & Disadvantages of using spices as preservatives ................ 65
1.2.8. Conclusions and future outlook................................................................. 67
1.2.9. Acknowledgements ................................................................................... 68
1.2.10. References ............................................................................................. 69
1.3. Hypothèse et objectifs du projet ....................................................................... 77
1.3.1. Hypothèse de l'étude.................................................................................. 77
1.3.2. Objectif général ......................................................................................... 77
Chapitre 2 Use of spices as alternative of nitrites and nitrates in meat-based products . 79
2.1. Résumé ............................................................................................................. 80
2.2. Abstract ............................................................................................................. 81
2.3. Introduction ...................................................................................................... 82
2.4. Materials and methods ...................................................................................... 84
2.4.1. Preparation of meat samples ..................................................................... 84
2.4.2. Determination of thiobarbituric acid (TBA). ............................................ 85
2.4.3. Determination of p-Anisidine value .......................................................... 86
2.4.4. Microbiological analysis ........................................................................... 87
2.4.5. Statistical analysis ..................................................................................... 87
2.5. Results and discussion ...................................................................................... 87
2.5.1. Determination of p-Anisidine value .......................................................... 87
2.5.2. Determination of TBA index ..................................................................... 89
2.5.3. Microbiological analysis ........................................................................... 91
2.6. Discussion ......................................................................................................... 93
2.7. Acknowledgements .......................................................................................... 93
2.8. References ........................................................................................................ 94
Chapitre 3 Optimization of spices as alternative of nitrites and nitrates in the meat-based products ........................................................................................................................... 97
3.1. Résumé ............................................................................................................. 98
3.2. Abstract ............................................................................................................. 99
3.3. Introduction .................................................................................................... 100
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3.4. Materials and methods ................................................................................... 101
3.4.1. Preparation of meat extract ..................................................................... 101
3.4.2. Determination of thiobarbituric acid ....................................................... 102
3.4.3. Determination of p-anisidine value ......................................................... 103
3.4.4. Microbiological analysis ......................................................................... 103
3.4.5. Statistical analysis ................................................................................... 104
3.4.6. Experimental design and optimization ................................................... 104
3.5. Results and discussion.................................................................................... 105
3.5.1. Effect of variables on physico-chemical properties of processed meat .. 105
Bold values: Significant (p < 0.05)....................................................................... 108
3.5.2. Effects of variables on biological properties of processed meat ............ 108
3.5.3. Determination of optimal conditions and optimal responses .................. 109
3.6. Conclusions .................................................................................................... 115
3.7. Acknowledgement .......................................................................................... 115
3.8. References ...................................................................................................... 116
Chapitre 4 Color Retention in Processed Meats by Using Natural Products and Tests of Organoleptic Properties ................................................................................................ 119
4.1. Résumé ........................................................................................................... 120
4.2. Abstract .......................................................................................................... 121
4.3. Introduction .................................................................................................... 122
4.4. Materials and Methods ................................................................................... 124
4.4.1. Preparation of meat extract for color ...................................................... 124
4.4.2. Color evaluation ...................................................................................... 125
4.4.3. Measurement of different parameters ..................................................... 125
4.4.4. Organoleptic tests ................................................................................... 126
4.4.5. Statistical analysis ................................................................................... 127
4.5. Results and discussion.................................................................................... 128
4.5.1. Effect of pH on color .............................................................................. 128
4.5.2. Solution for color .................................................................................... 129
4.5.3. Organoleptic test results .......................................................................... 133
4.5.4. Discussion ............................................................................................... 153
4.5.5. Aknowledge ............................................................................................ 154
4.5.6. References ............................................................................................... 155
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Chapitre 5 Analyse technico-économique du procédé de production des produits carnés (Utilisation des épices comme alternative aux nitrites et nitrates ................................. 157
5.1. Description des scénarios de simulation employés pour l'analyse technico-économique de la production des produits carnés ..................................................... 158
5.2. Estimation du coût des capitaux fixes ........................................................... 160
5.3. Estimation du coût d'exploitation annuel ..................................................... 165
5.4. Estimation du coût des matières premières ................................................... 166
5.5. Estimation du coût du produit ........................................................................ 169
5.6. Conclusion ...................................................................................................... 170
Conclusion générale ...................................................................................................... 171
Bibliographie ................................................................................................................. 175
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Liste des tableaux
Table 1.1 Summary of the properties of spices .............................................................. 33
Table 1.2 Main chemical characteristics of common spices .......................................... 49
Table 1.3 Spices Bioactivity ........................................................................................... 51
Table 1.4 List of bacterial strains inhibited by spices ..................................................... 54
Table 1.5 Use of spices as insecticides ........................................................................... 55
Table 2.1 Physicochemical properties of spices ............................................................. 83 Table 2.2 List of formulations ........................................................................................ 85
Table 3.1 Results of experimental plan by central composite design for ham and terrine ...................................................................................................................................... 102 Table 3.2 Experimental range of the three variables studied using CCD in terms of actual and coded factors ................................................................................................ 105
Table 3.3 Model coefficients estimated by central composite design and best selected prediction models .......................................................................................................... 108
Table 4.1 The 19 combinations of spices obtained by Statisticia with their pH ........... 124 Table 4.2 List of attributes for the organoleptic tests ................................................... 126
Table 4. 3 List of samples for organoleptic tests .......................................................... 127
Table 4.4 Effet of pH on colour meat ........................................................................... 128
Table 4.5 Color analysis of terrines containing different concentrations of red food chemical coloring agent ................................................................................................ 130
Table 4.6 Color analysis of terrines containing different fruits and vegetables used to improve red color .......................................................................................................... 131
Table 4.7 Analysis of variance of results of terrines' flavor ......................................... 134
Table 4.8 Analysis of variance of results of terrines' odor and texture ........................ 134
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Table 4.9 Results of Mean scores and Least Significant Difference for flavor of terrine ....................................................................................................................................... 135
Table 4.10 Results of Mean and Least Significant Difference for odor and texture of terrines ........................................................................................................................... 138
Table 4.11 Adjusted means of descriptors flavor, smell and texture of terrines ........... 141
Table 4.12 Analysis of variance of results of ham's flavor ........................................... 144
Table 4.13 Analysis of variance of results of ham's odor and texture .......................... 144
Table 4.14 Results of Mean and Least Significant Difference for flavor of ham ......... 145
Table 4.15 Results of Mean and Least Significant Difference for odor and texture of hams .............................................................................................................................. 148
Table 4.16 Adjusted means of descriptors flavor, smell and texture of hams .............. 151
Tableau 5.1 Capacité de production de l'entreprise des produits carnés ....................... 158 Table 5.2 Summary of cost data for food plants in 1986 (Bartholomai (1987); Clark (1997); adapted by Rouweler). ...................................................................................... 162
Table 5.3 Estimation des capitaux fixes ........................................................................ 165
Table 5.4 Coûts des matières premières utilisés dans les différents scénarios ............ 167 Table 5.5 Estimation du coût total des matières premières et du coût d'exploitation annuel ............................................................................................................................ 169
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Liste des figures
Figure 1.1 Endogenous biosynthesis of nitrate ............................................................... 13 Figure 1.2 Transformation of myoglobin in meat ........................................................... 17 Figure 1.3 Toxico- kinetics of nitrates and nitrites in human body ................................ 20 Figure 1.4 Nitrate cycle in water .................................................................................... 22 Figure 1.5 Nitrogen cycle ............................................................................................... 24 Figure 1.6 World Spice production in year 2010 (FAO, 2010) ...................................... 45 Figure 1.7 Spices classification (adapted from Peter and Shylaja, 2012, Sajilata and Singhal 2012) .................................................................................................................. 48 Figure 1.8 Ranges of bacterial inhibition data for different spices (adapted from Ceylan and Fung,2004; Holley and Patel, 2005; Naidu, 2000). ................................................. 53 Figure 1.9 Main medicinal uses for several spices (modified from Peter and Shylaja, 2012) ............................................................................................................................... 56 Figure 2.1 p-anisidine values of terrines preserved using different spice formulations at 4°C for 8 weeks............................................................................................................... 88 Figure 2.2 TBA index for terrines preserved using different spice formulations at 4°C for 8 weeks ...................................................................................................................... 90 Figure 2.3 Total viable counts for terrines preserved using different spice formulations at 4°C for 8 weeks ........................................................................................................... 92 Figure 3.1 Response surface of Log10 viability in terrine obtained by varying: a) the concentration of cloves (X1) and the concentration of cumin (X2) keeping the concentration of cinnamon (X3) constant ; b) concentration of cinnamon (X3) and the concentration of cloves (X1) and keeping the concentration of cumin (X2) constant: 0.2 % w/w ........................................................................................................................... 111 Figure 3.2 Response surface of p-anisidine in terrine obtained by varying: a) the concentration of cloves (X1) and the concentration of cumin (X2) keeping the concentration of cinnamon (X3) constant ; b) concentration of cinnamon (X3) and the concentration of cumin (X2) and keeping the concentration of cloves (X1) constant: 0.2 % w/w ........................................................................................................................... 112 Figure 3.3 Response surface of TBA in terrine obtained by varying: a) the concentration of cloves (X1) and the concentration of cumin (X2) keeping the concentration of cinnamon (X3) constant ; b) concentration of cinnamon (X3) and the concentration of cloves (X1) and keeping the concentration of cumin (X2) constant: 0.2 % w/w ......... 113
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Figure 4.1 Pictures of terrines uncooked and cooked at different pH ........................... 129 Figure 4.2 Pictures of coloured samples with differents percentages of fruits and vegetables ...................................................................................................................... 131 Figure 4.3 Effet of fruit powder concentration (w/w %) and associated terrines pictures: (a.) powder 1 bv (brazilian vine); (b.) powder 2 gp (grape); (c.) powder 3 st (strawberry). .................................................................................................................. 133 Figure 4.4 Sensory profile analysis of flavor sweetness, salty, bitterness, acidity and spicy of terrines ............................................................................................................. 136 Figure 4.5 Box plots of the 6 terrines following descriptors: sweet, salty, acid, bitter and spicy. A boxplot is lower than the M-1.5 (Q3-Q1) value, the first quartile (Q1), median (M) solid line, dotted average, the third quartile (Q3) and the highest value less than M +1.5 (Q3-Q1) ................................................................................................................. 137 Figure 4.6 Sensory profile analysis of odor -rancidity, aromaticity- and texture -tenderness, juiciness- of terrines ................................................................................... 139 Figure 4. 7 Box plots of odor and texture of terrines .................................................... 140 Figure 4.8 Score plot (a) and loading plot (b) of the PCA performed from the sensory analysis of flavor of terrines with nitrite (echNit), formulation 7(ech7), formulation 7+powder 3st (ech7fr), formulation 10 (ech10), formulation 12 (ech 12) and the blanc(blanc) ................................................................................................................... 142 Figure 4.9 Score plot (a) and loading plot (b) of the PCA performed from the sensory analysis of odor and texture of terrines with nitrite (echNit), formulation 7(ech7), formulation 7+powder 3st (ech7fr), formulation 10 (ech10), formulation 12 (ech 12) and the blanc(blanc) ............................................................................................................. 143 Figure 4. 10 Sensory profile analysis of flavor sweetness, salty, bitterness, acidity and spicy of tested hams ...................................................................................................... 146
Figure 4.11 The box plots of 5 tested hams following descriptors: sweet, salty, acid, bitter and spicy. ............................................................................................................. 147 Figure 4.12 Sensory profile analysis of odor -rancidity, aromaticity- and texture -tenderness, juiciness- of hams ....................................................................................... 149
Figure 4.13 The box plots of 5 tested hams with following descriptors: rancidity, aromaticity, tenderness and juiciness ............................................................................ 150 Figure 4.14 Score plot (a) and loading plot (b) of the PCA performed from the sensory analysis of flavor of hams with nitrites (hamNit), formulation 7(ham7), formulation 7+powder 3st (ham7fr), formulation 10 (ham10), formulation 12 (ham12) ................. 152 Figure 4.15 Score plot (a) and loading plot (b) of the PCA performed from the sensory analysis of odor and texture of hams with nitrites (hamNit), formulation 7(ham7),
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formulation 7+powder 3st (ham7fr), formulation 10 (ham10), formulation 12 (ham12) ...................................................................................................................................... 153 Figure 5.1 Scénarios 1 et 2 utilisés dans l’analyse technico-économique: Production de la terrine : pâté .............................................................................................................. 159 Figure 5.2 Scénarios 3 et 4 utilisés dans l’analyse technico-économique: Production du jambon .......................................................................................................................... 160 Figure 5.3 Estimation du coût de production des terrines de foie de porc et du jambon; Scénario1: terrines avec nitrites, Scénario2: terrines avec épices+poudre de fraise, Scénario3: jambon avec nitrites, Scénario1: jambon avec épices+poudre de fraise .... 170
xvii
Liste des abréviations
BHT-Butylated hydroxytoluene
BHA- Butylated hydroxyanisole
TBHQ- tertiary butyl hydroquinone
GRAS- Generally Recognized As Safe
w/w weight by weight
p/p poids par poids
UFC unité formant colonie
MDA malonaldehyd acid
TBA thio-barbituric acid
ppm partie par million
xix
Remerciements
Le projet d'étude arrive à son terme. A cette occasion, j'aimerais remercier et exprimer
ma gratitude à toutes les personnes qui m'ont aidées durant ces deux années d'étude.
Pour commencer, je voudrais remercier mon directeur de recherche, M. Khaled
Belkacemi, pour ses conseils, son soutien et pour m'avoir donné la chance de participer
à cette étude.
Je remercie ma co-directrice, Madame Satinder Kaur Brar, pour m'avoir accueillie dans
son équipe de recherche, pour ses conseils tant professionnels que personnels et sa
disponibilité tout au long du projet.
Ensuite, un gros merci à ma collègue de travail, mon amie, Fatma Gassara, pour sa
pédagogie, son dynamisme, ses observations, et ses conseils qui ont enrichit ces deux
années d'étude.
Je tiens à remercier également M. Mohammed Khelifi pour ses conseils et sa
convivialité pour le peu de fois où je l'ai rencontré.
J'adresse mes remerciements à toutes les personnes qui ont participé aux tests
organoleptiques, plus particulièrement à Madame Jocelyne Giasson, pour sa présence et
ses conseils.
Un merci tout particulier à Nasima Chorfa pour son aide non négligeable, à mon équipe
de recherche à l' INRS et aux stagiaires qui ont su apporter leur fraîcheur à ce projet.
Je tiens à remercier les Fonds de recherche du Québec – Nature et technologies
(FQRNT), le Ministère de l'Agriculture, des Pêcheries et de l'Alimenation (MAPAQ), le
Ministère du Développement économique, de l’Innovation et de l’Exportation
(MDEIE), Les Aliments Breton, La Maison du Gibier inc, Les Gibiers Canabec inc,
Yamaco (9138-9494 Québec inc.) et Les Spécialités Prodal (1975) ltée, pour le
financement de ce projet.
xx
Enfin, un merci et non des moindres, à ma famille, pour leur soutien indéfectible et à
mes amis, plus particulièrement Sandrine Tanoé et Ange Amon pour leur présence et
leur aide durant ce projet.
xxi
Avant propos
Ce mémoire comprend une revue de la littérature, un chapitre ``objectifs et
hypothèses`` et quatre chapitres principaux. La revue de la littérature est constituée de
deux revues qui ont été soumises, acceptées et en cours de publication. Je suis co-auteur
de la première revue et troisième auteur de la seconde revue.
La première revue : Fatma Gassara1, Anne Patricia Kouassi1 2, Satinder Kaur Brar2*,
Khaled Belkacemi1 (2013). Green Alternatives to Nitrates and Nitrites in Meat-Based
Products – A Review. (Acceptée). Elle va être publiée dans le journal Critical Reviews
in Food Science and Nutrition.
La deuxième revue : Jessica Elizabeth De La Torre Torres1, 2, Fatma Gassara1, Anne
Patricia Kouassi1, 3, Satinder Kaur Brar1*, Khaled Belkacemi3 . Properties and benefits
of spices in food. (Acceptée). Elle sera publiée également dans le journal Critical
Reviews in Food Science and Nutrition.
Les quatre chapitres principaux (2 à 5) comprennent 3 chapitres rédigés et présentés
sous forme de manuscrits dont deux ont été soumis et le troisième est en cours de
soumission. Je suis premier auteur du premier et du troisième article (chapitre 2 et 4); et
deuxième auteur du deuxième article (chapitre 3). J‘explique plus bas mes contributions
aux travaux pour lesquels je ne suis pas premier auteur.
Pour les chapitres dont je suis le premier auteur (2 et 4), j‘ai réalisé les expériences,
analysé les données et rédigé les manuscrits qui en découlent, avec l‘aide de ma
collègue associée de recherche, de mon directeur, et de ma co-directrice.
Chapitre 2: Anne Patricia Kouassi1, Fatma Gassara2, Satinder Kaur Brar2, Khaled
Belkacemi1(2014). Use of spices as alternative of nitrites and nitrates in the meat-based
products. Il a été soumis au Journal of Food Science and Technology.
Chapitre 3: Fatma Gassara, Anne Patricia Kouassi, Satinder Kaur Brar, Khaled
Belkacemi. (2014). Optimization of spices as alternatives of nitrites and nitrates in
meat-based products. J‘ai participé à la réalisation des expériences au laboratoire, j'ai
participé à la rédaction de plusieurs chapitres de l'article. Ce article est en cours de
soumission.
xxii
Le chapitre 4 prend la forme d‘un manuscrit pour une soumission prochaine : Anne
Patricia Kouassi1, Fatma Gassara2, Nasima Chorfa1, Satinder Kaur Brar2, Khaled
Belkacemi1. Color Retention in Processed Meats by Using Natural Products and Tests
of Organoleptic Properties.
1
Introduction
L'objectif principal de cette maîtrise était de développer des formulations capables de
remplacer les nitrites et nitrates dans les produits carnés tout en gardant la même durée
de conservation que ces derniers ainsi que leurs propriétés organoleptiques,
antibactériennes et antioxydantes. Un criblage qualitatif puis quantitatif de produits
naturels de faible coût comme les fruits (raisins) et les épices (la cannelle, les clous de
girofles, le cumin, le poivre noir, l’ail et le poivron rouge..) a été réalisé dans un premier
temps, afin de sélectionner les additifs assurant une meilleure activité antimicrobienne
et antioxydante dans les terrines de lapin et de porc, ainsi que dans celle du jambon. Les
résultats ont montré que les formulations 7 (Clou de girofle 0,3; Cumin 0,3; Cannelle
0,1),12 (Clou de girofle 0,2; Cumin 0,368; Cannelle 0,2) ont donné de bonnes
propriétés physico-chimiques et microbiologiques pour les terrines. De même, les
formulations 7 (Clou de girofle 0,3; Cumin 0,3; Cannelle 0,1), 10 (Clou de girofle
0,368; Cumin 0,2; Cannelle 0,2) ont été les meilleures formulations pour le jambon.
Cependant, aucune de ces formulation d'épices ne donnait de coloration rosée aux
jambons ni aux terrines, similaire à la coloration donnée par les nitrites. Ainsi, différents
agents de couleur, y compris les colorants alimentaires, les légumes (betterave) et les
fruits (fraise, raisin, melon..) ont été testés, donc ajoutés aux terrines et aux jambons
pour améliorer leur couleur. Les résultats du criblage des agents de couleur ont montré
que la poudre de fraise et la poudre de raisin sont des alternatives prometteuses aux
nitrites, et donnent une couleur rosée similaire à celle donnée par les nitrites. En outre,
l’effet des conditions avant emballage à savoir le pH, l’humidité sur les propriétés
microbiologiques, physicochimiques et la couleur des produits carnés ont été évalués.
Les résultats de ces analyses montrent que le pH alcalin donne une couleur rouge plus
prononcée pour la viande, mais une texture de la viande différente; nous avons gardé le
pH initial de la viande (qui était de 6). Par contre, la variation de l’humidité n'a pas
d'effet sur la couleur des produits carnés. Ayant obtenu des résultats satisfaisant tant
d'un point de vue physico-chimique que antimicrobien, des tests organoleptiques ont été
effectués sur un panel de dégustateur, avec les meilleures formulations d’épices
sélectionnées, étant donné que les facteurs sensoriels sont les principaux déterminants
des décisions ultérieures d'achat du consommateur. Les résultats des analyses
sensorielles ont permis de faire ressortir le caractère sucré de la poudre de fraise et les
2
propriétés très intéressantes de la formulation 7 par rapport à l'ajout des nitrites. Enfin,
une analyse technico-économique a été réalisée en vue d'évaluer l’intérêt de l'utilisation
des épices et de la poudre de fraise comme alternatives aux nitrites dans les produits
carnés, d'un point de vue industriel. Cette analyse, réalisée à partir d’une série de 4
scénarios, a montré une faible augmentation du coût de production des produits carnés
en utilisant les épices et la poudre de fraise, due à une légère augmentation du coût des
matières premières.
3
Chapitre 1: Revue de la littérature
4
1.1. Green Alternatives to Nitrates and Nitrites in Meat-Based Products – A Review
Fatma Gassara1, Anne Patricia Kouassi1 2, Satinder Kaur Brar2*, Khaled
Belkacemi1
1Département des sols et de génie agroalimentaire, Pavillon Paul-Comtois, Université
Laval, 2425, rue de l'Agriculture, Québec (Québec) G1V 0A6
2INRS-ETE, Université du Québec, 490, Rue de la Couronne, Québec, Canada G1K
9A9
5
1.1.1. Résumé
Il existe de nombreux additifs alimentaires qui, ajoutés dans les aliments, permettent
leur conservation ou encore ralentissent ou arrêtent la croissance des micro-organismes.
Les nitrites et les nitrates sont utilisés comme agents de conservation dans les viandes.
Les nitrites donnent un goût fumé à la viande, une couleur rosâtre et protégent les
consommateurs du risque de détérioration bactérienne. Leur ajout est toutefois très
limité car, à haute dose, ils peuvent être dangerux pour la santé humaine et
l'environnement. Ainsi, des alternatives aux nitrates et aux nitrites font l'objet de
nombreux travaux de recherche. En effet, les épices présentent de nombreuses
propriétés organoleptiques, anti oxydantes et anti microbiennes qui serait intéressant
d'étudier. Cette revue présente les différentes sources de nitrites et de nitrates, leur
utilisation comme additifs dans les produits alimentaires, leurs propriétés physico-
chimiques, leurs effets négatifs et l'utilisation d'alternatives dans la conservation des
produits à base de viande.
Mots-clés: nitrites, nitrates, viande, alternatives plus saines, conservation de la viande
6
1.1.2. Abstract
Several food additives are added in food for their preservation to maintain the
freshness of food (antioxidants) or to slow down or stop the growth of
microorganisms (preservative agents). Nitrites and nitrates are used as preservative
agents in meat. Nitrites give a smoked taste, a pinkish color in the meat and protect
the consumers against the risk of bacterial deterioration. Their addition is however
very limited as, in high dose, it can have risks on human health and the environment.
Thus, alternatives of nitrates and nitrites are the object of numerous research studies.
Alternatives, like spices are considered to have several organoleptic and anti-
microbial properties which would be interesting to study. This review discusses the
sources of nitrites and nitrates, their use as additives in food products, their
physicochemical properties, their negatives effects and the use of alternatives of
nitrites and nitrates in preserving meat products.
Keywords: nitrites, nitrates, meat, green alternatives, preservation
7
1.1.3. Introduction
Nitrates and nitrites are present everywhere in the environment, in water, soil, air,
plants and food. They are used as fertilizers, as explosives and also as preservative
agents in foods particularly against Clostridium botulinum. They are natural
chemical substances which are obtained from the oxidation of nitrogen by the
microorganisms. Oral reduction of nitrate is the most important source of nitrite,
accounting for approximately 70–80% of the human total nitrite exposure. About 5–
7% of all ingested nitrate is converted to nitrite at the base of the tongue, where
nitrate-reducing bacteria are present (Chan, 2011). For subjects with a high rate of
conversion, this figure may be up to 20% (Eisenbrand et al., 1980). Excessive use of
nitrates and nitrites not only presents a health hazard but may also result in nitrite
burn which is a green or white discoloration in the cured meat (Montana Meat
Processors Convention, 2001). The toxic effects of nitrates are due to its endogenous
conversion to nitrite. The Acceptable Daily Intake (ADI) for nitrate is 0-3.7 mg/kg
bw/day (expressed as nitrate ion) (Thomson, 2004). It can lead to risks to human
health and the environment. The health effect of most concern to the U.S. EPA for
children is the “blue baby syndrome” (methemoglobinemia) (Fan, Willhite & Book,
1987). The blue baby syndrome is named for the blue coloration of the skin of
babies who have high nitrate concentrations in their blood. The nitrate binds to
hemoglobin (the compound which carries oxygen in blood to tissues in the body),
and results in chemically-altered hemoglobin (methemoglobin) that impairs oxygen
delivery to tissues, resulting in the blue color of the skin (USEPA, 2007).
Nitrates and nitrites have been associated, at high level, with increased incidence of
cancer in adults, combining with secondary or tertiary amines to form N-nitroso
derivatives, and possible increased incidence of brain tumors, leukemia and
nasopharyngeal problems. The U.S. EPA concluded that there was conflicting
evidence in the literature as to whether exposure to nitrate or nitrites are associated
with cancer in adults and in children. The types of cancers studied included non-
Hodgkin’s lymphoma as well as stomach and gastric cancers in adults; and brain
tumors, leukemia, and nasopharyngeal cancers in children. Their addition is
however very limited (USEPA, 2006). Industries use nitrates and nitrites for the
8
stabilization of the red color of meats (Honikel, 2008), inhibition of the development
of toxic microorganisms, by decreasing the oxidation of lipids and to improve the
flavor (Pegg and Shahidi, 2000). Nitrates and nitrites are preferred as they are less
expensive for the properties that they offer. Meanwhile, the industries have not
found a better and economical substitute to these nitrites and nitrates. Due to the
pressure of the countries who want to establish stricter regulations, the industries are
obliged to find greener substitutes to nitrates and nitrites.
In this context, alternatives of nitrates and nitrites have been studied (Stoilova et al.,
2007; Shirin and Prakash, 2010; Stoilova et al., 2006; Grohs and Kunz, 2000). In
literature, there are chemical agents, such ascorbate and α–tocopherol, lactic-acid-
producing organisms, potassium sorbate, or treatments, such as irradiation (National
Academy of Sciences, 1982) that have been used as nitrite-nitrate substitutes. A
number of studies have been carried out to investigate the properties of aromatic
herbs, fruits, essential oils and spices and like clove, ginger, pepper or garlic
(Menon and Garg, 2001). Cinnamaldehyde, the major constituent of cinnamon
(Cinnamomum cassia) has been reported to possess antibacterial activity and
antioxidant properties (Chang et al., 2001). An essential oil is a complex mixture of
several volatile aroma compounds belonging to different classes of organic
chemistry: phenols (eg carvacrol), hydrocarbon (terpene compounds such as
limonene), alcohols (eg linalool), aldehydes (eg cinnamaldehyde), ketones (eg
menthone). Most of these compounds have antimicrobial properties, but they are
volatile compounds which contain the most important antimicrobial properties,
particularly phenols, alcohols and aldehydes: carvacrol (oregano, savory), eugenol
(sheet Ceylon cinnamon, clove), linalool (coriander), cinnamaldehyde (Chinese
cinnamon) (Oussalah, 2007). There are more than 1340 plants with defined
antimicrobial compounds, and over 30,000 components have been isolated from
phenol group-containing plant-oil compounds and used in the food industry
(Tajkarimi etal. ,2010). Researchers have found a positive linear correlation between
phenolic compounds, primarily phenolic acids and flavonoids, and the antioxidant
capacity of herbs and spices (Zheng, 2001).
The purpose of this review is to present nitrates and nitrites, their role in the
environment, later presence in natural products, their role in preservation of meat
9
and the reason why it is important to find alternatives. Some of them, such as spices
and natural products are also presented in this study, due to preservation, properties
and safety.
1.1.4. Definition of nitrates and nitrites
1.1.4.1. Nitrites and nitrates chemical’s composition Nitrates and nitrites occur naturally as compounds consisting of nitrogen and
oxygen, although in different chemical structures. The nitrogen cycle contains both
compounds. The chemical difference between nitrate and nitrite lies in one
additional oxygen atom. Nitrite is one part nitrogen and two parts oxygen. The
nitrogen cycle comprises oxidation of nitrite, NO2 into NO3, or nitrate.
1.1.4.2. Natural sources of nitrates and nitrites 1.1.4.2.1. Soil
Nitrate is a natural material in soils. Adequate supply of nitrate is necessary for good
plant growth. More than 90 percent of the nitrogen is probably absorbed by plants in the
nitrate form (Brown et al., 2007). Chemical nitrogen fertilizer is often in the
ammonium nitrogen (NH4+) form and is rapidly converted to nitrate (NO3
-) in the soil.
The amount of crop growth is essentially the same whether nitrogen fertilizer is applied
as ammonia (NH3), ammonium or nitrate (NO3-) (Brown et al., 2007). Chemical
fertilizers may be composed of ammonium nitrate, ammonium phosphates, ammonium
sulfate, various nitrate salts, urea and other organic forms of nitrogen. Soil organic
matter contains about 5 % of N. For each 1 percent of organic matter, 7-inch plow layer
of an acre (about 2,000,000 pounds of soil) contains about 1,000 pounds of N.
Microorganisms must change organic nitrogen to ammonium or nitrate before plants
can use it. Usual release of available N from soil organic matter is 1 to 4 percent
annually, depending on soil texture and weather conditions (Brown et al., 2007).
Nitrogen, present or added to the soil, is subject to several changes (transformations)
that dictate the availability of N to plants and influence the potential movement of NO3-
to water supplies (O'Leary, 1994). Animal manure is an excellent source of nitrogen
and can contribute significantly to soil improvement. Animal manure contains about 10
pounds of N per ton, poultry manure about 20 pounds; and legume residues 20 to 80
pounds. About half of this organic nitrogen may be converted to nitrate-nitrogen and
10
become available for plant use the year it is added to the soil. However, it is low in
phosphorus content. Excessive manure applications can result in toxic levels of nitrate
in forage crops the same as excessive use of chemical nitrogen fertilizer (Agbede and
Ojeniyi, 2009). Adding phosphate fertilizer to manure can reduce the nitrate content in
the crop produced. Effluent from animal waste treatment facilities may lose about 50
percent of its nitrogen to the atmosphere as it is applied to soils. However, applications
of large quantities of effluent or solid waste can add excessive amounts of nitrogen to
the soil. Applying large amounts per acre repeatedly to the same area may add more
nitrogen to the soil system than can be used. Using feed additives in livestock feeding
may contribute significant concentrations of certain elements such as copper, zinc,
arsenic or others to the solid animal waste collected in lagoons or similar facilities. Such
wastes continuously applied to soils may eventually result in soil levels toxic to plants
and possibly to animals that consume the crop.
1.1.4.2.2. The atmosphere
Nitrogen in our atmosphere is an inert molecule at ambient temperature. The nitrogen in
the air is also essential for life on Earth. It is incorporated into amino acids and proteins,
and is part of the nucleic acids, such as DNA and RNA (Encyclopedia of Earth, 2011).
However, the nitrogen atom itself is one of the chemical elements which can change its
state of oxidation widely. The outer shell of five electrons (s2p3) can take up three
additional electrons giving the nitrogen an ‘‘oxidation status’’ N3- as it exists in
ammonia (NH3) or amines or it can release five electrons forming N5+ as it exists in
nitrate NO3-. Atmospheric nitrate concentrations ranging from 0.1 to 0.4 μg/m3 have
been reported, the lowest concentrations being found in the South Pacific (Prospero and
Savoie, 1989; World Health Organization 2004). The highest levels of aerosol nitrate
measured at this northern Canadian location were about 0.40-0.55 µg/m3 between the
years 2000 and 2005 (Environment Canada, 2010). Higher concentrations ranging from
1 to 40 μg/m3 have also been reported, with annual means of 1–8 μg/m3. Mean monthly
nitrate concentrations in air in the Netherlands range from 1 to 14 μg/m3 (Janssen et al.,
1989; World Health Organization 2004). Indoor nitrate aerosol concentrations of 1.1–
5.6 μg/m3 were found to be related to outdoor concentrations (Yocom, 1982; World
Health Organization 2004).
11
1.1.4.2.3. Plant
Nitrates occur naturally in vegetables and plants. Nitrite and nitrates occur naturally in
vegetable as a consequence of the nitrogen cycle whereby nitrogen is fixed by bacteria.
Beetroot, broccoli, cabbage, celery, lettuce, radish and spinach have been reported to
contain high concentrations (greater than 1000 mg/kg) of nitrate. In contrast, nitrite
concentration in fresh vegetables is generally low (less than 1 mg/kg and not above 20
mg/kg) (Meah, 1994, Petersen and Stoltze 1999, Chung et al, 2003).
1.1.4.2.4. Water
Nitrates occur naturally in wastewater and drinking water. Nitrate is used mainly in
inorganic fertilizers. It is also used as an oxidizing agent and in the production of
explosives, and purified potassium nitrate is used for glass making. Sodium nitrite is
used as a food preservative, especially in cured meats. Nitrate is sometimes also added
to food to serve as a reservoir for nitrite. Nitrate can reach both surface water and
groundwater as a consequence of agricultural activity (including excess application of
inorganic nitrogenous fertilizers and manures), from wastewater treatment and from
oxidation of nitrogenous waste products in human and animal excreta, including septic
tanks (World Health Organization 2011). Nitrite can also be formed chemically in
distribution pipes by Nitrosomonas bacteria during stagnation of nitrate-containing and
oxygen-poor drinking-water in galvanized steel pipes or if chloramination is used to
provide a residual disinfectant and the process is not sufficiently well controlled.
1.1.4.2.5. Endogenous biosynthesis of nitrate
Nitrosating agents (NAs; NxOy form) can react under certain conditions with
nitrosatable compounds (NCs) to form N-nitrosamines and N-nitrosamides
(hereafter nitrosamines and nitrosamides), collectively called N-nitroso compounds
(NOCs): an equation showing the formation of N-Nitroso compounds (NAs + NCs
NOCs). This reaction is called N-nitrosation or simply nitrosation (Health
Canada, 2013). Human beings are exposed to various types of nitrosating agents
through diet, drinking water and tobacco smoke. These substances can also be
12
synthesised endogenously from ingested nitrate and nitrite (Bartsch et al., 1988;
Brambilla and Martelli, 2005).
Gastrointestinal infections greatly increase nitrate excretion via endogenous (non-
bacterial) nitrate synthesis, probably induced by activation of the mammalian
reticulo endothelial system (FAO/WHO, 1996; Lundberg et al., 2009). This
endogenous synthesis of nitrate, presented in figure 1.1, complicates the risk
assessment of nitrate. Increased endogenous synthesis of nitrate, as reported in
animals with induced infections and inflammatory reactions, was also observed in
humans. Infections and non-specific diarrhoea played a role in the increased
endogenous synthesis of nitrate (Gangolli et al., 1994; World Health Organization
2011). These observations are all consistent with the induction of one or more nitric
oxide synthases by inflammatory agents, analogous to the experiments described in
animals and macrophages. In humans, saliva is the major site for the formation of
nitrite. About 5% of dietary nitrate is converted to nitrite (Gangolli et al., 1994;
World Health Organization 2011). A direct correlation between gastric pH, bacterial
colonization, and gastric nitrite concentration has been observed in healthy people
with a range of pH values from 1 to 7 (Mueller et al., 1986, Viani et al., 2000;
Moigradean et al., 2008).
13
Figure 1.1 Endogenous biosynthesis of nitrate
1.1.5. Use of nitrates and nitrites as additives in food products
Nitrate and nitrite are used as food additives in processed food as preservatives and
colour fixatives in meat, poultry, fish and cheese (European Commission, 1995).
Sodium nitrite has been used extensively in curing meat and meat products, particularly
pork products, such as ham, bacon and frankfurters; certain fish and poultry products
are also cured with brines that contain sodium nitrite. The process may include dry
curing, immersion curing, or direct addition or injection of the curing ingredients.
Curing mixtures are typically composed of salt (sodium chloride), sodium or potassium
salts of nitrite and nitrate and seasonings. Sodium nitrite acts as a colour fixative and
inhibits the growth of bacteria, including Clostridium botulinum, which is the source of
botulism toxin. Nitrite is a relatively strong reducing agent that has antibacterial
properties; however, the preservation of foodstuffs can be attributed to a large degree to
the high concentration of salts (including nitrate) that are employed during the curing
endothelial cells
L- arginine +O2
NO-synthetase
NO
NO2
-
Oxidation
Urine (NO3
-;NO2-)
Oxidation
NO3-
Gastric gland
Salivary glands
NO3-
Oxidation
(Mouth) Bacterial production of NO2
-
(stomach) Bacterial production of NO2-
NO3-,NO2
-
NO3-,NO2-
Stool <0.01% (NO3
-.NO2-)
NO3-
exogenous (food)
14
process. In addition, nitrate can act as a reservoir whereby nitrite may be formed by
microbiological reduction (Pokorny et al., 2006).
1.1.6. Physicochemical properties of Nitrites and Nitrates
1.1.6.1. Role of nitrates and nitrites as antioxydant
One of the most important properties of nitrite is its ability to effectively delay the
development of oxidative rancidity. This prevention occurs even in the presence of salt,
which is a strong oxidant. Lipid oxidation is considered to be a major reason for the
deterioration of quality in meat and poultry products which often results in the
development of rancidity and subsequent warmed over flavors (Vasavada and
Cornforth, 2005). The oxidation of unsaturated fats occurs more quickly in uncured,
cooked meats than in cured meats because iron that is not bound to nitric oxide can act
as a catalyst for oxidation. The antioxidant effect of nitrite is likely due to the same
mechanisms responsible for cured color development involving reactions with heme
proteins and metal ions, chelating of free radicals by nitric oxide, and the formation of
nitriso- and nitrosyl compounds having antioxidant properties (Sebranek, 2009). The
antioxidant effect of nitrite has been well documented (Townsend and Olson, 1987;
Pearson and Gillett, 1996; Pegg and Shahidi, 2000; Honikel, 2004). Nitrite has been
shown to inhibit warmed over flavor development at relatively low levels. The addition
of nitrite to kavurma, a type of fried meat, could significantly reduce the level of
oxidation, measured by thiobarbituric acid, peroxide, and free fatty acids, as compared
to a control which did not have nitrite added (Yetim et al. (2006). Sato and Hegarty
(1971) reported significant inhibition of warmed over flavor development at a 50 ppm
nitrite level with complete inhibition at a 220 ppm level. Investigating the effect of
nitrite on lipid oxidation in various muscle systems, Morrissey and Tichivangana (1985)
reported as little as 20 ppm nitrite was sufficient to significantly (P < 0.01) inhibit
oxidation of lipid in fish, chicken, pork, and beef systems. In spite of the antioxidant
power of nitrites and nitrates, industries have to find healthier and greener alternatives
for these chemicals products
15
1.1.6.2. Antibacterial activity of nitrites and nitrates
Nitrites and nitrates play a key role in cured meat as a bacteriostatic and bacteriocidal
agent. Nitrite is strongly inhibitory to anaerobic bacteria, most importantly Clostridium
botulinum ( Sofos et al.,1979) and contributes to control of other microorganisms, such
as Listeria monocytogenes. The effect of nitrite and the likely inhibitory mechanism
differs in different bacterial species (Tompkin, 2005). The effectiveness of nitrite as an
antibotulinal agent is dependent on several environmental factors including pH, sodium
chloride concentration, reductants and iron content among others (Tompkin, 2005). The
reaction sequences involving nitric oxide are probably an important part of the
antimicrobial role of nitrite in cured meat. For example, some researchers have
suggested that nitrous acid (HNO2) and/or nitric oxide (NO) may be responsible for the
inhibitory effects of nitrite (Tompkin, 2005). As nitrite reactivity is key to microbial
inhibition (one indicator of this is the strong dependence on pH), there are questions
raised on the fact that whether ingoing or residual nitrite is most critical to antimicrobial
effects.
Tompkin (2005) concluded that residual nitrite at the time of product temperature abuse
is critical to antibotulinal effects and that depletion of residual nitrite during product
storage will reach some point at which inhibitory effects are also depleted. Other
compounds have the same antimicrobial properties as nitrites and nitrates; if the
industries use these compounds, it is also for their action on color and taste of meat.
1.1.6.3. Action of nitrites and nitrites on color and taste
The red colour of cured meat products is one of the important effects of nitrite in meat
products. The red colour develops in a number of complicated reaction steps until NO-
myglobin (Fe2+) is formed. Myoglobin exists in a muscle in three states, in which the
cofactor haem, a porphyrin ring with an iron ion in its centre binds different ligands or
in which the iron exists in the Fe2+ or Fe3+ state. In the native myoglobin, the porphyrin
moiety is supported in the ligand binding by amino acids of the protein in the
neighbourhood. In the ‘‘original’’ state, myoglobin with Fe2+ in the porphyrin cofactor
does not bind any ligand by a water molecule. In the presence of oxygen, the myoglobin
16
can bind an O2 molecule and it becomes bright red. The iron ion is in the Fe2+ state.
However, oxygen and other oxidizing agents, such asnitrite can oxidize the Fe2+ to Fe3+.
The formed metmyoglobin (MetMb) is brown. The ‘‘original’’ myoglobin (Mb),
oximyoglobin (MbO2) and the metmyoglobin occurrtogether in meat. In a muscle in a
live animal there is very little metmyoglobin which increases post-mortem with the
disappearance of oxygen except when meat is MAP-packed with high oxygen content
(Honikel, 2008).
Oxygen and NO are biatomic molecules. A similar biatomic molecule, CO also binds
very tightly to myoglobin. In some countries (e.g. USA and Norway) modified
atmospherepackaging (MAP) packaging of meat with 1–2% CO is permitted. By
reducing enzymes or chemical reactions with reducing agent, such asascorbate, Fe3+ is
reduced to Fe2+. The NO formed from N2O3 can bind to the myoglobin (Fe2+) and form
a heat stable nitrosylmyoglobin (NO-myoglobin). Oximyoglobin is not heat stable and
dissociates. The meat turns grey or brown. On heating the NO-myoglobin, the protein
moiety is denatured but the red NO-porphyrin ring system (often called nitroso-
myochromogen) still exists and is found in meat products heated to 120±1oC. This heat
stable red colour will change on bacterial spoilage and it fades in UV light. The
transformation of myoglobin have presented in details in Figure 1.2. The first one is
advantageous as the consumer recognizes spoilage in fresh meat which also changes
colour on spoilage. In most recent years, the riddle about the red colour of cured raw
hams such as Parma ham without added nitrite or nitrate has been solved. Various
authors proved that the Fe2+ in the porphyrin ring was exchanged with Zn2+ which gives
the products a pleasant red colour. Nitrite addition prevents the exchange (Adamsen,
Moller, Laursen, Olsen, & Skibsted, 2006; Moller, Adamsen, & Skibsted, 2003;
Parolari, Gabba, & Saccani, 2003; Wakamatsu, Nishimura, & Hattori, 2004;
Wakamatsu, Okui, Ikeda, Nishimura, & Hattori, 2004).
Nitrite is also responsible for the production of characteristic cured meat flavor, though
this is probably the least well understood aspect of nitrite chemistry (Pegg & Shahidi,
2000). It is easy to distinguish cooked, cured ham from fresh roast pork on the basis of
flavor but the chemical identity of distinguishing flavor components in cured meat has
eluded numerous researchers. Some of the flavor difference may be due to the
suppression of lipid oxidation by nitrite but other antioxidants do not produce cured
17
meat flavor. If nitrite does, in fact, form some volatile flavor factors, this would
represent yet another reaction product of nitrite in cured meat.
Figure 1.2 Transformation of myoglobin in meat
1.1.7. Regulation of nitrite and nitrate
Meat products manufactured in the United States are heavily regulated by the United
States Department of Agriculture (USDA) – Food Safety and Inspection Service (FSIS).
The amount of ingoing sodium or potassium nitrite in comminuted products
manufactured in the United States is 156 ppm. The amount of ingoing sodium or
potassium nitrite in comminuted products manufactured in the United States is 156 ppm
(Sebranek and Bacus, 2007). This is based on the green weight of the meat block (which
is different in other countries). According to regulations, dry cured products are
restricted to 625 ppm of ingoing sodium nitrite or potassium nitrite. Immersion cured
and massaged or pumped products are limited to 200 ppm ingoing sodium nitrite or
potassium nitrite. According to the USDA, a minimum of 120 ppm of ingoing sodium
nitrite is required for all cured “Keep Refrigerated” products. The only instance in
which the rule is not in effect is when “the establishment can demonstrate that safety is
assured by some other preservation process, such as thermal processing, pH or moisture
18
control” (Sebranek and Bacus, 2007). Neither nitrate nor nitrite is permitted in baby or
toddler food. Nevertheless, the food is safe for consumption due to the sterilization
processing to which all baby food is subjected. Health Canada has identified
maximum nitrites/nitrate levels in the Food and Drug Regulation. These levels are
maximum of 200 ppm in cured meat and meat by-products (except bacon) and maximun
of 100 ppm in bacon. These levels are well above those needed to stop the
growth Clostridum botulinum spores. A complete ban on the use of nitrates and nitrites
in foods has not been implemented because of the beneficial uses as preservatives and
particularly their prevention of Clostridium botulinum growth. There is also some
scientific evidence suggesting that low levels of nitrates and nitrites (below 200ppm)
pose no health concern (Health Canada, 2008).
Many countries have used others directives and regulations for the use of nitrites and
nitrates in meat products. The European Union (EU) have considered its regulation and
directives 2006/ 52/ DEC (Directive 2006). The use of nitrites and nitrates was limited.
In general, 150 mg nitrite/kg are allowed to be added to all meat products plus 150
mg/kg for unheated meat products. That is a maximum of 300 nitrites plus nitrates/kg. A
large number of exceptions such as dry cured bacon may have 425 mg of residual
nitrites plus nitrates / kg. The toxicity of nitrates and nitrites depend on their
concentration in meat. Due to regulations and the toxicity of nitrates and nitrites,
industries must be more stringent on their standard or find healthier alternatives for
nitrates and nitrates.
1.1.8. Toxico-kinetics of nitrates and nitrites
1.1.8.1. Absorption Nitrates and nitrites are present in the environment under ionic form, not volatile. There
are two major sources of nitrate and nitrite in the human system: endogenous l-
arginine–NO synthase pathway and the diet. The main pathway of absorption of these
substances is the ingestion. Dietary nitrate intake is considerable and nitrate is found as
a naturally occurring compound in foods such as vegetables, spinach and lettuce often
containing up to 2500 mg.kg−1 (World Health Organization 2011), fruit, cereals, fish,
milk, and walter also contain nitrate as a consequence of agricultural practices, such as
the use of nitrogen-containing fertilizers and from animal waste (Dennis and Wilson,
2003). Low levels are generally found from these sources, except in the case of some
19
vegetables. Nitrates and nitrites are also permitted as food additives in some foods,
primarily as protection against botulism.
1.1.8.2. Distribution
Concerning their distribution, nitrate is reduced to nitrite, in the oral cavity, by the
action of commensal bacteria found on the back of the tongue. The swallowed nitrite,
under the influence of the acidic conditions in the stomach, becomes protonated to
nitrous acid (NO2- + H+ HNO2). Once nitrite is formed, there are numerous
pathways in the body for its further reduction to NO, involving haemoglobin,
myoglobin, xanthine oxidoreductase ascorbate, polyphenols and protons (Lundberg et
al., 2008). After the ingestion of nitrate, either in its dietary or medicinal form, there is a
sharp rise in the salivary, plasma and urinary levels of nitrates and nitrites. In the
plasma, the levels of nitrate rise within 30 min and peak at 3 h and are sustained for up
to 24 h; In contrast, the levels of nitrites rise more gradually to a significant level by 1–
1.5 h and a plateau is reached at ∼2.5 h and remains significantly elevated for up to 6 h.
(Sami et al., 2012).
About 5% of the dietary nitrate is reduced to nitrites in the saliva and the
gastrointestinal tract. This value can reach 20% for individuals with a high rate of
conversion (Thomson et al., 2007).
Methemoglobinemia is another health hazard attributed to nitrites, a condition where
reduced iron (Fe2+) in haemoglobin is oxidized by nitrite to Fe3+, thus reducing the total
oxygen-carrying capacity of the blood (Santamaria, 2006).
Nitrites are able to be produced endogenously. Nitrites may also combine with
secondary or tertiary amines to form N-nitroso derivatives. Certain N-nitroso
compounds have been shown to produce cancers in a wide range of laboratory animals
(Codex, 1998).
1.1.8.3. Elimination
The elimination of nitrites is mainly carried out by urinary expression and maximal
urinary nitrite excretion occurred approximately 4 to 6 h after consumption of each
high-nitrate meal, with basal levels being reached by 24 h. Approximately 75% of the
total ingested nitrate was excreted via urine. The other major routes of nitrate excretion
21
The methaemoglobinaemia caused by nitrates in the drinking water was mainly
observed when the child's age was less than 3 months. Bacterial contamination of water,
gastrointestinal infections and inflammation with ensuing production of nitric oxide is
major factors that may contribute to methemoglobinemia (Fan, 2011). The blue baby
syndrome is named for the blue coloration of the skin of babies who have high nitrate
concentrations in their blood. The nitrate binds to hemoglobin (the compound which
carries oxygen in blood to tissues in the body), and results in chemically-altered
hemoglobin (methemoglobin) that impairs oxygen delivery to tissues, resulting in the
blue color of the skin. The blue coloration can be seen in the lips, nose, and ears in the
early stages of blue baby syndrome, and extend to peripheral tissues in more severe
cases (EPA, 2006).
Reduced oxygenation of the tissues can have numerous adverse implications for the
child, such as coma and death. Toxic doses of nitrites responsible for
methemoglobinemia range from 0.4 to more than 200 mg kg−1 of body weight. The
guideline value for nitrite ion in infants is 3 ppm (US EPA, 2006). Exposure to higher
levels of nitrates or nitrites has been associated with increased incidence of cancer in
adults, and possible increased incidence of brain tumors, leukemia, and nasopharyngeal
(nose and throat) tumors in children in some but not others. U.S. EPA concluded that
there was conflicting evidence in the literature as to whether exposures to nitrate or
nitrites are associated with cancer in adults and in children.
1.1.9.2. Effects on animal’s health
The effects of nitrates on animal’s health, livestock feeding contains nitrites and
nitrates. The nitrate is transformed into nitrite and in other N-nitroso compounds in the
saliva of most monogastrics or in ruminants’ rumen because of the microbial activity.
The unfavorable effects on the health of the livestock result from an acute exposure to
the nitrites due to the formation of methemoglobin in the blood. This can lead to the
cyanosis and death at very high levels.
Clinical signs of acute nitrite toxicity in a range of livestock associated with
methemoglobin are generally dose-dependent due to oxygen starvation and may include
accelerated pulse, dyspnoea, muscle tremors, weakness, vomiting, unstable gait, and
cyanosis leading to death. Symptoms of sub-chronic and chronic toxicity include
23
destruction of the ozone layer in the stratosphere. The nitrogen cycle is presented in
details in Figure 1.5.
Nitrates and nitrites are naturally occurring ions and are ubiquitous in the environment.
Nitrate contamination of surface- and groundwater is a pervasive, worldwide problem,
while nitrite is an important indicator of fecal pollution in water. According to the
National Primary Drinking Water Regulations, nitrates are highly mobile in soil and
have a higher potential to migrate to ground water due to higher solubility in water and
weak retention by soil. Nitrates and nitrites do not volatilize and therefore are likely to
remain in water until consumed by plants or other organisms. Ammonium nitrate is
taken up by bacteria, and nitrate degradation is fast under anaerobic conditions. Nitrite
is easily oxidized to nitrate, and nitrate is the more predominant compound of the two
forms detected in groundwater (EPA, 2006). Nitrates are essential for the growth of
vegetables, and are present in the composition of fertilizers natural as manure.
Fertilizers and badly used pesticides pollute subterranean waters (by infiltrating into the
ground by rainwater and watering) and surface (streaming). The excessive use of
fertilizers appreciably increases the quantity of nitrate in rivers and low depth ground
waters.
The nitrate is nevertheless a beneficial natural element integrated into the nitrogen cycle
and indispensable for the growth of vegetables. An excessive use of nitrates destabilizes
this process: the rainwater, after infiltration, entails the nitrate fertilizers, which plants
and grounds were not able to absorb, and then pollutes the fresh water.
Both nutrients, particularly in dissolved species such as nitrate, nitrite, ammonium and
phosphate, are easily assimilated by phytoplankton for growth and act as significant
factors in the regulation of primary productivity in water bodies. Nitrogen
concentrations in river and ground water increase as a response to increased agricultural
activity and point-source discharges and were one of the most implicated species in
eutrophication of water (Xinhai, 2010).
25
1.1.10.2. Vitamin
Several studies have shown that vitamins, such as -tocopherol could be an effective
alternative to nitrites and nitrates in meat products. In addition, it has been shown that
the -tocopherol can inhibit the growth of pathogens. However, vitamins are often
expensive which will make the meat process more expensive (Bhat et al., 2013).
1.1.10.3. Natural sources of nitrites and nitrates
It has been shown in the literature that there are certain products that naturally contain
nitrites such as celery, lettuce, spinach, radishes, among others. Some researchers have
used extracts of these natural products as substitutes of nitrite in meat products.
However, nitrites/nitrates, coming from natural sources or not, present a potential
harmful effect as chemical nitrites and nitrates, because even its origin is naturals, these
nitrites will be transformed to nitrosamines that have negatives effects.
1.1.10.4. Agents preventing the formation of nitrosamines
There are products that inhibit the formation of nitrosamines and nitrosamides from
nitrite and nitrate, such as ascorbate and ascorbic acid. These products can be used in
the presence of nitrites in meat products to inhibit their transformation into nitrosamines
and nitrosamides. The sequence of reactions of ascorbate preventing nitrosamine
formation has not been fully elucidated. It may be due to the reduction of residual nitrite
in meat products by ascorbate (EFSA, 2003) or the binding of NO to ascorbate and its
retarded release. In the last decades, ascorbic acid and isoascorbate (erythorbate) has
been used in cured meat batters. There is a reaction of ascorbate with oxygen forming
dehydroascorbate and thus reducing the amount of nitrite which could be oxidized to
nitrate (Bertelsen, and Quist, 1994). However, ascorbate also seems to react with nitrite
(nitrous acid or NO). Dahl, Loewe, and Bunton (1960), Fox and Ackerman (1968) and
Izumi et al. (1989) showed that ascorbate also reacts with ‘‘nitrite’’ and binding the
resulting NO. The bound NO seemed to react as NO with other meat ingredients.
Ascorbate is also added to reduce the formation of nitrosamines. The sequence of
reactions of ascorbate preventing nitrosamine formation has not been fully elucidated. It
may be due to the reduction of residual nitrite in meat products by ascorbate (EFSA,
2003) or the binding of NO to ascorbate and its retarded release together with nitrite and
26
salt. Nevertheless, it becomes clear that nitrite is a very reactive substance which
undergoes many reactions in meat products and thus its use has to be controlled. In
contrast, nitrites in meat products remain and pass through the stomach of the consumer
where they can transform under the action of acidic pH of the stomach to nitrosated
products. Similarly, nitrites themselves, without any transformation, are harmful to
consumers.
1.1.10.5. Spices and fruits
Spices are considered as good alternatives of nitrites due to their well known
antibacterial and antioxidant properties. Rosemary, mace, oregano and sage have
antioxidant properties that can delay the onset of rancidity in fats (Kim et al., 2011).
Black pepper, white pepper, garlic, mustard, nutmeg, allspice, ginger, cinnamon and red
pepper are known to stimulate the Lactobacillus bacteria producing lactic acid, which
increases the shelf life of meat products (Sallam et al. 2004). In this regard, the use of
spices and their volatile compounds as natural preservatives in food can be an
alternative to the use of chemical additives (Kim et al., 2011). The use of garlic powder
may be very important as garlic is known for its antioxidant and antimicrobial (Kim et
al., 2011). In addition, the use of garlic reduces the amount needed to ensure proper
conservation that will not alter the taste of the meat products. It has also been shown
that cloves and garlic in combination will inhibit all pathogenic bacteria
(Yadav and Singh 2004). The conjunction and the physical-chemical properties and
antimicrobial properties of garlic and cloves play an important in the use of these spices
as naturals alternatives of nitrites and nitrate in food. However, these spices if they will
be used in big quantity will damage the taste of food. Hence, the quantity of these spices
has to be minimized to maintain the organoleptic properties of food and their good
preservation in parallel.
1.1.11. Advantages in using spices as an alternative of nitrates and nitrites
Spices and herbs have been used since ancient times for different purposes. Originally,
most of them were used to preserve food. Within this group, there are spices, cinnamon,
cloves and turmeric. They possess bactericidal and fungicidal properties that can kill or
27
inhibit the growth of organisms which could spoil the food. Pepper and cinnamon are
reputed to be the best food preservatives.
Closely related to this function is the fact that the spices can disguise the bad taste or
smell of foods. This is evident especially in warm places, where the heat promotes
decomposition and is a leading cause of odors. Food processing technologies, such as
chemical preservatives cannot eliminate food pathogens, such as Listeria
monocytogenesis or delay totally the microbial spoilage (Gutierrez et al., 2009).
These constituents form the characteristic nature of the spice, and possess medicinal and
pharmacological properties with a possible impact on human health. India is known as
the “home” of spices, and is also a leading producer of major spices (Wealth of India,
2001). Among the spice-growing countries, such as India, Sri Lanka, Indonesia, and
Malaysia, these are used extensively as natural food flavorings. In addition to flavoring,
the spices help in protecting food from oxidative deterioration, thereby increasing shelf-
life, and also play a role in the body’s defenses against cardiovascular diseases, certain
cancers, and conditions, such as arthritis and asthma.
In fact, a part of the oxygen used by our body can produce “free radicals”. These
become fatal for a certain number of organic molecules as proteins, lipids and DNA of
cells. The free radicals are atoms which possess a free electron which can engender
chemical reactions found in the process of cellular oxidation. This oxidation can lead to
the development of diseases, such as cancer, cardiovascular diseases, diabetes.
Antioxidants, present in these natural plants, act to protect cells of degradation, reacting
with free radicals to make them harmless. Their properties are mainly derived from the
phenols or thiols present in these products
The first scientific studies of the preservation potential of spices, describing
antimicrobial activity of cinnamon oil against spores of anthrax bacilli, were reported in
the 1880s. Moreover, clove was used as a preservative to disguise spoilage in meat,
syrups, sauces and sweetmeats. In the 1910s, cinnamon and mustard were shown to be
effective in preserving applesauce. Since then, other spices, such as allspice, bay leaf,
caraway, coriander, cumin, oregano, rosemary, sage and thyme, have been reported to
have significant bacteriostatic properties (Burt, 2004; Ceylan and Fung, 2004; Gutierrez
et al., 2008a; Jayaprakasha et al., 2007; Tajkarimi et al., 2010).
28
These condiments can serve as disinfecting agents in the case of ground pepper and
clove, treatment of digestive disorders in the case of cinnamon, mustard, some cumin
and saffron, anti-inflammatory drug as in the case of cumin, aphrodisiac in the case of
the ginger and the hot pepper.
Natural antimicrobials have been identified in herbs and spices and several studies have
been reported on the preservative action of spices or their essential oils. Among these
natural antimicrobials are eugenol from cloves, thymol from thyme and oregano,
carvacrol from oregano, vanillin from vanilla, allicin from garlic, cinnamic aldehyde
from cinnamon, and allyl isothiocyanate from mustard (as reviewed by López-Malo et
al., 2006).
1.1.12. Different types of spices
1.1.12.1. Cinnamon (Cinnamomum zeylanicum) 1.1.12.1.1. Characteristic of Cinnamon
This spice is constituted by the internal bark of four main species of cinnamon tree
(Cinnamomum, Lauracée). C. verum allows to produce a cinnamon of very good quality
"Ceylon cinnamon". Others species lead to secondary lower-quality cinnamons called
"cassias". Cinnamomum zeylanicum (L.), commonly known as cinnamon is rich in
cinnamaldehyde as well as b-caryophyllene, linalool and other terpenes.
Cinnamaldehyde is the major constituent of cinnamon leaf oil and provides the
distinctive odor and flavor associated with cinnamon. It is used worldwide as a food
additive and flavoring agent, and the Food and Drug Administration lists it as
“Generally Recognized as Safe-GRAS. (Nikos G. Tzortzakis, 2008).
1.1.12.1.2. Role of cinnamon in preservation
This spice was found to exert antioxidant activity in the fermented meat sausage (Al-
Jalay et al., 1987), it possesses antibacterial properties against a large variety of
microorganisms. Some of them are pathogenic (Morozumi, 1978; Huhtanen 1980;
Hitokoto et al., 1980; Deans and Ritchie, 1987), inhibit growth and aflatoxin production
of molds (Bullerman et al., 1977), inhibit food spoilage by yeast (Conner and Beauchat,
29
1984) and delay acid production by the starter bacterium, Lactobacillus plantarum
(Zaika and Kissinger, 1979).
The antimicrobial and antifungal properties of cinnamon have also drawn great attention
from many researchers (Delespaul et al., 2000; Chang et al., 2001 and Kim et al., 2004).
Cinnamon possesses notable anti-allergenic, anti-inflammatory, anti-ulcerogenic, anti-
pyretic, and anaesthetic activities (Kurokawa et al., 1998; Qin et al., 2009). Some
evidence suggests that cinnamon may be effective in the supportive treatment of cancer
(Ka et al., 2003), infectious diseases, and complaints associated with modern lifestyle
due to its antioxidant (Lin et al., 2003; Okawa et al., 2001; Toda, 2003), anti-microbial
(Inouye et al., 2001; Smith-Palmer et al., 1998), and anti-inflammatory effects.
For preservation of food, cinnamon is known as an aperitif and digestion stimulant.
Cinnamaldehyde has been reported to possess antibacterial activity against a wide range
of bacteria (Chang et al., 2001), antioxidant properties (Gurdip et al., 2007).
A recent study investigated the effect of dietary supplementation with cinnamon and
garlic powder as growth promoter agents on performance, carcass traits, immune
responses, serum biochemistry, haematological parameters and thigh meat sensory
evaluation in broilers (Toghyani et al., 2010). The study concluded that cinnamon as
antimicrobial substances may inhibit intestinal pathogenic organisms and improve
digestion and absorption; particularly inclusion of 2 g/kg cinnamon proved satisfactory
results on performance indices and may have the potential to be applied as an alternative
for antibiotics growth promoter in broiler’s diet.
In another study (Tabak et al., 1999), dealing with the inhibitory effect on the growth
and urease activity of extracts from stem bark of Cinnamonum cassia on Helicobacter
pylori, it was shown that cinnamaldehyde at 200 mg/disk had the strongest inhibitory
effect (\90 mm) followed by eugenol 200 mg/disk (68 mm) and carvacrole 2000
mg/disk (66 mm). Cinnamaldehyde seems to be the main inhibitory component of
cinnamon and the utilization of cinnamon extract to inhibit both growth and urease
activity of H. pylori in vitro has proved to be more effective than the actual thyme
extract. The efficiency of cinnamon extracts in liquid medium and its resistance to lower
pH levelsmay enhance its effect in the human stomach.
30
1.1.12.2. Clove (Eugenia caryophyllus) 1.1.12.2.1. Caracteristic of cloves
The dried flower buds (Caryophylli floss) are used for medicinal and culinary purposes
and an essential oil is also distilled. The taste is hot, intense, fresh, with a peppery taste
and a touch of oriental fragrance. Not soluble in water but can be dissolved in alcohol.
Cloves control nausea and vomiting, improve digestion, protect against internal
parasites, cause uterine contractions and are strongly antiseptic. The major flavor
component is eugenol.
1.1.12.2.2. Role of cloves in preservation
The eugenol inhibits prostaglandin formation, which explains the anti-inflammatory and
analgesic effect, but the herb has further antiseptic, antispasmodic and carminative
properties. Among the many properties, it is antiplatelet, antiviral, fragrant and
flavoring properties, with a bacterial inhibition of 75-100% (Tajkarimi, 2010).
During conservation, essential oils are well known inhibitors of microorganisms
(Burt, 2004). Clove oil showed its antimicrobial activity in a study based on the
inhibitory effect of clove oil on Listeria monocytogenes in meat and cheese (Vrinda
Menon and Garg, 2001). Clove oil managed to limit the proliferation of Listeria
monocytogenes with 0.5% and 1 %, 30°C and 7°C.
Soliman & Badeaa (2002) found that 500 ppm of cinnamon oil can inhibit A. flavus, A.
parasiticus, A. ochraceus and Fusarium moniliforme on PDA. August (1978) reported
that higher concentrations of cinnamon oil and clove oil could also inhibit the asexual
spores of fungi. Mixtures of cinnamon and clove oils are therefore an interesting
alternative to use other chemical preservatives and appear well suited to use in active
packaging systems (Matan, 2006).
Many studies have shown the efficiency of clove associated with other spices, such as
cinnamon. The efficiency of cinnamon oil and clove oil, as an antibacterial agent, was
reported by Ouattara et al. (1997) and Pradsad and Seenayya (2000). The main
inhibitory components of cinnamon oil and clove oil are believed to be cinnamaldehyde
31
and eugenol, respectively (Jayatilaka et al., 1995; Porta et al., 1998), and their
effectiveness against molds and yeasts has been reported by López-Malo et al. (2002).
1.1.12.3. Cumin (Cuminum cyminum L.) 1.1.12.3.1. Caracteristic of Cumin
Sometimes spelled cumin (Cuminum cyminum L.) is a flowering annual plant of the
Umbelliferae family. This plant, which is one of the most important spices in the world,
is native to India, Iran, the Mediterranean, and Egypt. There are two varieties of cumin:
the white one (more common) and the black one. The cumin is a biennial plant, which
grows in light soils. It is sown in autumn or at the beginning of the year. The seeds
of cumin can contain 3 to 7 % of essential oil. Cumin, the aromatic seed spice, finds
wide applications in foods, beverages, and traditional medicine, and is known to possess
bioactive properties.
The cumin, pricked, spiced, flavor of hazelnut with a strong aromatic odor, has
anti-spasmodic virtues and helps to digest the heavy food. Its aroma matches with
the meat, the autumn vegetables, the salad, the cheese or the diverse pastries. It is a
carminative, an astringent, a stomachic, and is useful in dyspepsia and diarrhea.
1.1.12.3.2. Role of Cumin in preservation
Studies have illustrated its application as a preservative in foods, as well as its
antibacterial, antioxidant, hypoglycemic, hypolipidemic activities, in addition to its use
in perfumery and flavoring liquors. Cumin is traded as whole product, in the ground
form, or as an essential oil (Kanagal Sahana, 2011). Cuminaldehyde was found to be the
main component at concentrations of 53.6% for seed oil and 40.5% for herb oil (El-
Sawi et al., 2002).
Takahashi, Muraki and Yoshida (1881) reported that cumin oil contained mint sulfide as
a trace constituent. Twelve years later, (Anon, 1993) and (Shaath and Azzo, 1993)
reported that the main constituents of Egyptian cumin seed oil were cumin aldehyde, β-
pinene, γ-terpinene, ρ-mentha-1,3-dien-7-al, ρ-mentha-1,4-dien-7-al and p-cymene.
Black cumin seeds have many biological properties including anti-tumor (El-Daly,
32
1998), anti-diabetic (Al-Hader et al., 1993), diuretic (Zaoui et al., 2000) and anti-
bacterial (Kamali et al., 1998) activity.
1.1.12.4. Black pepper (P. nigrum) 1.1.12.4.1. Caracterictic of black pepper
Berry stemming from a plant native of India, called pepper plant, pepper is a part of the
family of Piperaceae and appears under the shape of clusters. Black pepper (P. nigrum)
is a perennial plant and derives its name Piper, perhaps, from the Greek name for black
pepper, Piperi (Rosengarten, 1973) and most of the European names for black pepper
were derived from the ancient Indian language, Sanskrit, such as Pippali, the name for
long pepper (P. longum). It was the great botanist Linnaeus (1753) who established the
genus Piper in his Species Plantarum.
The essential oil in the berry contributes to the aroma, while the alkaloid piperine
imparts the unique pungency. Oleoresin is extracted from the dry powdered berries by
solvents, and is the product that imparts the unique aroma, flavor and pungency in
pepper (Lewis et al, 1976) attributed blackening of pepper berries due to enzymatic
oxidation of polyphenolic substrates present in the skin of green pepper.
Black pepper is valued mostly for its spicy aroma and piquant pungent taste. Oleoresin,
produced by solvent extraction of dried powdered pepper, contains both aroma and
pungency principles (Premi, 2000). The active constituent of pepper, piperine is
sensitive to light and oxygen. Different products from black pepper available are ground
pepper, pepper oil and oleoresin (Ravindran & Johny, 2001).
Pepper is associated with a number of functional properties, such as analgesic and
antipyretic properties, antioxidant effects and antimicrobial properties (Kapoor et al.,
1993). Piperine, an active ingredient in pepper, exerts substantial analgesic and
antipyretic effects (Lee et al., 1984).
1.1.12.4.2. Role of black pepper in preservation
Cinnamon seems to have a lot of good properties for preservation in food. Nevertheless,
studies on the organoleptic properties are still required; the cinnamon having a
pronounced taste and a rather present smell. It would be necessary to measure in that
case the proportion of this spice in the formulations without changing the properties
33
looked for the preservation of meats. The organoleptic analyses are also appropriate
with cloves. Cloves, on its own, have good anti-inflammatory, analgesic and
antimicrobial properties. It can be associated with cinnamon, as mentioned above, and
maybe with other spices, due to antioxidant properties, to improve this property.
It is obvious that organoleptic analyses are important for all the formulations of spices;
in the case of black pepper, it has a spicy and strong aroma. Finally, it will be interesting
to see if the cumin powder has the same properties than cumin oil given that most of
articles deal only with cumin oil. Table 1.1 presents the summary of various properties
of spices which help to understand their eventual applications.
Table 1.1 Summary of the properties of spices
Major constituent properties References
Cinnamon
(Cinnamomum
zeylanicum)
Cinnamaldehyde
O
H
antimicrobial,
antifungal
anti-inflammatory,
anti-allergenic,
anti-ulcerogenic,
anti-pyretic,
anaesthetic
(Delespaul et al.,
2000), (Chang et
al., 2001), (Kim
et al., 2004),
(Kurokawa et al.,
1998), (Qin et al.,
2009), (Lin et al.,
2003), (Okawa et
al., 2001), (Toda,
2003), (Inouye et
al.,
2001), (Smith-
Palmer et al.,
1998)
Cloves
(Eugenia
Caryophyllus)
4-allyl-2-methoxyphenol (eugenol)
CH2
OCH3
OH
anti-inflammatory
analgesic effect,
antiplatelet,
antiviral, flavoring
properties
bacterial inhibition
antimicrobial
activity
(Tajkarimi, 2010)
(Menon and
Garg, 2001)
Cumin aldehyde anti-bacterial (Sahana, 2011)
34
1.1.13. Grapes
The grapes are good alternatives of nitrites and nitrates due to their important
antioxidant properties and presence of higher concentration of phenolic compounds
(Jayaprakasha et al., 2003), such as gallic acid, chlorogenic and caffeic acids, catechin,
etc. In addition, according to studies conducted at Oregon State University, raisins
showed conservation properties similar to those of sodium nitrite in meat products and
sausages. The combination of antioxidants, sugars and acids found in grapes proved to
be as effective as sodium nitrite in maintaining food security. The use of grapes to
replace sodium nitrite in meat has many health benefits. Firstly, while nitrite can form
carcinogenic nitrosamines, grapes do not form. Secondly, unlike sodium nitrite, the
addition of grape is not accompanied by the addition of sodium. This is important for
those who are dieting sodium hypo knowing that the addition of sodium nitrite
concentration can cause problems with hypertension. Thirdly, raisins can improve the
overall nutritional profile of meat products, because they are rich in antioxidants
(Perumall et al., 2011) and help to maintain the taste of products in addition to being
low fat. In addition, the grape is a fruit that is produced in large quantities and low cost.
Its use as an additive for preservation of meat products could be interesting especially if
Cumin
(Cuminum
cyminum L.)
CH3CH3
O
antioxidant,
hypoglycemic,
hypolipidemic
activities
anti-tumor
anti-diabetic
diuretic
anti-bacterial
(El-Daly, 1998)
(Al-Hader et al.,
1993),
(Zaoui et al.,
2000)
(Kamali et al.,
1998)
Black pepper
(Pepper
Nigrum)
Piperine
O
O
N
O
analgesic
antipyretic
antioxidant
antimicrobial
(Kapoor et al., 1993), (Lee et al., 1984)
35
it is added in the dry and ground form, which should facilitate its use and also reduce
the amount used to maintain the organoleptic properties of meat products.
1.1.14. Conclusion
Curing of meat is a process known since ancient times with the intention to prolong the
shelf-life of meat. The curing agents, nitrite and nitrate, react due to easily varying
oxidation status of nitrogen into many derivatives with meat ingredients (Honikel,
2007). Nitrites and nitrates have antioxidant, anti microbial properties, preserve the red
color of meat and are very cheap.
Nitrates have been measured in foods, and have been detected in vegetables, in
preserved meats and baby foods. Because of stricter regulations, industries have to find
a healthier alternative for these products. When ingested, they can be dangerous in high
dose and in long term for humans, animals and can also be dangerous to environment.
Among the various alternatives, the addition of spices is a very promising one; this
alternative seems healthier and could be very interesting given the numerous properties
of spices: antioxidants, antibacterial, among others. In fact, spices and herbs, which
were originally added for improving taste, can also naturally and safely improve shelf
life of food products. However, studies show that the use of essential oil is more
effective than the powder but nevertheless more expensive. Numerous spices, others
than presented here, are also being studied such as ginger, red pepper, coriander and
many others; additional analyses are necessary, such as the organoleptic, chemical and
microbiological analyses, to prove the reliability of these alternatives.
1.1.15. Acknowledgements
The authors are sincerely thankful to the Natural Sciences and Engineering Research
Council of Canada (Discovery Grant 355254), FQRNT (Programme de recherche en
partenariat visant le développement d'alternatives santé à l'ajout des nitrites et des
nitrates dans les produits carnés) for financial support. The views or opinions expressed
in this article are those of the authors.
36
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Kurokawa, M., A Kumeda, C., Yamamura, J.-i., Kamiyama, T., and Shiraki, K. (1998). Antipyretic activity of cinnamyl derivatives and related compounds in influenza virus-infected mice Original Research Article. Eur. J. Pharmacol. 348 : 45-51 Lee, E. B., Shin, K. H., Woo, W. S. (1984). Pharmacological Study of Piperine. Arch. Pharm. Res. 7 : 127–132
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42
1.2. Spice use in food: Properties and benefits –A review
Jessica Elizabeth De La Torre Torres1, 2, Fatma Gassara1, Anne Patricia Kouassi1,
3, Satinder Kaur Brar1*, Khaled Belkacemi3
1 INRS-ETE, Université du Québec, 490, Rue de la Couronne, Québec, Canada G1K
9A9
2 Instituto Tecnológico y de Estudios Superiores de Monterrey (ITESM)
3 Département des sols et de génie agroalimentaire, Pavillon Paul-Comtois, Université
Laval, 2425, rue de l'Agriculture, Québec (Québec) G1V 0A6
*Corresponding Author: Tel.: (418) 654 3116 Fax : (418) 654 2600 ; E-mail
43
1.2.1. Résumé
Les épices sont tirées des plantes qui, en raison de leurs propriétés sont utilisées comme
colorants, agents conservateurs ou médicaments. L'intérêt qu'est porté aux épices est
remarquable en raison de leur composition chimique, comprenant les
phénylpropanoïdes, les terpènes, les flavonoïdes et les anthocyanines. Les épices, telles
que le cumin (cuminaldehyde), les clous de girofle (eugénol), entre autres, sont connues
et étudiées pour leurs propriétés antimicrobiennes et antioxydantes. Ces épices sont
susceptibles d'être utilisées comme conservateurs dans de nombreux aliments à savoir
dans la viande traitée, afin de remplacer les conservateurs chimiques. Les épices ont des
effets bénéfiques, tels que lutter contre l'oxydation et sont comparables à des
antioxydants chimiques utilisés régulièrement afin d'être utilisés comme alternative
naturelle aux conservateurs de synthèse. Dans cette revue, les principales
caractéristiques des épices seront décrites ainsi que leurs différentes propriétés
chimiques, leur diverses applications et les avantages et inconvénients de leur
utilisation.
Mots-clés: Antioxidant, Antimicrobien, épices, Colorants, conservation, Propriétés.
44
1.2.2. Abstract
Spices are parts of plants that due to their properties are used as colorants, preservatives
or medicine. The interest in the potential of spices is remarkable due to their chemical
composition, as phenylpropanoids, terpenes, flavonoids and anthocyanins. Spices, such
as cumin (cuminaldehyde), clove (eugenol), among others, are known and studied for
their antimicrobial and antioxidant properties due to their main chemical compounds.
These spices have the potential to be used as preservatives in many foods namely in
processed meat to replace chemical preservatives. Spices provide beneficial effects,
such as antioxidant activity levels that are comparable to regular chemical antioxidants
used so they can be used as a natural alternative to synthetic preservatives. In this
review, the main characteristics of spices will be described as well as their chemical
properties, different applications of these spices and the advantages and disadvantages
of their use.
Key words: Antioxidant, Antimicrobial, Spices, Colorants, Preservatives, Properties.
45
1.2.3. Introduction
Spices have been used since the ancient civilization; their flavor and properties make
them important for culinary and medicinal uses (Parthasarathy et al., 2008). During
early travels to India and Africa to explore new countries, arrivals at The Spice Islands
made possible the discovery of new species into Europe and the development of trading
networks of spices. For a long time ago, several countries had fought for the control of
spice trade but the strongest nations were the ones who succeded. (World Trade
Organization, 2012). World Spice production is almost entirely achieved in India but
there are other countries that also have major spice production such as Bangladesh,
Turkey and China. Figure 1.6 presents in details the main spice producers countries
(FAO, 2010).
Figure 1.6 World Spice production in year 2010 (FAO, 2010)
Due to their important properties, spices have become essential for culinary and
medicinal proposes in several regions around the world, the trading of these spices has
been an important commercial activity since ancient times and a mean of economic
development (Tufail, 1990).
Asia is the super producer of all kinds of spices; cinnamon, pepper, nutmeg, clove and
ginger are found. There are also Latin American countries that have the leadership in
production of some trade spices, such as Brazil which stands as the major supplier of
pepper or Guatemala as the leading producer of cardamom (Parthasarathy et al., 2008).
68%
8%
7%
5%
4% 1%
1% 1% 1% 4%
World spice production
India
Bangladesh
Turkey
China
Pakistan
Nepal
Colombia
46
Due to spices properties and large applications, they have become an important
economical activity. Between the year 2000 and 2004, the value of spice imports
increased by 1.9% per year and the volume increased by 5.9%. In the year 2004, the
trade of spices was around 1.547 millions of tons with a value of US 2.97 billions. This
reflects the importance and demand of spices around the globe (International Trade
Centre, 2006).
Spices have many applications, namely as flavoring, medicinal, preservative and
coloring agent. Spices and their extracts possess preservative and natural antioxidant
properties, these extracts are popular and some of them have antibacterial, antifungal
and antiviral activities (Hernández et al., 2011). Due to the different applications
discovered in spices, research has been done over the most popular spices to determine
the chemical components that confer their properties. Main chemical compound actives
have been identified in several spices, such as cinammaldehyde in cinnamon, eugenol in
clove and cuminaldehyde in cumin which have proven to prevent food from spoilage
and inhibit the growth of pathogenic microorganisms (Carlos and Harrison, 1999).
Spice phenolic compounds are responsible for the majority of antimicrobial and
antioxidant properties, these compounds grant properties that make spices useful for
medicinal and preservative uses (Bozin et al., 2008). Nowadays, food preservation is a
main concern since most of the existing preservatives are based on synthetic chemicals.
Since spices are natural sources, the application of some as preservatives in food has
been evaluated in order to determine its efficiency and offer an opportunity to replace
synthetic preservatives such as nitrates, which have been claimed to possess negative
effect on human health (Anand and Sati, 2013). Along with this review, the main
characteristics of spices are examined as well the chemical compounds in spices, which
posses several properties that lead to wide ranging applications of spices. Finally, a brief
discussion is presented about the advantages and disadvantages of use of spices
regarding their potential.
1.2.4. General description of spices
The Geneva International Organization for Standardization defines Spices as “vegetable
products or mixtures thereof, free from extraneous matter, used for flavoring, seasoning
and imparting aroma to foods” (ISO, 1995). Spices have special properties that make
47
them useful for several proposes. Among these spices there are special characteristics
that give them distinct features, which have been given in details in Figure 1.7.
Spices incorporate leaves as mint or rosemary flowers as clove; bulbs as garlic or onion,
fruits, such as cumin or red chili, stems as cinnamon and rhizomes as ginger. Since all
spices are coming from plants they have been generally recognized as safe (GRAS).
Plants synthesize via a secondary metabolism, many compounds with complex
molecular structures. Among these metabolites are found alkaloids, flavonoids,
isoflavonoids, tannins, cumarins, glycosides, terpenes and phenolic compounds which
confer most of the properties of spices, such as flavoring, antimicrobial activity (Ceylan
and Fung, 2004), and antioxidant activity (Shobana and Akhilender, 2000; Souza et al.,
2005). Spices are well known due to their medicinal (Shan et al., 2007), preservative
and antioxidant (Burt, 2004) properties but they have been currently used for flavoring
proposes rather than for extending shelf-life of comestibles.
All spices are considered as different dried plant organs and they reside among different
taxonomical categories that correspond to several vegetal species. The wider
classification corresponds to spices that come from monocotyledoneae plants, such as
garlic, ginger, turmeric and vanilla or from dicotyledoneae plants, such as paprika,
pepper, nutmeg, cinnamon and clove (Spices Board, 2013). A more informal but
common classification of spices refers to their sensorial properties and classifies spices
within their flavor intensity or aromatic properties; for example, chili, pepper and ginger
belonging to hot spices or cinnamon clove and cumin belonging to aromatic spices
(Peter and Shylaja, 2012). Spices are defined as useful for different proposes, such as
flavoring and preserving food, these properties are due to several chemical compounds
contained in spices, namely phenylpropanoids, terpenes, flavonoids and anthocyans
(Sajilata and Singhal, 2012). All these compounds confer different properties to spices
such as antimicrobial and antioxidant activity that will be explained further in this
review.
48
Figure 1.7 Spices classification (adapted from Peter and Shylaja, 2012, Sajilata and Singhal 2012)
Spices description
Taxonomy
Angiospermae
Monocotyledoneae
Liliiflorae
Scitamineae
Orchidales
Dicotyledoneae
Archichlamydaeae
Sympetalae
Classification
Hot spices
Mild spices
Aromatic spices
Herbs
Aromatic vegetables
Main plant organs as spices
Aril
Barks
Berries
Buds
Bulbs
Pistil
Kernel
Leaf
Rhizome
Latex
Roots
Seeds
Major chemical constituents of spice essential oil
Phenylpropanoids
Terpenes
Flavonoids
Coumarins
Anthocyans
Aliphatic aldehydes
Aliphatic esters
Aliphatic ketones
Aliphatic acids
Aromatic compounds
49
1.2.5. Chemical properties of spices
There are many properties in spices that make them unique, such as their aroma but
amongst all, chemical characteristics allow spices to be used as preservatives in food.
Due to several chemical compounds, spices present antimicrobial activity and inhibit the
growth of pathogens in meat and other foods. Table 1.2 presents main chemical
characteristics that have been identified in several common spices.
Table 1.2 Main chemical characteristics of common spices
Spice Chemical profile References
Clove
Eugenia
caryophyllata
carvacrol, thymol, eugenol, cinnamaldehyde Chaieb et al., 2007
Coriander
Coriandrum sativum
linalool ,oxygenated monoterpenes ,monoterpene
hydrocarbons
Coriander seed: 60%-70% linalool 20 %
hydrocarbons
Essential oil of leaves and fruits: 2-decenoic acid
(30.8 %), E-11-tetradecenoic acid
(13.4 %), capric acid (12.7 %), undecyl alcohol
(6.4 %), tridecanoic acid (5.5 %),
undecanoic acid (7.1 %)
Coleman and Lawrence,
1992
Leung and Foster, 1996
Guenther, 1950
Bhuiyan et al., 2009
cinammon
Cinnamomum
zeylanicum
Leaves oil: eugenol (76.10 %), trans-β
caryophyllene (6.7 %),linalool (3.7 %), eugenol
acetate (2.8 %) benzyl benzoate (1.9 %).
Branches oil: linalool (10.6 %), α-pinene (9.9 %),
α-phellandrene (9.2 %)
Trajano et al., 2010
Lima et al., 2005
Indan babyleaf
Cinnamomum tejpata
Linalool (50 %) is the major compound; α-pinene,
p-cymene, β-pinene, limonene 5–10 %
Sajilata and Singhal, 2012
Nutmeg
Myristica fragrans
Nutmeg oil a-pinene, βb-pinene,
and sabinene(77.83%) in general 76.8 %
Mullavarapu and Ramesh,
1998
50
monoterpenes, 12.1 % oxygenated monoterpenes,
9.8 % phenyl propanoid ether
Gopalakrishnan, 1992
Origan
origanum vulgare
Leaf essential oil carvacrol (18.06 %)
thymol (7.36 %), g-terpinene (5.25 %), p-cymene
(5.02 %), limonene (4.68 %), caryophylene (4.12
%), cymene (3.56 %), ledene (3.41 %), linalool
(2.47 %), α-pinene (2.15 %), g-terpineol (2.10 %),
and germacrene (2.08 %).
Derwich et al., 2010
Rosemary
Rosmarinus
officinalis
a-pinene (18.25 %), followed by camphor (6.02
%), 1.8-cineole (5.25 %), camphene (5.02 %), b-
pinene (4.58 %), bornyl acetate
(4.35 %), limonene (3.56 %), borneol (3.10 %), a-
terpineol (2.89 %), and cymene
(2.02 %)
Derwich et al., 2011
The main bioactive components of all spices are mostly phenolic compounds,
flavonoids and terpenes. Table 1.3 summarizes the active compounds of the main spices
and their bioactivity. For example eugenol and cinnamaldehyde in clove are related to
their antimicrobial and antibacterial activity; however, these compounds are not
exclusive from clove, cinnamon also contains cinnamaldehyde and possesses the
antimicrobial activity but it also contains other chemical compounds, such as pinene
which confers antioxidant activity. There are a variety of phenolic compounds that hold
these properties and some of them are common among spices (Chaieb et al., 2007).
51
Table 1.3 Spices Bioactivity
Spice Active compounds Bioactivity References
Blackpepper Piperine Stimulates the digestive
enzymes of the pancreas,
enhances digestive capacity,
reduces gastrointestinal
food transit time, enhance
bioavailability of
therapeutic drugs and
phytochemicals, inhibits
hepatic and intestinal aryl
hydrocarbon hydroxylase
and glucuronyl transferase,
lower lipid peroxidation in
vivo, improves antioxidant
status, anti-mutagenic and
anti-tumor influences
Srinivasan K.,
2007
Aggarwal et al.,
2009
Han H.K., 2011
Liu Y. et al.,
2010
Cinnamon cinnamaldehyde Insulin potentiating
properties, cyclooxygenase-
2 inhibitor, reduces blood
glucose and lipids, restores
activities of plasma
enzymes, anesthetic,
antibacterial, anti-
inflammatory, anticancer,
antioxidant, antiviral,
inhibits systolic blood
pressure
Qin B. et al.,
2003
Akilen R. et al.,
2013
Aggarwal et al.,
2009
Clove Eugenol/ Isoeugenol Antioxidant, anti-
inflammatory, cytotoxic
activities, inhibition by
eugenol-related compounds
of lipopolysaccharide LPS
stimulated cyclooxygenase-
2, antimicrobial, inhibition
of platelet aggregation and
Kim S.S. et al.,
2003
Raghavendra
R.H. and Naidu
K.A., 2009
Aggarwal et al.,
2009
52
thromboxane syntheisis.
Ginger 6-gingerol Antioxidant, anti-
inflammatory, antiemetic,
antiulcer, cardiotonic,
antihypertensive,
hypoglycemic,
immunostimulant, activate
cell regulatory signals.
Ghayur M.N.
and Gilani A.H.,
2005
Nya E.J. and
Austin B., 2009
Aggarwal et al.,
2009
Rosemary Rosmarinic acid,
Carnosic acid
Antimicrobial, antioxidant,
inhibits lipoxygenase and
cyclooxygenase activity,
inhibit the Ca+2 dependent
pathways, anti-
inflammatory ,
immunomodulatory,
neuroprotective,
antiallergic, scavenge of
reactive oxygen radicals.
Kayashima T.
and Matsubara
K., 2012
Cheung S. and
Tai J., 2007
Aggarwal et al.,
2009
Different spices provide antimicrobial activity; this is due to certain chemical
compounds with the capacity to inhibit the growth of microorganisms. Figure 1.6
summarizes the range of inhibition achieved by different spices. Cinnamon clove,
rosemary and oregano have achieved levels of bacterial inhibition between 75% and
100% due to their chemical compounds, such as pinenes, eugenol and cinnamaldehyde
(Holley and Patel 2005; Naidu, 2000; Ceylan and Fung, 2004).
0
20
40
60
80
100
Perc
enta
ge
Ranges of bacterial inhibition
min
max
54
Table 1.4 List of bacterial strains inhibited by spices
Bacteria Spice with inhibitory effect Reference
Listeria monocytogenes Nisin, Origan, thymine, origan
with marjoram, thyme with sage
Clove oil, coriander, eugenol,
origan, rosemary
Burt, 2004
Du and Li, 2008
Hayouni al., 2008
Escherichia coli O157:H7 Clove,tea tree Origan, thymine,
origan with marjoram, thyme with
sage, pepper, origan with pepper
Moriera et al., 2007
Du and Li, 2008
Mosqueda-Melgar et al.,
2008
Oussalah et al., 2004
B.aereus and P. aeruginosa Origan, thymine, origan with
marjoram, thyme with sage
Du and Li, 2008
Pseudomonas ssp. Origan, pepper and origan with
pepper
Mosqueda-Melgar et al.,
2008
Oussalah et al., 2004
Aeromonas hydrophila Eugenol Burt, 2004
Salmonella typhimurium Carvacrol, citral, geraniol Burt, 2004
Photobacterium phosphoreum Oregano oil Burt, 2004
Salmonella enteritidis Mint oil Burt, 2004
Antimicrobial activity of spices depends on several factors, which include the type of
spices, composition and concentration of spices; microbial species and its occurrence
level, the substrate composition and the processing conditions and storage. Spices
stabilize foods from microbial deterioration by making the microbial growth
progressively slower and leading to complete suppression. (Souza et al., 2005).
1.2.6. Main applications of spices
Nowadays, all given properties of spices lead to numerous uses, from coloring to
flavoring spices that have been used since ancient times. The main uses of spices are
their natural colorants (Ravindran et al., 2006), flavoring, antioxidants (Shobana and
Akhilender, 2000) and antimicrobials (Ceylan and Fung, 2004). The application of
spices corresponds mainly with the food industry, but they are also used for medicine
(Shan et al., 2007), cosmetics, perfumery and nutraceuticals industry (Peter and Shylaja,
2012).
55
1.2.6.1. Insecticides Some spices have been used as insecticides as they have the potential of killing insects
in several life stages. Plants have been used as botanical insecticides as long time
traditions, for example neem is commonly applied to grain and act as a repellent and
insecticide, pyrethrum is used in flowers to control stored produced insects. These
plants, which possess insecticidal uses, contain essential oils, which have specific
chemical structures that confer this insecticidal property. Sometimes, these essential oils
are secondary metabolites that the plants produce for defense against herbivores or
disease (Suthisut et al., 2011).
The compounds that confer insecticidal properties are mostly complex mixtures of low
molecular weight, such as terpenoid compounds that give characteristic odor and flavor
to leaves, flowers, fruit, seeds bark and rhizomes (Bakkali et al., 2008). Many essential
oils of plants as spices are toxic to insects and act as fumigants, contact insecticides,
anti-feedants or repellents. It is important to mention that these essential oils are toxic to
insects but due to their low toxicity to warm blooded mammals they can be used as
sources to control produced insects (Suthisut et al., 2011). Table 1.5 summarizes the
main insecticidal uses of spices. The use of some spices as insecticides provides an
opportunity to replace the synthetic compounds from insecticides to natural alternatives,
which creates a sustainable market for this kind of products. Several chemical
insecticides in the market are claimed to have toxic compounds adverse to human
health. With the use of spices as insecticidal natural products, this problem can be
solved by the substitution of the synthetic compounds related to harmful health effects
with the main active compound of spices which are safe (Eddleston et al., 2006).
Table 1.5 Use of spices as insecticides
Spice Insects species Reference
Cardamom Tetropium castaneum,
Sitophilus zeamais,
Huang et al., 2000
Cinammon Acanthoscelides oblectus
Ceratitis capitata
Parthasarathy et al., 2008
Nutmeg/Mace Toxocara canis Nakamura et al., 1988
Curry Rhizopus stolonifer
Gloeosporium psidii
Dwivedi et al., 2002
Analgesic
coriander
peppermint
Antipyretic
dill
Anise
Anti inflammatory
Coriander
Celery
Parsley
Cumin
Ginger oil
Anticarcinogenic
Cumin
basil
57
Spices may also be used as bio enhancers; for example, piperine in black pepper has
been reported to possess bioavailability by enhancing activity with various structurally
and therapeutically diverse drugs (Singh et al., 2011). The use of spices is related to an
increased absorption of a drug in the organism due to alteration in membrane lipid
dynamics and enzymatic changes in the intestine, both of them being directly related to
several chemical structures of spices (Parthasarathy et al., 2008). Medicinal
applications of spices are important and the active compounds that provide these
properties should be further investigated to create natural based medicinal products with
a variety of uses, such as analgesic or anti-inflammatory.
1.2.6.3. Colorants
Spices are used as natural sourced colorants, bringing the advantage as against chemical
or synthetic colorants. Spices tint in different colors from yellow and orange to different
variations of red (except chlorophyll from herbs). The most common spices used for
coloring are paprika, red pepper, mustard, parsley, ginger and turmeric (Ravindran et
al., 2006).
The coloring properties of spices is due to several already mentioned chemical
compounds in spices, the principal compound responsible for the color are the
carotenoids, such as beta carotene, lutin and neoxanthin (Bartley and Scolnik, 1995).
Other compounds that provide these coloring properties to spices are flavonoids with
yellow colors, curcumin with orange and chlorophyllwith green (Ravindran et al., 2006;
Peter and Shylaja, 2012). Spices provide strong color pigments commonly between
orange, yellow and red; this can be advantageous since spices can be used as natural
colorants especially for food. Using spices as colorants in food is a natural alternative
that avoids the use of conventional synthetic colorants.
1.2.6.4. Natural flavors
Flavoring food is one of the most common uses for spices, almost each spice is related
to a specific flavor and they are basic for culinary proposes around the world.
Depending on the region, different spices are used for flavoring foods bringing a
distinguished flavor to each food style that even gives culinary identity. For example,
58
Mexico is known for its use of these flavors from cinnamon, vanilla, dried chilies and
cocoa. England uses ginger, mustard seeds, cloves, coriander and allspice. France is
known for different flavors in their foods, such as tarragon, savory marjoram, rosemary
and thyme flavor. The Arabian Peninsula is known to use a variety of spices for
flavoring proposes which include black peppercorn, caraway seed, whole cumin,
cardamom seed, fresh hot pepper garlic and coriander (Exploratorium, 2013).
Flavors given by spices are due to the certain families of chemicals, such as
phenylpropanoids, monoterpenes and other phenol compounds. Some important
chemical compounds for the flavoring potential of spices are eugenol, apiol, sufranol,
vanillin, piperine, beta caryophyllene, alfa pinene, carvacol, thymol, sabinene,
cinnamaldehyde and gingerol (Peter and Shylaja, 2012).
1.2.6.5. Natural Antioxidants
Spices are considered natural antioxidants for food. In order to preserve lipid
components from deterioration antioxidants are necessary in food. There are several
studies that consider antioxidants as defense mechanisms in the body against
cardiovascular diseases, cancer, arthritis, asthma and diabetes. Synthetic antioxidants
used nowadays in food, such as propyl gallate and hydroxyl toluene have been related to
carcinogenesis promoters so there is a strong tendency for the use of natural sources of
antioxidants (Peter and Shylaja, 2012).
The antioxidant properties of spices are due to their chemical compounds especially to
phenolic compounds, in fact there is a linear relationship between the phenolic content
and the antioxidant activity of a spice. Essential oils, oleorosin and other spice extracts
contain important antioxidant activity which can be profited by the food industry
(Wojdyło et al., 2007). Among the most important spices with antioxidant properties,
plants, such as lamiaceae, rosemary, oregano, thyme, sage, marjoram, basil, coriander
and pimento are predominant. The most common chemical compounds that provide
antioxidant properties to spices are eugenol, curcumin, gingerol, carcavol, thymol,
pimento and capsaicin (Peter and Shylaja, 2012).
59
1.2.6.6. Preservation of food
Foods most susceptible to microbial contamination are dairy products, such as
processed meat and chicken. These foods are a common vehicle for diseases and
pathogens, among them we find Escherichia coli, Salmonella, Listeria monocytogenes,
Yersinia enterocolitica, Campylobacter jejuni, Clostridium perfringen, Staphylococcus
aureus and Toxoplasma Gondi which have been isolated from meat (Reuben et al.,
2003). The meat processing industry is trying to find antimicrobial treatments to inhibit
the pathogens or decontaminate their products, these treatments can be synthetic
chemicals or antibiotics but also natural sources of antimicrobials (Hernández et al.,
2011).
Optimal microbial growth occurs at pH values between 6.5 and 7 although most
microorganisms continue to grow within the pH range of 4 and 9.5, for fresh meat, pH
varies around 5.0 and 6.5, hence microorganisms can easily grow into the meat (Tarté,
2009). Temperature is also an important factor for microorganisms and processed
meats, mesophylls replicate at temperatures between 20ºC and 40ºC, psychrotrophs
have the ability to survive and slowly replicate under refrigeration, with their optimal
growth occurring between 20ºC and 30ºC and for thermophiles, the optimal conditions
of growth are between 55-65ºC so almost at any temperature in which food can be
processed, there is a risk of contamination by one of these types of microorganisms
(Ercolini et al., 2009).
Obtaining antimicrobials from natural sources is a good alternative for preservatives in
meat products, other kinds of preservatives, such as synthetic chemicals have been
claimed to cause several adverse effects and preservatives as antibiotics produce
consequences, such as antibiotic resistance. Some natural antimicrobials studied in meat
products include bacteriocins, lactoferrin, lysozyme species, essential oils and a variety
of plant extracts. Species, such as clove cinnamon, cumin and oregano are effective
against inoculated microorganisms on meat, particularly against gram-positive and
gram-negative bacteria (Souza et al., 2006; Sema et al., 2007; Celikel and Kavas, 2008).
1.2.6.6.1. Cumin as preservative
60
Cumin (Cuminum cyminum) is a spice traditionally used as an antiseptic agent and it has
powerful antimicrobial activity in different kinds of bacteria, pathogenic and non-
pathogenic fungi for humans (Haloci et al.,2012). The cumin essential oil contains
cuminaldehyde, β-pinene, p-cymene and γ-terpinene as major chemical compounds
(Hajhashemi et al., 2004;Heinz and Varo, 1970). The main compound of the cumin’s
essential oil is cuminaldehyde which provides the antimicrobial properties. (Hernández
et al., 2011)
The alcoholic extract of cumin has been proven to present a significant inhibition of
microorganisms, such as Bacillus subtilis, Escherichia coli and Saccharomyces
cerevisiae with an outstanding antimicrobial activity for species, such as A.tumefaciens,
B. subtilis, Bacillus licheniformis, Pseudomonas oleovorans, Trichophyton rubrum, S.
cerevisiae and Saccharomyces pombe (De et al., 2003).
The antifungal properties of cumin oil have been proven in recent studies. Whole cumin
oil inhibit Aspergillus flavus and Aspergillus niger by over 90% when aldehyde fraction
of the oil containing the antimicrobial chemical compound cuminaldehyde was tested
(Balacs, 1993; Pawar and Thaker, 2006).
1.2.6.6.2. Clove as preservative
Clove (Eugenia caryophyllata) is a common spice used around the world for culinary
proposes but it also poses different properties that make cloves a potential preservative.
Clove essential oil compounds are eugenol and beta caryophyllene, both compounds
have antibacterial activity against Escherichia coli, Listeria monocytogenes, Salmonella
enterica, Campylobacter jejuni and Staphylococcus aureus (Chaieb et al., 2007).
Clove essential oil has a high concentration of eugenol of around 88.58% and it has
been proved to have diverse antimicrobial activity. Clove oil treatment in concentrations
from 1% to 2% has shown a reduction in growth rates of Listeria monocytogenes strains
(Mytle et al., 2006). Clove leaf oil has been found to inhibit Bacillus cereus with a MIC
of 39 µg/mL (Ogunwande et al., 2005).
Sensitivity of different bacterial strains to cloves essential oil have been tested and the
highest level of sensitivity was observed against five strains of Staphylococcus
epidermidis with an inhibition zone greater than 16 mm (Chaeib et al., 2007). Clove
also has fungicidal activity and their chemical compounds, such as carvacrol and
61
eugenol are known to possess fungicidal characteristics against Candida albicans and
Trichophyton mentagrophytes (Tampieri et al., 2005).
Antioxidant capacity of cloves is due to eugenol as the main chemical compound. The
main mechanisms of antioxidant activity are scavenging the radicals and chelating metal
ions and eugenol participates in photochemical reactions displaying strong antioxidant
activity (Ogata et al., 2000). Chelating potential of clove essential oil has been proven
resulting in the prevention of the hydroxyl radicals due to the eugenol in clove oil
(Jirovetz et al., 2006).
1.2.6.6.3. Cinammon as preservative
Cinnamon (Cinnamomum verum) is considered a preservative because it is an effective
antimicrobial and antibacterial which can inhibit bacterial growth, especially gram-
positive bacteria. Cinnamon oil is composed of different chemicals; amongst them the
most important are cynammyldehyde, cynammyl alcohol and eugenol (Herwita and
Idris, 2007).
Antimicrobial capacity of cinnamon has been tested against Staphylococus aureus
proving its capacity to inhibit S.aureus growth with an optimum inhibiting effort of
0.09% this result is mainly attributed to the chemical compound in cinnamon called
cynammyldehyde (Winias et al., 2011).
Cynammyldehyde inhibition to bacterial growth can be caused by inhibition of the
synthesis of cell walls, inhibition of the cell membrane function, inhibition of protein
synthesis or inhibition of the synthesis of nucleic acids (Winias et al., 2011).
Cinnamon extracts have antioxidant effects by scavenging activity against superoxide
and it has shown excellent antioxidant activities in enzymatic and non enzymatic liver
tissue oxidative systems as well as inhibition on FeCl(2)-ascorbic acid induced lipid
peroxidation of rat liver homogenate in vitro. (Aggarwal et al., 2009).
1.2.6.6.4. Black pepper as preservative
Black pepper (Piper nigrum) is a spice native from India and it’s volatile oil has been
proven to have antimicrobial activity (Dorman and Deans, 2000). The phenolic
compounds of black pepper have claimed to be responsible for the antimicrobial activity
62
by damaging the membrane of bacteria avoiding its growth (Karsha and Lakshmi,
2010).
Analysis using GC-MS has showed that black pepper essential oil contains main
chemical compounds, such as piperine, pierolein B and piperamide. This essential oil
was obtained based on acetone extraction and has proven to be effective in controlling
the mycelial growth of some fungi, such as Fusarium graminearum and Penicillum
viridcatum (Singh et al., 2004).
Most of its bioactivity is due to the main chemical compound, the alkaloid piperine.
This compound has been proven by in vitro studies to protect against oxidative damage
by inhibiting or quenching reactive oxygen species. Table 1.3 describes most of the
bioactivity of piperine in blackpepper. (Aggarwal et al., 2009)
Black pepper has been proven to have antibacterial activity with reported minimum
inhibitory concentrations of around 50-500 ppm demonstrating excellent inhibition on
the growth of gram positive bacteria, such as Staphylococcus aureus, followed by
Bacillus cereus and Streptococcus faecalis and also demonstrated inhibition against
some gram negative bacteria, such as Pseudomonas aeruginosa (Karsha and Lakshmi,
2010).
1.2.6.6.5. Rosemary as preservative
Rosemary (Rosmarinus officinalis) has been shown to possess preservative properties
for their use in foods since it’s antioxidant activity has been tested in pork products,
such as patties (Chen et al., 1999). The antioxidant properties of rosemary have been
attributed to the variety of phenolic compounds in this spice, such as carnosol, carnosic
acid, rosmarinic acid, rosmanol and tosemaridiphenol (Shahidi et al., 2003).
Carnosic acid is the main compound found in rosemary followed by other phenolic
compounds, such as carnosol. Rosemary chemical compounds are classified into three
groups, the phenolic diterpenes related to abietic acid structure, the flavonoids and the
phenolic acids (Almela et al., 2006). The main preservative properties are due to
carnosic acid in rosemary, which have a high antioxidant. The antioxidant activity of
this carnosic acid has been compared to the antioxidant activity of substances, such as
butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA) and tertiary butyl
63
hydroquinone (TBHQ) and the results showed that this acid has antioxidant activity
higher than BHT and BHA (Steven et al., 1996).
Carnosic acid, one of the main active compounds of rosemary, has originated from
isopentenyl diphosphate via methylerythritol phosphate and is located in chloroplasts
and intracellular membranes, such as carnosol (Almela et al., 2006). The rosemary has
been compared with other chemical preservatives and antioxidant compounds proving
efficiency that is comparable to the currently used preservatives so that the rosemary
can be used as a natural green alternative to some chemical antioxidants with
comparable results. Rosemary can be used as natural antioxidant in many foods as it
does not have a strong flavor akin to the majority of spices, namely cloves, cumin and
cinnamon among others. Hence, the use of rosemary as antioxidant will not damage the
organoleptic properties of foods.
1.2.6.6.6. Ginger as preservative
Ginger (Zingiber officinale) is a commonly used spice that contains polyphenolic
compounds, among them the 6-gingerol and its derivatives, these chemical compounds
made ginger a potent antioxidant (Stoilova et al., 2007). Fresh ginger contains moisture,
proteins, fats, fiber carbohydrates and some minerals like iron or calcium
(Govindarajan, 1982).
Ginger CO2 extracts have been proven to contain high polyphenol content and found to
have an enhanced efficiency as an antioxidant preservative at an earlier stage of fat
oxidation. The antioxidant effect of ginger is comparable to BHT, which is a chemical
antioxidant, inhibiting peroxidation in temperature range from 37°C to 80°C (Stoilova
et al., 2007).
Ginger has been shown to inhibit the multiplication of colon bacteria (Gupta and
Ravishankar, 2005) and other microorganisms, such as Escherichia coli, Proteus sp,
Staphylococci, Streptococci and Salmonella (Ernst and Pittler, 2000;White, 2007).
Ginger also has antifungal activity against some species, such as Aspergillus (Nanir and
Kadu, 1987).
The phenolic compounds in ginger are denaturing agents that avoid microbial growth by
changing the cell permeability leading to rupture of bacterial cells. Most of the phenolic
compounds are metal chelators and attach to active sites of metabolic enzymes reducing
enzyme activities and bacterial metabolism and reproduction (Ho et al., 1992).
64
Studies have showed that ginger extracts at concentrations of 0.4 mg/ml have better
antimicrobial activity than commercial antibiotics, such as Gentamicin against
Klebsiella pneumonieae, Proteus vulgaris, Streptococcus pyogenes and Staphylococcus
aureus (Ahmed et al., 2012). Ginger root extracts have been shown to be more effective
than extracts from other parts of plants, such as leaves and has been able to inhibit the
growth of Staphylococcus species with better results than common antibiotics, such
aschloramphenicol, ampicillin and tetracycline (Sebiomo et al., 2011).
1.2.6.6.7. Curry as preservative
Curry is a traditional spice used in conventional foods, the origin of curry is found in
India but nowadays, it is one of the most popular spices in the world with a
characteristic flavor and aroma (Sathaye et al., 2011). Curry has been shown to have an
important antimicrobial activity. Antimicrobial assays of coumarin extracts performed
with petroleum ether and chloroform exhibited prominent antibacterial and antifungal
activity. Chloroform extract of curry showed a good inhibitory property being effective
in species, such as Aspergillus niger and P. aeruginosa (Vats et al., 2011).
Curry contains a variety of carbazole alkaloids and coumarins that confer antimicrobial
activity. Minimum inhibitory concentrations of curry compounds have been found to be
between the range of 3.13-100µg/ml. (Rahman et al., 2005)
The antimicrobial activity of curry extracts is proportional to the concentration used and
growth inhibition has been reported against species, such as Bacillus subtilis,
Pseudomonas aeruginosa and Escherichia coli with a less minimum inhibitory
concentration (MIC) than compared to other species such as Staphylococcus aureus and
Micrococcus luteus. From these studies, E.coli has been determined as the most
resistant microorganism and higher concentrations of curry are required for its
inhibition (Vats et al., 2011).
Curry has been studied as a natural antimicrobial food preservative and also as a
detoxifying food preserving agent. Curry has been proven to be an antifungal and
antiaflatoxigenic (Murugan et al., 2013), these characteristics have set curry as an
important natural preservative with a high potential for becoming a replacement for
other types of unnatural preservatives.
65
Whole spices by themselves can be used as preservatives but their essential oils can also
be isolated and their properties determined. Essential oils from spices are homogeneous
mixtures of organic chemical compounds from the same chemical family; they are
composed of terpenoids, monoterpenes and sesquiterpenes. The antibacterial activity of
essential oils is not attributed to a specific mechanism but to several attack mechanisms
to cells with different targets (Burt, 2004). It is known that substances act on the cell’s
cytoplasmic membrane, in several cases, the presence of a hydroxyl group is related to
the deactivation of enzymes and it is probable that this group causes cell component
losses, a change in fatty acids and phospholipids and prevents energy metabolism and
genetic material synthesis (Di Pascua et al., 2005).
Antioxidant properties are important for conservation of processed meats. Nowadays,
there are several synthetic antioxidants, such as BHA, BHT and alfa tocopherol. It has
been proved that antioxidant properties from different essential oils from black pepper,
clove, geranium, Melissa, nutmeg, oregano and others show superior antioxidant
capacity to tocopherol analogue Trolox. Between all the species proven in the trial,
clove and oregano were exceptionally potent in the assay (Dorman, 2000).
In recent studies, essential oils from cumin and clove at concentrations from 500mg/L to
750 mg/L were used on meat samples at three different concentrations, 750, 1500 and
2250 microliters. The cumin essential oil produced a reduction of 3.78 log UFC/g with
the application of 750 microliters and the clove essential oil produced a reduction of
3.78 l of UFC/g with the application of 2,250 microliter, clove and cumin extracts got a
reduction of 3.6 log UFC/g demonstrating the antibacterial potential of these essential
oils (Hernández et al., 2011).
1.2.7. Advantages & Disadvantages of using spices as preservatives
Antioxidant and antimicrobial activity has been found in spices proving an important
preservative activity for food but several aspects need to be studied before assuring the
effectiveness of spices as preservatives. As it has been reviewed, spices have different
levels of aroma and flavor but most of them are characterized as being strong. If spices
are used in high quantities in order to achieve a good antioxidant or antimicrobial
activity, they can interfere with the original flavor of the food and products could be not
66
useful in the market since they would interfere with commercial desired characteristics
for several foods.
Essential oils from spice extracts are a good alternative for preservatives in meat
products but the main concern is that, when essential oils are used in meat, their
antimicrobial effect is lower because high-fat and protein levels contained in meat
protect bacteria from the essential oils' action, the essential oil is dissolved into the food
fatty phase being less available to act against the microorganisms (Rasooli, 2007).
Encapsulated rosemary essential oil has an improved antimicrobial effect than standard
rosemary essential oil against L.monocytogenes in pork liver sausage and this is
associated with the interaction of essential oils with the fatty phase of meat
(Carraminana et al., 2008). Thus, higher concentration of spices might be needed to
assure an antioxidant and antimicrobial activity but the strong flavor of spices can affect
the flavor of meat and its commercial value.
Essential oils from spices also require a process of extraction, which can make the
whole process pricier and would not result in a higher antimicrobial activity since; for
example, these essential oils can dissolve in the fatty phase of meat. Therefore, the use
of the whole spices might be a better solution for preservation in meat and other foods
since they present less complexity, less expenses and equivalent antimicrobial activity.
Another important aspect is that spice formulation effectiveness against microorganisms
differs depending on the food or media; same formulation can be effective for a specific
type of meat but not for another. A combination of clove and oregano in broth culture
showed inhibitory effect for L.monocytogenes but did not show the desired effect in
meat slurry (Lis-Balchin et al., 2003). Different spices formulations have to be tested in
vitro and in vivo in order to prove their antimicrobial effect for each type of meat.
Spices are provided from natural herbs and plants and thus do not have a synthetic
origin, essential oils of cinnamon and clove and their main active chemical compound
cinnamaldehyde and eugenol have been recognized as safe consumption products
(GRAS) by regulatory agencies of U.S. (Raybaudi et al., 2008; Turgis et al, 2009). In
contrast, nitrate and nitrite preservatives used nowadays in meat products have been
found to produce carcinogenic N-nitroso compounds, such as nitrosamines and this has
caused concerns about possible adverse health effects. (Assembly of Life Sciences U.S.,
67
1982; Anand and Sati, 2013). Therefore, spices are a safe alternative for preservatives in
food approved by regulation and with no adverse health effects reported.
1.2.8. Conclusions and future outlook
Spice uses vary from flavoring, coloring, medicinal or preservative uses and their trade
is a significant economic activity in the world. The unique properties of spices have
created a huge demand for several common spices around the world making the spices a
niche of research and economical benefits.
Several spices have been proved to have microbial growth inhibition potential to some
of the most common bacteria in food, such as L.monocytogenes, E.colli and Salmonella.
Thus, it is possible to use spices as preservatives but is necessary to prove its
antimicrobial effect on different foods, such as meat, poultry, dairy products, vegetables
and fruit to guarantee a preservative effect comparable to the conventional synthetic
preservative effect for each food prior to settle the use of spices as preservatives for
industrial or commercial proposes.
Albeit, whole spices and their essential oil have proven good antimicrobial activity, but
the use of the whole spice or essential oil is in debate due to the high purification costs
that can be involved without necessarily having an improving efficiency in the
antimicrobial or antioxidant activity. As whole spices owe this properties they can be
settled as natural preservatives and adapted to the industry for this propose.
Finally, the antimicrobial and antioxidant properties of several spices such as black
pepper, clove, nutmeg, turmeric, cumin, cinnamon among others leads to a research
field in order to use them as preservatives in food. Spices used in foods, such as meats
have a high possibility of success and potential antimicrobial activity that is comparable
with the effect of preservatives based on nitrites that are used nowadays and which
have been claimed to own negative health effects, making possible to research a way to
substitute chemical based preservatives with natural based ones for food preservation.
68
1.2.9. Acknowledgements
The authors are sincerely thankful to the Natural Sciences and Engineering Research
Council of Canada (Discovery Grant 355254), FQRNT (Programme de recherche en
partenariat visant le développement d'alternatives santé à l'ajout des nitrites et des
nitrates dans les produits carnés) for financial support. We specially thank MITACS
Globalink program for the prestigious funded internship opportunity given to Miss
Jessica Elizabeth De La Torre Torres, which made it possible for her to accomplish this
review. The views or opinions expressed in this article are those of the authors.
69
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1.3. Hypothèse et objectifs du projet
1.3.1. Hypothèse de l'étude
L'utilisation des épices peut être vue comme une alternative à l’utilisation des nitrates et
des nitrites pour la conservation des produits carnés, tout en garantissant les mêmes
propriétés anti-fongiques, antioxydantes et anti-bactériennes ainsi que la texture et les
propriétés organoleptiques allouées à ces produits chimiques de conservation, avec un
coût relativement abordable.
1.3.2. Objectif général
Plusieurs alternatives aux nitrites et nitrates ont été suggérées par des scientifiques à
savoir des produits chimiques comme le dioxyde de soufre, l’acide éthylène diamine
tétracétique, l’hydroxyanisole butylé, les esters fumarates, l’hypophosphite de sodium et
des produits naturels contenant les nitrites comme le céleri, la laitue, les épinards.
L’objectif principal de ce projet est de développer des alternatives vertes à l’utilisation
des nitrites et des nitrates comme agents de conservation dans les produits carnés
(jambon, terrines, produits saumurés, saucissons et produits fumés à chaud et à froid)
qui sont à la fois efficaces, économiques et sécuritaires pour la santé humaine étant
donné que l'on ajoute que des épices, tout en gardant la même durée de conservation et
les mêmes qualités organoleptiques, antibactériennes et antioxydantes que les nitrites.
1.3.2.1. Objectifs spécifiques
Spécifiquement, il s’agira de réaliser :
1) Un criblage qualitatif puis quantitatif des additifs alimentaires naturels de faible coût
comme les épices (la cannelle, les clous de girofles, le cumin, le poivre noir, l’ail et le
poivron rouge...) afin de sélectionner celles assurant une meilleure activité
antimicrobienne et antioxydante grâce à des analyses physico-chimiques et
microbiologiques.
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2) Une optimisation des conditions de production et de traitement avant emballage des
produits carnés ainsi qu'une analyse sensorielle; L'optimisation se basera sur une
méthode de surfaces des réponses, un plan composite centré donnant 19 combinaisons
des trois meilleures épices criblées avec des concentrations allant de 0, 1 % à 0, 3%
(m/m).
3) Application des alternatives à l’échelle industrielle avec la réalisation d’une analyse
technico-économique qui prendra en compte l'ajout des épices dans nos viandes.
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Chapitre 2 Use of spices as alternative of nitrites and nitrates in meat-based products
Anne Patricia Kouassi1,2, Fatma Gassara2, Satinder Kaur Brar2, Khaled
Belkacemi1
1Département des sols et de génie agroalimentaire, Pavillon Paul-Comtois, Université
Laval, 2325, rue de l'Université, Québec (Québec) G1V 0A6
2INRS-ETE, Université du Québec, 490, Rue de la Couronne, Québec, Canada G1K
9A9
(*Corresponding author, Phone: 1 418 654 3116; Fax: 1 418 654 2600; E-mail:
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2.1. Résumé
L'activité antimicrobienne et antioxydante de certaines formulations d'épices utilisés
dans les produits à base de viande (les terrines) ont été étudiés. A cet effet, les valeurs
de l'indice de TBA et p-anisidine ainsi que les comptages totaux viables ont été réalisés
sur les terrines préservées avec ces formulations d'épices. Leurs activités
antimicrobiennes et anti-oxydantes sont comparées à celles des nitrites. Les résultats ont
montré que les clous de girofle, le cumin, les clous de girofle + cumin, la cannelle, les
clous de girofle + cannelle ont des valeurs de TBA et de p-anisidine inférieures à celles
des nitrites. De même, les terrines de neuf (9) formulations d'épices ont une durée de vie
égale, voire supérieure aux terrines contenant les nitrites (7 semaines). Par conséquent,
ces formulations pourraient être d'excellents agents de conservation, et ainsi empêcher
l'oxydation des lipides et prévenir la contamination microbienne des produits à base de
viande.
Mots-clés: formulations d'épices, nitrites, terrine, antimicrobien, antioxidant
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2.2. Abstract
The antimicrobial and antioxidant activity of some spice formulations used in meat
products (terrines) were investigated. For this purpose, terrines were preserved using
these spice formulations and TBA index and p-anisidine values and total viable counts
were performed. The antimicrobial and antioxidant activities of these spices were
compared with those of nitrites. The results showed that cloves, cumin, cloves + cumin,
cinnamon, cinnamon + cloves formulations showed a TBA index and p-anisidine values
lower than those of nitrites. Similarly, eight (9) formulations of spices (cloves, cumin,
cloves + cumin, cinnamon, cloves + cumin, curry, red pepper, curry + cloves, red
pepper + cloves) gave a shelf life of terrine equal to or higher than the terrines of nitrites
(7 weeks). Hence, these formulations could be an excellent preservative that can prevent
the oxidation of lipids and prevent microbial spoilage of meat product. Spices can thus
provide an important nitrite-nitrate alternative for processed meats.
Key words: spice formulations, nitrites, terrine, antimicrobial, antioxidant
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2.3. Introduction
Nitrates and nitrites are present everywhere in the environment. They are used as
fertilizers, explosives and preservative agents in food particularly against Clostridium
botulinum. They are natural chemical substances which are obtained from the oxidation
of nitrogen by the microorganisms in plants, soils or water. The toxic effects of nitrate
are attributed to its endogenous conversion to nitrite. At its 44th meeting (2002), JECFA
(Joint FAO/WHO Expert Committee on Food Additives) concluded that the range of
nitrate conversion is 5-7% for normal individuals and 20% for individuals with a high
rate of conversion. The acceptable daily intake (ADI)for nitrate is 0-3.7 mg/kg bw/day
(expressed as nitrate ions) (Thomson 2004). It can lead to risks to human health and the
environment. The health effect of most concern to the U.S. EPA for children is the
“blue baby syndrome” (methemoglobinemia) (Fan and others 1987). The blue baby
syndrome is named for the blue coloration of the skin of babies who have high nitrate
concentrations in their blood. The nitrate binds to hemoglobin (the compound which
carries oxygen in blood to tissues in the body), and results in chemically-altered
hemoglobin (methemoglobin) that impairs oxygen delivery to tissues, resulting in the
blue color of the skin (USEPA 2007).
At higher level, nitrates and nitrites have been associated with increased incidence of
cancer in adults, combining with secondary or tertiary amines to form N-nitroso
derivatives, and possible increased incidence of brain tumors, leukemia and
nasopharyngeal problems. Their addition in food is however very limited (USEPA,
2006). Industries use nitrates and nitrites for the stabilization of the red color of meats
(Honikel, 2008), inhibition of the development of toxic microorganisms, decreasing the
oxidation of lipids and to improve the flavor (Pegg and Shahidi, 2000). Nitrates and
nitrites are preferred as they are less expensive for the properties that they offer.
Meanwhile, the industries have not found a better and economical substitute to these
nitrites and nitrates. Due to the possibility of establishment of stricter regulations by
various countries, the industries are obliged to find greener substitutes to nitrates and
nitrites.
In this context, alternatives of nitrates and nitrites have been the subject of numerous
research studies (Stoilova et al., 2007). In literature, there are chemical agents, such
ascorbate and α–tocopherol, lactic-acid-producing organisms, potassium sorbate, or
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treatments, such as irradiation (National Academy of Sciences, 1982) that have been
used as nitrite-nitrate substitutes. A number of studies have been carried out to
investigate the properties of aromatic herbs and spices, such as clove, ginger, pepper or
garlic (Menon and Garg, 2001). Cinnamaldehyde, the major constituent of cinnamon
(Cinnamomum cassia) has been reported to possess antibacterial activity and
antioxidant properties (Chen and Chang, 2001).
To the best of our knowledge, there is no systematic study on use of spices. With the
emergence of green alternatives and strict environmental regulations, this study is
important and aims at evaluating the best spices among 18 which can be used as
alternatives to nitrates and nitrites in terrines and ham, with chemical and
microbiological properties identical or better than nitrates and nitrites. The objective of
this study is the quantitative screening of the natural food additives, in order to choose
the best spices with good antioxidant and antimicrobial properties. Some
physicochemical properties of spices are listed in Table 2.1.
Table 2.1 Physicochemical properties of spices
Spice Physic-chemical properties References
Clove carvacrol, thymol, eugenol, cinnamaldehyde Chaieb et al., 2007
Coriander
linalool ,oxygenated mono terpenes
,monoterpene hydrocarbons
Coriander seed: 60%-70% linalool 20 %
hydrocarbons
Essential oil of leaves and fruits: 2-decenoic
acid (30.8 %), E-11-tetradecenoic acid
(13.4 %), capric acid (12.7 %), undecyl alcohol
(6.4 %), tridecanoic acid (5.5 %),
undecanoic acid (7.1 %)
Coleman and Lawrence,
1992
Leung and Foster, 1996
Guenther, 1950
Bhuiyan et al., 2009
cinammon Leaves oil: eugenol (76.10 %), trans-β
caryophyllene (6.7 %), linalool (3.7 %), eugenol
acetate (2.8 %) benzyl benzoate (1.9 %).
Branches oil: linalool (10.6 %), α-pinene (9.9
%), α-phellandrene (9.2 %)
Trajano et al., 2010
Lima et al., 2005
84
Red pepper Ascorbic acid 108.65±6.481 (mg/100 g DW)
Licopene 123.89±0.170 (mg/ Kg DW)
Carotene 2282.45±5.362 (mg/ Kg DW)
Total phenols 89.82±4.721( mg GAE g DW -1)
Ozgur et al, 2011
Garlic total phenolic compounds (6.5 mg GAE/g of
dw)
Caffeic acid ( 2.9 mg/kg of dw)
Ferulic acid (2.6 mg/kg of dw)
vanillic, p-hydroxybenzoic,
p-coumaric acids
Beato et al., 2011
Ginger
Phenols (5.69 %)
Oleoresin content (2.93 %)
Eleazu and Eleazu, 2012
Curry Antioxidant activity: Koenigine; Mukonicine;
Mahanimbinine; Murrayacinine;
Mahanimboline; Isomahanine
Antimicrobial activity : Murrayanol;
Mahanimboline; Mahanimbinine;
Murrayacinine
Ganesan et al., 2013
Cumin Total phenolic compounds (24.66 mg GAE/g)
α-pinene (0.5%), Myrcene (0.3%), limonene (0.5%), 1-8-cineole (0.2%), p-menth-3-en-7-ol (0.7%), p-mentha-1, 3-dien-7-ol (5.6%), caryophyllene (0.8%), β-bisabolene (0.9%), β-pinene (13.0%), P-cymene (8.5%), β-phellandrene (0.3%), D-terpinene (29.5%), cuminic aldehyde (32.4%), cuminyl alcohol (2.8%), β-farnesene (1.1%)
Nadeem and Riaz., 2012
2.4. Materials and methods
2.4.1. Preparation of meat samples
Samples of 200g of terrines were prepared using simple recipe of terrine of pork and
rabbit, by adding 1% w/w of various spices to be tested. The list of spices tested is
presented in details in Table 2.2. Samples were cooked in a microwave oven (Danby,
Quebec, Canada; power= 700 watts) for 30 min and then cooled in a refrigerator for 2
85
days. Each terrine was cut into several pieces using sterilized knife to avoid cross-
contamination of samples and placed under vacuum by using a vacuum food storage
system. Terrines were stored in the refrigerator during 8 weeks and samples were taken
for the analysis every week.
2.4.2. Determination of thiobarbituric acid (TBA).
The lipid oxidation was determined by 2-thiobarbituric acid according to the procedure
of Schmedes and Holmer (1989). Terrine sample (2g) was mixed with 5 ml of
trichloroacetic acid (TCA) solution (200 g/l of TCA in 135ml/l of phosphoric acid
solution) and homogenized in a blender for 30 s. After filtration with a filter paper (0.45
µm), 2 ml of the filtrate was mixed with 2ml of a solution of TBA (3g/l) in a test tube.
The tubes were incubated at room temperature in the dark for 20h. The absorbance was
measured at 532 nm using UV-Vis spectrophotometer (model UV-1200, Shimadzu,
Kyoto, Japan). TBA value was expressed as mg malonaldehyde per kg of sample and
determined by the following equation 1:
10-2 (1)
Where, A532nm is the measured absorbance, VTCA denotes the extraction solvent volume
(5ml), M is the molar mass of malonaldehyde (72g/mol) and m is the mass of the
analyzed sample (2g). TBA value was measured in triplicates and means value are
determined.
Table 2.2 List of formulations
Formulations Spices Concentration
1 Nitrite 1 % w/w
2 Ginger 1 % w/w
3 Black Pepper 1 % w/w
4 Cumin 1 % w/w
5 Coriander 1 % w/w
6 Garlic 1 % w/w
7 Cinnamon 1 % w/w
8 Curry 1 % w/w
9 Chili 1 % w/w
10 Grapes 1 % w/w
86
11 Cloves 1 % w/w
12 Cloves 1 % w/w
Ginger 1 % w/w
13 Cloves 1 % w/w
Black Pepper 1 % w/w
14 Cloves 1 % w/w
Cumin 1 % w/w
15 Cloves 1 % w/w
Coriander 1 % w/w
16 Cloves 1 % w/w
Cinnamon 1 % w/w
17 Cloves 1 % w/w
Curry 1 % w/w
18 Cloves 1 % w/w
Red Pepper 1 % w/w
2.4.3. Determination of p-Anisidine value
The hydroperoxide decomposition was determined by p-anisidine value (p-Anv)
according to IUPAC standard method (1986). Terrine sample (0.5g) was mixed with
5ml of isooctane solution. The absorbance (Ab) of this sample was measured at 350 nm
using isooctane solution as a blank reagent. Later, 1ml of p-anisidine solution (0.25
(v/v) % in glacial acetic acid) was added to the sample tube and to 5ml of isooctane test
tube. After 10 minutes, the absorbance (As) of samples was read using isooctane as
blank. The p-anisidine values were calculated using the following equation 2:
(2)
Where, As is the absorbance of the fatty solution after reaction with
p-anisidine solution, Ab is the absorbance of the fatty solution and m is the sample mass
(g).
87
2.4.4. Microbiological analysis
Total viable count (TVC) of bacteria is one of the most important indexes in evaluation
of quality and safety of meat products. The total viable count of bacteria (TVC) ufc/g on
meat product sets a limit to its shelf-life. Meat will “spoil” with TVC at 107/g because
of off-odours. Slime and discoloration appear at 108/g. The main factors determining the
time taken for the TVC to reach these levels are the initial count due to contamination
during slaughtering and processing, further contamination during storage, temperature,
pH, relative humidity and food preservative added during process.
To estimate the TVC, samples (1 g) from meat were weighed aseptically, chopped and
harvested properly and then added to 25 ml NaCl sterile solution, 0.9 % v/w (25 ml),
and homogenized for 60 s at room temperature. Decimal dilutions in NaCl sterile
solution (0.9 % v/w) were plated on plate count agar (PCA; Quelab Laboratories Inc.,
Montreal, QC, CANADA) and incubated at 30 °C for 48 h for enumerating total viable
counts (TVC). Enumeration of TVC was performed on these duplicate samples and
results are displayed as the mean of both measurements. Each sample was analyzed in
duplicate (coefficient of variation of samples from the same experiment).
2.4.5. Statistical analysis
All the experiments were assayed in replicates and an average of 3 replicates was
calculated along with the standard deviation. Data means were analyzed using
individual Student's t-tests to distinguish differences among spices formulations. The
test was performed at the level of P-value < 0.05 to determine the significance of the
difference between spices formulation for meat products preservation (SPSS 1999).
Standard errors and error bars presented in the tables and figures, respectively were
calculated using untransformed data in ANOVA. Standard error for a single proportion
was estimated as described by Gottelli and Ellison (2004).
2.5. Results and discussion
2.5.1. Determination of p-Anisidine value
88
Generally, it is possible to measure either the precursors of oxidation, such as free
radicals, hydroperoxides, or oxidation products, such as aldehydes or ketones. The p-
anisidine value essentially measures 2-alkenal. In the presence of acetic acid, p-
anisidine reacts with aldehydes producing a yellowish color and an increase in
absorbance when the aldehyde contains a double bond. The p-anisidine value is
dependent on the oxidation of fatty acids including polyunsaturated fatty acids. The p-
anisidine values measured during 8 weeks of meat product storage at 4°C are presented
in Fig. 2.1. For all the formulations tested, the value of p-anisidine increased during
storage time. This is due to the growth of lipolytic bacteria that can oxidize fatty acids
during storage. In addition, the value of p-anisidine was dependent on the spice
formulations used in terrines. From the results shown in Fig.2.1, the values of p-
anisidine in terrines preserved using cloves, cumin, cinnamon, cinnamon + cloves were
comparable and sometimes lower than the terrines conserved using nitrite (150 ppm).
This is explained by the richness of these spices with polyphenols that protect against
fatty acids oxidation contained in terrine (Kim and others 2011). The results of p-
anisidine values were consistent with the values of TBA index that showed that these
formulations had excellent antioxidant activities. Hence, cloves, cumin, cinnamon, and
the mix of these formulations could be excellent preservatives that prevent the oxidation
of lipid contained in meat products, such as terrine, bacon, salami, ham, among others.
Figure 2.1 p-anisidine values of terrines preserved using different spice formulations at
4°C for 8 weeks
0
20
40
60
80
100
120
P an
isid
ine
inde
x
5 week6 week7 week8 week
89
2.5.2. Determination of TBA index
Polyunsaturated fatty acids, such as linoleic acid are easily oxidized by the oxygen in
the air. This auto-oxidation leads to the occurrence of chain reactions with the formation
of coupled double bonds, and at a later stage also to obtain secondary products, such as
aldehydes, ketones, and alcohols. In order to assay the antioxidant effect of the spices
formulations, the experiments for inhibiting the peroxidation of the polyunsaturated
fatty acids were performed. During storage of meat products at 4°C for two months, the
lipids are oxidized and malondialdehyde (MDA), a secondary component fatty acids is
formed due to the degradation of polyunsaturated fatty acids. TBA index is able to
measure the formation of malondialdehyde. The results of the analysis of the TBA
index are presented in Fig 2.2. Black pepper, black pepper + cloves and coriander
showed a weaker effect in inhibiting lipid peroxidation. Black pepper formulation used
in this study is a commercial formulation that contains starch. Starch could be used by
microorganisms as carbon source to grow, to damage meat products by the oxidation of
lipid compounds. The presence of starch in black pepper formulation could be the cause
of weaker antioxidant activity of these formulations. Moreover, the total flavonoid
content in decreasing order was: thyme > rosemary > marjoram and oregano > basil >
cumin > clove > caraway and fennel > savory > turmeric, mace and coriander (p <
0.001) (Kim and others 2011). A significant correlation was observed between
antioxidant activity and phenol content in the plant foods studied (IJFSN 2007). Hence,
the coriander has a weaker antioxidant activity due its low content on polyphenolic
compounds. Most efficient spice formulations to inhibit lipid oxidation were cloves,
cumin, cinnamon, cumin + cloves, cinnamon + cloves. This was due to the high total
phenolic content in cloves (108.28 μg catechin equivalent (CE)/g) (Kim and others
2011), cinnamon (Muchuweti and others 2007) and cumin. Moreover, the DPPH
radical scavenging ability of these spices extracts were very important (Kim and others
2011). 2,2-diphenyl-1-picrylhydrazyl (DPPH) is widely used to test the ability of
compounds to act as free radical scavengers or hydrogen donors, and to evaluate
antioxidant activity of foods ( Frankel and Meyer 2000). In addition, a significant and
linear relationship existed between the DPPH scavenging activity and phenolic content
indicating that phenolic compounds are major contributors to antioxidant activity (Kim
and others 2011). Hence, the most efficient antioxidant activities of these spice
90
formulations were due to the high phenol content that prevented the polyunsaturated
fatty acids oxidation and the release of malonaldehyde.
Figure 2.2 TBA index for terrines preserved using different spice formulations at 4°C
for 8 weeks
0
1
2
3
4
5
6
7
TBA
Inde
x (m
g M
A/k
g)
1 week 2 week 3 week 4 week
0
2
4
6
8
10
12
TBA
Inde
x (m
g M
A/kg
)
5 week 6 week
7 week 8 week
91
2.5.3. Microbiological analysis
The enumeration of total viable counts (TVC) in terrines preserved by different
formulations of spices during 8 weeks was performed and the results of this study were
presented in Fig. 2.3. Storage time, spices formulations used for terrines preservation
and meat type had a statistically significant (P > 0.05) effect on the development of the
microbial groups determined in this study. Large microbiological changes due to
different biomass developments in the terrines resulted in higher standard deviations of
bacterial counts. TVC values will be used to determinate the shelf life of terrine. A meat
product is non-consumable if the TVC is greater than 107 UFC/g (ICMSF 1986).
Through its antimicrobial and antioxidant properties, nitrite is able to extend the shelf-
life of meat products (Jackson 2010). The results of this study have shown that the shelf
life of terrine preserved using 150 ppm of nitrates was 7 weeks. Terrines preserved by
black pepper had the shortest shelf life (5 weeks). Black pepper did not show any
antimicrobial activity. However, black pepper showed a good antimicrobial activity in
previous studies (Ghori and Ahmad 2009). The weaker antimicrobial activity of black
pepper found in our study could be due to the presence of starch in black pepper
formulations that enhanced microbial growth. Terrines preserved by, cloves (>8 weeks),
cumin (8 weeks), cloves + cumin (>8 weeks), cinnamon (7 weeks), cloves + cumin (8
weeks), curry (7 weeks), red pepper (>8 weeks), curry + cloves (8 weeks), red pepper +
cloves (>8 weeks) had a shelf life equal or higher than terrines preserved by nitrites.
These spice formulations have good antimicrobial activities compared to nitrites. The
antimicrobial potency of plants and spices is believed to be due to tannins, saponins,
phenolic compounds, essential oils and flavonoids (Aboaba and Efuwape 2001).
Moreover, naturally occurring compounds in spices, such as sulphur compounds,
terpenes and terpene derivatives, phenols, esters, aldehydes, alcohols and glycosides
have been shown to exhibit antimicrobial functions (Russel and Gold 1991; Davidson
and Baren 1993; Deis 1999). However, the inhibitory effects of spices are mostly due to
the volatile oils present in their composition (Arora-Dlijit and Kaur 1999). The result of
this study showed that cumin, cloves, cinnamon, red pepper and curry had good
antimicrobial activities. These results were also well correlated with the results of
Rahman and others (2010). Rahman and others (2010) found that cinnamon, cloves and
cumin were found to have important antimicrobial activity against Staphylococcus
aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Enterococcus faecalis,
92
Micrococcus luteus, Escherichia coli and Candida albicans. In this regard, the use of
these spices and their volatile compounds as natural preservatives in food products can
offer an alternative to the use of chemical additives, such as nitrites.
Figure 2.3 Total viable counts for terrines preserved using different spice formulations
at 4°C for 8 weeks
1,E+00
1,E+01
1,E+02
1,E+03
1,E+04
1,E+05
1,E+06
1,E+07
black p
epper
red pep
per
ginger
clove
scu
min
cinnam
oncu
rry
coria
ndergarl
ic
red pep
per + c
loves
cumin +
clove
s
grapes
cinnam
on + clo
ves
coria
nder + c
loves
black
pepper
+clove
s
curry
+ clove
s
ginger+ cl
oves
Nitrite
s
Formulations
TVC
(UFC
/g)
1 week 2 week 3 week 4 week
1,0E+001,0E+011,0E+021,0E+031,0E+041,0E+051,0E+061,0E+071,0E+081,0E+091,0E+101,0E+11
black p
epper
red pep
per
ginger
clove
scu
min
cinnam
oncu
rry
coria
ndergarl
ic
red pep
per + c
loves
cumin +
clove
s
grapes
cinnam
on + clo
ves
coria
nder + c
loves
black
pepper
+clove
s
curry
+ clove
s
ginger+ cl
oves
Nitrite
s
TVC
(ufc
/g)
5 week 6 week 7 week 8 week
93
2.6. Discussion
In view of the results obtained for the antimicrobial and antioxidant activity of some
spices formulations and their use in meat products (terrines) preservation, following
conclusions can be drawn:
1) The values of p anisidine in terrines preserved using cloves, cumin, cinnamon,
cinnamon + cloves were comparable and sometimes lower than nitrite conserved
terrines (150 ppm).
2) Spices formulations that gave lowest TBA index were cloves, cumin, cinnamon,
cumin + cloves, cinnamon + cloves.
3) Terrines preserved by cloves (>8 weeks), cumin (8 weeks), cloves + cumin (>8
weeks), cinnamon (7 weeks), cloves + cumin (8 weeks), curry (7 weeks), red pepper (>8
weeks), curry + cloves (8 weeks), red pepper + cloves (>8 weeks) had a shelf life equal
or higher than terrines preserved by nitrites.
4) The use of cloves, cumin cinnamon spices and their volatile compounds as natural
preservatives in food products may be an alternative to the use of chemical additives,
such as nitrites.
2.7. Acknowledgements
The authors sincerely thank the FQRNT (programme special) in collaboration with four
adro-industries, MDEIE and MAPAQ for their financial support. We express our
gratitude to Mr. Boiteau of Aliments Breton for supplying us the raw meat to carry
out various tests. The views and the opinions expressed in this article are those of the
authors.
94
2.8. References
Aboaba O, Efuwape BM. 2001. Antibacterial Properties of Some Nigerian Species. Biol Res Commun 13:183-188. Arora-Daljit S, Kaur J. 1999. Antimicrobial activity of spices. J Antimicrob Agents 12: 257-262. Chang ST, Chen PF and Chang SC. 2001. Antibacterial activity of leaf essential oil and their constituents from Cinnamomum osmophloeum. J Ethopharmacol 77:123-127. Davidson PM, Baren AL. 1993. Antimicrobials in Foods. Marcel Dekker, New York. pp. 1-9 Deis RD. 1999. Secret world of spices. Food Product Design 5:1-7. Fan AM, Willhite CC, and Book SA. 1987. Evaluation of the nitrate drinking water standard with reference to infant methemoglobinemia and potential reproductive toxicity. Regul Toxicol Pharmacol 7(2):135–148. Frankel EN, Meyer AS. 2000. The problems of using one-dimensional methods to evaluate multifunctional food and biological antioxidants. J. Sci. Food Agric 80:1925–1941. Ghori I, Ahmad S S. 2009. Antibacterial activities of honey, sandal oil and black pepper. Pak. J. Bot 41:461-466, 2009. Gotelli N J, Ellison AM. 2004. A Primer of Ecological Statistics Sinauer Associates. Sunderland, MA, 510 pp. Honikel KO. 2008. The use and control of nitrate and nitrite for the processing of meat products. Meat Sci 78:68-76. International Commission on Microbiological Specifications for foods Sampling plans for fish and shellfish.1986. In: ICMSF (Eds.), Microorganisms in Foods. Sampling for Microbiological Analysis, Principles and Scientific Applications.Vol. 2, (2nd ed.). University of Toronto Press, Toronto, Canada. Jackson AL. 2010. Investigating the microbiological safety of uncured no nitrate or nitrite added processed meat products. Graduate Theses and Dissertations. Iowa State University. JUPAC.1986. Standard Methods for the Anal vsis of Oils, Fats and Derivatives, 7th ed. Oxford, Blackwell Scientific Publications. Kim IS, Yang MR, Lee OK, Suk-Nam Kang SN. 2011. Antioxidant Activities of Hot Water Extracts from Various Spices. Int J Mol Sci 12:4120-4131.
95
Menon KV, Garg SR. 2001. Inhibitory effect of clove oil on Listeria monocytogenes in meat and cheese. Food Microbiol 18:647-650. Muchuweti M, Kativu E, Mupure CH, Chidewe C, Ndhlala AR and Benhura MAN.2007). Phenolic composition and antioxidant properties of some spices. Am J Food Technol 2:414-420. National Academy of Sciences, Assembly of Life Sciences.1986. Alternatives to the current use of nitrite in foods. National Academy Press, Washington, D. C., p 1-3 through 1-9. Pegg RB, Shahidi F. 2000. Nitrite curing of meat. The n-nitrosamine problem and nitrite alternatives. Trumbull, CT: Food and Nutrition Press, Inc. Rahman MSA, Thangaraj S, Salique SM, Khan KF, and Natheer SE. 2010. Antimicrobial and Biochemical Analysis of Some Spices Extract against Food Spoilage Pathogens. Int J Food Nutr Saf 12:71-75. Russel RJ, Gould GW. 1991. Food Preservatives. Van Nostrand Reinhold Co., New York. Schmedes A, Holmer GA. 1989. New thiobarbituric acid (TBA) method for determination of free malonaldehyde (MDA) and hydroperoxides selectivity as a measure of lipid peroxidation. J Am Oil Chem Soc 66:813–817. Stoilova I, Krastanov A, Stoyanova A, Denev P, and Gargova S. 2007. Antioxidant activity of a ginger extract (Zingiber officinale). Food Chem:102: 764-770. SPSS Base 10.0 Application Guide SPSS, Chicago, IL (1999) Thomson B. 2004. Nitrates and nitrites dietary exposure and risk assessment. Available from: http://www.nzfsa.govt.nz/consumers/food-safety-topics/chemicals-infood/ residues-in-food/consumer-research/nitrite-nitrate-report.pdf. Accessed 2012 USEPA. 2007. National Water Quality Inventory: Report to Congress; 2002 Reporting Cycle. Document No. EPA-841-R-07-001. Washington, D.C: U.S. Environmental Protection Agency U.S. Environmental Protection Agency Ground Water and Drinking Water. 2006. Consumer Factsheet on Nitrates/Nitrites. Available from : http://www.epa.gov/safewater/dwh/c-ioc/nitrates.html. Accessed 2012
97
Chapitre 3 Optimization of spices as alternative of nitrites and nitrates in the meat-based products
Fatma Gassara1, Anne Patricia Kouassi2, Satinder Kaur Brar1*, Khaled
Belkacemi2
1INRS-ETE, Université du Québec, 490, Rue de la Couronne, Québec, Canada G1K
9A9
2Département des sols et de génie agroalimentaire, Pavillon Paul-Comtois, Université
Laval, 2325, rue de l'Université, Québec (Québec) G1V 0A6
(*Corresponding author, Phone: 1 418 654 3116; Fax: 1 418 654 2600; E-mail:
98
3.1. Résumé
L'utilisation des clous de girofle, du cumin et de cannelle contre l'oxydation des lipides
et la contamination microbienne a été étudiée par la méthode de réponse de surface sur
les échantillons de jambon et terrines en considérant l'effet des concentrations de ces
trois épices sur les propriétés physico-chimiques (indice de TBA et de la valeur de p-
anisidine) et les propriétés microbiologiques (TVC). Parmi elles, la concentration de
clous de girofle et de cumin ont eu un effet positif significatif sur les propriétés
microbiennes et les propriétés physico-chimiques pour les échantillons de viande (p
<0,05). Cependant, la concentration de la cannelle n'a pas eu d'effet significatif sur la
TVC, la TBA et les valeurs de p-anisidine dans les produits à base de viande (p> 0,05).
Les clous de girofle et le cumin ont joué un rôle important dans la prévention de la
croissance microbienne et l'oxydation des lipides et peuvent servir d'alternatives
intéressantes aux nitrites-nitrates dans les viandes transformées.
Mots-clés: clous de girofle, cumin, cannelle, réponse de surface, oxidation des lipides,
altération microbienne
99
3.2. Abstract
The use of three spices (cloves, cumin, and cinnamon) in the preservation of meat
products from lipid oxidation and microbial spoilage was investigated through response
surface method by using ham and terrine. The effect of concentration of cloves,
concentration of cumin and concentration of cinnamon on the physico-chemical
properties (TBA index and p-anisidine value) and microbiological properties (TVC) of
ham and terrine process were thoroughly investigated. Among them, the respective
concentration of cloves and cumin had significant positive effect on microbial (total
viable cells, TVC) and physico-chemical properties for terrine and ham (p < 0.05).
However, the concentration of cinnamon did not have a significant effect on TVC, TBA
and p-anisidine values in meat products (p>0.05). Thus, cloves and cumin played an
important role in the prevention of microbial growth and lipid oxidation and can serve
as good alternatives to nitrites-nitrates in processed meats.
Keywords: cloves, cumin, cinnamon, response surface, lipid oxidation, microbial
spoilage.
100
3.3. Introduction
Nitrites are present in environment, fertilizers, plants and soils. Nitrates and nitrites are
chemical substances also used in preservation of food. Industries use nitrates and nitrites
for the stabilization of the red color of meats (Honikel, 2008), inhibition of the
development of toxic microorganisms, by decreasing the oxidation of lipids and to
improve the flavor (Pegg & Shahidi, 2000). At higher level, nitrates and nitrites have
been associated with increased incidence of cancer in adults, combining with secondary
or tertiary amines to form N-nitroso derivatives, and possible increased incidence of
brain tumors. For all these reasons and also due to governmental restrictions, adoption
and research of a green alternative such as the addition of spice instead of nitrites, has
been carried out. It seemed important to investigate the properties of aromatic herbs and
spices, such as clove, ginger, pepper or garlic (Menon & Garg, 2001). It has been
mentioned that spices, such as cinnamon could be efficient in treatment of cancer (Ka et
al, 2003) because of its antioxidant properties (Lin et al, 2003; Okawa et al, 2001; Toda,
2003). Despite the usefulness and important physical-chemical properties of the spices,
no systematic study has been carried out so far on their use as nitrite alternatives, to the
best of our knowledge.
In this context, a qualitative analysis was carried out with terrines and ham, and three
spices were selected based on the physical-chemical and microbiological properties
imparted by them (Tajkarimi, 2010; El-Daly, 1998 ; Kamali et al., 1998; Delespaul et
al., 2000; Chang et al., 2001). In order to quantify them, the application of statistical
methodologies is helpful in defining the effects and interactions of the physiological
factors that play a role in biotechnological processes, such as in agro food production.
Response Surface Methodology (RSM) is a collection of statistical and mathematical
techniques useful for developing; improving and optimizing processes. There are
different types of RSM designs, such as 3-level factorial design, central composite
design (CCD) (Boza et. al, 2000) which was used in this study, Box-Behnken design
(Singh et. al, 1995, and D-optimal design (Sanchez-Lafuente et. al, 2002).
The aim of the present study is to evaluate the influence of the different spices which
have been chosen for the quantitative analysis using response-surface method. Response
surface methodology has been utilized to optimize the quantity of spices in terrines and
101
ham in order to find a better alternative to nitrites using the optimum quantity of
spices.To the best of our knowledge, there is no study reported on optimization, using
an experimental design, of the quantity of spices for the replacement of nitrites in meat
based product. The design used in this study is CCD which is a first-order (2N) design
augmented by additional centre and axial points to allow estimation of the tuning
parameters of a second-order model; the parameters used was p-anisidine value, TBA
index and TVC value.
3.4. Materials and methods
3.4.1. Preparation of meat extract
Samples of 200g of terrines were prepared using simple recipe of terrine of pork and
rabbit, by adding a percentage, as provided by STATISTICA 6 of STATSOFT Inc.
(Thulsa, U.S.), of various spices to be tested. Samples were cooked in a microwave for
6 min and then cooled in a refrigerator for 2 days. Each terrine was cut into several
pieces and placed under vacuum by using a vacuum food storage system.
Samples of pork were also prepared as ham with different concentrations of spices. The
respective concentrations are provided in Table 3.1.
102
Table 3.1 Results of experimental plan by central composite design for ham and terrine
3.4.2. Determination of thiobarbituric acid
The lipid oxidation was determined by 2-thiobarbituric acid according to the procedure
of Schmedes & Holmer (1989). Terrine sample (2g) was mixed with 5 ml of
trichloroacetic acid (TCA) solution (200 g / l of TCA in 135 ml / l of phosphoric acid
solution) and homogenized in a blender for 30 s. After filtration with a filter paper (0.45
µm), 2ml of the filtrate was mixed with 2ml of a solution of TBA (3g/l) in a test tube.
The tubes were incubated at room temperature in the dark for 20h. The absorbance was
measured at 532 nm using UV-Vis spectrophotometer (model UV-1200, Shimadzu,
Trial X1 X2 X3 log10 (TVC) p anisidine TBA (mg MDA/kg)
Cloves Cumin Cinnamon Terrine Ham Terrine Ham Terrine Ham
1 0.1 0.1 0.1 8.28 8.86 2.10 1.97 1.72 1.230
2 0.1 0.1 0.3 7.02 8.80 1.85 1.59 1.44 1.400
3 0.1 0.3 0.1 6.16 7.47 1.10 1.13 0.71 0.450
4 0.1 0.3 0.3 5.56 7.45 1.34 1.25 0.68 0.532
5 0.3 0.1 0.1 4.82 7.83 0.97 0.83 0.59 0.286
6 0.3 0.1 0.3 5.62 7.74 1.01 0.91 0.65 0.572
7 0.3 0.3 0.1 2.30 6.99 0.32 0.61 0.37 0.226
8 0.3 0.3 0.3 3.85 7.47 0.44 0.74 0.55 0.134
9 0.031 0.2 0.2 5.75 8.47 1.74 1.66 0.86 0.917
10 0.368 0.2 0.2 2 7.37 0.78 0.7 0.47 0.201
11 0.2 0.031 0.2 6.15 9.04 1.4 1.59 0.88 1.212
12 0.2 0.368 0.2 2 7.10 0.6 0.77 0.49 0.255
13 0.2 0.2 0.031 3.17 7.93 0.90 0.85 0.65 0.315
14 0.2 0.2 0.368 5.77 9.01 1.23 0.99 0.67 0.457
15 0.2 0.2 0.2 4.19 8.11 1.27 0.95 0.74 0.302
16 0.2 0.2 0.2 4.54 8.13 1.24 0.94 0.70 0.348
17 0.2 0.2 0.2 4.86 8.06 1.23 0.96 0.71 0.314
18 0.2 0.2 0.2 4.23 8.14 1.21 0.9 0.72 0.316
19 0.2 0.2 0.2 4.65 8.14 1.23 0.96 0.73 0.311
103
Kyoto, Japan). TBA value was expressed as mg malonaldehyde per kg of sample and
determined by the following equation 1:
10-2 (1)
Where, A532nm is the measured absorbance, VTCA denotes the extraction solvent volume
(5ml), M is the molar mass of malonaldehyde (72g/mol) and m is the mass of the
analyzed sample (2g).
3.4.3. Determination of p-anisidine value
The hydroperoxide decomposition was determined by p-anisidine value (p-Anv)
according to IUPAC standard method (1986). Terrine sample (0.5g) was mixed with
5ml of isooctane solution. The absorbance (Ab) of this sample was measured at 350 nm
using isooctane solution as a blank reagent. Later, 1ml of p-anisidine solution (0.25
(v/v) % in glacial acetic acid) was added to the sample tube and to 5ml of isooctane test
tube. After 10 minutes, the absorbance (As) of samples was read using isooctane as
blank. The p-anisidine values were calculated using the following equation 2:
(2)
Where, As is the absorbance of the fatty solution after reaction with
p-anisidine solution, Ab is the absorbance of the fatty solution and m is the sample mass
(g).
3.4.4. Microbiological analysis
Samples (1 g) from meat were weighed aseptically, added to 25 ml NaCl sterile
solution, 0.9 % v/w (25 ml), and homogenized for 60 s at room temperature. Decimal
dilutions in NaCl sterile solution (0.9 % v/w) were plated on plate count agar (PCA;
Quelab Laboratories Inc., Montreal, QC, CANADA) and incubated at 30 °C for 48 h for
enumerating total viable count (TVC). Enumeration of TVC was performed on these
duplicate samples and results are displayed as the mean of both measurements. Each
sample was analyzed in duplicate (coefficient of variation of samples from the same
experiment).
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3.4.5. Statistical analysis
Data means were analyzed with individual Student's t-tests to distinguish differences
among spice formulations. The test was performed at P-value < 0.05 to determine the
significance of the difference between spices formulation for meat products preservation
(SPSS, 1999). Standard error for a single proportion was estimated as described by
Gottelli & Ellison (2004).
3.4.6. Experimental design and optimization
In order to identify the significant factors that affect the responses, an attempt was
made to improve the quantity of spices comparing different levels of several factors that
were found to have more influence on the antioxidant and antimicrobial properties of
spices in meat based products. The impact of three independent quantitative variables,
including cloves (X1), cumin (X2) and cinnamon (X3), was evaluated by a factorial
central composite design (CCD) to find the optimal concentrations of these three
factors. In this regard, a set of 19 experiments including, seven center points or
repetitions, six axial points (α = 1.68) and 8 points corresponding to a matrix of 23
which incorporates 8 experiments including 3 variables (+1, -1, 0), were carried out.
Each variable was studied at three different levels (−1, 0, +1) and center point (0) which
is the midpoint of each factor range. The minimum and maximum range of variables
investigated and the full experimental plan with respect to their actual and coded values
are listed in Table 3.2. A multiple regression analysis of the data was carried out by
STATISTICA 6 of STATSOFT Inc. (Thulsa, U.S.) by surface response methodology
and the second-order polynomial equation that defines predicted responses (Yi) in terms
of the independent variables (X1, X2, and X3):
Yi = b0i + b1iX1 + b2iX2 + b3iX3 + b11iX2+ b22iX2 + b33iX2 +b12iX1X2 +
b23iX2X3 + b13iX1X3 (1)
Where, Yi = predicted response, boi is intercept term, b1i, b2i, b3i linear coefficients,
b11i, b22i, b33i squared coefficients and b12i, b23i, b13i are interaction coefficients
and i refer to the response. Combination of factors (such as X1X2) represents an
interaction between the individual factors in the respective term. There are 3 different
105
responses, TBA index p-anisidine value and TVC value. These responses are a function
of the level of factors. The response surface graphs indicate the effect of variables
individually and in combination and determine their optimum levels for quantity of each
spice.
Table 3.2 Experimental range of the three variables studied using CCD in terms of
actual and coded factors Variables Symbol Coded levels
−1.682 Low (−1) Mid (0) High (1) +1. 682
Cloves (% w/w) X1 0.031 0.1 0.2 0.3 0.368
Cumin (% w/w) X2 0.031 0.1 0.2 0.3 0.368
Cinnamon(%w/w) X3 0.031 0.1 0.2 0.3 0.368
3.5. Results and discussion
3.5.1. Effect of variables on physico-chemical properties of processed meat
The central composite design was used to find the suitable values of the variables on the
physico-chemicals properties of meat products: terrine and ham. The results of CCD
experiments consisted of experimental data for studying the effects of three independent
variables; concentration of cloves, concentration of cumin and concentration of
cinnamon on the physico-chemical properties (TBA index and p-anisidine value) and
microbiological properties (TVC) of ham and terrine are presented in Table 1. The
physico-chemical properties (TBA index and p-anisidine value) and microbiological
properties (TVC) exhibited different responses in the two meat products assayed,
namely terrine and ham. The data were fitted into a second-order polynomial function
(Eq. (1)). Linear, quadratic and interaction coefficients of variables under study that
were found to be significant at p < 0.05 were retained in reduced models. Data were
best fitted by a first-order polynomial equation as it can be inferred from the good
agreement of experimental data with those predicted by the model. The quality of the
model fit was evaluated by the coefficient R2 and its statistical significance was
determined by an F-test. R2 represents the proportion of variation in the response data
106
that can be explained by the fitted model. High R2 was considered as an evidence for the
applicability of the model in the range of variables included. It should be noted that a R2
value greater than 0.75 indicates the aptness of the model. The coefficients of
determination (R2) are presented in Table 3.3. In all models, R2 is higher than 0.84 and
indicated that the model fitted well into the experimental results. The analysis of
variance (Table 3) indicated that the linear model terms of X1(the concentration of
cloves) and X2 (the concentration of cinnamon) had significant positive effect on the
physico-chemicals (TBA index and p-anisidine value) in both terrine and ham. The
values of linear coefficient related to X1 and X2 were negatives, hence when X1 and X2
increased, p anisidine, TBA values decreased. TBA index is a physico-chemical
parameter that is able to measure the formation of malondialdehyde, a secondary
product that is formed due to the degradation of polyunsaturated fatty acids.
Polyunsaturated fatty acids, such as linoleic acid are easily oxidized by the oxygen in
the air. This auto-oxidation leads to the occurrence of chain reactions with the formation
of coupled double bonds, and at a later stage also to obtaining secondary products, such
as aldehydes, ketones, and alcohols. The p-anisidine value measures also a secondary
product (adehydes) formed by secondary oxidation of polyunsaturated acid. In order to
optimise the concentrations of three spices (cloves, cumin and cinnamon), giving the
optimal antioxidant on meat product, namely terrine and ham, the experiments for
inhibiting the peroxidation of the polyunsaturated fatty acids were performed.
The concentration of cloves had a significant positive effect on physicochemical
properties of meat products. The increase of clove concentration prevents lipid
oxidation. This is due to high levels of phenolic compounds, which have antioxidant,
anti-inflammatory, and anti-clotting properties (Parle & Gurditta, 2011). The level of
phenolic compounds was a major factor in labeling cloves as the best natural
antioxidant. Clove and eugenol possess strong antioxidant activity, which is comparable
to the activities of the synthetic antioxidant, BHA (butylated hydroxyl anisole) and
Pyrogallol (Dorman et al., 2000). Clove has the highest capacity to release hydrogen
and reduce lipid peroxidation. With respect to the lipid peroxidation, the inhibitory
activity of clove oil determined using a linolenic acid emulsion system indicated a
higher antioxidant activity than the standard BHT (Butylated hydroxyl toluene). It also
showed a significant inhibitory effect against hydroxyl radicals and acts as an iron
chelator (Gulcin et al, 2004). The metal chelating activity, bleomycin dependent DNA
oxidation, diphenyl -p- picryl hydrazyl (DPPH) radical scavenging activity and the
107
ferric reducing antioxidant power (FRAP) of different spices were measured in rat
liver homogenate. Cloves showed the highest DPPH radical scavenging activity and
highest FRAP values (Yadav & Bhatnagar, 2007). The antioxidant activity of clove and
its major aroma components, eugenol and eugenol acetate were comparable to that of
the natural antioxidant α-tocopherol (Lee & Shibamoto, 2001). Moreover, cumin has a
good antioxidant potential as it contains appreciable amounts of antioxidant compounds
and its non-volatile extracts also have good inhibition properties against the free
radicals. There is also a good correlation between the total phenolic content in cumin
and its antioxidant activities and this spice can be used to produce novel natural
antioxidants as well as flavoring agents that can be used in various food products
(Annie et al., 2006). For this reason, the concentration of cumin had significant positive
effect on antioxidant activities in meat products, such as terrine and ham (p-value <
0.05).
However, the results shown in Table 3.3 indicated that the linear model terms of
X3(the concentration of cinnamon) did not have a significant effect on - p-anisidine
value in both terrine and ham and on TBA index in terrine but have significant negative
effect on TBA index in ham. These results showed that antioxidant capacities of cumin
and cloves were higher than those of cinnamon. These findings are not well correlated
with the results of Ho et al. (2008) who showed that antioxidant capacity ranked the
spices (in decreasing order): cloves > rosemary, cinnamon, turmeric > nutmeg > cumin
> paprika and cardamom.
The interactive effect of the spices on antioxidant activities has not been
described earlier in literature. The results shown in this study indicated the absence of
interactive effects of variables on p-anisidine in terrine.
108
Table 3.3 Model coefficients estimated by central composite design and best selected
prediction models
Coefficients log10 (TVC) p anisidine TBA (mg MDA/kg) Terrine Ham Terrine Ham Terrine Ham
Constant 4.44 8.13 1.23 0.94 0.72 0.31
Linear
Cloves (X1) -2.45 -0.73 -0.77 -0.65 -0.45 -0.52
Cumin (X2) -2.17 -0.95 -0.58 -0.43 -0.40 -0.55
Cinnamon (X3) 0.71 0.39 0.10
0.03
-0.003
0.10
Interactions
Cloves x Cumin -0.18 0.56 0.07 0.197 0.36 0.28 Cloves x
Cinnamon 1.054 0.27 0.05 0.117 0.14 -0.01
Cumin X Cinnamon 0.35 -0.047 0.14 0.137 0.09 -0.12
Quadratic
Cloves 0.13
-0.29
0.026
0.18 0.034
0.18
Cumin 0.27 -0.22 -0.11
0.18
0.04 0.31
Cinnamon 0.54
0.06 -0.11 -0.004 0.031153
0.060
R2 0.84 0.88 0.95 0.98 0.85 0.98 Bold values: Significant (p < 0.05).
Reduced equations for physico-chemicals (TBA index and p-anisidine value) and
microbiological properties (TVC) of ham and terrine : best selected models: Log10
TVC (terrine) = 4.44 - 2.45 X1 - 2.17 X2; Log10 TVC (ham) = 8.13 - 0.73 X1 - 0.95 X2
+ 0.39 X3 + 0.56 X1 x X2 ; p anisidine (terrine) = 1.23 - 0.77 X1 - 0.58 X2; p anisidine
(ham) = 0.94 - 0.65 X1 - 0.43 X2 + 0.18 X12 + 0.18 X2
2 + 0.197 X1 x X2 + 0.137 X2 x X3;
TBA (terrine) = 0.72 - 0.45 X1 - 0.40 X2 + 0.36 X1 x X2; TBA (ham) = 0.31 - 0.52 X1 -
0.55 X2 + 0. 10 X3 + 0.18 X12 + 0.31 X2
2 + 0.28 X1 x X2 - 0.12X2 x X3;
3.5.2. Effects of variables on biological properties of processed meat
The analysis of variance (Table 3.3) indicated that the linear model terms of
X1(concentration of cloves) and X2 (concentration of cumin) had significant positive
effect on microbial properties (TVC value) in terrine and ham. The values of linear
109
coefficient for X1 and X2 were negative, hence when X1 and X2 increased, p anisidine,
TVC values decreased. The enumeration of total viable count, TVC is an important
indicator to determinate the shelf-life of meat products. A meat product is non-
consumable, if the TVC is greater than 107 UFC/g (ICMSF, 1986).
The concentrations of cloves and cumin had a significant positive effect on microbial
properties of meat products (terrine and ham). The increase of clove concentration
prevents the microbial spoilage of meat products. This is due to high antimicrobial
activity of cloves and cumin (Parle et al., 2011). The antimicrobial potency of plants
and cumin and cloves is believed to be due to tannins, saponins, phenolic compounds,
essential oils and flavonoids (Aboaba and Efuwape, 2001). Moreover, naturally
occurring compounds in spices, such as sulphur compounds, terpenes and terpene
derivatives, phenols, esters, aldehydes, alcohols and glycosides have shown
antimicrobial functions (Russel and Gold, 1991; Davidson and Baren, 1993; Deis,
1999). However, the inhibitory effects of cumin and cloves are mostly due to the
volatile oils present in their composition (Arora-Daljit and Kaur, 1999).
The results shown in Table 3.3 indicated that the linear model terms of X3 (the
concentration of cinnamon) did not have a significant effect on TVC values in terrine,
but have significant negative effect on TVC values in ham. These results showed that
antimicrobial capacities of cumin and cloves were higher than those of cinnamon. These
findings are not well correlated with the results of Rahman et al. (2008) who showed
that cloves, cumin and cinnamon have good antimicrobial activities.
3.5.3. Determination of optimal conditions and optimal responses
By using the method of experimental factorial design and response surface analysis, the
optimal spice concentrations to obtain the best microbial and physico-chemical
properties of meat products, namely terrine and ham were determined. The validity of
the model was proved by fitting different values of the variables into the model equation
and by carrying out experiments at these values of the variables. The results of
experimental plan by central composite design for ham and terrine are presented in
details in Table 3.1. The optimal concentrations of spices giving the lowest, p-anisidine,
TBA and TVC values are determined using 3D plots presented in Figures 3.1, 3.2 and
3.3. The optimal concentrations of spices giving the lowest p-anisidine index in both
110
ham and terrine were 0.3 , 0.3 and 0.1 % w/w for cloves, cumin and cinnamon,
respectively (Table 3.2). This spice formulation rendered lowest p-anisidine values
(0.32 in terrine and 0.61 in ham) and TBA index in terrine (0.37 mg MDA/kg).
However, the optimal concentration of spices giving the lowest TBA index in ham
(0.134 mg MDA/kg) was 0.3 % w/w of cloves, cumin and cinnamon. The lowest p-
anisidine value and TBA index in terrine and ham were obtained using higher
concentration of cloves and cumin. This is due the high antioxidant potential of cumin
and cloves because it contains appreciable amounts of antioxidant compounds and
inhibition properties against the free radicals (Dorman et al., 2000; Annie et al., 2006).
However, the minimal values of TBA and p-anisidine were obtained using 0.1 % w/w
of cinnamon in terrine, but lowest TBA index in ham was obtained using 0.3 % w/w of
cinnamon. The concentration of cinnamon did not change the physicochemical
properties of meat products despite the higher anti-oxidant capacity of cinnamon
compared with cloves and cumin (Ho et al., 2008). This could be due the inefficiency of
polyphenols inside the cinnamon, that are not available in the meat products. In this
case, the use of oil or polyphenols extract could enhance the antioxydant activity of
cinnamon. The extraction of spices or the use of cinnamon oil could ameliorate the anti-
oxidant activity by the liberation of polyphenolics compounds from the spices.
111
(a)
(b)
Figure 3.1 Response surface of Log10 viability in terrine obtained by varying: a) the
concentration of cloves (X1) and the concentration of cumin (X2) keeping the
concentration of cinnamon (X3) constant ; b) concentration of cinnamon (X3) and the
concentration of cloves (X1) and keeping the concentration of cumin (X2) constant: 0.2
% w/w
112
(a)
(b)
Figure 3.2 Response surface of p-anisidine in terrine obtained by varying: a) the
concentration of cloves (X1) and the concentration of cumin (X2) keeping the
concentration of cinnamon (X3) constant ; b) concentration of cinnamon (X3) and the
concentration of cumin (X2) and keeping the concentration of cloves (X1) constant: 0.2
% w/w
113
(a)
(b)
Figure 3.3 Response surface of TBA in terrine obtained by varying: a) the
concentration of cloves (X1) and the concentration of cumin (X2) keeping the
concentration of cinnamon (X3) constant ; b) concentration of cinnamon (X3) and the
concentration of cloves (X1) and keeping the concentration of cumin (X2) constant: 0.2
% w/w
The best concentration of spices giving the lowest microbial growth in terrine
(log TVC= 2) was 0.368, 0.2 and 0.2 % w/w of cloves, cumin and cinnamon,
respectively. However, the optimal concentration of spices giving the lowest microbial
114
growth in ham (log TVC= 6.99) was 0.3, 0.3 and 0.1 % w/w of cloves, cumin and
cinnamon, respectively. The lowest TVC values have been obtained using high
concentration of cloves. Hence, cloves have the most important antimicrobial activity
when compared with cinnamon and cumin. Cloves represent one of the Mother Nature’s
antiseptic (Parle and Gurditta., 2011). Clove oil was found to be more effective than
sodium propionate (standard food preservative) against some food borne microbes.
Clove oil was found to be very effective against Staphylococcus species (Parle and
Gurditta, 2011). Clove oil showed antimicrobial activity against some human
pathogenic bacteria resistant to certain antibiotics (Lopez et al., 2005). The
antimicrobial potency of plants and spices is believed to be due to tannins, saponins,
phenolic compounds, essential oils and flavonoids (Aboaba and Efuwape, 2001).
Moreover, naturally occurring compounds in cloves, such as sulphur compounds,
terpenes and terpene derivatives, phenols, esters, aldehydes, alcohols and glycosides
have shown antimicrobial functions (Russel and Gold, 1991; Davidson and Baren,
1993; Deis, 1999). Furthermore, the inhibitory effects of spices namely cloves are
mostly due to the volatile oils present in their composition (Arora-Dlijit and Kaur,
1999). Moreover, the 3 D plot on response surface of Log10 viability presented in
Figure 1 a and Figure 3.4 a showed that when cloves and cumin concentration
increased, log 10 TVC decreased. Hence, both cumin and cloves concentration have
high antimicrobial activities. These findings are well correlated with the results of
Rahman et al (2010) who found that cloves and cumin had important antimicrobial
activity against Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas
aeruginosa, Enterococcus faecalis, Micrococcus luteus, Escherichia coli and Candida
albicans. However, the 3D plot presented in Figure 3.1a and 3.1b showed that lowest
values of TVC were obtained when the concentration of cinnamon decreased. These
results are not correlated with the results of Rahman et al (2010) who found that
cinnamon showed that cinnamon had a very good antimicrobial activity. This could be
due to the non-liberation of the components responsible for anti-microbial activity from
cinnamon. The extraction of these components or the use of cinnamon oil could increase
the antimicrobial activity of cinnamon.
115
3.6. Conclusions
Use of response surface methodology for optimization of spices concentrations giving
the best anti-oxidant and microbial activities led to following conclusions:
1) The optimal concentrations of spices giving the lowest p-anisidine index in both ham
and terrine and TBA index in ham were 0.3, 0.3 and 0.1 % w/w for cloves, cumin and
cinnamon, respectively.
2) The optimal concentration of spices giving the lowest TBA index in ham (0.37 mg
MDA/kg) was 0.3 % w/w for cloves, cumin and cinnamon.
3) The best concentration of spices giving the lowest microbial growth in terrine (log
TVC= 2) was 0.368 , 0.2 and 0.2 % w/w of cloves, cumin and cinnamon, respectively
and the optimal concentration of spices giving the lowest microbial growth in ham (log
TVC= 6.99) was 0.3, 0.3 and 0.1 % w/w of cloves, cumin and cinnamon, respectively.
4) The concentration of cloves and cumin had significant positive effect on microbial
(TVC value) and physico-chemical properties in both terrine and ham (p < 0.05).
5) The concentration of cinnamon did not have a significant effect on TVC, TBA and p-
anisidine values in meat products (p < 0.05).
3.7. Acknowledgement
The authors are sincerely thankful to the Natural Sciences and Engineering Research
Council of Canada (Discovery Grant), FQRNT (partenariat) and MAPAQ (No. 809051)
for financial support. The views or opinions expressed in this article are those of the
authors. We express our gratitude to Mr. Boiteau of Aliments Breton and La Maison du
Gibier for supplying us the raw meat to carry out various tests. The views or opinions
expressed in this article are those of the authors.
116
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Boza, A., De la Cruz, Y., Jordan, G., Jauregui-Haza, U., Aleman, A., & Caraballo, I. (2000) Statistical optimization of a sustained-release matrix tablet of lobenzarit disodium. Drug Development and Industrial Pharmacy, 26, 1303-1307.
Chang, S. T.; Chen, P. F., and Chang, S. C. (2001). Antibacterial activity of leaf essential oils and their constituents from Cinnamomum osmophloeum. Journal of Ethnopharmacology, 77, 123–127.
Davidson, P. M., & Baren, A. L. (1993). Antimicrobials in Foods. Marcel Dekker, New York. pp. 1-9 Deis, R. D. (1999). Secret world of spices. Food Product Design, 5, 1-7. Dorman, H. J. D., Surai, D., & Deans, S. G. (2000). In vitro antioxidant activity of a number of plant essential oils and Phytoconstituents. Journal of Essential Oil Research, 12, 241–248. Gottelli, N. J., & Ellison, A. M. (2004). A Primer of Ecological Statistics Sinauer Associates. Sunderland, MA, 510 pp. Gulcin, I., Sat, I. G., Bey demir, S., Elmastas, M., & Kufrevioglu, O. I. (2004). Comparison of antioxidant activityof clove (Eugenia caryophyllata Thunb) buds and lavender (Lavandula stoechas L.). Food Chemistry, 87, 393-400. International Commission on Microbiological Specifications for foods Sampling plans for fish and shellfish (1986). In ICMSF (Eds.), Microorganisms in Foods. Sampling for Microbiological Analysis, Principles and Scientific Applications,Vol. 2, (2nd ed.). University of Toronto Press, Toronto, Canada. pp 139-147. Ka, H., Park, H-J., Jung, H-J., Choi, J-W., Cho, K-S., Ha, J., & Lee, K-T. (2003). Cinnamaldehyde induces apoptosis by ROS-mediated mitochondrial permeability transition in human promyelocytic leukemia HL-60 cells. Cancer Letters, 196, 143-152. Lee, K. G., & Shibamoto, T. (2001). Antioxidant property of aroma extract isolated from clove buds [ Syzygium aromaticum (L.) Merr. et Perry ]. Food Chemistry, 74, 443 – 448. Lopez, P., Sanchez, C., Batle, B., & Nerin, C. (2005). Solid and vapour phase antimicrobial activities of six essential oils: susceptibility of selected food borne bacterial and fungal strains. Journal of Agriculture and Food Chemistry,53, 6338 – 6346.
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Honikel, K. O. (2008). The use and control of nitrate and nitrite for the processing of meat products. Meat Science, 78, 68-76.
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Okawa, M., Kinjo, J., Nohara, T., & Ono, M. (2001). DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging activity of flavonoids obtained from some medicinal plants. Biological & Pharmaceutical Bulletin, 24, 1202-1205. Parle, M., & Gurditta (2011). Basketful benefits of papaya. International Research Journal of Pharmacy, 2, 6-12.
Pegg, R. B., & Shahidi, F. (2000). Nitrite curing of meat. The n-nitrosamine problem and nitrite alternatives. Trumbull, CT: Food and Nutrition Press, Inc.
Rag havenra, H., Diwakr, B. T., Lokesh, B. R., & Naidu, K. A. (2006). Eugenol, the active principle from cloves inhibits 5 - lipoxygenase activity and leukotriene - C4 in human PMNL cells. Prostaglandins, Leukotrienes and Essential Fatty Acids, 74, 23 –27.
Rahman, M. S. A., Thangaraj, S., Salique, S. M., Khan. K. F., & Natheer. S. E. (2010). Antimicrobial and Biochemical Analysis of Some Spices Extract against Food Spoilage Pathogens. Internet Journal of Food Safety, 12, 71-75. Russel, R.J., & Gould, G. W. (1991). Food Preservatives. Van Nostrand Reinhold Co., New York.
Sanchez-Lafuente, C., Furlanetto, S., & Fernandez-Arevalo, M. (2002). Didanosine extended-release matrix tablets: optimization of formulation variables using statistical experimental design. International Journal of Pharmaceutics, 237, 107-118.
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Chapitre 4 Color Retention in Processed Meats by Using Natural Products and Tests of Organoleptic Properties
Anne Patricia Kouassi1, Fatma Gassara2, Nasima Chorfa1, Satinder Kaur Brar2*,
Khaled Belkacemi1
1Département des sols et de génie agroalimentaire, Pavillon Paul-Comtois, Université
Laval, 2325, rue de l'Université, Québec (Québec) G1V 0A6
2INRS-ETE, Université du Québec, 490, Rue de la Couronne, Québec, Canada G1K
9A9
(*Corresponding author, Phone: 1 418 654 3116; Fax: 1 418 654 2600; E-mail: [email protected])
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4.1. Résumé
Dans la présente étude, la couleur et l'analyse sensorielle des terrines de porc et de lapin
et des échantillons de jambon ont été étudiées. Afin d'obtenir une coloration semblable
aux terrines et aux jambons industriels, deux colorants naturels nommés poudre de
raisin (2 gp) et poudre de fraise (3 st), ont donné de bons résultats proches de ceux des
nitrites pour la da (rougeur), le db (jaunissement) et le dl (luminosité). Pour voir
l'ìmpact de ces ajouts sur les propriétés organoleptiques, une analyse sensorielle a été
menée et a montré qu'il n'y avait pas de différence significative pour l'attribut épicé
entre les terrines contenant les nitrites et terrines avec la formulation 7 (0.3% clous de
girofle, 0.3% cumin, 0.1% cannelle p/p) à p <0,05, ainsi que pour les jambons. Le
tableau de moyennes ajustées et l'analyse du composant principal ont montré le
caractère sucré de la poudre de fraise.
Mot clés: Couleur, test organoleptic, épices, analyse en composantes principales,
nitrites, terrine, jambon
121
4.2. Abstract
In the present study, color and sensory analysis of pork and rabbit terrines and ham
were investigated. In order to achieve visual color appearance to the industrial product
without significantly impairing the taste of the samples, two natural coloring agents
named grape powder (2 gp) and strawberry powder (3 st) were found and gave good
results for the da (redness), db (yellowing) and dl (brightness), very closed to the
control, which is the nitrite. For sensorial analysis, nine attributes were analyzed and
according to the Tukey t-test, there was no significant difference for the spicy attribute
between the terrines containing nitrites and terrines with formulations 7 (0.3% cloves,
0.3% cumin, 0.1% cinnamon w/w) at p <0.05, neither for hams. The table of ajusted
means and analysis of Principal Component Analysis showed that the terrine with
formulation 12 has a similar profile to the one with nitrites.
Keywords: Color, organoleptic test, spices, principal component analysis , nitrites,
terrine, ham
122
4.3. Introduction
Nitrates and nitrites are used in meat-based products not only because of their
antioxidant and antimicrobial properties but also for their action on color and meat taste
(Honikel, 2008). Many alternatives, such as spices, were found to exhibit the
antioxidant and antimicrobial properties in exchange of nitrites which have toxic effects
on human health (Fan et al., 1987; USEPA, 2006). Cinnamaldehyde, for example, the
major constituent of cinnamon (Cinnamomum cassia) has been reported to possess
antibacterial activity and antioxidant properties (Chang et al., 2001); Pepper is also
associated with a number of functional properties, such as analgesic and antipyretic
properties, antioxidant effects and antimicrobial properties (Kapoor et al., 1993); clove
oil showed its antimicrobial activity in a study based on the inhibitory effect of clove oil
on Listeria monocytogenes in meat and cheese (Vrinda et al., 2001). However,
coloration of spiced meat is still a big challenge. Color has a major role in the
acceptability of the product and is related to the consumer perception of flavor,
sweetness, scents and other physical properties in relation to the quality of the product
(Roth et al., 1988; Calvo et al., 2001; MacDougall, 2002 ). Meat color is important for
industries and consumers. Thus, it necessitates a study to find a natural way to maintain
the red color of meat. In fact, studies have already been carried out to stabilize a bright
red color of beef during frozen display, on beef fed Vitamin E, beef exposed to pure
oxygen to fully bloom the meat, as well as on exposure to carbon monoxide (Huffman
et al., 1975; Liu et al., 1995; Jeong and Claus, 2011). However, none of these
approaches improved the color and shelf-life to the point of producing a commercial
viable extension (Claus and Du, 2013).
The pigment responsible for the characteristic pink color of cured meat is a ferrous
complex of myoglobin containing nitric oxide (NO), namely, nitrosylmyoglobin or NO-
Mb. The complex is formed by the reaction of myoglobin with nitrite generated NO
(Morita et al., 1998). Nitrosohemachrome is a denatured, stable form of NO-Mb in
cooked and cured meats (Martin, 2001). The chemical reactions leading to the cured
meat pigment are a complex series of processes involving microbial, enzymatic and/or
chemical catalyzed steps, which depend on pH, pigment concentration, redox potential,
curing agent distribution, temperature and relative humidity (Chasco et al. 1996). Thus,
to find a solution for the red color, fruits and vegetables were used to color the meat.
123
To measure color of different materials, various color spaces have been reported. Two
frequently used color spaces are RGB (Red, Green and Blue) and CIE Lab. RGB color
space consists of a three-dimensional rectangular coordinate system with R, G and B
axes. A color image is represented in RGB format with these three components per
pixel in the range 0–255 and their intensities are electronically combined to produce a
digital color picture (Afshari-Jouybari and Farahnaky, 2011). In this study, the CIE
L*a*b* color space was used for the measures of color of terrines and hams. It is the
most popular numerical colour space systems in food industry, which is also referred as
the CIELAB system, originally defined by the CIE in 1976 (MacCaig, 2002; CIE, 1986.
It aspires to perceptual uniformity, and its L component closely matches human
perception of lightness. CIE L*a*b* (CIELAB) is a complete color space specified by
the International Commission on Illumination (French Commission internationale de
l'éclairage, CIE). It describes all the colors visible to the human eye and was created to
serve as a device-independent model to be used as a reference.
The organoleptic tests were carried out on a trained panel in order to know if the
formulations of spices were approved and taste well. Descriptive analysis by a panel of
trained judges (also called panelists) is probably the best way to objectively assess and
compare the sensory properties of food products. The sensory quality of meat is
influenced by several factors that act before and during eating and which are often
mutually interacting (Gasperi et al., 2005). In the case of meat, the literature provides
very few useful indications about standardized references (for texture, tenderness,
juiciness and flavour) (Gorraiz et al., 2000; Byrne et al., 2001).
To the best of our knowledge, there are not many systematic studies on use extracts of
fruits and vegetables to color spicy meats. This study aims at finding the best
combination of extracts of fruits and vegetables to improve color of meat samples
containing spices. Fruits and vegetables are natural color agents, healthy products.
Moreover, the benefits of fruits have been attributed to their high phenolic compound
content, which act as antioxidants (Zuo et al., 2002). Subsequently, a trained sensory
panel was subjected to various descriptors (e.g. spicy, bitter, acid, sweet, salty, rancidity
and juiciness, among others) to judge the suitability of the finished formulations for the
industry and hence its commercialization.
124
4.4. Materials and Methods
4.4.1. Preparation of meat extract for color
In a previous study by our research group, a qualitative analysis has been made with
terrines and ham. Three spices (clove, cumin and cinnamon) were selected for their
optimal anti-microbial and anti-oxidant properties in the effective concentrations of
0.368% , 0.2% and 0.2 % w/w respectively, according to the microbial growth) (Fatma
Gassara et al., 2014). Samples of 200 g of terrines were prepared using simple recipe of
terrine of pork and rabbit, by adding a percentage, given by the statistics, of the three
spices to be tested. These concentrations are given in Table 4.1.
Table 4.1 The 19 combinations of spices obtained by Statisticia with their pH
Formulations Cloves Cumin Cinnamon pH
1 0.1 0.1 0.1 6,5
2 0.1 0.1 0.3 6,21
3 0.1 0.3 0.1 5,86
4 0.1 0.3 0.3 6,06
5 0.3 0.1 0.1 6,42
6 0.3 0.1 0.3 6,32
7 0.3 0.3 0.1 6,35
8 0.3 0.3 0.3 6,37
9 0.031 0.2 0.2 6,2
10 0.368 0.2 0.2 6,02
11 0.2 0.031 0.2 6,35
12 0.2 0.368 0.2 6,12
13 0.2 0.2 0.031 6,22
14 0.2 0.2 0.368 6,26
15 0.2 0.2 0.2 6,19
16 0.2 0.2 0.2 6,02
17 0.2 0.2 0.2 6,26
18 0.2 0.2 0.2 6,26
19 0.2 0.2 0.2 6,14
ctrl + Cloves Cumin Cinnamon 6,14
With ctrl + : terrine + nitrites
125
For color experiments, samples of 50-200 g of terrines were prepared using simple
recipe of terrine of pork and rabbit, with different percentage of red coloring agent
(Table 4.5), fruit powder, flower powders and vegetables, with nitrite as blank. The fruit
powder was mixed with 5 mL of distilled water and added to samples. Samples are
cooked in a microwave for 6 min and then cooled in a refrigerator for 2 days.
4.4.2. Color evaluation
Colorimetry is the science of measuring color. This allows separating the different color
settings: chromatic tone, chroma and lightness. Several systems for expressing color
numerically were developed by an international organization concerned with issues of
lighting and color, the International Commission on Illumination (CIE). One of the best
known of these systems is the L*a*b color space (also referred to as CIELAB) devised
in 1976 (CIE, 2007). The CIE recommends a particular illuminant/observer
combination and color spaces (CIEXYZ, CIE L*a*b) to standardize the definition of
color and provide more uniform color differences in relation to visual differences
(Korifi et al., 2013). In this system, each color can be located with its rectangular
coordinates: L* for axis of brightness, a* represent the red/green axis and b* represent
the yellow/blue axis.
4.4.3. Measurement of different parameters
4.4.3.1. Color
Colorimetric analysis on freshly cooked terrines and hams were performed using a
colorimeter , Minolta Chroma Meter Mesure CR-310 ). It is a compact tristimulus color
analyzer for measuring reflective color of surfaces. The measuring head of the chroma
meter uses wide-area illumination, a 0 degree viewing angle and a has a 50 mm-
diameter measuring area to average the reading over a wide area for measuring textured
surfaces.
4.4.3.2. Effect of color on pH
The pH value determines the quality of the meat, especially the color, tenderness,
flavor, water retention and conservation. The pH of cooked and uncooked hams was
126
determined by blending 1 g of sample with 10 ml distilled water. The pH values were
measured using an electrode attached to a digital pH meter Metler Toledo. To know the
influence of pH on terrines and hams, tests on samples without spice were made, over a
range of pH 3.5 to 11.5. The pH of all 20 terrines was taken in the first two weeks
(Table 4.1).
4.4.4. Organoleptic tests
4.4.4.1. Training
Nine panelists (6 males and 3 females) were selected from spontaneous candidature
mostly from Université Laval and INRS (Institut National de Recherche Scientifique),
for the detection, recognition and assessment of tastes, odors, flavors, and physical
characteristics, as well as for the use of classification scales. In order to train candidates
to recognize and memorize the attributes, the training was done by providing five basic
tastes, two odors and two large textures in the samples of meat. The nine attributes are
listed in Table 4.2. All of these attributes were extensively examined during group
discussions in order to reach a consensus about their meaning and to establish an
evaluation procedure and chose suitable reference standards (STONE et al. 1974). Table 4.2 List of attributes for the organoleptic tests
Attributes Adding Quantity % (w/w)
Sweety
Sugar
1
Salty Salt 0,2
Bitter Caffeine 0,05
Acid citric acid 0,04
Spicy Clove 1
Rancidity linseed oil heated
Aromaticity liquid vanilla
Tenderness Easiness with which the meat is divided into
fine particles during mastication
Juiciness Amount of wetness/juiciness released from
sample
127
4.4.4.2. Sensory evaluation
Four sessions were organized over a period of month. During each session, the panel
evaluated the 6 terrines of rabbit and pork and 5 samples of pork with our spices. All
samples were prepared with the same method to eliminate potential effects of the
preparation. Samples submitted for the tests are listed in table 4.3.
Table 4. 3 List of samples for organoleptic tests
*Powder 3st (strawberry) for coloration due to quantificationof powders (Figure 4.3)
4.4.5. Statistical analysis
The intensity of each attribute was rated in scale from 1 (not detected) to 4 (strongest).
At the end of the organoleptic test, the scores were analyzed for mean scores and
variance ratios. The least significant difference (LSD) of the Tukey test was calculated
for product comparison of each attributes. Principal Component Analysis (PCA)
highlighted similarities and differences among spices formulation for meat products
preservation. Data are presented as means±standard errors of the mean. P≤0.005 was
considered statistically significant. All calculations were performed using Sigmaplot
and SensomineR statistical package.
Nitrite
(ppm)
clove
(% w/w)
cumin
(%w/w)
cinnamon
(% w/w)
Powder 3
st (%w/w)
Sample 1 White terrine 0 0 0 0 0
Sample 2 terrine with nitrite 150 0 0 0 0
Sample 3 terrine +
formulation 7
0 0,3 0,3 0,1 0
Sample 4 terrine formulation 7 +
powder 3st*
0 0,3 0,3 0,1 0,9
Sample 5 terrine formulation 10 0 0,368 0,2 0,2 0
Sample 6 terrine formulation 12 0 0,2 0,368 0,2 0
Sample 7 ham with nitrite 150 0 0 0 0
Sample 8 ham form 7 0 0,3 0,3 0,1 0
Sample 9 Ham fomr 7 + powder 3
st*
0 0,3 0,3 0,1 0,9
Sample 10 Ham formulation 10 0 0,368 0,2 0,2 0
Sample 11 Ham formulation 12 0 0,2 0,368 0,2 0
128
In order to have a visualization of the most discriminating attributes, the box plot were
made by SigmaPlot. A comparison of several populations quickly is possible with box
plots which also allow to visually appreciate the asymmetry and the presence of atypical
individuals parameters. Each population is represented by a box whose lower bound is
the first quartile and the upper bound on the third quartile. These diagrams also allow a
glance to appreciate the essential elements of the distribution of each group and thus a
comparison.
4.5. Results and discussion
4.5.1. Effect of pH on color
Effect of pH on color was tested on ham without spice within a pH range of 3.50 to
11.5. These tests were carried out on cooked and uncooked samples as presented in
Figure 4.1 and Table 4.4. As seen in Figure 4.1, there was a change of color and texture
of meat. With the higher pH, change in meat color was more. Further away from neutral
pH to the two extremes, the texture becomes crumbly (acid pH) and elastic basic pH).
Table 4.4 Effet of pH on colour meat
Ph Sample Uncooked sample Cooked sample
de da dl db de da dl db
3,05 13,6 -5 11,2 -5,9 8,7 -3,7 4,9 -6,1
4,04 15 -2,9 14,4 -2,7 13,3 -4,9 9,7 -7,7
4,9 13 -2,3 11,8 -5 11,9 -3,6 11 -2,7
6,02 4,4 1,8 2,1 -3,4 14,4 -7,4 10,5 -6,7
7 4,7 4,1 -0,3 -2,3 15,4 -6,9 13,1 -4,2
7,97 4,4 0,5 -0,6 -4,3 9 -5,7 5,3 -3,7
9 6,6 -0,5 -2,9 -5,9 5,7 -4,7 -2,3 -2,2
10,18 3,2 2,4 1 -1,8 5 -4 -2,5 -1,6
11,5 9,7 -1,6 9,5 -0,1 4,1 -1,5 -3,7 1
Ctrl + 150
ppm
17,1 7,2 15,4 -1,2 16,6 1,8 14,3 -8,3
L* for axis of brightness, a* represent the red/green axis and b* represent the yellow/blue axis.
129
Uncooked Sample
Cooked Sample
Figure 4.1 Pictures of terrines uncooked and cooked at different pH
4.5.2. Solution for color
Out of the 19 formulations prepared, 3 formulations (formulations 7, 10 and 12
presented in Table 4.3) gave best physical and microbiological results in research of
alternatives of nitrites in meat-based products. These terrines samples were colored by
the spices. To find a solution to the coloring terrines, red coloring chemical food agent
was added at different concentrations (Table 4.5). The results showed a strong power of
red agent on terrines (without spices). It gave a "da" close to nitrites samples with one
drop per 100 g of sample. However, for greener alternatives, it was removed because it
contains chemical compounds.
130
Table 4.5 Color analysis of terrines containing different concentrations of red food
chemical coloring agent
Sample(g) Lab Color pictures
Nitrite 150 ppm dE 16,2
da 0,1
dl 13,1
db -7
4 drops (50g) dE 28,3
da 27
dl -3,4
db -7,8
1 drop (50g) dE 11,9
da 7,1
dl 3,8
db -8,7
1 drop (100g) dE 11,6
da 0,4
dl 10,5
db -4,8
Later, the coloring efficiency of red fruits powder such as strawberry, raspberry, and
vegetables such as beets, and flowers like hibiscus were tested (Table 4.6, Figure 4.2).
Beet exhibited best results in terms of meat color; however, it could not be used because
of its high nitrite content (3300 mg/kg) (IPCS).
131
Table 4.6 Color analysis of terrines containing different fruits and vegetables used to
improve red color
Sample Cooked sample
de Da dl Db
Beet 22,6 19,4 -2,5 -11,3
Hibiscus (15mL) 17,9 -3,6 15,2 -8,7
Hibiscus powder
(0.5g)
17,6 -4,4 9,7 -14
Strawberry puree 1% 21,8 -6,7 18,5 -9,5
2% raspberry
strawberry puree
(prepared in the lab)
21,4 -6,9 19,6 -5
2% raspberry
strawberry pulp
(prepared in the lab)
20,6 -6,3 19 -5,1
5% raspberry
strawberry pulp
22,9 -6,2 21,3 -5,7
10% raspberry
strawberry pulp
22,4 -5,5 20,6 -6,7
Nitrite 150ppm 16,2 0,1 13,1 -7
Figure 4.2 Pictures of coloured samples with differents percentages of fruits and
vegetables
Nitrite 150 ppm 5% raspberry strawberry pulp
10% raspberry strawberry pulp
Strawberry puree Hibiscus Hibiscus powder Beet
2% raspberry strawberry pulp
134
Table 4.7 Analysis of variance of results of terrines' flavor
Attributes
Sweetness Salty Bitterness Acidity spicy
Source of
Variation DF1 F2 P3 F P F P F P F P
Panelist 8 2.953 0.004 1.742 0.092 2.421 0.017 5.904 <0.001 5.543 <0.001
Sample 5 7.285** <0.001 3.246** 0.008 1.685 0.141 0.996 0.422 4.202** 0.001
panelist x
sample
40 0.745 0.862 0.901 0.641 0.520 0.991 0.571 0.980 0.484 0.996
Residual 162 F 5% 2.21
F 1% 3.02 Total 215
*indicates a significant difference at 5% **indicates a significant difference at 1% according to statistical tables 1Degree of freedom 2Variance ratio 3Probability
For odor and texture of terrines
The results of the ANOVA of odor and texture, presented in Table 4.8 , showed that
there was a significant difference between the samples, for the attributes aromaticity and
juiciness at 5%; and rancidity at 1%.
Table 4.8 Analysis of variance of results of terrines' odor and texture Attributes
Rancidity aromaticity tenderness juiciness
Source of
Variation DF F P F P F P F P
Panelist 8 4.129 <0.001 3.122 0.003 2.995 0.004 1.465 0.174
Sample 5 2.619* 0.026 19.02** <0.001 1.655 0.148 4.511** <0.001
panelist x
sample 40 0.681 0.923 0.848 0.724 0.240 1.000 0.461 0.997
Residual 162 F 5% 2.21
F 1% 3.02 Total 215
*indicates a significant difference at 5% **indicates a significant difference at 1%
135
4.5.3.2. Tuckey test and descriptive analysis for terrines
For flavor of terrines
The difference of sweetness, salty, acidity, bitterness and spicy in terrine containing
nitrite was not significantly different from other terrines made with different spices
according to the calculation of scores mean and LSD, presented in Table 4.9.
Table 4.9 Results of Mean scores and Least Significant Difference for flavor of terrine
Terrines Attributes1
Sweetness Salty Bitterness Acid Spicy
White (without nothing) 1.3330a 2.5830a 1.2500a 1.3610a 2.0830a
Terrine with nitrite 1.4440a 2.5830a 1.5000a 1.5830a 1.972a
Terrine with formulation 7 1.5000a 2.7780a 1.3330a 1.6390a 2.7690a
Terrine with formulation 7 +
powder 3 st
2.1670a 2.2130a 1.6110a 1.6940a 2.3330a
Terrine with formulation 10 1.6390a 2.4720a 1.3330a 1.5280a 2.4720a
Terrine with formulation 12 1.4440a 2.7410a 1.4440a 1.5000a 2.4440a
Standard error2 0.1110 0.1130 0.1020 0.1170 0.1410
LSD3 0.898 0.09027 1.120 0.818 0.943 1Mean of 9 observations 2Mean standard error of mean score based on 162 df 3Least Significant Difference
However, thanks to the descriptive analysis we can see which is the most discriminating
descriptors. The Figure 4.4 showed the descriptive analysis for flavor of our 6 terrines.
The terrine with the formulation 7 shows a barely higher salty and spicy taste than the
other terrines; the terrine with formulation 7 + powder 3 st seems to have a more
pronounced sweetness and bitterness than other terrines. There was no significant
difference of acidity on all terrines. Terrines formulation 10 and 12 showed no huge
difference in flavor compared to terrine with nitrites.
136
sweetness
salty
bitterness
acidity
spicy
0.0 0.5 1.0 1.5 2.0 2.5
blanc vs attributes terrine+nitrite vs attributes terrine 7 vs attributes terrine 7 + powder 3st vs attributes terrine 10 vs attributes terrine 12 vs attributes
Figure 4.4 Sensory profile analysis of flavor sweetness, salty, bitterness, acidity and spicy of terrines
To complete the first graph of descriptive analysis, the box plot of flavor was presented
in Figure 4.5. The 6 terrines selected were divided into 3 sub-groups (group 1: 3
<median (M) ≤ 4 in dark gray; group 2: 2 <M ≤ 3in less dark gray and hatched; Group
3: 1 <M ≤ 2 in light gray) for the descriptors tested . Indeed, the figure 4.5 shows
boxplots of the descriptor spicy is discriminant, in accordance with Figure 4.4.
137
Box plot for sweetness
score
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5
ter7+powder3st
nitrite
ter 7
ter 12
blanc
ter 10
Box plot for salty
score
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5
ter12
ter7+powder3st
ter7
blanc
nitrite
ter10
Box plot for bitterness
score
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5
ter7+powder3st
ter12
nitrite
ter7
ter10
blanc
Box plot for acidity
score
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5
nitrite
ter 7
ter7+powder3st
ter10
blanc
ter12
Box plot for spicy
score
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5
ter7+powder3st
ter7
ter10
ter12
nitrite
blanc
Figure 4.5 Box plots of the 6 terrines following descriptors: sweet, salty, acid, bitter
and spicy. A boxplot is lower than the M-1.5 (Q3-Q1) value, the first quartile (Q1),
median (M) solid line, dotted average, the third quartile (Q3) and the highest value less
than M +1.5 (Q3-Q1)
138
Terrines with formulation 7, 7 + powder 3 st, 10 and 12 were in group 2 (2 <M ≤ 3)
according to figure 4.5, and were therefore spicier than terrines containing nitrites,
which is quite normal. According to the figure 4.5, among the 4 terrrines containing
spices, the formulation 7 and sample 7 + powder 3st are spiciy.
For odor and Texture
For the odor and texture, the results presented in Table 4.10 showed that there was no
difference between the terrines for the attributes rancidity, tenderness and juiciness.
However, the aromaticity of terrines with formulation 7, 7 + powder 3 st, 10 and 12 is
significantly different from the one with nitrite.
Table 4.10 Results of Mean and Least Significant Difference for odor and texture of
terrines
Terrines Attributes1
rancidity aromaticity tenderness Juiciness
Blanc 1.2220a 2.2220ab 2.9170a 2.4440a
Terrine with Nitrite 1.2220a 2.0000b 3.1110a 2.9440a
Terrine with formulation 7 1.3890a 3.3330a 3.4170a 3.3060a
Terrine with formulation 7 + powder 3st 1.6940a 2.9440ab 3.2500a 2.5090a
Terrine with formulation 10 1.2500a 2.9440ab 3.0560a 2.9170a
Terrine with formulation 12 1.3610a 2.8330ab 3.1110a 3.1110a
Standard error2 0.1050 0.1150 0.1330 0.160
LSD3 0.842 0.925 1.074 1.269 1Mean of 9 observations 2Mean standard error of mean scare on 160 ddl 3Least Significant Difference
In the descriptive analysis for odor and texture of our 6 terrines (Figure 4.7), terrine
with formulation 7 was more aromatic, slightly tender and juicy than other terrines; the
one with formulation 7 + powder 3st seems less juicy and slightly rancid compared to
that of nitrites.
139
rancidity
aromaticity
tenderness
juiciness
0.0 0.5 1.0 1.5 2.0 2.5 3.0
terrine blanc vs attributesterrine+nitrite vs attributes terrine 7 vs attributes terrine 7 + powder 3st vs attributesterrine 10 vs attributes terrine 12 vs attributes
Figure 4.6 Sensory profile analysis of odor -rancidity, aromaticity- and texture -
tenderness, juiciness- of terrines
The box plots of odor and texture (Figure 4.8) confirmed the results of Figure 4.7 and
showed that the descriptors aromaticity, juiciness and tenderness are discriminating.
Terrine with formulation 7 has a more pronounced aromaticity than other terrines,
thereafter comes formulation 10. All terrines are relatively juicy, since they are from the
group 1 and 2.
140
Box plot for rancidity
score
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5
ter7+powder3st
ter7
nitrite
ter12
blanc
ter10
Box plot for aromaticity
score
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5
ter7
ter10
ter7+powder3st
ter12
blanc
nitrite
Box plot for juiciness
score
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5
ter7
ter10
ter12
nitrite
ter7+powder3st
blanc
Box plot for tenderness
score
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5
ter12
nitrite
ter7
ter7+powder3st
ter10
blanc
Figure 4. 7 Box plots of odor and texture of terrines
141
The table 4.11 shows the adjusted means of descriptors flavor, smell and texture of
terrines, using an analysis of variance model with descriptor. This test, performed by all
products and all descriptors was introduced in the table of adjusted means via a color-
coded: blue when the coefficient is significantly larger than mean, red when it is
significantly lowerand white when it is not significantly different from zero.
Table 4.11 Adjusted means of descriptors flavor, smell and texture of terrines
Attributes
Sample Salty sweety acidity Bitter spicy Ranci
d
aromatic tender Juicy
Blanc 2.583 1.333 1.361 1.25 2.083 1.222 2.222 2.917 2.444
Nitrite 2.583 1.444 1.583 1.5 1.972 1.333 2 3.111 2.944
Sample
7 2.778 1.5 1.639 1.333 2.778 1.389 3.333 3.417 3.306
Sample
7+ 3st 2.222 2.167 1.694 1.611 2.333 1.694 2.944 3.25 2.556
Sample
10 2.583 1.639 1.528 1.333 2.472 1.25 2.944 3.056 2.917
Sample
12 2.75 1.444 1.5 1.444 2.444 1.361 2.833 3.111 3.111
4.5.3.3. Principal Component Analysis (PCA) for terrines
For flavor of terrines
The SensoMineR software made a Principal Component Analysis (PCA) with the
adjusted means by terrines and descriptors. The PCA was applied as an unsupervised
pattern recognition method to observe trends in the data and to indicate relationships
between the 6 terrines and/or between the sensory attributes. Fig. 4.8 shows the
projection of the terrines samples on the plane defined by the first and second principal
components (Fig.4.8a) and also the corresponding loading plot (Fig.4.8 b). The first and
second principal components (Dim.) describe 88.2% of the variability (60.73% Dim 1
and 27.47% Dim 2, respectively. The first dimension, highly correlated with the
sweetness of the terrines and the second dimension with the spiciness of the terrines.
142
Terrine 7+ powder 3st is is characterized by a sweeter taste and terrine with formulation
7, by a spicier taste, compared to other terrines. Terrines 10 and 12 were closer to the
terrine with nitrites in terms of flavor and were not significantly more spicy or sweet as
the nitrite one.
a)
b)
Figure 4.8 Score plot (a) and loading plot (b) of the PCA performed from the sensory
analysis of flavor of terrines with nitrite (echNit), formulation 7(ech7), formulation
7+powder 3st (ech7fr), formulation 10 (ech10), formulation 12 (ech 12) and the
blanc(blanc)
143
For odor and texture
The same analysis were made for the attributes of odor and texture (Figure 4.9). The
first and second principal components (Dim.) describe 88.55% of the variability
(59.71% Dim 1 and 28.84% Dim 2, respectively. The results showed that there was a
difference of aromaticity, especially for the terrine with formulation 7. Terrine with
formulation 12 were closer to the nitrites one in terms of smell and texture
a)
b)
Figure 4.9 Score plot (a) and loading plot (b) of the PCA performed from the sensory
analysis of odor and texture of terrines with nitrite (echNit), formulation 7(ech7),
formulation 7+powder 3st (ech7fr), formulation 10 (ech10), formulation 12 (ech 12) and
the blanc(blanc)
144
4.5.3.4. Variance analysis for hams
For flavor of hams
The same analysis were made for ham. The results of the ANOVA are presented in
Table 4.12 . Regarding the variance ratio F of samples, for a probability p <0.005, there
is no significant difference in taste between the 6 tested hams.
Table 4.12 Analysis of variance of results of ham's flavor
Attributes
sweetness salty Bitterness Acidity spicy
Source of
Variation DF F P F P F P F P F P
panelist 8 8.39 <0.001 5.701 <0.001 1.561 0.17 8.471 <0.001 9.194 <0.001
sample 4 2.648 0.051 1.054 0.395 1.626 0.19 0.471 0.757 1.500 0.226
Residual 32 F 5% 2.69
F 1% 4.02 Total 44
*indicates a significant difference at 5% **indicates a significant difference at 1%
For odor and texture of hams
Table 4.13 showed a significant difference of aromaticity and tenderness between 6
hams tested with a probability of 1% and 5% respectively.
Table 4.13 Analysis of variance of results of ham's odor and texture
Attributes rancidity aromaticity tenderness juiciness Source of
Variation DF F P F P F P F P
panelist 8 12.623 <0.001 4.894 <0.001 2.293 0.046 11.25 <0.001 sample 4 1.351 0.273 4.663** 0.004 2.862* 0.039 2.350 0.075 Residual 32 F 5% 2.69
F 1% 4.02 Total 44
*indicates a significant difference at 5% **indicates a significant difference at 1%
145
4.5.3.5. Tukey test and descriptive analysis for hams
For flavor of hams
The sweet taste of ham with formulation 7 was lower and significantly different from
other hams. There was no significant difference from the spiciness of 5 hams, although
the ham with the formulation 10 is the higher average score (1.7780). The same applies
to the bitter and acid taste, although the ham with the formulation 10 has the highest
score (1.4440) in both cases. (Table 4.14)
Table 4.14 Results of Mean and Least Significant Difference for flavor of ham
Hams Attributes1
sweetness Salty Bitterness Acid Spicy
Ham with Nitrite 1.6670a 1.5560a 1.1110a 1.3330a 1.3330a
Ham with formulation 7 1.3330b 1.7780a 1.0000a 1.2220a 1.6670a
Ham with formulation 7 + powder 3st 2.0000a 1.3330a 1.4440a 1.3330a 1.6670a
Ham with formulation 10 1.7780a 1.6670a 1.4440a 1.4440a 1.7780a
Ham with formulation 12 1.6670a 1.5560a 1.4440a 1.3330a 1.7780a
Standard error2 0.1480 0.1610 0.1700 0.1150 0.1490
LSD3 0.56 0.606 0.640 0.432 0.564 1Mean of 9 observations. 2Mean standard error of mean scare on 32 df. 3Least significant difference
The descriptive analysis presented in Fig.4.10 showed the most discriminating
descriptors of flavor for the 5 hams. There was no significant difference between the
various flavors of hams according to the Tukey test. However, the descriptive analysis
shows that the ham with formulation 7 + powder 3st appears to have a slightly
pronounced sweetness taste than other hams. Ham with formulation 12 had the same
sweetness profile as nitrites one and ham with formulation 10 and 12 seemed to be a
little bit spicier than other hams.
146
sweetness
salty
bitterness
acidity
spicy
0.0 0.5 1.0 1.5 2.0
ham 7 vs attributesham 7 + powder3st vs attributes ham Nit vs attributesham 10 vs attributes ham 12 vs attributes
Figure 4. 10 Sensory profile analysis of flavor sweetness, salty, bitterness, acidity and
spicy of tested hams
The box plot of ham's flavor , which is another representation of Tukey test, was
presented in Figure 4.11 . The 5 hams selected were divided into the same 3 sub-groups
as terrines. No descriptor is discriminative in our case. They are all from the group 3.
148
For odor and Texture
For the odor and texture, the results presented in Table 4.15 showed that hams with
formulation 10 and 7+ powder 3st are significantly different from ham with nitrite in
term of aromaticity. There is a significant difference of juiciness between the ham
formulation 7 and 10. There is no significant difference of tenderness between the 5
hams, while the ham with formulation 7 has the highest average score (2.4440). The
rancid smell of ham is not significantly different between the 5 hams, although the ham
with formulation 7 + powder 3st has the highest score (1.4440).
Table 4.15 Results of Mean and Least Significant Difference for odor and texture of
hams
Hams Attributes1
rancidity aromaticity tenderness Juiciness
Ham with Nitrite 1.3330a 1.5560b 1.7780a 1.8890ab Ham with formulation 7 1.2220a 2.1110ab 2.4440a 2.2220a Ham with formulation 7 + powder 3st 1.4440a 2.5560ac 2.1110a 1.8890ab Ham with formulation 10 1.1110a 3.0000a 2.2220a 1.5560b Ham with formulation 12 1.2220a 2.3330ab 1.6670a 1.7780ab
Standard error2 0.1090 0.2480 0.1890 0.1570
LSD3 0.413 0.936 1.353 0.595 1Mean of 9 observations. 2Mean standard error of mean scare on 32 df. 3Least significant difference
The descriptive analysis presented in Fig. 4.12 showed the most discriminating
descriptors of odor and texture for the 5 hams. According to the results, there was no
significant difference between hams containing nitrites and other hams, except for
juiciness and aromaticity descriptors. Ham formulation 7 is juicier, tenderer and more
aromatic.
150
Box plot for aromaticity of ham
score
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5
ham7fr
ham10
ham12
ham 7
hamNit
Box plot for rancidity of ham
score
0.5 1.0 1.5 2.0 2.5 3.0 3.5
ham7fr
ham12
hamNit
ham 7
ham10
Box plot for juiciness of ham
score
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5
ham 7
ham7fr
ham12
ham10
hamNit
Box plot for tenderness of ham
score
0.5 1.0 1.5 2.0 2.5 3.0 3.5
ham10
ham 7
ham7fr
hamNit
ham12
Figure 4.13 The box plots of 5 tested hams with following descriptors: rancidity,
aromaticity, tenderness and juiciness
The table 4.15 shows the adjusted means of descriptors flavor, smell and texture of
hams, using an analysis of variance model with descriptor thanks to the software
SensoMineR (products * descriptors). This table 16 is a summary in the scores
presented above.
151
Table 4.16 Adjusted means of descriptors flavor, smell and texture of hams
Attributes
Ham
sample
salty sweety acid bitter spicy Rancid aromatic tender juicy
Nitrite 1.556 1.667 1.333 1.111 1.333 1.333 1.556 1.778 1.889
Sample 7 1.778 1.333 1.222 1 1.667 1.222 2.111 2.444 2.222
Sample
7+3st 1.333 2 1.333 1.444 1.667 1.444 2.556 2.111 1.889
Sample 10 1.667 1.778 1.444 1.444 1.778 1.111 3 2.222 1.556
Sample 12 1.556 1.667 1.333 1.444 1.778 1.222 2.333 1.667 1.778
4.5.3.6. Principal Component Analysis (PCA)
For flavor of terrines
The first and second principal components (Dim.) describe 100% of the variability
(89.63% Dim 1 and 10.37% Dim 2, respectively). It is the reason why there are only
two attributes, sweetness and bitterness. Hams are not significantly different between
them and between the one with nitrites. However, ham 7 + powder 3st is the sweetest
because of the strawberry powder.
152
a)
b)
Figure 4.14 Score plot (a) and loading plot (b) of the PCA performed from the sensory
analysis of flavor of hams with nitrites (hamNit), formulation 7(ham7), formulation
7+powder 3st (ham7fr), formulation 10 (ham10), formulation 12 (ham12)
For odor and texture
The results of analysis of PCA for odor and texture of ham showed, in Fig.4.16 the first
and second principal components (Dim.) describe 96.34% of the variability (52.65%
Dim 1 and 43.69% Dim 2, respectively). The absence of the attribute rancidity shows
that rancidity is not significant. Ham with the formulation 12 has a profile close enough
with ham with nitrite. Ham 7, nevertheless, seems more tender and juicy and 10 more
aromatic.
153
a)
b)
Figure 4.15 Score plot (a) and loading plot (b) of the PCA performed from the sensory
analysis of odor and texture of hams with nitrites (hamNit), formulation 7(ham7),
formulation 7+powder 3st (ham7fr), formulation 10 (ham10), formulation 12 (ham12)
4.5.4. Discussion
Terrines with the best anti microbial and antioxidant results were selected for this study:
the formulation 7 (clove 0,3%; cumin 0,3%; cinnamon 0,1%), 10 (clove 0,368%; cumin
0,2%; cinnamon 0,2%) and 12 (clove 0,2%; cumin 0,368%; cinnamon 0,2%). The
results for the quantification of the addition of strawberry powder in the terrine
formulation 7 gave very satisfactory results in terms of color, as well as close to the
values of nitrite. Regarding the organoleptic tests, the Tukey test for flavor, showed that
154
there was no significant difference between the different terrines in terms of taste.
Analysis of variance and descriptive analyzes terrines showed the most discriminating
attributes which are spicy for terrine with formulation 7 and sweetness for terrine with
formulation 7 + powder 3st. The aromaticity is present in terrines containing spices,
which is normal. However, the table of ajusted means and analysis of PCA showed that
the terrine with formulation 12 has a similar profile to the one with nitrites. Moreover,
the rancid smell noticed in terrine with formulation 7 + powder 3st is probably due to
transport of samples and the composition of the strawberry powder. There has been a
breakdown in the cold chain. More cautiously, terrine with formulation 7 + strawberry
would surely have a closer nitrites profile taste. The slightly bitter taste of this terrine is
probably comes from the variety of strawberry powder incorporated into the sample.
For hams, there was also no difference in terms of taste but more in terms of texture
according to the Tukey test. Hams with spices do not seem spicier than those containing
nitrites, but more aromatic like ham containing the formulation 10. On rancidity of
hams, compared to the terrines, there was no significant difference with nitrites; this is
due to the nature of the fatty acids present in. There are more satured fatty acids in ham
than in terrine. Ham with formulation 7 is very interesting in terms of its sensory
properties compared with ham + nitrites. Terrine with formulation 7 tended to be juicier
in both terrines and hams. To quantify the addition of strawberry powder or other
natural coloring agents, it would be advisable for future studies to use the formulation
10 and 12, with average values close to the nitrites one. The strawberry powder played a
relatively important role in this organoleptic tests. It would be wise, for a future study,
to analyze the concentration of strawberry on sensory effects and thus a compromise
between color samples and sweet setting.
4.5.5. Aknowledge
The authors sincerely thank the FQRNT (programme special) in collaboration with four
agro-industries, MDEIE and MAPAQ for their financial support. We express our
gratitude to the panelists and to Mr. Boiteau of Aliments Breton for supplying us
the raw meat to carry out various tests. The views and the opinions expressed in this
article are those of the authors.
155
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157
Chapitre 5 Analyse technico-économique du procédé de production des produits carnés (Utilisation des épices comme alternative aux nitrites et nitrates
158
5.1. Description des scénarios de simulation employés pour l'analyse technico-économique de la production des produits carnés
Dans le cadre de cette étude, l’évaluation de l’intérêt technico-économique de
l'utilisation des épices et de la poudre de fraise comme alternative aux nitrites dans les
produits carnés, été réalisée à partir d’une série de 4 scénarios. Les 4 scénarios sont
présentés en détails dans les figures 5.1 et 5.2. Les deux premiers scénarios tiennent
compte de la production de 350.000 kg/an de terrines et les deux autres scénarios
tiennent compte de la production de 1.500.000 kg/ an et de jambon. Le premier
scénario, est le scénario de référence pour la production des terrines (contenant les
nitrites) et le deuxième scénario tient compte de la production des terrines en utilisant
les épices comme agent de conservation . De même, Le troisième scénario, est le
scénario de référence pour la production des jambons (contenant les nitrites) et le
quatrième scénario tient compte de la production des jambons en utilisant les épices
comme agent de conservation. Les paramètres d’opération et les hypothèses de base
utilisés dans les 4 scénarios ont été présentés en détails dans le Tableau 5.1.
Tableau 5.1 Capacité de production de l'entreprise des produits carnés
Scénario 1 Scénario 2 Scénario 3 Scénario 4
Produits Terrines Terrines Jambon Jambon
Production 350.000 kg/an 350.000 kg/an 1.500.000 kg/ an 1.500.000 kg/ an
160
Figure 5.2 Scénarios 3 et 4 utilisés dans l’analyse technico-économique: Production du
jambon
5.2. Estimation du coût des capitaux fixes
La méthode factorielle d'estimation des capitaux fixes (Cfx) est exprimée par l'équation:
Cfx =fL *Ceq
fL : le facteur de Lang ( Sinnott, 1996) .
Ceq= coût d' achat des équipements (M $)
Le facteur de Lang dépend du type de l'industrie . Pour les entreprises alimentaires, le
facteur de Lang est de 1,8 (Maroulis & Saravacos, 2003 ).
Des divisions plus sophistiquées des capitaux fixes sont prises en compte dans la
littérature (Clark, 1997; Peters & Timmerhaus, 1991). D'après ces travaux, les capitaux
fixes sont estimés en utilisant l'équation 3.
161
Cfx = Ceq + Ccv +Cme (1)
Avec
Ccv : Coût des travaux de génie civil, y compris les aménagements, les bâtiments et les
structures.
Cme : Coût des travaux mécaniques et électriques, y compris l'installation des
équipements, la tuyauterie, l' instrumentation et le contrôle, les équipements électriques,
l'ingénierie et la supervision.
Ccv =fcv *C (2)
Cme =fme *Ceq (3)
fcv : facteur des travaux de génie civil
fme : facteur des travaux mécaniques et électriques
Cfx = (1 + fcv + fme)* Ceq (4)
Pour estimer les capitaux fixes totaux, nous étions basés sur les données de Bartholomai
(1987), qui a déterminé les capitaux fixes des différentes entreprises alimentaires y
compris l'entreprise des produis carnés. L'estimation des capitaux fixes par Bartholomai
(1987) a été présentée en détaille dans le tableau 5.3. En revanche, il est primordial de
faire la mise à jour des coûts donnés par Bartholomai (1987). Pour cette raison, nous
avons adopté l'équation 6 pour calculer les coûts des capitaux fixes d'une entreprise de
produits carnés pour l'année 2013.
Coût des capitaux fixes (2013)= Coût des capitaux fixes (1987)• (CE index 2013/CE
1987) (5)
CE index : 593,2
CE index 1987: 318,4
Coût des capitaux fixes (1987): 2000000 $: Cette valeur correspond aux coûts des
capitaux fixes d'une entreprise de produits carnés.
Les résultats de l'estimation des capitaux fixes relatifs aux différents scénarios d'analyse
ont été présentés en détail dans le Tableau 5.3.
162
Table 5.2 Summary of cost data for food plants in 1986 (Bartholomai (1987); Clark (1997); adapted by Rouweler).
Total invest-
ment cost
Raw
mate-
rials
Finis-
hed
pro-
duct
capac.
Area
(land)
Area
(land)
Equipment
cost
Wor king
hours
Investment
cost
Food production
plant
(excl. land) (excl. land)
US$ TON/h TON/h m2 ft2 US$ h/year $/ton prod/h
for details: (1986) (1986) (1986)
see Bartholomai
(1987)
[<-use for 0.6 exponent [<--Do NOT use in 0.6 exponent
calculation-->] calculation->]
1 Apple processing
(slices/sauce)
$2 923 000,00 5 4 1 860 20 000 $2006 000,00 1000 $731 000,00
2 Cannery
(community; small
scale)
$204 000,00 5 4.8 140 1 500 $123 000,00 1500 $42 500,00
3 Fruit puree
(aseptic; bag in box)
$1 100 000,00 4 3 470 5 000 $782 000,00 2000 $367 000,00
4 Multi purpose
fruit (jam; preserv)
3 2.4 95 1 000 $402 000,00
5 Orange juice
(concentration)
$2 057 000,00 20 1.6 1 860 20 000 $1116 000,00 2000 $1286 000,00
6 Baby food (glass
jars)
$200 000,00 6.6 5.5 280 3 000 $184 000,00 2000 $36 000,00
7 Tomato paste $1 837 000,00 15 2.5 $1087 000,00 2000 $735 000,00
8 Frozen vegetable $1 340 000,00 4.8 2.2 2 800 30 000 $852 000,00 2000 $609 000,00
9 Mushroom farm $116 000,00 0.04 5 600 60 000 $41 000,00 $2 900000,00
163
10 Mozzarella
cheese
$842 000,00 5.6 0.7 1 400 15 000 $350 000,00 3000 $1 203000,00
11 Blue cheese $4 093 000,00 11.5 2.1 10
500
113
000
$2908 000,00 2560 $1949 000,00
12 Dairy (fresh
milk; UHT; etc)
$13530000,00 20.8 18.1 6 510 70 000 $6400 000,00 2400 $748 000,00
13 Modular dairy $1 210 000,00 2 2 9 300 10 000 $760 000,00 250days/y $605 000,00
14 Powder milk $4 000 000,00 18 1.7 1 400 15 000 $2835 000,00 7200 $2352 000,00
15 Dried whole egg $2 287 000,00 2.5 0.25 1 120 12 000 $1357 000,00 2000 $9148 000,00
16 Yoghurt $4 286 000,00 8 8 7 900 85 000 $3 477000,00 3125 $536 000,00
17 Ice cream $2 515 000,00 2 2 1 600 17 000 $1530 000,00 2000 $1258 000,00
18 Parboiled rice $1 379 000,00 5 5 560 6 000 $1004 000,00 7200 $276 000,00
19 Corn starch $30298
000,00
8.3 5.3 2 240 24 000 $14169000,00 7920 $5717 000,00
20 Pasta $2 353 000,00 0.73 0.71 1 860 20 000 $1 760 000,00 5500 $3314 000,00
21 Precooked
lasagna
$3 261 000,00 0.7 0.67 1 070 11 500 $2 305 000,00 5700 $4867 000,00
22 Tofu 0.38 1.32 140 1 440 $350 000,00
23 Baker's yeast $26550
000,00
2.5 1.14 7 500 80 000 $11049000,00 7200 $23289000,00
24 Vinegar $750 000,00 0.033 0.31 350 3 750 $525 000,00 7200 $2 419 000,00
25 Quenelles $985 000,00 0.66 0.66 325 3 500 $600 000,00 2000 $1 492 000,00
26 Tortilla chip $1 688 000,00 0.5 0.55 900 9 500 $1 270 000,00 3500 $3 069 000,00
27 Corn snacks $310 000,00 0.18 0.27 470 5 000 $131 000,00 2000 $1 148 000,00
28 Catfish $2 400 000,00 3.2 1.8 1 120 12 000 $1 040 000,00 2000 $1 333 000,00
29 Shrimp $431 000,00 0.5 0.25 560 6 000 $204 000,00 1500 $1 724 000,00
30 Surumi $10 000
000,00
15 5.7 10
700
115
000
$6 080 000,00 3528 $1 754 000,00
31 Cattle slaughter $3 660 000,00 40 16 4 830 52 000 $930 000,00 1800 $228 000,00
32 Co-extruded
sausage
$2 000 000,00 1 1 930 10 000 $1 049 000,00 1600 $2 000 000,00
33 Protein recovery $2 671 000,00 12 6 930 10 000 $1 972 000,00 4000 $445 000,00
34 Soybean oil $24 900 00,00 42 42 2 330 25 000 $6 200 000,00 7200 $593 000,00
164
extraction
35 Vegetable oil
refinery
$2 359 000,00 2 1.8 930 10 000 $1 456 000,00 6000 $1 311 000,00
36 Pan bread $2 803 000,00 1.5 2.2 2 600 28 000 $1 795 000,00 5000 $1 274 000,00
37 Arabic bread $1 272 000,00 1.2 1.7 930 10 000 $702 000,00 6240 $748 000,00
38 Half-baked
frozen baquette
$1 953 000,00 0.48 0.54 981 10 560 $1 228 000,00 4000 $3 617 000,00
39 Seawater
desalination
$18
433000,00
3100 417 105 1 100 $9 262 000,00 7200 $44 200,00
40 Fruit juices from
concentrate
$809 000,00 2 2 675 7 240 $514 000,00 2000 $405 000,00
Soymilk 0.15 1 300 3 150 $910 000,00
Orange juice
concentrate
$2 424 000,00 20 1.5 $890 000,00 $1 616 000,00
Sausage $10
323000,00
1 1 $3 219 000,00 $10323000,00
165
Table 5.3 Estimation des capitaux fixes
Scénario 1 Scénario 2 Scénario 3 Scénario 4
Capacité (ton /
2013)
350 350 1200 1200
Capitaux fixes
(2013)
1497018 1497018 3135414 3135414
5.3. Estimation du coût d'exploitation annuel
De la même manière, le coût d'exploitation annuel peut être estimé sur la base des coûts
des matières premières et des services publics, en utilisant des facteurs appropriés. Les
coûts des matières premières et des services publics peuvent être calculés avec précision
en utilisant les bilans massiques et les bilans énergétiques. Pour le cas des entreprises
alimentaires, le coût des matériaux d'emballage est important (Clark, 1997) et il
devrait être inclus dans le coût des matières premières.
La méthode à un seul facteur pour l'estimation du coût d'exploitation annuel
est exprimée par l'équation ci-dessous:
Cop =fop *Cmu (6)
fop : le facteur de coût d'exploitation
La méthode factorielle détaillée pour l'estimation du coût d'exploitation annuel (Cop) est
résumée par les équations suivantes:
Cop = Cmu + Clabour +Cmisc (7)
Clabour =flabour *Cmu (8)
Cmisc=fmisc *Cmu (9)
Avec
Cop = Coût d'exploitation annuel
Cmu : Coût des matières premières et des services publics
166
Clabour : Coût de la main d'œuvre
Cmisc : Coût divers (entretien, les réparations, les redevances et brevets)
flabour :facteur du coût de la main d'œuvre
fmisc : facteur des coûts divers
Le modèle ci-dessus est équivalent à ce qui suit:
Cop = (1 + flabour + fmisc)* Cmu (10)
D' après les travaux de Mouralis et Mouralis (2004), dans le cas des entreprises
agroalimentaires :
Cop =1,1 *Cmu
Pour estimer le coût d'exploitation annuel, nous avons déterminé d'abord le coût des
matières premières, puis les résultats de l'estimation du coût des matières premières et
des services publics et l'estimation du coût d'exploitation annuel. Ces résultats sont
présentés dans le tableau 5.5.
5.4. Estimation du coût des matières premières
Les matières premières nécessaires pour la production des terrines et des jambons
constituent une fraction notable du coût de production. Le tableau 5.4 représente la
consommation et les coûts des matières premières utilisés dans les différents scénarios.
167
Table 5.4 Coûts des matières premières utilisés dans les différents scénarios
Scénario Matières premières quantité
utilisé (%)
Prix par tonne Références
Scénario 1 Fois de porc 25 700 Canadian $ Alibaba.com
Gras mou 40 700 Canadian $ Alibaba.com
Oeufs 5 1,92 $ / 12 gros
œufs
Agriculture
Canada, Nov 2013
Lait entier 25 958 $/ 1000 L Centre canadien
d'information
laitière, 2013
Sucre 0,4 550-700 $ Alibaba.com
Sel ordinaire 0,9 155 $ Alibaba.com
Sel nitrité 1 2000 $ Alibaba.com
Oignons 2 500 $ Agriculture canada,
2013
Ail frais 0,5 2000 $ Alibaba.com
Poivre blanc 0,15 $ 1000 $ Alibaba.com
Assaisonnement 0,05 1000- 2500 $ Alibaba.com
Scénario 2 Fois de porc 25 700 Canadian $ Alibaba.com
Gras mou 40 700 Canadian $ Alibaba.com
Œufs 5 1,92 $ / 12 gros
œufs- 2689 $
/tonne
Agriculture
Canada, Nov 2013
Lait entier 25 958 $/ 1000 L Centre canadien
d'information
laitière, 2013
168
Sucre 0,4 550-700 $ Alibaba.com
Sel ordinaire 1 $ 155 $ Alibaba.com
Oignons 2 $ 500 $ Agriculture canada,
2013
Ail frais 0,5 2000 $ Alibaba.com
Poivre blanc 0,015 1000 $ Alibaba.com
Assaisonnement 0,05 1000- 2500 $ Alibaba.com
Cumin 0,3 2000 $ Alibaba.com
Clous de girofles 0,3 8000$ Alibaba.com
Cannelle 0,1 1800$ Alibaba.com
Poudre de fraise 0,9 25000$ Alibaba.com
Scénario 3 Jambon 100 2680 $ Agriculture et
agroalimentaire
Canada, Novembre
2013
Sel nitrite 1 2000 $ Alibaba.com
Sel ordinaire 0,9
155 $ 1,395
Scénario 4 Jambon 100 2680 $ Agriculture et
agroalimentaire
Canada, Novembre
2013
Cumin 0,3 2000 $ Alibaba.com
Clous de girofles 0,3 8000$ Alibaba.com
Cannelle 0,1 1800$ Alibaba.com
Poudre de fraise 0,9 25000$ Alibaba.com
Sel ordinaire 0,9
155 $ Alibaba.com
169
Remarque: Le poids moyen de l'œuf gros est de 56 à 63 g (Fédération des
producteurs d'œufs de consommation du Québec)
Table 5.5 Estimation du coût total des matières premières et du coût d'exploitation
annuel
Scénario
Coût des matières
premières/ tonne de
produit finis ($)
Productivité
(tonne)
coût total annuel
des matières
premières ($)
coût d'exploitation
annuel ($)
1 875,22
350 306327 336959,7
2 1112,02
350 389207 428127,7
3 2701,395
1500 4052093 4457302
4 2938,195
1500 4407293 4848022
5.5. Estimation du coût du produit
L'analyse téchnico-économique réalisée dans cette étude est basée sur le model de
(Marouli et Maroulis, 2005). Le but de cette nalyse est de déterminer le coût du produit,
qui est la terrine, dans les scénarios 1 et 2, et le jambon dans les scénarios 3 et 4. Le
coût du produit est calculé en se basant sur la formule 11.
C= (e * Cfx + Cop)/F (11)
Avec
C: cout du produit ($/kg)
e : Facteur de recouvrement du capital: 5 ans
Cfx: Coût des capitaux fixes (M $ / an)
Cop: coût annuel de fonctionnement (M $ / an)
F: capacité de production annuelle (1 Gg = 106 kg)
170
Figure 5.3 Estimation du coût de production des terrines de foie de porc et du jambon;
Scénario1: terrines avec nitrites, Scénario2: terrines avec épices+poudre de fraise,
Scénario3: jambon avec nitrites, Scénario1: jambon avec épices+poudre de fraise
5.6. Conclusion
D’après les résultats trouvés dans la Figure 5.3 , l'utilisation des épices et de la poudre
de fraise comme alternatives aux nitrites à augmenté le coût de production des produits
carnés. Cette augmentation est faible dans le cas des terrines de foie de porc (8,42%) et
du jambon (7,51%). Cette faible augmentation du coût de production des produits
carnés en utilisant les épices et la poudre de fraise comme alternatives aux nitrites est
expliqué par une légère augmentation du coût des matières premières. Ceci est
principalement dû aux coûts élevés des épices, notamment les clous de girofle
(8000$/tonne) et de la poudre de fraise (25000$/tonne). Néanmoins, le coût des sels
nitrites, habituellement utilisés pour la conservation des produits carnés est relativement
faible (2000$/tonne). Par contre, cette légère augmentation est expliquée par la bonne
qualité des produits carnés contenant les épices et la poudre de fraise comme
alternatives aux épices. Ces alternatives sont sécuritaires pour la santé et leurs coûts ne
sont pas très élevés par apport à d'autres produits qui pourraient constituer un danger
pour la santé des consommateurs (cancer. etc).
0
500
1000
1500
2000
2500
3000
3500
4000
Scénario 1 Scénario 2 Scénario 3 Scénario 4
Coû
t du
prod
uit (
$/to
nne)
171
Conclusion générale
Les nitrates et les nitrites sont des substances chimiques naturelles présentes partout
dans l’environnement. Ils prolongent la durée de conservation des viandes transformées,
stabilisent la couleur des viandes rouges, ralentissent le processus d’oxydation des
lipides et inhibent le développement de microorganismes toxiques comme expliqué
dans le chapitre 1. Cependant, les nitrites, à forte dose peuvent être dangereux pour la
santé et pour l'environnement.
L'objectif général de cette étude était de trouver des alternatives vertes, sans présence de
nitrites et de nitrates au départ, dans les produits carnés, tels que la terrine faite à base
de viande de porc et de lapin et le jambon, en utilisant des épices, connues pour leurs
nombreuses propriétés antibactériennes et antioxydantes. Le premier objectif était le
criblage qualitatif puis quantitatif des additifs alimentaires naturels. Par des analyses
physico chimique et microbienne, effectuée sur une vingtaine d'épices, cinq épices ont
été sélectionnées. Les épices et combinaison d'épices ayant à la fois la meilleure activité
antimicrobienne, les meilleures propriétés anti-oxydantes et antimicrobiennes étaient : le
clou de girofle, le cumin, la cannelle, la cannelle et le clou de girofle, le cumin et le clou
de girofle. Les échantillons formulées avec ces épices avaient un durée de conservation
égale voir supérieure aux échantillons contenant les nitrites.
Dans la deuxième partie de l'étude, une optimisation quantitative a été réalisée en se
basant sur la méthode de réponse de surfaces, qui a founit un plan composite centré de
19 combinaisons de trois épices, les clous de girofle, la cannelle et le cumin, avec des
concentrations allant de 0, 1 % jusqu’au 0, 3% (m/m). Les résultats de ces
expérimentations ont montré que les activités antioxydants de la formulation 7 (clous de
girofle (0,3%) , cannelle (0,1%), cumin (0,3%)) et 15 (clous de girofle (0,2%) , cannelle
(0,2%), cumin (0,2%)) sont les plus importantes. Par contre, les formulations ayant de
très bonnes activités antimicrobiennes tant pour les terrines que le jambon, étaient la
formulation 7 (clous de girofle (0,3%) , cannelle (0,1%), cumin (0,3%)) et la
formulation 13 (clou de girofle (0,2%), cumin (0,2%), cannelle (0,031%)). De plus,
plus les concentrations de clous de girofle et de cumin étaient élevés, plus les activités
antimicrobiennes et les activités antioxydants l'étaient également. Les quantités
172
optimales des épices sélectionnées sont clous de girofle (0,3% p/p) , cannelle (0,1%
p/p), cumin (0,3% p/p) car elles assurent de bonnes qualités microbiologiques et
physico-chimiques des terrines. Ainsi, trois formulations d'épices qui donnaient les
meilleures résultats physico chimiques et microbiologiques, tant pour le jambon que
pour la terrine ,ont été sélectionnées pour la suite de l'etude: la formulation 7, la
formulation 10 (clous de girofle 0,368%, cumin 0,2% cannelle 0,2%) et la formulation
12 (clou de girofle 0,2% cumin 0,368% Cannelle 0,2%).
Voulant garder des propriétés organoleptiques semblables à celles des nitrites, la
coloration des échantillons par les épices étaient l'une des problématiques majeures de
cette étude. Pour palier à ce problème, une poudre de fraise a été ajouté à la formulation
7 . La concentration de la poudre de fraise a été quantifiée, en terme de colorimétrie par
rapport aux nitrites (0.9% p/p), et a servi pour nos tests sensoriels. Les résultats de
l'ANOVA ont montré qu'il n'y avait pas de différence significative entre les goûts des
terrines et des jambons par rapport aux nitrites. Pour obtenir des données plus
spécifiques, des tests de Tukey et une analyse en composante a fait ressortir le caractère
sucré de l'échantillon contenant la poudre de fraise. Cette analyse a révélé une légère
amertume et une certaine rancidité pour les terrined contenant cette poudre de fraise.
Ceci s'explique par un transport peu précautionneux des échantillons lors des
dégustations, une rupture de la chaîne de froid et surement à la variété de fraise utilisées
dans cette poudre. Les terrines et les jambons n'ont pas semblé très épicés dans
l'ensemble, ce qui est un résultat assez encourageant. Les échantillons contenant la
formulation 7 ont même semblés plus tendres et juteux que ceux des nitrites.
Enfin, le dernier chapitre a été consacré à l'analyse technico-économique de cette étude
avec 4 scénarios, les deux premiers concernant la terrine et les deux derniers, le jambon.
Le premier scénario, était le scénario de référence pour la production des terrines
(contenant les nitrites) et le deuxième scénario, de la production des terrines en utilisant
les épices comme agent de conservation . De même, Le troisième scénario, était le
scénario de référence pour la production des jambons (contenant les nitrites) et le
quatrième scénario, de la production des jambons en utilisant les épices comme agent de
conservation. Il en résulte une faible augmentation du coût de production des produits
carnés, 8,42% dans le cas des terrines de foie de porc et 7,51% dans le cas du jambon
étant donné le coût élevé des épices (8000$/tonne pour les clous de girofle) et de la
poudre de fraise (25000$/tonne) par rapport aux nitrites (2000$/tonne). Cette
173
augmentation serait le prix à payer pour avoir des aliments plus sains et sécuritaire pour
la santé.
D'autres études devraient être menées sur l'ajout de poudre de fruits pour la coloration
des viandes, notamment une analyse de l'impact de la concentration de fraise sur les
effets sensoriels des panélistes. Il faudrait de plus rechercher une autre variété de fraise
et faire attention lors du transport des échantillons pour obtenir des résultats plus
satisfaisants. Enfin, à des fins industrielles, une étude antibactérienne sur des bactéries
comme Listeria monocytogenes devrait être menée, étant donné le caractère assez
résistant de ces bactéries, aux traitements de nettoyage-désinfection dans les ateliers de
production de l’industrie agro-alimentaire.
175
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