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PRODUCTION OF TRANS-FREE MARGARINE FAT BY ENZYMATIC
INTERESTERIFICATION OF RICE BRAN OIL AND HARD PALM STEARIN
By
Pimwalan Ornla-ied
A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree
Master of Science Program in Food Technology
Department of Food Technology
Graduate School, Silpakorn University
Academic Year 2014
Copyright of Graduate School, Silpakorn University
PRODUCTION OF TRANS-FREE MARGARINE FAT BY ENZYMATIC
INTERESTERIFICATION OF RICE BRAN OIL AND HARD PALM STEARIN
By
Pimwalan Ornla-ied
A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree
Master of Science Program in Food Technology
Department of Food Technology
Graduate School, Silpakorn University
Academic Year 2014
Copyright of Graduate School, Silpakorn University
The Graduate School, Silpakorn University has approved and accredited the
Thesis title of "Production of trans-free margarine fat by enzymatic interesterification of
rice bran oil and hard palm stearin" submitted by Miss Pimwalan Ornla-ied as a partial
fulfillment of the requirements for the degree of Master of Science in Food Technology.
(Associate Professor Panjai Tantatsanawong, Ph.D.)
Dean of Graduate School
.......... ./ ................... ./ ........... .
The Thesis Advisor
Assistant Professor Sopark Sonwai, Ph.D.
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56403213: MAJOR: FOOD TECHNOLOGY
KEY WORDS: MARGARINE FAT I TRANS-FREE I ENZYMATIC INTERESTERIFICATION I
RICE BRAN OIL I HARD PALM STEARIN
PIMWALAN ORNLA-IED: PRODUCTION OF TRANS-FREE MARGARINE FAT BY
ENZYMATIC INTERESTERIFICATION OF RICE BRAN OIL AND HARD PALM STEARIN.
THESIS ADVISOR: ASST.PROF.SOPARK SONWAI, Ph.D. 114 pp.
This research studied production of trans free margarine fat by enzymatic
interesterification of rice bran oil and hard palm stearin. lnteresterified blends of hard
palm stearin and rice bran oil (hPSIRBO) in ratios of 20:80, 30:70, 40:60, 50:50, 60:40,
70:30 and 80:20 were prepared using Lipozyme RM IM (immobilized Mucor miehei
lipase) at 60°C and mixing speed was 300 rpm. Changes in triacylglycerol compositions
during enzymatic interesterification was studied after the reaction time at 2, 4, 6, 8, 12,
16 and 24 h. A reaction time of 6 h was chosen as an optimal reaction time for
enzymatic interesterification for all fat blends and would be used for the scale-up
experiments. Crystallization and melting profiles, solid fat content, polymorphism and
crystal morphology of the interesterified blends were investigated by different
techniques and compared with margarine fats extracted from commercial margarines.
The Lipozyme RM 1M-catalyzed interesterification significantly modified the physical
properties of the hPSIRBO blends. The slip melting point and solid fat content results of
all the interesterified blends had lower than their unreacted blends. The interesterified
blend of hPSIRBO 40:60 had SFC curves and crystallization and melting behavior most
similar to commercial margarine fats. Mostly 13' crystal form was found in the
interesterified fats, which is a desirable property for margarines, it provides smooth
texture to the products. Therefore, the interesterified blend of hPSIRBO 40:60 is suitable
for use as a margarine fats without trans fatty acid.
Department of Food Technology Graduate School, Silpakorn University
' . ~y VV\ V'J Q \ \.l \A_ Students signature .......... ~. ............................. , Academic Year 2014
Th · Ad · • · t n dQ/YfN~ eSIS VISOrS Signa ure ...... :::;).~ .. : ............ .
ACKNOWLEDGEMENTS
I would like to express my special thanks to my supervisor, Dr. Sopark Sonwai
who gave me the golden opportunity to do this wonderful project and thanks for his
grateful patience, guidance and valuable suggestion during doing this project from the
beginning to its finish. I believe without his unflagging opinions and proofread of my
work, I could not complete this project.
In addition, I am grateful for funding from Thailand Research Fund (TRF) and
Lam Soon (Thailand) PCL.
Finally, I would also like to thanks my parents, my friends and all staffs for all
their support and encouragement throughout the period of this research during my
postgraduate studies in Silpakorn University.
iii
CONTENTS
Page
ENGLISH ABSTRACT ........................................................................................................... i
THAI ABSTRACT ........................................................................................................... ii
ACKNOWLEDGEMENTS ................................................................................................ iii
LIST OF TABLES ............................................................................................................ vii
LIST OF FIGURES .......................................................................................................... ix
Chapter ........................................................................................................................... 1
Introduction ........................................................................................................... 1
State of the problem .......................................................................................... 1
Objectives of research ...................................................................................... 3
Research Hypothesis ........................................................................................ 3
Scope of the study ............................................................................................ 3
2 Literature review .................................................................................................... 4
Margarine ......................................................................................................... 4
Trans fatty acids ............................................................................................... 9
lnteresterification ............................................................................................ 21
Rice Bran Oil. .................................................................................................. 32
Palm Stearin .................................................................................................... 37
Crystallization of fats and oils .......................................................................... 40
Fractionation ................................................................................................... 46
3 Methodology ....................................................................................................... 48
iv
Materials and reagents ................................................................................... 48
Material preparation ........................................................................................ 51
Study of triacylglycerol and fatty acid compositions of the initial fat and oil and
the physical blends ................................................................................... 51
Study of changes in triacylglycerol compositions during enzymatic
interesterification ....................................................................................... 52
Study of physical properties and crystallization behavior of interesterified fat
at different weight ratio and compare with commercial margarine fats ...... 53
Summary of optimal condition for producing trans-free margarine fat.. ........... 54
Statistical analysis ........................................................................................... 55
4 Results and Discussion ....................................................................................... 56
TAG Composition changes during interesterification ...................................... 56
Triacylglycerol composition ............................................................................ 58
Fatty acid composition .................................................................................... 59
Slip melting point ............................................................................................ 61
Solid fat contents ............................................................................................ 62
Crystallization and melting profile ................................................................... 65
Polymorphism ................................................................................................. 69
Crystal microstructure ..................................................................................... 73
5 Conclusion .......................................................................................................... 77
References .................................................................................................................... 78
Appendix I Laboratory data ........................................................................................... 97
v
Appendix II Preparation of fatty acid methyl esters (FAMEs); Boron trifluoride method 104
Appendix Ill Free Fatty Acid Removing ....................................................................... 105
Appendix IV Determination of solid fat content (SFC) .................................................. 106
Appendix V Study of crystallization and melting behavior of fat... ................................ 107
by differential scanning calorimeter (DSC) .............................................. 107
Appendix VI Determination of slip melting point (SMP) ................................................ 108
Appendix VII Study of polymorphism by XRD .............................................................. 109
Appendix VII Study crystal microstructure by PLM ...................................................... 110
Appendix IX Commercial margarine fat extraction ...................................................... 111
Appendix X Pictures of enzymatic interesterification process ...................................... 112
Appendix XI Pictures of commercial margarine fat extraction process ........................ 113
Bibliography ................................................................................................................ 114
vi
LIST OF TABLES
Table Page
Solid fat content of table margarine ....................................................................... 6
2 TFAs in partially hydrogenated vegetable oil ....................................................... 12
3 Typical trans fatty acid content of foods produced or prepared with partially
hydrogenated vegetable oils in the United States ......................................... 13
4 Techniques of fat modification ............................................................................. 22
5 Comparison of chemical and enzymatic interesterification processes ................. 24
6 The typical composition of crude rice bran oil ..................................................... 33
7 Fatty acid composition of refined rice bran oil ..................................................... 34
8 Selected bioactive compounds in rice bran evaluated for their properties with
regard to prevention of chronic disease ....................................................... 35
9 Fatty acid compositions of palm olein and palm stearin (%) ................................ 38
10 Melting and solidification characteristics of palm olein and palm stearin ............. 38
11 TAG composition (%) of palm oil, palm stearin .................................................... 39
12 Three typical polymorphic forms of fats and their main physical properties ......... 44
13 Identification of polymorphic forms of fats Based on X-ray analysis of short
spacings ....................................................................................................... 44
14 Triacylglycerol profile (area%) of hPS/RBO blends before and after
interesterification .......................................................................................... 58
15 Fatty acid compostion of hPS/RBO blends before and after interesterification with
Li pozyme RM I M ........................................................................................... 60
16 Slip melting point of the hPS/RBO blends before and after interesterification with
Lipozyme RM 1M ........................................................................................... 51
vii
17 Crystal polymorphic forms of hPS/RBO blends before and after interesterification,
commercial margarine fats and hPS at 4 ac and 25aC .................................. 73
18 Solid fat content of hPS, RBO and hPS/RBO blends before interesterification ..... 99
19 Solid fat content of hPS/RBO blends after interesterification at 6 h .................... 100
20 Solid fat content of commercial margarine fats .................................................. 100
21 Crystallization temperature of hPS/RBO blends before interesterification .......... 101
22 Crystallization temperature of hPS/RBO blends after interesterification at 6 h .... 101
23 Crystallization temperature of commercial margarine fats ................................. 101
24 Melting temperature of hPS/RBO blends before interesterification ..................... 102
25 Melting temperature of hPS/RBO blends after interesterification at 6 h .............. 102
26 Melting temperature of commercial margarine fats ............................................ 102
viii
LIST OF FIGURES
Figure Page
Fatty acids position in molecule ofT AG ................................................................ 9
2 Structure of cis, trans and saturated fatty acids .................................................. 10
3 Total trans fatty acid content of new survey products compared with
existing data for similar products ................................................................. 16
4 Reaction schemes for the lipase-catalyzed interesterification between two
triacylglycerols with chemical catalysts and sn-1 ,3 specific lipases ............. 25
5 General scheme of the full hydrolysis of triglycerides catalyzed by
Rhizomucor miehei (RML) to produce free fatty acids .................................. 26
6 General scheme of interesterification catalyzed by Rhizomucor miehei (RML) ... 27
7 Reactions catalyzed by non-specific and 1 ,3 specific lipases ............................ 30
8 Structure of Rice with different layers .................................................................. 33
9 Polymorphic structures of three typical forms of triacylglycerol (TAG).
(a) Subcell structures and (b) chain length structures ................................ .45
10 Evolution of the relative quantity of TAG species obtained at different reaction
times during enzymatic interesterification of hPS/RBO (a) blend 20:80, and
(b) blend 40:60 (P = palmitic, 0 = oleic, L = linoleic acids) ......................... 57
11 SFC profile of hPS/RBO blends before interesterification .................................... 62
12 SFC profile of hPS/RBO blends after interesterification ....................................... 64
13 SFC profile of the interesterified hPS/RBO blends compared with commercial
margarine fats .............................................................................................. 64
14 DSC crystallization thermograms of hPS, RBO and hPS/RBO blends before
interesterification ......................................................................................... 66
ix
15 DSC melting thermograms of hPS, RBO and hPS/RBO blends before
interesterification ......................................................................................... 66
16 DSC crystallization thermograms of hPS, RBO and hPS/RBO blends after
interesterification compared with commercial margaine fats ........................ 67
17 DSC melting thermograms of hPS/RBO blends after interesterification compared
with commercial margaine fats .................................................................... 68
18 X-ray diffraction spectra of hPS/RBO blends before and after interesterification
and commercial margarine fats stored at 4 oc 24 h ...................................... 70
19 X-ray diffraction spectra of hPS/RBO blends before and after interesterification
and commercial margarine fats stored at 25°C 24 h .................................... 71
20 Crystal microstructure of fat blend at different ratio of hPS/RBO blends before
and after enzymatic interesterification and commercial margarine fats
obtained after static crystallization at 4°C and then left at 25oc for 24 h using
PLM ............................................................................................................. 75
21 Evolution of the relative proportions of TAG species of hPS/RBO blends (30:70) 97
22 Evolution of the relative proportions ofT AG species of hPS/RBO blends (50: 50) 97
23 Evolution of the relative proportions of TAG species of hPS/RBO blends (60:40) 98
24 Evolution of the relative proportions ofT AG species of hPS/RBO blends (70:30) 98
25 Evolution of the relative proportions of TAG species of hPS/RBO blends (80:20) 99
26 DSC crystallization and melting thermograms of extracted commercial margarine
fats ............................................................................................................ 103
X
1 .1 State of the problem
Chapter 1
Introduction
Margarine is a popular table spreads that substitute for butter. Margarine is a
water-in-oil emulsion comprising at least 80% lipid phase. The lipid phase consists of
fat, antioxidant, vitamins, and emulsifier, whereas the aqueous phase may contain water,
preservatives and salt. The traditional margarine fats contain trans fatty acids (TFAs),
which are formed during a partial hydrogenation process used to impart desirable
physical and textural properties to margarines (Kim et al., 2008). The adverse health
effects of trans fatty acids produced during hydrogenation of vegetable oils are well
known. Health concerns began to arise in the 1990s following reports that diets high in
total TFA were associated with increased risk of coronary heart disease (CHD) through
increases in serum LDL cholesterol and decreases in HDL cholesterol (Willet et al.,
1993). A recent systematic review and meta-analysis of cohort studies revealed that
increased total TFA intake, ranging from 2.8 to 10 g per day, was associated with a 22%
increase in risk of CHD events with a similar increased risk of fatal CHD (Bendsen et al.,
2011).
Because of this, in recent years, many food manufacturers and retailers have
voluntarily decreased trans fat levels in many foods and products they sell. In the U.S.,
since trans fat content information began appearing in the Nutrition Facts label of foods
in 2006, trans fat intake among American consumers has declined from 4.6 grams per
day in 2003 to about 1 gram per day in 2012 and consumption of trans fat should be as
low as possible while consuming a nutritionally adequate diet (FDA, 2013). Concurrently,
the demand for healthy trans-free fat products is rapidly increasing. One of the best
methods for production of trans-free fats is the interesterification process.
lnteresterification changes the position of fatty acids in the structure of triacylglycerol
2
molecules and, consequently, the physical properties of fat. Unlike hydrogenation,
which can only increase melting point and solid fat content (SFC), interesterification can
decrease or increase the melting point and SFC of fat blends. Furthermore, if trans-free
base stocks are used, the interesterification product will be trans-free (Lee et al., 2008).
lnteresterification is divided into two categories: chemical interesterification which
involves high temperature and catalysts and enzymetic interesterification (EI) which
uses enzyme as biocatalyst.
El has been known as an effective way to modify the physical and chemical
properties of fats and oils without producing the undesirable trans fatty acids (Rodrigues
and Gioielli, 2003). The technique has been widely used because of its mild, region-and
stereo-specific and easily controlled reaction. El catalyzed ester exchange reaction
between two acyl groups, thereby altering the overall chemical composition and
physical properties of the interesterified fats (Mukherjee, 2000). Nowadays, interest in
applications of enzyme technology for the production of plastic fats for food uses is
increasing in both academia and industry but currently it is limited since the price of
commercial lipase is too high for industry (Zhang et al., 2001 ).
This research studied the production of trans-free margarine fat from rice bran
oil (RBO) and hard palm stearin (hPS). Rice bran oil (RBO) is one of the healthy edible
oils, containing relatively high amount of bioactive phytochemicals with balanced fatty
acid composition. It is reported that RBO has hypocholesterolemic effect in both animals
and humans, in which RBO contained limited level of polyunsaturated fatty acids
(Rukmini, 1988; Raghuram et al., 1989). Palm stearin (PS) is an important co-product
obtained during palm oil fractionation. It is the harder fraction of palm oil containing
higher proportion of saturated fatty acids and a natural source of solid fat that provides
strength and structure to the product. In this work PS was solvent fractionated and only
the solid fraction (hard palm stearin: hPS) was used. RBO and hPS were blended into
different proportions and the structure of the blends was modified via El. The melting
and crystallization profiles, SFC, polymorphism and crystal morphology of the
interesterified blends were investigated by different techniques and compared with the
3
physical blends and the margarine fats extracted from commercial margarines. Then,
the best ratio of the blends was selected to be used as a margarine fat for the
production of trans-free margarine.
1.2 Objectives of research
1. To study fatty acid and triacylglycerol composition of rice bran oil (RBO), hard
palm stearin (hPS) and physical blends between RBO and hPS at different weight ratio.
2. To study changing of triacylglycerol composition of interesterified blends
during enzymatic interesterification at different reaction time to find optimal reaction time
at each weight ratio of interesterfied blend.
3. To study physical properties and crystallization behavior of interesterified fats
at different weight ratio and compare with commercial margarine fats.
1.3 Research Hypothesis
Trans-free margarine fats have physical properties and crystallization behavior
similar or same as commercial margarine fat and suitable for producing trans-free
margarine.
1 .4 Scope of the study
1. Study of triacylglycerol and fatty acid composition of initial fat and oil and
physical blends.
2. Study of triacylglycerol composition changing during enzymatic
interesterification.
3. Study of physical properties and crystallization behavior of interesterified fat at
different weight ratio and compare with commercial margarine fat.
4. Summary of optimal condition for producing trans-free margarine fat.
2.1 Margarine
Chapter 2
Literature review
Margarine (butter-like product) is water-in-oil (w/o) emulsion and must contain at
least 80% fat by federal regulation. Worldwide margarine production was estimated to
13.5 millions of tons in 2013. United States of America is the largest margarine
producing country followed by Pakistan. In Thailand, the margarine production was
estimated to 8.5 thousands of tons in 2005 and increase continuously to 20.9 thousands
of tons in 2013 (FAOSTAT, 2013). Margarine consists of two phase. The aqueous phase
consists of water, salt and preservatives. The fatty phase, which contributes to the
polymorphic behavior of margarine, is a blend of oils and fats. Lecithin, distilled
monoacyl glycerol and diacylglycerol are common emulsifiers added together with
flavouring, colouring agents and antioxidants (Miskandar et al., 2005). Structurally,
margarine consists of a continuous liquid fat phase with fat globules, crystalline fats and
aqueous phase dispersed in it (Juriaanse and Heertje, 1988). Under certain conditions,
fat globules are capable of extreme elongations without rupture (indicating the elastic
nature of the membrane that surrounds the globule and liquid fat surrounding the
globule acts as a viscous fluid and flows on application of stress (Diener and Heldmand,
1968). Thus, margarine exhibits viscoelastic charateristics. Usually margarine obtained
from combination of various edible fats and oils. It contains appropriate ratios of hard
vegetable fats and liquid vegetable oils from coconut, palm kernel, interesterified
vegetable oils and/or hydrogenated vegetable oils. (Kandhro et al., 2008).
A good margarine should not suffer oil separation, discoloration, hardening,
sandiness. graininess and water separation (Chateris and Keogh, 1991 ). The oils and
fats, process conditions and handling methods used should be selected so as not to
produce a strong crystal network (Miskandar et al., 2002a), crystal migration and
4
5
transformation of 13'- to ~-crystals. The major effort in margarine production should be
for the product to be in the 13'-crystal form as it would then be smooth, creamy and
homogenous. The ~-crystal form, on the other hand, will produce a margarine that is
post-hardened, brittle, grainy, sandy, oiled out and greasy (Miskandar et al., 2002b).
2.1 .1 Category of margarines
Margarines have various types based on their texture and application.
Margarines can be categorized by their hardness and melting point of their fats hard
and medium plastic margarines for baking (bakery margarine), and medium plastic and
soft margarines for the table (table margarine).
2.1.1.1 Bakery margarine
Bakery margarine is used like shortening - as bakery fat, and in short pastry,
cakes, cookies, breads and pastries. Bakery margarine, like its counterpart, shortening,
has a wide plastic range. Thus, within limits, the products are intersubstitutable.
Generally, however, bakery margarine is firmer and requires no refrigeration. It is
formulated to withstand dough working and, at the same time, provide lubrication for
cake leavening (Manley, 1983). Premium bakery margarines produce a high cake
volume and stable cream. Palm kernel oil (both unhydrogenated and hydrogenated) is
commonly used in the products.
2.1.1.2 Table margarine
Table margarines are further divided into two group base on their texture and
storage; tub and brick margarine or refrigerated and non-refrigerated margarine.
Tub and brick margarines: Tub margarine has low solid fat content (SFC) at low
temperature, thus enabling it to be spreadable direct from the refrigerator. Brick
margarine have properties similar to tub margarine, but with higher SFC (Pande et al.,
2013) caused they can not to be spreadable immediately after bring them out from
refrigerator.
One more group is refrigerated and non-refrigerated margarines: both
spreadable at room temperature. Refrigerated margarine is either soft or block type. The
soft type margarine is spreadable straight from the refrigerator and should not suffer any
6
oiling out, while the block type should be sufficiently firm to retain its shape in packets
(Nawar, 1985). Table 1 shows solid fat content (SFC) of both margarines at different
temperature, indicate that tub margarine has lower in SFC but packet margarine has
higher in SFC due to it is used in tropical country (ambient temperature >30°C) (Rasid et
al., 1996).
Table 1 Solid fat content of table margarine
Margarine Type Solid Fat Content (%SFC)
1 ooc 21.1 oc 26. rc 33.3oc 37.8oc
Packet
Tub
28
13
Source: Gunstone and Norris ( 1983)
2.1.2 Characteristics of margarine
16
8
12
6
2-3
2
0
0
Margarine can be considered a bacteriologically safe product. The only
concerns are the chemical and/or physical defects caused during or after production.
The possible defects during production are lumpiness, watering, hardening,
discoloration, mottling, saltiness and greasiness, while the defects in storage include
sandiness, discoloration, hardening, oil separation and greasiness (Nawar, 1985). For
margarines made with good raw materials, the defects can only be due to processing
and handling. The overall product quality summarizes the product characteristics of
spreadability, texture and consistency and polymorphic behavior (Berger, 1986).
2.1.2.1 Spreadability
This is probably the most important attribute for table margarines and spreads.
To the consumer, spreadability is the ease with which the margarine can be applied in a
thin, even layer on bread. According to deMan et al. (1989) the penetration yield
correlates very well with spreadability. To produce a spreadable margarine, three
conditions are necessary: 1) the two phases of liquid and solid oils must co-exist, 2) the
solid crystals must be sufficiently finely dispersed throughout the entire mass to be
7
effectively held together in the crystal matrix by internal cohesive force. In turn, the
matrix should be able to prevent the entrapped liquid from seeping away, and 3) the
proper proportions of solid and liquid should be at a certain temperature and the
crystals should melt at below body temperature (Chateris and Keogh, 1991; Greenwell,
1981). A product with 10-20% SFI at the serving temperature will have good
spreadability.
2.1 .2.2 Consistency and texture
Consistency is the measure of smoothness, evenness and plastic state in
margarine. It can range from very soft, like petroleum jelly, to soft, medium, firm, tough,
hard and brittle. Texture is a measure of the structure. It varies from smooth to mealy or
floury, grassy, granular or sandy and, finally, coarse and lumpy. According to Greenwell
(1981 ), the consistency and texture of margarine is principally dependent on the
processing techniques and the oil and fats used in its manufacture.
In cases where the oils and fats crystallize slowly, the margarine hardens during
storage. This phenomenon is known as post-hardening. Oils and fats having more than
30% of their TAGs as POP are slow to crystallize, and therefore susceptible to post
hardening (deMan et al, 1989). Although POP has a melting point of 30°C, it crystallizes
only at 20°C. The big difference between the melting and crystallizing points makes for a
long crystallizing time (slow crystallization rate).
Post-hardening is caused by inappropriate processing conditions. A product
over-stirred during supercooling produces excessive small crystals. The structure will
be too compact, reducing the size of the capillaries in between the solids (Greenwell,
1981 ). This increases interlocking of the framework, restricting movement of the solids.
The viscosity then increases leading to an increase in brittleness (deMan et al, 1989).
Mechanical work applied during fast cooling produces margarine with better
consistency and stability. However, if the margarine has not had sufficient work, a large
number of its primary bonds will still be intact and it will undergo considerable post
hardening, becoming brittle and hard-textured (Berger, 1986; Greenwell, 1981 ).
Crystallizing the product as much as possible in the scraped surface heat exchanger
(SSHE) will contribute greatly to good spreadability and consistency.
2.1.2.3 Oil separation
8
Oil separation occurs when the crystal matrix is inadequate to entrap the liquid
oil. This occurs because of transformation of the crystals to the 13-form. The 13-crystals
continuously grow bigger (causing sandiness) until the network can no longer retain its
lattice structure to entrap the liquid oil. The liquid oil then exudes from the product and
the aqueous phase coalesces. The problem is serious for stick margarine but not for
soft tub margarine. The susceptibility of a margarine to oil separation can be determined
by placing a sample of defined shape and weight on a wire screen or filter paper at
27"C for 24-48 hours and then measuring the oil exuded (Berger, 1986). Oil separation
at a particular temperature has strong relation to the SFC.
2.1.2.4 Sandiness
According to Nawar (1985), deMan and deMan (1994) and Timms (1994), the 13'
polymorph is the desired form in margarine and shortening. deMan and deMan stated
that 13' has very small crystals so that it can incorporate a large volume of liquid oil in the
crystal network giving a smooth, continuous and homogeneous product. It was also
pointed out that the 13'-crystal form gives the margarine surface a gloss in contrast to 13,
which produces a dull and mottled look. The 13'-crystals not only provide a good texture
to margarine but also contribute to good whipping characteristics. According to Nawar,
the small crystals help to trap and hold air during whipping.
In general, the 13-polymorph is not desired in margarine, although desired in
pastry margarine for pie-crust (deMan and deMan, 1994). The 13-crystals have the
tendency to grow bigger and bigger into needle-like agglomerates. This is more
common in homogenous TAGs. The large crystals impart a sensation of sandiness in the
mouth (deMan et al, 1989). Soft margarines are more prone to polymorphic transition
than the hard ones because of the low SFCs in the soft oils used (deMan and deMan,
1994). This usually occurs in formulations with oils and fats that favor the 13-crystal form,
inappropriate oil: solid ratios and processing conditions (Nawar, 1985). The 13-crystal
9
polymorph crystallizes at a higher temperature than the 13' -crystals and produces
margarine of stiff and hard consistency, which is ideal for piecrust and desirable in
pourable shortening. The 13-crystal polymorph will support formation of a solid
suspension in liquid oil, making the product pourable.
2.2 Trans fatty acids
Both nutritional and technological properties of fats depend on their fatty acids
composition and position in molecules of triacylglycerols (TAG) (Figure 1 ). Unsaturated
fatty acids show two types of isomerism: geometric and positional (changes in the
position of the double bond).
R2COO - C - H sn-2
I CH2COOR3 sn-3
Figure 1 Fatty acids position in molecule of TAG.
Source: zbikowska (2010)
All natural fats and oils are a combination of monounsaturated, polyunsaturated
and saturated fatty acids. Trans fatty acids (TFAs) are unsaturated fatty acids in which
the carbon moieties on the two sides of a double bond point in opposite directions, as in
elaidic acid (trans-~9-C18: 1 ). Most natural fats and oils contain only cis double bonds,
such as oleic acid (cis-~9-C18:1), in which the carbon moieties lie on the same side of
the hydrocarbon chain (Figure 2)
HO
elaidlc add
HO
oleic acid
HO
stearic acid
Figure 2 Structure of cis, trans and saturated fatty acids
Source: Doyle ( 1997)
10
The configuration of the hydrogen atoms in cis fatty acids causes a bend in the
carbon chain, whereas in saturated fatty acids such as stearic acid the carbon chain is
straight. In a trans fatty acid, the carbon chain is relatively straight, so that in this respect
the trans isomer resembles the corresponding saturated fatty acid (in this case, stearic
acid, C18:0); but the chain is twisted slightly, which affects its sectional area and
therefore its space requirements. These differences in geometry allow saturated fatty
acids to be packed together more tightly than the trans monounsaturated isomer and
produce a more rigid configuration, while the cis isomer will be the most loosely packed
11
and tend to be liquids or oils at room temperature. As a consequence, reported melting
points of these substances vary from 72 oc (stearic acid) to 44 oc (elaidic acid) to 13 oc
(oleic acid) (Stender et al., 1995). Trans fatty acids, then, appear to have characteristics
intermediate between those of the corresponding saturated and cis fatty acids.
2.2.1 Definition of trans fatty acids
TFA have been defined by different organizations, for instance Codex
Alimentarius (1985) and European Food Safety Authority or EFSA (201 0). Although they
are consistently the same, small differences may be found between them. According to
Codex Alimentarius, TFA are "all the geometrical isomers of monounsaturated and
polyunsaturated fatty acids having non-conjugated, interrupted by at least one
methylene group, carbon-carbon double bonds in the trans configuration". EFSA
specifies that trans polyunsaturated fatty acids "have at least one trans double bond
and may therefore also have double bonds in the cis configuration". TFA definition
excludes conjugated FA such as conjugated linoleic acid (CLA) (Kim et al., 2008).
2.2.2 Sources of trans fatty acids
There are three sources for the origin of trans fats. The first source is the partial
hydrogenation process which converts liquid vegetable oils into solid or semi-solid fats
with appropriate melting properties suitable for products such as shortening and
margarines and increase oxidation stability and the shelf-life of the oil. The second
source is the natural presence in fats from bacteria transformation of unsaturated fatty
acids in the rumen of ruminant animals. This second origin is responsible for low TFA
levels found in meat and dairy products (Richter et al., 2009); consequently it is
impossible to completely eliminate trans fats form a balanced diet (Remig et al., 201 0).
Ruminant and industrial fats contain the same TFA isomers, but the proportions differ
(Weiland et al., 1999). The third source of TFA is heat treatment (Richter et al., 2009).
TFA can be produced from heating oils above 180°C and deodorization of oils (Wolff,
1994). However, Tsuzuki (201 0) has reported that trans fats are not formed during frying
12
process. An ordinary frying process using unhydrogenated oils has little impact on TFAs
intake from edible oils. No trans fatty acids were formed in unhydrogenated and
hydrogenated soya bean oil during heating at 160, 180 or 200oC for 24 h, implying that
trans fatty acid can only be formed under drastic heating conditions i.e. heating the oil at
high temperatures or reusing the oil many times (Liu et al., 2007). Deodorization of
vegetable oils, a process utilizing high temperatures to drive off compounds with
undesirable flavors or odors, also induces the cis-to-trans isomerization of fatty acids.
Isomerization begins at 220 oc, and as much as 10% of fatty acids may be converted to
the trans isomer after exposure to 280 °C. Under the usual conditions for deodorization,
3-6% trans fatty acids would be formed (Pudel and Denecke, 1997).
The primary dietary TFA are vaccenic acid and elaidic acid. Vaccenic acid
(18:1, trans-11) is the major ruminant TFA, whereas elaidic acid (18:1, trans-9) is the
main TFA isomer in industrial hydrogenation (Dijkstra et al., 2008; Mensink, 2005;
Weiland et al., 1999).
The level of TFAs in partially hydrogenated vegetable oil shown in Table 2. Trans
fatty acids are found in small amounts in the fats of ruminants; for example, 100 g of milk
fat contains 4 to 8 g of trans fatty acids (Parodi, 1976). Much larger amounts are found
in certain types of margarines, margarine-based products, shortenings, and fats used
for frying (Slover, 1985).
Table 2 TFAs in partially hydrogenated vegetable oil
TFA Level (% total TFA)
Trans C18: 1 85-95
Trans C18:2
Trans C18:3
Trans C16:1
Source: i:bikowska (201 0)
8-22
< 1-7
0.04
13
Hydrogenated fats account for an estimated 80-90% of the intake of trans fatty
acids by Americans. Margarines contain 11-49% trans fatty acids, while some cooking
fats have even higher percentages. Soft margarines packaged in tubs have lower levels
of trans fats than the harder, stick margarines. Other major contributors to trans fatty
acid intake include doughnuts, pastries, fried chicken, French fried potatoes, snack
chips, and imitation cheese; these foods contain 35-38% trans fatty acids (Feldman et
al., 1996).
The highest TFA values are found in products with partially hydrogenated
vegetable oils. These oils contain approximately 30% of TFA and may account for up to
60% of TFA content, whereas animal fats and dairy products are considerably lower and
account for 2%-5% of TFA (Daniel et al., 2005; Weggemans et al., 2004).
The average consumption of industrially produced trans fatty acids in the United
states is 2 to 3 percent of total calories consumed (Allison et al., 1999). Major source of
trans fats are deep-fried fast foods, bakery products, packaged snack foods,
margarines and crackers (Table 3)
Table 3 Typical trans fatty acid content of foods produced or prepared with partially
hydrogenated vegetable oils in the United States.
Trans Fatty Acid Content*
Type of Food %of Total % of Daily Energy g/Typical Serving g/100 g
Fatty Acids Intake for 2000-kcal Diet
Fast or frozen foods
French friest 4.7-6.1 4.2-5.8 28-36 2.1-2.7
Breaded fish burger:j: 5.6 3.4 28 2.5
Breaded chicken nuggets:j: 5.0 4.9 25 2.3
French fries, frozent 2.8 2.5 30 1.3
Enchilada:j: 2.1 1.1 12 0.9
Burrito:j: 1.1 0.9 12 0.5
Pizzat 1.1 0.5 9 0.5
Packaged snacks
Tortilla (corn) chipst 1.6 5.8 22 0.7
14
Popcorn, microwavet 1.2 3.0 11 0.5
Granola bart 1.0 3.7 18 0.5
Breakfast bart 0.6 1.3 15 0.3
Bakery products
Piet 3.9 3.1 28 1.8
Danish or sweet rollt 3.3 4.7 25 1.5
Doughnutst 2.7 5.7 25 1.2
Cookiest 1.8 5.9 26 0.8
Caket 1.7 2.7 16 0.8
Browniet 1.0 3.4 21 0.5
Muffint 0.7 1.3 14 0.3
Margarines
Vegetable shorteningt 2.7§ 19.2 19 1.2§
Hard (stick)t 0.9-2.5 6.2-16.8 15-23 0.4-1.1
Soft (tub)t 0.3-1.4 1.9-10.2 5-14 0.1-0.6
Other
Pancakest 3.1 2.0 21 1.4
Crackerst 2.1 7.1 34 0.9
Tortillast 0.5 1.8 25 0.2
Chocolate bart 0.2 0.6 2 0.1
Peanut buttert 0.1 0.4 0.05
*Values are based on national brands that contain partially hydrogenated vegetable oils and that were sold in the
United States in 2002. These same foods will contain little or no trans fatty acids if produced or prepared with other
fats or oils, such as unhydrogenated vegetable oils or fully (not partially) hydrogenated vegetable oils. Very small
amounts of trans fatty acids (less than 2 percent) may be formed during deodorization of vegetable oil, a process
unrelated to partial hydrogenation. Dairy and meat products from cows, sheep, and other ruminants contain small
amounts (1 to 8 percent of total fatty acids) of naturally occurring trans fatty acids produced by the action of bacteria
in the ruminant stomach. Data are from the Harvard Food Composition Database.
t For french fries and margarines, the top national brands were analyzed individually for fatty acid composition with
the use of gas chromatography (Harvard Biomarker Laboratory, Dr. Hannia Campos); the ranges are shown. For the
other foods, the top national brands were combined into one sample for analysis. The proportion of the total sample
that each brand constituted was determined by the brand's percent market share in the national marketplace, as
determined from market-share and consumption statistics (TableBase, Responsive Database Services) and from
Harvard cohort pilot consumption data representing a national sample.
15
+The contents and types of partially hydrogenated oils were determined from recipe information (Harvard Food Com
position Database, based on packaging and USDA sources), and the trans fatty acid content of these oils was
calculated on the basis of gas-chromatography analysis of similar foods. For the fast or frozen foods, the trans fat
content of the oils was calculated from the fatty acid composition of the oils in french fries; for granola bars, from a mix
of partially hydrogenated soybean oil and household vegetable shortening; and for breakfast bars, from household
vegetable shortening.
§Values are per tablespoon (given that there is no typical serving size)
Source: Mozaffarian et al. (2006)
In New Zealand, the TFA content of margarines and table spreads appears to
have declined significantly over the past decade. In 1995 the mean margarine TFA
content was 14.9 g/1 00 g fatty acids (Lake et al., 1996). In 1998 two separate groups of
margarines could be identified, one having up to 1 g/1 00 g fatty acids TFA, and the
other having a mean of 14.5 g/100 g fatty acids. Results from this 2006 survey,
augmented by label data collated by Consumer (2005) magazine, indicate that the
higher TFA content group of margarines has undergone a formulation change to reduce
their TFA content to no more than 8 g/1 00 g fatty acids. Margarine products and
formulations change regularly, but where products with the same brand name could be
compared between the 1998 and 2006 information, the TFA content had declined by
approximately half. Although comparisons cannot be made between brands, New
Zealand table spread products appear to have lower TFA content than similar products
available in U.S.A (Mozaffarian et al., 2006). In 2006, Saunders et al., (2008) survey TFA
content of New Zealand foods the results found declines in the TFA content of New
Zealand foods between 1995, 1998 and the present 2006 survey. Consequently, it
seems reasonable to conclude that the intake of TFA by New Zealanders has also
declined over the same period.
In United Kingdom, Roe et al. (2013) survey to determine the trans fatty acid
content of a range of processed foods. The foods analysed included pizza, garlic bread,
breakfast cereals, quiche, fat spreads, a range of fish and meat products, chips, savory
snacks, confectionery and ice cream. The result of comparison between total TFA from
this survey and previous existing analytical data held for comparable foods given in
16
Figure 3. The results show that levels of 1-TFA in processed foods have reduced
considerably compared with previous analyses of similar foods carried out over the last
20-30 years. Therefore, levels of trans fatty acids were reduced considerably compared
with previous UK analyses of similar foods where comparisons are possible.
Butter, spreadable (75-80% fat)
Mayonna1se
Cream of tomato soup, canned
Chocolate-covered bar with caramel and cereal
Milk chocolate
Toffees
Potato crisps
Chicken pieces, coated, takeaway
Fat spread (41-62%), not polyunsaturated, with olive oil
Ghee, vegetable
Quiche lorraine
Garlic and herb bread
0
• Current Survey ll Previous value
1 2 3 4 s 6
TFA content (g/100g Food)
Figure 3 Total trans fatty acid content of new survey products compared with existing
data for similar products.
Source: Roe et al. (2013)
2.2.3 Consumption and levels of trans fatty acids
7
The dietary consumption of TFA was mainly from two sources: Natural fats,
which occur in dairy and other animal fats (milk, butter and meat of ruminants) as well as
products made of them, and industrially partial hydrogenated edible oils, which are
used for the production of hardened margarine spreads and industrial fats as well as
products made of them (Zbikowska, 201 0). The former is considered to be harmless or
even benefit for preventing against CVDs, while the latter is famous for its palatability
and long shelf time which make it be widely used in fried food, snacks, fast food and
baked food (Bendsen et al., 2011).
The consumption of TFA is different in each country. Previous data reported
average intakes of trans fatty acids is estimated to range from 2.6 to 12.8 g/day in the
17
U.S.A. (Lichtenstein, 1997). The TRANSFAIR study average consumption of TFA in 14
countries of European intakes and sampling was done between June 1995 and April
1996. TFA were found in either the fast food or the pre-fired or home-fired products more
than 10% in all of the countries (Aro er al., 1998) and they estimated that average
consumption of TFA was 0.5-2.1% of total energy intake in men and 0.8-1.9% in women
(Hulshof et al., 1999).
In 2006, Craig-Schmidt has compared the consumption of TFA in several
countries and found that in North America the daily intake was about 3-4g/person and in
Europe (Germany, Finland, Denmark, Sweden, France, United Kingdom, Belgium,
Norway, The Netherlands and Iceland), the daily intake was between 2.1 and 5.4g/day,
while in Mediterranean countries (Italy, Portugal, Greece and Spain), the intake of TFA
was lower (1.4-2.1 g/day) due to the use of olive oil.
According to Saunders et al. (2008), the TFA levels in snack bars,
margarines/table spreads, biscuits and cakes, pies and pastries were all below 10
g/1 00 g of FA (less than 3.5 g/1 00 g of product). Moreover, TFA content of foods, in New
Zealand, has declined over the previous decade, with a likely decrease in consumption
of these lipids by New Zealanders.
In 2010, Roe et al. (2013) has collected UK processed foods for analyze fatty
acids and a range of nutrients. TFA were detected in all samples with total TFA ranging
from 0.04-2.40% of fatty acid methyl esters (FAME). The highest concentrations were
found in cod in batter (2.40 g/1 00 g FAME) and potato chips from fish and chip shops
(2.05 g/100 g FAME), dairy ice cream (1.99 g/100 g FAME), garlic and herb baguette
(1.93 g/100 g FAME), spreadable butter (1.81 g/100 g FAME) and pizza (1.22 g/100 g
FAME).
The current consumption of TFA varies significantly in different countries
throughout the world, from less than 1 g/person/day in Asian and Pacific countries to
10-20 g/person/day in subpopulations of some Western countries (Chung et al., 2013).
According to recent studies, it is estimated that the average intake of TFA is 0.39
g/person/day in China, 0.6 g/person/day in Korea, 0.99 g/person/day in India and 1.3
18
g/person/day in the U.S.A. (Craig-Schmidt, 2006; Dixit and Das, 2012; Doell, 2012),
which has declined over the previous decade. The intake of TFA has decreased
progressively in Western countries in recent years but its trend is increasing in Asian
countries. Due to the public and scientific discussion, TFAs content of foods has been
reduced worldwide thereby also reducing the intake of TFAs (Craig-Schmidt, 2006;
Wagner et al. 2008).
In sum, TFA content of foods presents great variability among different food
groups and countries and this is reflected on the variability of TFA intakes. However, it is
evident that TFA food content is declining due to the progress of food industry in
reducing the trans-fat content in a wide range of products and due to more aware
consumer food choices (Albuquerque et al., 2011 ).
2.2.4 Health implications
Results of numerous studies show that the intake of either saturated or TFAs
caused many adverse health diseases in human. Following risks are reported to be
associated with the intake of trans fatty acids on human health.
Cardiovascular diseases
Cardiovascular diseases (CVDs) are now the leading cause of death worldwide
and refer to a group of disorders of heart and blood vessels, which mainly include
coronary heart disease (CHD), peripheral arterial disease, cerebrovascular disease,
rheumatic heart disease and so on. In general, the major risk factors of CVDs include
abnormal serum lipids and lipoproteins, diabetes, hypertension and obesity (WHO,
2011 ). More people die annually from CVDs than from any other cause. An estimated
17.5 million people died from CVDs in 2012, representing 31% of all global deaths. Of
these deaths, an estimated 7.4 million were due to coronary heart disease and 6.7
million were due to stroke (WHO, 2015)
Health concerns began to arise in the 1990s following reports that diets high in
total TFA were associated with increased risk of coronary heart disease through
increases in serum LDL cholesterol and decreases in HDL cholesterol (Willet et al.,
19
1993). TFA intake of ranging from 2.8 to 10 g per day was associated with a 22%
increase in risk of CHD events with a similar increased risk of fatal CHD (Bendsen et al.,
2011). Many years of epidemiological research have shown that populations consuming
diets high in saturated fatty acids show relatively high levels of serum cholesterol and
carry a high prevalence of coronary heart disease (Caggiula and Mustad 1997;
Kromhout and Lezenne 1984). Mensink and Katan (1990) reported that a diet with 10%
of energy from TFAs versus a diet with 10% of energy from oleic acid significantly (P<
0.001) increased LDL cholesterol (LDL-C) (14 mg/dl) and significantly lowered (P<
0.001) HDL cholesterol (HDL-C) (7 mg/dl). Saturated fat (10% of energy) also increased
LDL-C (18 mg/dl), but had no effect on HDL-C compared with oleic acid. The authors
concluded that the effect of TFAs on the serum lipoprotein profile is at least as
unfavorable as that of the cholesterol-raising saturated fatty acids (SFAs) because of
their similar LDL-C raising effects. Moreover, TFAs may be more detrimental because
they also lower HDL-C versus SFAs and it was established that TFA could be worse than
the saturated fatty acids. Soon after, Mozaffarian et al. (2006) reported that saturated fat
and TFA had similar effects on LDL-C. However, when compared with either saturated
or unsaturated fat, TFA reduced HDL-C and increased the ratio of total cholesterol to
HDL-C. TFA consumption also increased serum triglyceride and lipoprotein levels and
reduced LDL-C particle size in controlled trials indicating higher risk of coronary heart
disease. Although it is established that TFAs increase LDL levels and decrease HDL
levels (markers of coronary heart disease), little is known about the mechanisms by
which TFAs actually work at the cellular level. Moreover observational studies by
Mozaffarian et al. (2009) showed that a higher CHD risk is related to TFA from industrial
sources. Because ruminant fat contains low levels of TFA (<6% of fatty acids), the
amounts of ruminant TFA consumed are low in most countries studied (generally < 1
energy %). Thus, even when the total ruminant fat intake is relatively high, the potential
amount of TFA from this source is still quite modest. In the amounts actually consumed,
ruminant TFA is not a contributor to CHD risk.
20
Diabetes
Analysis of the Nurses' Health Study after 14 years obseNation showed that the
risk of the development of type-11 diabetes was associated with trans fatty acid intake.
Thus, an increase in polyunsaturated fatty acid intake and a decrease in TFA will
substantially reduce the risk of developing type-11 diabetes in women (Salmeron et al.,
2001 ). It was obseNed that as the intake of industrially produced trans fatty acids in the
USA is on average 3% energy, the replacement of 2% of energy from TFA could reduce
the incidence of type-11 diabetes by 40% if the fats containing the trans fatty acids were
consumed in their original unhydrogenated form (Meyer et al. 2001 ). However, the
effects of TFA were greater than SFA in increasing insulin resistance. Increasing dietary
linoleic acid did not prevented TFA induced increase in insulin resistance; it becomes
necessary to reduce the absolute intake of TFA (Ghafoorunissa, 2008). But the
prospective studies on male health professionals (Meyer et al., 2001; Van Dam et al.,
2002) reported that they did not find that consumption of TFA was significantly
associated with the risk of diabetes.
Cancer
Cancers of the breast, colon, rectum and prostate are the most common types of
cancer that have been extensively studied and related with fat intake. There is
conflicting evidence concerning the possible role of TFA in breast cancer. Kohlmeier et
al. ( 1997) reported the analysis of trans and cis fatty acids levels in blood serum of
women showed that breast cancer risk increased with the increase in trans fatty acid
level, reflecting processed food consumption. It was reported that women with elevated
serum levels of trans fatty acid have double the risk of developing breast cancer as
compared to women with the lower levels.
2.2.5 Regulation
Associations with adverse health effects have led to efforts to decrease the
intake of TFA by consumers, including labeling of food products for TFA content, most
recently in the United States and Canada (Department of Health, Canada, 2003; Food
21
and Drug Administration, U.S.A., 2003). Denmark is the first country banned partially
hydrogenated oils in 2003, and several other countries followed suit; in the United
States, New York City passed such a ban for restaurant foods in 2006, and the state of
California did the same in 2008 (Brownell et al., 2014). More than a decade after the first
ban, the Food and Drug Administration (FDA) has proposed a regulation that would
declare partially hydrogenated oils unsafe and allow only a small amount of remaining
artificial trans fats in foods sold in the United States (Department of Health and Human
Services, 2013). FDA required the food industry to declare the amount of trans fat in
food on the Nutrition Facts label since January 2006. The FDA requires that the Nutrition
Facts panel list the amount of trans fat in a serving of food if a serving contains 0.5 g or
more of trans fatty acids but trans fats with less than 0.5 g/serving can be listed as
containing 0 g. FDA data indicate that many processed foods have been reformulated to
reduce the amount of trans fat since the requirement was instituted, but a substantial
number of products still contain PHOs and in November 2013, based on a thorough
review of the scientific evidence, the FDA made a preliminary determination that PHOs
are not "generally recognized as safe" (GRAS) for use in food. This action is expected to
reduce coronary heart disease and to prevent thousands of fatal heart attacks each
year. Now, FDA has set a compliance period of three years. This will allow food
companies to either reformulate products without PHOs and/or petition the FDA to
permit specific uses of PHOs. (FDA, 2015).
2.3 lnteresterification
lnteresterification (IE) has been used for many years to modify edible fats and
oils. IE was introduced in the 1940s and commercialized in the 1950s (Hamm, 2000).
lnteresterified fats have been an ingredient in foods in the U.S. since the 1950s and
have been in widespread use there for the last 15 years as a substitute for partially
hydrogenated oils (Mercola, 2009). IE is one of the four modification processes to alter
the physical properties of oils and fats, the other three modification processes are
blending, fractionation and hydrogenation (ldris and Mat Dian, 2005). Techniques and
22
principles of fat modification are given in Table 4. Nowadays, IE has received much
interest in the edible oil industry due to the regulation mentioned above. IE is an acyl
rearrangement reaction on the triacylglycerol (TAG) molecules that involves fatty acids
redistribution between and within TAG molecules, the fatty acids of TAG exchange
positions from one glyceride to another, until reached a thermodynamic equilibrium thus
the fatty acid distribution is altered, but the fatty acid composition remains unchanged
and is an alternative process that alters the physical properties of fats without producing
the undesirable trans fatty acid (da Silva Lanners and Ignacio, 2013; ldris and Mat Dian,
2005; Rodrigues and Gioielli, 2003).
Table 4 Techniques of fat modification
Technique
Blending
Fractionation
Hydrogenation
Chemical interesterification (CIE)
Enzymatic interesterification (EIE)
Source: Cowan (201 0)
Principle
Mixing two or more different kind of fats
together to obtain the required melting
profile
Uses the different melting points of the fat
components to separate fractions with
significantly altered SFC by a process of
fractional crystallization
Uses a catalytic hydrogenation reaction to
increase saturation of the fat
Uses a chemical catalyst operating at high
temperature to promote random exchange
of fatty acids between two glycerides
Uses an enzyme catalyst to promote either
a random or a 1,3-specific (depending on
lipase type) exchange of fatty acids
between two glycerides
23
There are two types of interesterification currently in commercial use: chemical
interesterification (CI E) and enz ymatic interesterification (EI E).
2.3.1 Chemical interesterification
CIE has long been used to modify oils and fats into functional products. CIE is a
process which produces complete positional randomization of the acyl groups in the
triacylglycerols by use sodium methoxide or sodium ethoxylate as a catalyst (ldris and
Mat Dian, 2005). This process operates at moderately high temperature and occurs very
rapidly once started thus the equilibrium of process is reached very fast but uses a
catalyst which is non-specific therefore CIE is usually impossible for the producing of
such kind of TAGs due to the lack of positional specificity (steriospecificity) (Xu, 2000).
Although the reaction time is short, washing, bleaching and deodorization are required
after Cl E. The disadvantage of CIE is that a violent reaction, the catalysts are very
reactive and must be handled with extreme care to prevent contact with the skin or
eyes. The level of CIE cannot be controlled. The process produced by-product and yield
loss due to the post treatments (Asif, 2011; Cowan, 201 0).
2.3.2 Enzymatic interesterification
EIE has been used by microbiallipases as the catalyst to modification of oils and
fats for the production of specific-structured triacylglycerols (SSTs). EIE induces a
rearrangement of fatty acid within and between TAGs producing in new altered TAG
molecules same as CIE but EIE is more specific especially for 1 ,3-specific lipase, much
simpler, requires less severe reaction conditions, no requirement for any post treatment
of the interesterified oil afterward and produces less waste than CIE (Asif, 2011; ldris
and Mat Dian, 2005; Lee et al., 2008). Each type of interesterification has its advantages
and disadvantages that are summarized in Table 5.
24
Table 5 Comparison of chemical and enzymatic interesterification processes
Chemical interesterification
• Non-selective
• High temperature and high pressure
conditions
• Problem of environmental pollution
• High levels of reaction by-product
(monoacylglycerol; MAG,
diacylglycerol; DAG, glycerol)
• Require post treatment of interesterified
oil
• Low processing cost
• Use sodium methoxide as a catalyst
• Degrade minor compounds present in
the oil or fat as tocopherol
Enzymatic interesterification
• Selective (regia- and sterio- specificity)
• Mild reaction conditions
• Reduced environmental pollution
• Low levels of reaction by-product
• No require post treatment of
interesterified oil
• High processing cost
• Use lipases as a catalyst
• Decreased degradation of minor
compounds present in the oil or fat as
tocopherol
• Immobilized lipases can be used many
times
• No use of chemical or solvents
Source: Summarize from many studies: As if, 2011; Bornscheuer et al., 2003; Criado et
al., 2007; ldris and Mat Dian, 2005.
Two types of lipase were used for the production of margarine fats: sn-1,3-
specific and non-specific lipases. The reaction catalyzed by these two types of lipase
produce similar TAG component, but the proportion of each TAG is different because
non-specific lipase will result in a fully randomized product. The two reaction are
showed in Figure 4. The reaction between two triacylglycerols by sn-1,3 specific lipases
will result in similar triacylglycerol species to those produced by randomization, even
though their contents may not be totally the same (Mohamed et al., 1993). Chemical
25
interesterification produces randomized products in which fatty acids are randomly
distributed in the three positions of the glycerol backbone. Sn-1 ,3 specific lipase-
catalyzed interesterification produces similar triacylglycerol species, but the proportions
of each triacylglycerol specie are not the same as those of the randomized products
even at the same substrate molar ratios (XXXIYYY). Triacylglycerol species in each
rectangle should have the same content for randomized products and also for sn-3
specific lipase-catalyzed products provided that the sn-1 ,3 specific lipase has the same
specificity towards sn-1 and -3 positions and also different fatty acids. X and Y are
different fatty acids.
E: [ sodium methoxide E: IE:. E . E: +
lL_1' _____ 1' ___
~E~l· E: y
E [ sn-1.3 sped fie E ~E E: [ + + + lipases
[ E E E: y + + +
y
Figure 4 Reaction schemes for the lipase-catalyzed interesterification between two
triacylglycerols with chemical catalysts and sn-1 ,3 specific lipases.
Source: Xu (2000)
+
+
For the lipase-catalyzed interesterification, the first step of the reaction is the
hydrolysis that produced MAGs, DAGs and FFAs; the second step is the esterification
between the MAGs, DAGs and FFAs that forms new TAGs (Zhang et al., 2000).
26
( 1) Hydrolysis:
Hydrolysis of triglycerides is an essential reaction for the production of fatty
acids or special glycerides. Hydrolysis reaction involve an attack on the ester bond of
fats in the presence of water to produce glycerol, fatty acids (FA), and partial
acylglycerols, depending on the positional specificity of the enzyme (Akoh et al., 2002).
Hydrolysis of the oil blend requires water to produced MAGs, DAGs and FFAs. Although
using sn-1 ,3-specific lipase, the natural acyl migration from position 2 to positions 1 or 3
allows the full hydrolysis of the glycerides but this situation usually occurs in extreme
condition reaction (Figure 5). In other cases, the target of the reaction is the production
of di- or monoglycerides with a special composition (Rodrigues and Fernandez-
Lafuente, 201 0).
Figure 5 General scheme of the full hydrolysis of triglycerides catalyzed by Rhizomucor
miehei (RML) to produce free fatty acids
Source: Rodrigues and Fernandez-Lafuente (201 O)
(2) Esterification:
Esterification is the opposite of hydrolysis. The shift in equilibrium between the
Forward reaction (hydrolysis) and the reverse reaction (esterification) is affected by a
number of variables such as the water content of the reaction mixture. lnteresterification
involves the exchange of acyl groups or radicals between an ester (ester-ester
interchange) and two esters (two TAGs) (Akoh et al., 2002).
27
lnteresterification may be realized using a blend composed by several oils, just
one oil having different fatty acids or one oil and determined esters (Figure 6). It is one
of the most widely employed strategies to produce structured lipids using Rhizomucor
miehei (RML). In the case of glycerides, if we want to introduce a new fatty acid into the
glycerol moiety, the ester bond between the native fatty acid residue (the original
substituent group) and the glycerol moiety must first be hydrolyzed. This reaction
liberates the native fatty acid and produces a lower (less substituted) glyceride
containing at least one hydroxyl group. The hydrolysis step is followed by the formation
of a new ester bond by reaction of the newly created hydroxyl group with the incoming
replacing fatty acid (that needs to be also released from the ester) (Reyes and Hill,
1994)
RML
Figure 6 General scheme of interesterification catalyzed by Rhizomucor miehei (RML).
Source: Reyes and Hill (1994)
In many studies, the interesterification was used for producing trans-free
margarine and shortening fats in which fully hydrogenated vegetable oil, fractioned oil,
or vegetable oil were used as substrates. In the studies of Lee et al. (2008), they
produced interesterified plastic fats with trans-free substrates of fully hydrogenated
soybean oil, extra virgin olive oil and palm stearin in weight ratio of 10:20:70, 10:40:50
and 10:50:40, respectively, by lipase catalysis. The results showed that the produced
fats containing no trans fatty acids could be used as an alternative to partially
hydrogenated bakery fats. Siew et al. (2007) studied production of value-added
products by an enzymatic interesterification process from hard palm stearin (hPS) and
canola oil (CO) in weight ratio of 20:80, 30:70, 40:60, 50:50, 60:40 and 70:30 by using
28
immobilized Thermomyces /anuginosus lipase (Lipozyme TL IM). The results showed
that the interesterified blend of hPS/CO 40:60 is expected to be suitable for use as
vanaspati and as stick margarine. The interesterified blend of hPS/CO 50:50 is suitable
for use in margarines, puff pastry margarine and shortening applications while the
interesterified blend of hPS/CO 60:40 is suitable for use in shortenings, bakery and puff
pastry margarines applications. Adhikari et al. (201 Oa) studied production of trans-free
margarine stock by enzymatic interesterification of rice bran oil (RBO) and palm stearin
(PS) at three molar ratios (RBO:PS 1:1, 1:2 and 1:3), and coconut oil (CO) (40 wt% on
total weight of RBO and PS). The interesterified fat in molar ratios of RBO:PS 1 :3 showed
desirable physical properties and suitable crystal form (13' polymorph) for possible use
as a spreadable margarine stock. Adhikari et al. (201 Ob) produced interesterified
structured lipid from pine nut oil (PN) and palm stearin (PS) at two weight ratios (PN:PS
30:70 and 40:60) using lipase (Lipozyme TL IM, 30 wt%) as a catalyst at 65oC for 24h.
The results showed both different weight ratios of interesterified products contained
mostly 13' polymorphic form which is desirable property of margarine fat stock without
trans fatty acids. In another study, the studies of Ming et al. (1999) and Zhang et al.
(2004), the interesterified plastic fats of palm stearin and sunflower oil or coconut oil
catalyzed by lipases from Pseudomonas sp. or Rhizomucor miehei were suitable for
table margarine or shortening preparation.
2.3.2.1 Lipase
Lipases (E.C. 3.1.1.3; systematic name: triacylglycerol hydrolases) are a class of
enzymes that natural function is the hydrolysis of lipids to diacylglycerol,
monoacylglycerol, glycerol and free fatty acids during digestion. The reaction reverses
under anhydrous conditions and the enzyme is able to synthesize new molecules by
esterification, alcoholysis and transesterification. All reactions can be performed with
high regia- and enantioselectivity under mild reaction conditions. Lipases have been
used on a variety of substrates and show very broad substrate specificity due to the
ubiquity in nature and the heterogeneity of lipases from different sources. There are
29
widely distributed in animals, plants, and microorganisms. Lipases from these sources
vary in their specificity and stability even though they have a common reaction pattern
and substrate (lipids) (Akoh et al., 2002; Bornscheuer et al., 2003). Lipases are among
the most widely used enzymes in enzyme technology, because they recognize a wide
variety of substrates and may catalyze many different reactions: hydrolysis or synthesis
of esters bonds, alcoholysis, aminolysis, peroxidations, epoxidations, interesterification,
etc. The apparent promiscuity of these enzymes made lipases useful in many different
reactions, finding application in pharmaceuticals and drugs production, in the
production of biodiesel or in food modification.
The lipase from Rhizomucor miehei (RML), formerly Mucor miehei, is a
commercially available enzyme in both soluble and immobilized forms with very high
activity and good stability under diverse experimental conditions (anhydrous organic
solvents, supercritical fluids, etc.). Immobilized lipases are usually preferred for
interesterification processes, because immobilization generally improves lipase stability
and immobilized enzyme catalyst can be easily recovered for reuse. The uses of the
enzyme were initially oriented towards food industry, that way the enzyme has found a
broad application in this area. (Rodrigues and Fernandez-Lafuente, 201 0). The majority
of lipases are either nonspecific or sn-1 ,3 specific. Lipozyme RM IM from Rhizomucor
miehei is an immobilized sn-1 ,3 specific lipase and was used in this study. The widely
studied Lipozyme RM IM lipase (immobilized onto a weak anion exchange resin)
preferentially hydrolyzes short-chain acids relative to medium and long chains from
triacylglycerols. The sn-1 ,3 specific lipase allows the retention of the fatty acid s at the
sn-2 position while modifying at sn-1 ,3 positions (Akoh et al., 2002). Hydrolysis at the sn-
1 position is somewhat faster than at sn-3, and hydrolysis at sn-2 is very slow (Xu, 2000).
According to Barros et al. (2010), Macrae and Hammond (1985), Sonnet (1988)
and Villeneuve (2003), lipase specificities can be divided into three main groups as
follows:
30
1. Substrate specificity: the natural substrates are glycerol esters. Not only are
these enzymes able to catalyze the hydrolysis of triacylglycerols (TAGs), but also di
and monoacylglycerols and even phospholipids, in the case of phospholipases.
2. Regioselective- subdivided into:
I. Non-specific lipases: catalyze the complete hydrolysis of triacylglycerols into
fatty acids and glycerol in a random way, mono- and diacylglycerols being intermediate
products (Figure 7).
II. Specific 1.3 lipases: only hydrolyse triacylglycerols at the C1 and C3 glycerol
bonds, producing fatty acids, 2-monoacylglycerols and 1.2- or 2.3-diacylglycerols, the
latter two being chemically unstable, promoting migration of the acyl group producing
1.3-diacylglycerol and 1- or 3-monoacylglycerols (Figure 7).
Ill. Specific or selective type fatty acid: lipases can be specific for a particular
type of fatty acid or, more frequently, for a specific group of fatty acids. They hydrolyze
fatty acid esters located at any triacylglycerol position.
3. Enantioselective: the ability to discriminate enantiomers in a racemic mixture.
The enantio specificities of lipases can vary according to the substrate and this variation
can be connected to the chemical nature of the ester (de Castro and Anderson, 1995).
E~x ~y ~X
E~' ~y ~X
EOHo E~x o~x
non-specific lipase ~ ~ E -----• o 0 v ou o v ou OHo
~x OH oAx 1 ,3-specific lipase
Figure 7 Reactions catalyzed by non-specific and 1,3 specific lipases
Source: adapted from Paques and Macedo (2006)
2.3.2.2 Effects of different parameters on the enzymatic interesterification
2.3.2.2.1 Water content
31
The amount of water present in a reaction mixture influences biocatalysis in
several ways. In interesterification reactions, water is needed to initiate hydrolysis before
synthesis before synthesis of TAG can occur, but it has been proven that too much
water will discourage the esterification and instead promote hydrolysis (Ghazali et al.,
1995) resulting to decrease in the yield of synthesis and leading to more DAGs and
FFAs in the final products. Lipozyme RM IM contained sufficient water in the original
lipase preparation for efficient interesterification, so it is not necessary to add water to
the system. The hydrolysis reaction consumed not only the water from the lipase, but
also the water from the oil (Zhang et al., 2000). In esterification reactions, water is
produced and must be removed to enable the reaction to proceed toward synthesis of
esters. Removal of water from the reaction mixture can be achieved by use of molecular
sieves. The presence of water also causes acyl migration to occur, leading to formation
of a high level of MAGs as opposed to ester formation (Akoh et al., 2002).
2.3.2.2.2 Temperature
Temperature assist in solubilizing reactants for biocatalysis, it reduces the
viscosity of the reaction mixture which related to mass transfer and the overall reaction
rate. However, high temperatures cause denaturation and do not favor enzyme activity.
The optimum temperature for most immobilized lipases falls within the range of 30°C to
75°C, whereas it tends to be slightly lower (30-40°C) for free lipases (Malcata et al.,
1992). Lipozyme RM IM was relatively stable under varying temperatures in the range of
50-75°C. The degree of interesterification did not depend on temperature. In addition,
the higher temperature had a slight influence on increasing FFA contents. Therefore,
should to use a lower temperature in the above temperature range to favor a lower FFA
content in the products (Akoh et al., 2002; Zhang et al., 2000).
32
2.3.2.2.3 Enzyme load
The amount of the lipase and increased with increase of lipase load affect the
rate of interesterification. In the studies of Zhang et al. (2000) mentioned less than 6%
lipase load was not enough to reach a stabile degree of interesterification for the 6 h
reaction at 60°C. When the lipase load reached a certain level (more than 6%), the
reaction rate reached a steady state in which the degree of interesterification was only
little increased with an increasing enzyme load. However, the contents of FFA and DAG
were greatly increased by the increase of enzyme load because more water from
enzyme causes more hydrolysis.
2.4 Rice Bran Oil
Rice (Oryza sativa L.) is a major source of nourishment for the world's
population, especially in Asia. World production of rice is estimated at around 740
million tons. China is the largest rice producing country followed by India (FAOSTAT,
2013). Rice bran is the cuticle between the paddy husk and the rice grain and is
obtained as a by-product of rice processing. Rice is harvest from fields in the form of
paddy rice in which each kernel is fully enveloped by a tough, fibrous hull. After the rice
is dried, the first stage of the milling process is removal of the hull, which yields brown
rice. Next, the outer layer is removed from the brown rice kernels to yield white rice. The
separated outer (brown) layer is designated as rice bran (Figure. B)(Ryan, 2011 ). Rice
bran is a rich source of oil, protein, fiber and nutrients essential to life. Rice bran
contains 12-23% oil, which is high in polyunsaturates, monounsaturates and
extraordinary in heat stability. Furthermore, natural antioxidants such as tocopherol and
oryzanol are also present in RBO. Rice bran oil is widely used in pharmaceutical, food
and chemical industries due to its unique properties and high medicinal value. It is ideal
oil for margarine and shortening. The flavor gives the good palatability and the desired
prime form crystal provides smooth plasticity and spreading qualities (Mishra, 2013). All
these constituents contribute to the lowering blood serum cholesterol, have anti-cancer
properties and protect the body from free-radical damage. Rice bran also contains a
33
high level of dietary fibers and B-complex vitamins (Moreau and Powell, 1999; Yu et al.,
2006; Ani I Kumar et al., 2006; Anderson and Guraya, 2001; Kadam and Bhowmick,
2006; Gopala Krishna et al., 2005).
RICEHl.'SK 120"ol
RICE GEIDf U-2"o)
RICEBR.\X 17-S"ol
RICE GR.-\IX 170"ol
Figure 8 Structure of Rice with different layers
Source: Mishra (2013)
Rice bran oil (RBO) has many properties that contribute to its appeal as a
functional food ingredient. Rice brain oil contains approximately 85% TAGs, with the
remaining fraction consisting of waxes, mono- and diglycerides, free fatty acids, and
other compounds such as tocopherols, tocotrienols, and phytosterols (Hemavathy and
Prabhakar, 1987; Hu et al., 1996; Prasad et al., 2011). Crude oils of vegetable origin
contain impurities of varying types. These impurity levels are affected by storage and
handling as well as extraction processes. The typical composition of crude rice bran oil
is given in Table 6.
Table 6 The typical composition of crude rice bran oil
Composition Amount(%)
Triacylglycerides (TAG) 81-84
Diacylglycerides (DAG) 2-3
Monoacylglycerides (MAG) 1-2
Free fatty acids 2-6
Wax 3-4
Glycolipids 0.8
Source: Ghosh (2007)
Phospholipids
Unsaponifiable matters
1-2
4
34
The main fatty acids of rice bran oil are oleic acids and linoleic acids (Table 7)
and the major triglyceride are PLO, LOO, POO, LLO and 000 (P:Palmitic acid, O:Oieic
acid, L:Linoleic acid) (Adhikari et al., 2010a; Adhikari and Hu, 2012).
Table 7 Fatty acid composition of refined rice bran oil
Fatty acid Amount(%)
C14:0 0.4-0.6
C16:0 14-22
C18:0 0.9-2.5
C18:1 38-46
C18:2 33-40
C18:3 0.2-2.9
C20:0 ND-0.5
C20:1 ND-0.5
C22:0 ND-0.5
Source: Mishra (2013)
Rice bran oil is not only a nutritious vegetable oil, but also a special oil with
unique properties and many health benefits. Good stability, appealing flavor and long
fry-life enable rice bran oil be used for frying and also to make margarine and shortening
and advanced nutritional oils. More importantly, rice bran oil has been reported to have
a high potential for making pharmaceuticals and cosmeceuticals. Rice bran oil has
surprisingly high levels of nutraceutical components, such as oryzanol, fat-soluble
vitamins, sitosterol, other plant sterols and other nutrients (Liang et al., 2014).
35
2.4.1 Benefits of rice bran oil
The nutraceutical potential of tocotrienol, sterol, and y-oryzanol in rice bran oil
(RBO) aswell as its flavor, oxidative stability, and frying performance contributes to the
rising interest in RBO product in US markets (McCaskill and Zhang, 1999). RBO is the
main natural rich source of oryzanols (Patel and Naik, 2004). The y-oryzanol in rice bran
oil is a mixture of ferulic acid esters of sterols (campesterol, stigmasterol, and 13-
sitosterol) and terpene alcohols (cycloartanol, cycloartenol, 24-methylenecycloartanol
and cyclobranol). Selected compounds from rice bran have been investigated for
preventive and control of chronic disease via multiple mechanisms (Table 8).
Table 8 Selected bioactive compounds in rice bran evaluated for their properties with
regard to prevention of chronic disease
Rice bran compound
Ferulic acid y-Oryzanol Inositol hexaphosphate Campesterol B-Sitosterol linoleic acid
a-Tocopherol Tocotrienol Salicylic acid Caffeic acid Coumaric acid Tricin
Source: Ryan (2011)
Disease prevention activity
Antioxidant. chemopreventive, anti-inflammatory, and lipid-lowering effects Antioxidant, chemopreventive, anti-inflammatory, and lipid-lowering effects Blocks cancer growth and signaling Antiangiogenic Blocks cholesterol Anti-inflammatory
Inhibits lipid peroxidation and intracellular signaling Inhibits lipid peroxidation and intracellular signaling Anti-inflammatory Gastrointestinal microbe interactions Antimutagenic, inhibits the cell cycle, antioxidant, and chemopreventive Antimutagenic, inhibits the cell cycle, antioxidant, and chemopreventive
These compounds are of special interest to researchers because of the health
benefits they offer. The benefits include the ability to lower plasma cholesterol (Yoshino
et al., 1989), lower serum cholesterol concentration (Gerhardt and Gallo, 1998), reduce
cholesterol absorption and decrease early atherosclerosis, inhibit platelet aggregation,
and decrease fecal bile excretion (Seetharamaiah et al., 1990; Bucci et al., 2003).
Oryzanol has been used to treat hyperlipidemia (Nakayama et al., 1987), disorders of
menopause (Murase and Lishima, 1963) and also to increase the muscle mass (Boner
36
et al., 1991 ). Tocotrienols and tocopherols are natural forms of vitamin E, which together
with oryzanol in rice bran oil have antioxidant effects (Liang et al., 2014).
Following risks are reported to be associated with the intake of y-oryzanol on
human health.
Hyperlipidemia
One of the most important properties of y-oryzanol is its cholesterol lowering
properties and it is also considered effective in the treatment of hyperlipidemia because
of its potential to decrease cholesterol and triglyceride levels, as well as boost the ratio
of high-density lipoprotein (HDL) level. There are several studies on humans and
animals (Sharma and Rukmini, 1986; Rukmini, 1988; Nicolosi et al., 1991; Kahlon et al.,
1992; Hegsted and Windhuser, 1993) showing that the RBO has the property of lowering
low density lipoprotein cholesterol and total serum cholesterol and increasing the high
density lipoprotein cholesterol to some extent either by influencing absorption of dietary
cholesterol or by enhancing the conversion of cholesterol to fecal bile acids and sterols.
Cholesterol-lowering action of y-oryzanol appears to involve a combination of the
following effects: y-oryzanol increases the conversion of cholesterol to bile acids,
increases bile acid excretion, and inhibits the absorption of cholesterol. Further studies
confirm that the y-oryzanol component of RBO is responsible for the
hypocholesterolemia (Sasaki et al., 1990; Lichtenstein et al., 1994; Sugano and Tsuji,
1997). Several studies indicate that y-oryzanol is quite effective in lowering blood
cholesterol and triglyceride levels as follows.
Hata et al. (1981) reported that y-oryzanol decreased 62.7% of serum
cholesterol and 51 %of serum triglyceride by administration of 300 mg per day for 4
months. Go to et al. ( 1983) reported that y-oryzanol decreased 6 to 38% of serum
cholesterol and 14 to 39% of serum triglyceride by administration of 75 mg per day for 4
months, and decreased 24 to 64% of serum cholesterol and 23 to 61% of serum
triglyceride by administration of 300 mg per day for 4 months. Moreover, Cicero and
Gaddi (2001) have studied the effect of rice bran oil and y-oryzanol in the treatment of
hyperlipoproteinamia. When added to a high cholesterol diet it also inhibits platelet
aggregation, preventing heart attacks and strokes (Kaimal, 1999).
2.5 Palm Stearin
37
Palm oil is extracted from the mesocarp of the fruit of oil palm, Elaeis guineensis.
It is one of the traditional fats that have been widely used throughout the world in the
human diet. Global palm oil production was estimated to 54.3 millions of tons in 2013.
Malaysia is the largest palm oil producing country followed by Indonesia (FAOSTAT,
2013). Palm oil exhibits several excellent properties such as high productivity, low
price, ease of the formation of 13' type crystal, high thermal oxidative stability and
plasticity at room temperature conditions (Hodate et al., 1997). Therefore, palm oil is
considered to be the most economical and abundant edible oil worldwide in the near
future (Tan et al., 2009) and has been widely used as cooking oil, margarine, shortening
in cooking, confectionery, bakery etc. However, the application of palm oils in their
original form is limited due to their specific chemical composition. Palm oil consists of
high melting point triacylglycerols (TAGs) and low melting point TAGs, resulting in a
broad melting range. In practice, the modification operation of hydrogenation or
interesterification (chemical or enzymatic) or fractionation on the physicochemical
properties of palm oil would extend its use in food industry (Zhang et al., 2013). Palm oil
products and their fractions have become important raw materials in food products as
such or after modifications. Palm oil has unique fatty acid and triacylglycerol (TAG)
compositions which makes it suitable for numerous food applications. It could be
physically modified by fractionation while hydrogenation and interesterification will
modify its chemical characteristics (Aini and Miskandar, 2007; Sahri and ldris, 201 0).
Palm oil contains a mixture of high and low melting points triacylglycerols. It is
high in palmitc acid (44%) and crystallizes in the 13' form which is desirable in
margarines and shortenings (Aini and Miskandar, 2007). Palm oil can be separated
under controlled thermal condition into two fractions, namely olein (liquid fraction) and
stearin (solid fraction) (Lai et al., 2000). These fractions have distinct physical and
38
chemical properties, yet keeping the chemical and physical properties of the olein to
within a very narrow range of values as shown in Tables 9 and 10 and TAG composition
of palm oil and palm stearin also showed in Table 11. Palm stearin, obtained by
fractionation of palm oil after crystallization at a controlled temperature, can be used in
formulation of margarines and shortenings as it provides strength and structure to the
product (Aini and Miskandar, 2007).
Table 9 Fatty acid compositions of palm olein and palm stearin (%)
Fatty Acids Oleins Stearin
12:0 0.1-0.5 0.1-0.6
14:0 0.9-1.4 1.1-1.9
16:0 37.9-41.7 47.2-73.8
16:1 0.1-0.4 0.05-0.2
18:0 4.0-4.8 4.4-5.6
18:1 40.7-43.9 15.6-37.0
18:2 10.4-13.4 3.2-9.8
18:3 0.1-0.6 0.1-0.6
20:0 0.2-0.5 0.1-0.6
Source: Tan and Oh (1981)
Table 10 Melting and solidification characteristics of palm olein and palm stearin
Tests
Iodine value
Slip melting point (°C)
Cloud point (OC, crude)
Source: Tan and Oh (1981)
Ole ins
56.1-60.6
19.4-23.5
6.6-14.3
Stearin
21.6-49.4
44.5-56.2
Table 11 TAG composition(%) of palm oil, palm stearin
Palm oil Palm stearin TAG species
IV52.3 IV 34.3
LLL 0.5 0.3
PLLIMOL 2.7 1.5
OOL 1.9 1.1
PLO/SLL 10.7 5.9
PLP/MOP 10.4 7.5
MPP 0.4 2.0
POO 22.7 12.9
POP/PLSt 30.3 27.5
ppp 6.1 26.5
StOO 2.5 1.5
POSt 5.5 4.8
PPSt 1.2 5.3
StOSt 0.7 0.5
PStSt 0.1 0.6
uuu 6.0 3.4
SUU-USU 38.6 21.8
SSU-SUS 47.5 40.7
sss 7.9 34.1
Abbreviation: M: myristic; P: palmitic; St: stearic; 0: oleic; L: linoleic; S: saturated fatty
acid; U: unsaturated fatty acid; IV: iodine value
Source: Kellens et al., 2007
39
Palm stearin, the cheaper high-melting fraction from palm oil, can be used as a
source of fully natural hard component in the manufacture of solid fat products such as
shortenings, margarines and fat spreads. Palm stearin is available in a wide range of
melting points and iodine values. However, because of its high melting point (44°C-
40
56°C), palm stearin poses problems in the manufacture of the solid fat products as it
confers low plasticity to the products and does not completely melt at body temperature
(Pantzaris, 2000). Nevertheless, palm stearin deserves attention as a potential hard fat
of vegetable origin to replace hydrogenated lipids. It might be appropriately blended
and interesterified with liquid oils in order to modify the physical characteristics of the
mixture to meet the functional properties and the quality required for margarine
preparation (Sellami et al., 2012). Palm stearin is known to promote the formation of
desirable plastic fats when it is interesterified with vegetable oils (Ghotra et al., 2002)
and has also been used with several other oils such as rice bran oil and coconut oil
(Adhikari et al., 201 Oa), pine nut oil (Adhikari et al., 201 Ob), canola oil and palm kernel
oil (Kim et al., 2008) in enzymatic interesterification to produce trans-free margarine fat
analogs. In addition, During handling and storage where there are fluctuations in
temperature, palm stearin prevents the products from melting and thus helps maintain
the product's structure and shape so that they will not collapse and are able to
withstand the temperature fluctuations (Aini and Miskandar, 2007) ..
2.6 Crystallization of fats and oils
The physical properties of the food fats (gloss, hardness/softness, melting,
texture, rheology, and spreadability) are influenced primarily by three factors: ( 1)
polymorphism (structural, crystallization and transformation behavior); (2) the phase
behavior of fat mixtures; and (3) the rheological and textural properties exhibited by fat
crystal networks (Sa to and Ueno, 2014).
TAG crystallization forms the basis for the development of a fat crystal network
that is directly related to the macroscopic properties of the end products such as
spreadability, hardness and mouth feel. As such, the TAG composition of a fat system is
of primary importance for the consumers' perception of the end products (Narine and
Marangoni, 1999; Marangoni, 2002; Sato, 2001 b). Small changes in the TAG
composition can have a big impact on the crystallization and polymorphic properties of
the fats, as demonstrated in the important reviews of Timms (1984), Sato (2001 b) and
41
Himawan et al. (2006). In food systems, fat crystal number, size (both the mean size and
the distribution) and polymorph all affect the physical and textural properties, and
subsequent sensory properties (e.g. appearance, mouthfeel or flavour release), of the
final product. Crystallization consists of successive stages: super cooling of the melt,
nucleation, and crystal growth; crystals normally do not remain single but tend to
agglomerate (Kellens et al., 2007).
2.6.1 Nucleation and growth
Crystallization is a two stage process, involving the formation of nuclei followed
by crystal growth. Crystallization occurs providing that the phase is supersaturated (i.e.
the concentration of dissolved species is greater than the equilibrium concentration, i.e.
the concentration where molecules are in the equilibrium state). This can be achieved
by cooling (i.e. cooling below the equilibrium point) until a free-energy barrier is
overcome and a stable nuclei is formed. When nucleation does occur there is a release
of energy (latent heat of fusion) as the molecules assume the lower energy state in the
crystal lattice.
Nucleation can either be as primary (homogeneous or heterogeneous) or
secondary nucleation. Primary nucleation occurs in the absence of crystals (i.e. straight
from liquid state to crystal formation), whilst secondary nucleation occurs if crystals of
the same species are already present. Homogeneous nucleation occurs when there is
an accumulation of molecules in the liquid state until a nucleus forms, whereas in
heterogeneous nucleation foreign nucleating sites (i.e. impurities) catalyst nucleation
(reducing the free-energy required for nucleation). Secondary nucleation occurs when
new nuclei are formed in the presence of exist in crystals, typically due to fragments of
growing crystals that are chipped off and act as nuclei, collisions between two crystals,
or between a crystal and a surface, such as a stirrer or vessel walls. In industrial settings
nucleation can be promoted with the addition of crystal seeds.
Once nuclei have formed they grow by the incorporation of other TAG molecules
from the liquid phase into the surface of the crystal lattice. In order for growth to occur,
42
molecules must migrate to the surface of the crystal (diffusion), and orient to an
appropriate site for incorporation into the lattice (deposition). This leads to a release of
latent heat; this energy must be transported away from the crystal surface to prevent
temperature increase, or no further growth can occur. Crystal growth continues, typically
in an exponential manner, until there is a phase equilibrium or the entire system is
crystallized. The morphology of the crystal is determined by the composition of the
mixture, and by growth rate. The structure is modified as the size of the crystals
increases overtime due to Ostwald ripening: small crystals dissolve in solution, while
larger crystals grow as they are energetically favored.
Whilst in the liquid state TAGs are miscible, on crystallization the different TAG
species can come together to form 1) solid-solution mixtures (when the TAGs are very
similar in melting temperature, molecular volume and polymorphism; the melting point of
the mixture is between that of the pure components), 2) eutectic mixtures (when the
TAGs differ in chain-length, molecular volume, shape and polymorph, but have similar
melting points; the melting point of the blend is lower than the melting point of the
individual components) or 3) compound crystals (synergistic compatibility between two
TAGs, improving packing ability) (Sato and Ueno, 2005). In TAG mixtures phase
behavior is affected by chain length, degree of saturation, nature of any double bonds,
and arrangement of the fatty acids on the glycerol backbone.
Cooling rate has a dramatic effect on crystallization, affecting both crystallization
rate (as a result of time to rearrange into a crystal lattice), and nucleation rate, which
governs crystal size; rapid cooling to a low temperature promotes a higher nucleation
rate, resulting in many small crystals. Shear also affects nucleation and crystal growth,
due to mechanical disturbance that supplies energy to overcome the energy barrier for
nucleation, and promotes secondary nucleation. Post-crystallization processes include
the bridging between crystals to form a network (Douaire et al., 2014).
43
2.6.2 Polymorphism
The TAG molecule can crystallise into different crystalline forms in the crystal
lattice (i.e. conformation and arrangement), depending on processing conditions (e.g.
cooling rate and shear), a phenomenon termed polymorphism. In lipids, differences in
hydrocarbon chain packing and variations in the angle of tilt of the hydrocarbon chain
packing differentiate polymorphic forms. The arrangement of the molecules into the
crystalline state depends on such factors as the cooling rate, the temperature at which
crystallization occurs, the agitation rate, and the composition of the lipid phase (Metin
and Hartel, 2005). Rapid cooling of liquid fat results in the formation of a diffuse
crystalline phase (low-energy polymorph), whereas slower cooling means that the
molecules have time to organize into lamellae to form consistent, three-dimensional
crystals. Polymorphic behavior, such as melting point, is affected by the chain length of
the fatty acids within the TAG (Acevedo et al., 2011; Himawan et al., 2007).
The crystallization behavior of TAG, including crystallization rate, crystal size,
morphology, and total crystallinity, are affected by polymorphism. The molecular
structure of the TAG and several external factors such as temperature, pressure, rate of
crystallization, impurities, and shear rate were influence polymorphism (Sato, 2001a).
Liquid molecules in lipid move around at random. Once the lipid is cooled,
crystals occur in polymorphs which can be distributed to the different patterns of
molecular packing (Lee et al., 2008). The basic physical properties of the three typical
polymorphic modifications of a, 13' and 13 are summarized in Table 12. Polymorph a is
least stable, easily transforming to either the 13' form or the 13 form, depending on the
thermal treatment. Polymorph 13', the meta-stable form and intermediate in melting point,
is used in margarine and shortening because of its optimal crystal morphology and fat
crystal networks, which give rise to optimal rheological and texture properties. The most
stable 13 form and has the highest melting point tends to form large and plate-like crystal
shapes, resulting in poor macroscopic properties in shortening and margarine (Timms,
1984; Sato and Ueno, 2014). A conversion from one form to another (i.e., 13' to 13)
generally occurs in the direction that favors more stable forms.
44
Table 12 Three typical polymorphic forms of fats and their main physical properties
Form Stability Density Melting point Morphology
a Least stable Lowest Lowest Amorphous-like
13' Metastable Intermediate Intermediate Rectangular
13 Most stable Highest Highest Needle-shaped
Source: Sato and Ueno (2014)
The three main polymorphs of fats, a, 13' and 13. are defined in accordance with
subcell structure. Based on the unique configuration of the molecules within the crystal
lattice, each polymorph has a different crystallographic unit cell: a polymorphs have a
hexagonal subcell (H); 13' polymorphs have an orthorhombic-perpendicular subcell
(O.L); and 13 polymorphs have a triclinic-parallel subcell (T;;) (Larsson, 1966; see Figure 9
(a)). The subcell structures can be determined most clearly by measuring X-ray
diffraction (XRD) short spacing patterns of poly-crystalline samples. Each polymorphic
form has distinct short spacings (the distances between parallel acyl groups on the
TAG) that are used to distinguish the polymorphic forms based on their X-ray diffraction
patterns, as summarized in Table 13.
Table 13 Identification of polymorphic forms of fats Based on X-ray analysis of short
spacings
Polymorphic Form
a
Unit Cell
Hexagonal
Orthorhombic
Triclinic
Source: Larsson ( 1994)
Lines and Short Spacings (A)
A single strong and very broad at 4.15
Two strong lines at 4.2 and 3.8
A strong line at 4.6
A TAG molecule
Hexagonal (H)
double
(a)
Orthorhombic perpendicular (0_)
(b)
Triclinic parallel (T )
triple quatro hexa
Long spacing
Figure 9 Polymorphic structures of three typical forms of triacylglycerol (TAG). (a)
Subcell structures and (b) chain length structures.
Source: Sato and Ueno (2014)
45
Figure 9 (b) shows the chain-length structure, illustrating the repetitive sequence
of the acyl chains involved in a unit cell lamella along the long-chain axis (Larsson,
1972). A double chain-length structure (DCL) is formed when the chemical properties of
the three acid moieties are the same or very similar. In contrast, when the chemical
properties of one or two of the three chain moieties are largely different from those of the
moieties, a triple chain-length (TCL) structure is formed because of chain sorting. The
relevance of the chain-length structure is revealed in the mixing phase behavior of the
different types of the TAGs in the solid phase: when the DCL fats are mixed with the TCL
fats, phase separation readily occurs. The chain length structures can be determined
only by measuring the XRD long spacing patterns of the poly-crystalline samples.
46
2. 7 Fractionation
Fractionation (also called fractional crystallization) refers to a separation process
in which edible fats are separated into solid and liquid phases based on the solubility of
triacylglycerol species with different degree of unsaturation, at a controlled temperature.
Industrial-scale fractionation is classified into three main groups, such as dry
fractionation, detergent fractionation, and solvent fractionation (Kellens et al., 2007). The
main advantage of solvent fractionation is the great separation efficiency and the
enhanced yield of the targeted phase compared to that with the other methods (Kang et
al., 2013).
2.7.1 Solvent fractionation
In solvent fractionation, crystallization is performed in dilute solutions, thereby
reducing viscosity. Mainly acetone or hexane is used as a solvent. The process is
characterized by a short crystallization time and easy filterability. The main advantage of
solvent fractionation is the high separation efficiency and hence improved yield and
higher purity of the finished products. The major reason for this can be explained quite
simply by the fact that with any type of separation technique, it is not possible to remove
all liquid from the solid portion. Part of the liquid will always remain entrapped within the
solid phase. The amount of liquid is strongly dependent on the origin of the fatty matter
as well as on the crystallization conditions and the separation technique applied. In a
dilute solution, as is the case in solvent fractionation, this liquid portion consists of a
considerable quantity of solvent that hinders oil occlusion. After separation, the solvent
is evaporated, leaving behind a much lower quantity of liquid oil in the solid phase
(Kellens et al., 2007).
2. 7.2 Dry fractionation
Dry fractionation is the simplest and cheapest fractional crystallization process,
qualified as "natural" or "green" technology (no effluent, no chemicals, no losses). It
simply consists in a controlled crystallization of the melted oil, conducted according to a
47
specific cooling program followed by separation of the solid from the liquid fraction. Due
to the continuous developments of the dry fractionation process, a whole variety of
fractions normally produced by solvent or detergent fractionation can now be obtained
with a high degree of selectivity with dry fractionation (multistage).
In dry fractionation, crystallization operates in the bulk and for this reason
viscosity problems limit the degree of crystallization of the fat. Multi-step operations are
therefore used, with the advantage of producing a wide range of fractions suitable for
different applications.
The process consists in two steps: the crystallization stage which produces solid
crystals in a liquid matrix and the separation stage where the liquid phase (olein) is
separated from the crystals (stearin) (Kellens et al., 2007).
3.1 Materials and reagents
3.1.1 Main materials
Chapter 3
Methodology
3.1.1.1 Palm stearin (PS) was obtained from Lam Soon (Thailand) PCL.
3.1.1.2 Rice bran oil (RBO) was obtained from Lam Soon (Thailand)
PCL.
3.1.1.3 Lipozyme RM IM, an sn-1 ,3-selective lipase from Mucor miehei,
was purchased from Sigma Aldrich (Denmark).
3.1.1.4 Molecular sieves (3A, beads, 4-8 mesh) were purchased from
Aldrich Chemical Company (Milwaukee, WI, USA).
3.1.1.5 Six different commercial margarines were purchased from local
supermarkets in Bangkok, Thailand.
3.1.1.6 Triacylglycerol standard; PLL, PPP, POP, POO, 000 were
purchased from Sigma Aldrich (Denmark) and TRC (Canada).
3.1 .2 Chemical and reagents
3.1.2.1 Acetone AR grade (Labscan Asia Co. Ltd., Bangkok, Thailand)
3.1.2.2 Acetone HPLC grade (Labscan Asia Co. Ltd., Bangkok, Thailand)
3.1.2.3 Acetonitrile HPLC grade (Labscan Asia Co. Ltd., Bangkok,
Thailand)
3.1.2.4 Hexane (Macron TM Fine Chemicals, Center Valley, PA, USA)
3.1.2.5 Lithium Chloride anhydrous (Ajax Finechem Pty Ltd)
3.1.2.6 Potassium hydroxide (Merck, Darmstadt Germany)
3.1.2.7 Ethanol 95%(v/v)
3.1.2.8 Phenolphthalein (Ajax Finechem Pty Ltd)
3.1.2.9 Sodium hydroxide (Ajax Finechem Pty Ltd)
48
3.1.2.1 0 Sodium chloride
3.1.2.11 n-Heptane
3.1.2.12 Boron trifluoride (Sigma Aldrich, Denmark)
3.1.2.13 Distilled water (V Unique, Better Syndicate Co., Ltd)
3.1 .3 Equipment and analytical machine
3.1.3.1 Beaker 25, 50, 100, 250, 500, 600 and 1000 ml
3.1.3.2 Spatula
3.1.3.3 Forcep
3.1.3.4 Stirring rod
3.1.3.5 Separating funnel
3.1.3.6 Pipette 1, 2, 5, 10 and 25 ml
3.1.3.7 Burette
3.1.3.8 NMR tube
3.1.3.9 Magnetic bar
3.1.3.1 0 Glass syringe and syringe filter 0.45 !Jm
3.1.3.11 Glass slide and cover slip
3.1.3.12 Clear glass vial and amber glass vial
3.1.3.13 Clear glass bottle and amber glass bottle
3.1.3.14 Hot plate (IKA ® C-MAG HS 7)
3.1.3.15 Hot plate with magnetic stirrer (Heidolph, MR Hei-Standard)
3.1.3.16 Weigh balance (Denver S-4002)
3.1.3.17 Weigh balance 4 decimal point (BP221 S, Sartorius, Germany)
3.1.3.18 Thermometer
3.1.3.19 Dessicator
3.1.3.20 Water bath (Memmert, Germany)
3.1.3.21 Heating circulator bath (Thermo Scientific HAAKE DC1 0)
3.1.3.22 Cooling circulator bath (Thermo Scientific HAAKE DC10)
3.1.3.23 Water-jacketed reactor 100 ml (Lenz)
3.1.3.24 Magnetic stirrer (IKA ®color squid IKAMAG® white)
49
3.1.3.25 Hot air oven (Memmert, Germany)
3.1.3.26 Flat-bottomed flask 50, 100 and 250 ml
3.1.3.27 Rotary evaporator (Buchi V-805 and R-805, Switzerland)
3.1.3.28 Vacuum pump (ME 1 C, Becthai Bangkok Equipment &
Chemical Co., Ltd)
50
3.1.3.29 High performance liquid chromatography- UV detector (HPLC)
(Shimadzu, Japan SPD-M20A)
3.1.3.30 Polarized Light Microscopy (PLM) (OiympusCH-2, Japan)
3.1.3.31 Digital camera with adapter for connect with PLM Olympus
CAMEDIA digital camera (model NO. C-7070 Wide Zoom,
7.1 Megapixel, Japan)
3.1.3.32 Pulse-Nuclear Magnetic Resonance (p-NMR) (BRUKER, the
minispec mq20)
3.1.3.33 X-ray Diffractometer (XRD) (Rigaku TTRAX Ill, Rigaku
Corporation, Tokyo, Japan)
3.1.3.34 Differential Scanning Calorimeter (DSC) (Perkin Elmer Pyris
Diamond, DSC 8000)
3.1.3.35 Gas chromatography with flame ionization detector (GC-FID)
(Shimadzu, Japan)
51
3.2 Methods
3.2.1 Material preparation
3.2.1.1 Fractionation of palm stearin
Palm stearin is solid fraction of palm oil which was obtained from Lam Soon
(Thailand) PCL. Palm stearin was solvent-fractionated using acetone (5 mllg palm
stearin) at 35°C for 3 h (Ghosh and Bhattacharyya, 1997) to obtain 19.8% wt. of solid
fraction (called hard palm stearin: hPS) and 81.2% wt. of liquid fraction.
3.2.1.2 Substrate preparation
hPS was melted and then blended with RBO into seven physical blends of
hPS/RBO: 20/80, 30/70, 40/60, 50/50, 60/40, 70/30 and 80/20 (% wt.). Immobilized
Lipozyme RM IM was conditioned in a desiccator using lithium chloride (LiCI) to reduce
the water activity (aw) content of the enzyme to 0.12.
3.2.2 Study of triacylglycerol and fatty acid compositions of the initial fat and oil
and the physical blends
The initial fat and oil and the physical blends were analyzed the compositions of
triacylglycerol (TAG) and fatty acid as followed
3.2.2.1 Analysis of triacylglycerol composition
The TAG compositions of the samples were determined by with system
controller CBM-20A and diode array detector SPD-M20A. Two reverse C-18 columns
(lnertsil ODS-3; 4.6 x 250 mm; 5 iJm particle size; by GL Sciences Inc., Japan) were
used in series. The mobile phase consisted of acetone and acetonitrile (63.4 : 36.4,
vol/vol) with a flow rate of 1.00 ml/min. The sample was dissolved in acetone at
concentration of 10 mg/ml before injection. The column temperature was set at 25oC
with a column heater (Shimadzu CT0-1 OAS column oven). The injection volume was 20
iJL. Peak identification for TAGs was performed by comparing their retention times with
those of authentic TAG standards. All TAG contents were given in percentage area.
3.2.2.2 Analysis of fatty acid compositions
The samples were converted into fatty acid methyl esters following AOAC official
method 969.33 (see the details in Appendix II). The fatty acid methyl esters analysis was
52
performed in a Shimadzu GC with flame ionization detector (GC-FID). The system had
an ATTM-WAX capillary column (50 m long, 0.25 mm internal diameter and 0.20 ~m film
thickness). Compound identification was carried out using extemal standards of fatty
acids methyl esters. Helium was used as a carrier gas with a flow rate of 0.5 ml/min and
with a controlled initial pressure of 93.2 kPa at 120°C. N2 and air were makeup gases.
The injection temperature was 21 ooc. and the oven temperature program was holding at
120°C for 3 minutes before increasing at a rate of 1 ooc/min to 220°C, holding at this
temperature for 30 minutes, increasing at a rate of soc/min to 240°C, followed by
holding at 240°C for 30 minutes. The split ratio was 100:1, the injection volume was 1 ~1.
and the detector temperature was 280°C. All fatty acid contents were given based on
percentage area.
3.2.3 Study of changes in triacylglycerol compositions during enzymatic
interesterification
The small-scale lipase-catalyzed interesterification reactions were performed in
glass vials to obtain optimal reaction time which rendered the highest conversion ofT AG
for each ratio of the fat blends. The melted physical blend of hPS and RBO was reacted
with 10% wt. of conditioned immobilized Lipozyme RM IM and 10% wt. of molecular
sieves for 2, 4, 6, 8, 12, 16 and 24 h at 60°C and the mixing speed was set at 300 rpm
using a magnetic stirrer. After the reaction, the immobilized enzyme and the molecular
sieves were removed from the mixture by filtration. Free fatty acids formed during the
reaction were removed using the procedure describe in Appendix Ill (Aiim et al., 2008).
The TAG profile of reacted samples at various reaction times was then analyzed by
HPLC. The enzymatic reactions were carried out in duplicate for each ratio of hPS/RBO
blends. Changes in quantity of the main TAG components in the blends were plotted
against the reaction time and the optimum reaction time for each blend was determined
from the time when the changes in TAG compositions reached an equilibrium or a
plateau. Then, a scale-up reaction was performed for each blend in a double-jacket
batch type reactor using the pre-determined optimal reaction time with the same amount
53
of enzyme, amount of molecular sieves, reaction temperature and mixing speed. After
that, the interesterified blends together with the physical blends and the fats extracted
from the commercial margarines were characterized using various techniques as
mentioned below.
3.2.4 Study of physical properties and crystallization behavior of interesterified
fat at different weight ratio and compare with commercial margarine fats
lnteresterified fats at different weight ratio were analyze physical properties and
crystallization behavior followed by
3.2.4.1 Characterization of solid fat content
Changes in the solid fat content (SFC) of the physical and interesterified blends
and the commercial margarine fats (extraction method showed in Appendix IX) as a
function of temperature between 15oc and 40oc and melting behavior were determined
by pulse-nuclear magnetic resonance (p-NMR) spectrometer (Minispec-mq20,
BRUKER, Karlsruhe, Germany) following an AOCS official method Cd 16b-93. See the
details in Appendix IV.
3.2.4.2 Characterization of crystallization and melting profiles
The melting and crystallization characteristics of the physical and interesterified
blends and the commercial margarine fats were determined using a Perkin-Elmer
differential scanning calorimeter (DSC) (Model 08000, Perkin-Elmer Co., Shelton, CT,
USA) following an AOCS recommended procedure Cj 1-94 (Appendix V). The profiles
were analyzed by the software provided with the DSC (Pyris software, Perkin-Elmer,
Shelton, CT, USA). The crystallization onset (T co) and melting completion temperatures
(T Mc) were obtained from the peaks located at the highest temperature of each fat
sample. T co and T Mc were considered to be the temperatures at which the crystallization
began and the melting ended, respectively. Slip melting point (SMP) also was
determined using method describe in Appendix VI.
54
3.2.4.3 Polymorphic characterization
The crystal polymorphic forms of the interesterified blends and the commercial
margarine fats were determined by an x-ray diffractometer (Rigaku TTRAX Ill, Rigaku
Corporation, Tokyo, Japan). Scans were performed in wide angle x-ray scattering
(WAXS) from 3° 28 to 35° 28 with a scan speed and a step width of 4 o 28/min and 0.01 o
28, respectively. The samples were melted at 800C for 10 min and pores into rectangular
plastic moulds (20 mm x 25 mm x 3 mm) and then left to crystallize at 4°C and 25oC for
24 h, after which they were analyzed (Kim et al., 2008). Generally, short spacings for the
13' form of fats are identified with two strong spacings at 3.88 and 4.20 A or three strong
spacings at 3.71, 3.97 and 4.27 A (D'Souza et al., 1990). In addition, a strong short
spacing at 4.36 A has also been reported for the ~· polymorph of palm oil and palm
stearin (Yap et al., 1989). In contrast, ~ polymorph always displays a strong d-spacing
at 4.6 A (D'Souza et al., 1990). In this work, the content of~· and ~structures in the
samples was estimated by the relative intensity of the short spacings of ~· form at 4.20 A
and 3.80 A and that of ~ form at 4.60 A following a method described by Kim et al.
(2008) (see the details in Appendix VII).
3.2.4.4 Crystal microstructure
Crystal network microstructure of the physical and interesterified blends and the
commercial margarine fats crystallized under static conditions at 4 oc and 25°C was
observed by polarized light microscopy (PLM) (Olympus BX51, Olympus Optical Co.,
Ltd., Tokyo, Japan) equipped with a digital camera (Olympus C-7070, Olympus Optical
Co., Ltd., Tokyo, Japan). See the method in Appendix VIII.
3.2.5 Summary of optimal condition for producing trans-free margarine fat
Criteria for selection of the best interesterified fat to produce trans-free margarine fat:
0 Physical properties and crystallization behavior same as or similar to extracted
fat from commercial margarine
0 Tend to crystallize in 13' form > ~form
0 Small crystals and with needle-shaped crystals
Trans-free margarine fats were determined trans-free fatty acid content and summary
optimal weight ratio and optimal reaction time for producing trans-free margarine fat
3.2.6 Statistical analysis
55
All experiments were done in duplicate and the results were analyzed by
Analysis of Variance with Least Significant Difference (ANOVA/LSD) and compared the
results data by Duncan at 95% confidence interval by SPSS version 16.0
Chapter 4
Results and Discussion
4.1 TAG Composition changes during interesterification
Figure 10 shows changes in the relative quantity (in area percent) of different
Triacylglycerol (TAG) species as a function of reaction time for hPS/RBO blends 20:80
(Figure 1 Oa) and 40:60 (Figure 1 Ob). Increasing the reaction time resulted in an increase
in the amount of PLO, PLP and POP and a concurrent decrease in the quantity of LLO,
LOO, PLL, PPP and 000. This indicated that the exchange of palmitic, oleic and linoleic
acids in TAG species of hPS and RBO was taking place at each other's TAG backbone
to form new TAG. In most blends, TAG species changed during enzymatic
interesterification and reached equilibrium after 2 h of reaction time (e.g. blend 40:60 as
shown in Figure 10b). However, for some blends, the exchange of fatty acids in TAG
species took 4-6 h before the equilibrium was reached (e.g. blend 20:80 as shown in
Figure 1 Oa). The results from the enzymatic interesterification for the rest of the fat
blends are given in Appendix I (Figures 21-25). Therefore, in this work, a reaction time of
6 h was chosen as an optimal reaction time for enzymatic interesterification for all fat
blends and would be used for the scale-up experiments in the next sections to ensure
the highest conversion of all fatty acids into new TAG. Then, the interesterified fats from
the scale-up experiments were analyzed using different techniques as explained in
Chapter 3 and the results are showed in the next sections.
56
30
a 25
20
ro
~ 15
0 2 4 6 8 10 12 14 16 18 20 22 24
Reaction time (h)
30
b 25
20
10
5
0
0 2 4 6 8 10 12 14 16 18 20 22 24
Reaction time (h)
-tr-LLO
~PLL
-+-LOO
-PLO
---e-PLP
---.-ooo ~POO
~POP
~ppp
-tr-LLO
~PLL
-+-LOO
-PLO
---e-PLP
_._ooo ~POO
~POP
~ppp
57
Figure 10 Evolution of the relative quantity ofT AG species obtained at different reaction
times during enzymatic interesterification of hPS/RBO (a) blend 20:80, and (b) blend
40:60 (P = palmitic, 0 = oleic, L = linoleic acids).
hPS/RBO
(wtlwt)
Unreacted
0-100
20-80
30-70
40-60
50-50
60-40
70-30
80-20
100-0
Reacted
20-80
30-70
40-60
50-50
60-40
70-30
80-20
58
4.2 Triacylglycerol composition
TAG compositions of RBO, hPS, hPS/RBO blends before and after the scale-up
interesterification with Lipozyme RM IM at 6 h are given in Table 14. The main TAG
species in RBO were PLO (18.3%), PLL (17.5%), LLO (17.4%) and LOO (15.5%) and the
main TAG species in hPS were PPP (41.3%) and POP (17.7%). Before interesterification,
the quantity of PLO, PLL, LLO and LOO decreased and, on the other hand, the quantity
of PPP and POP increased as the amount of hPS in the unreacted or physical blends
increased.
Table 14 Triacylglycerol profile (area%) of hPS/RBO blends before and after
interesterification
Area%
LLO PLL LOO PLO PLP 000 POO POP
17.4 ± o.o' 17.5 ± o.o' 15.5 ± o.o' 18.3 ± o.o· 6.2 ± o.o' 3.3 ± 0.1' 4.0 ± 0.0' o.o ± o.o'
16.9 ± 0.4b 16.6 ± 0.4bo 14.6 ± 0.3'b 17.7 ± 0.3· 6.3 ± 0.7' 3.2 ± 0.2' 5.0 ± 0.2'bod 2.0 ± 0.1b
16.6 ± 0.2b 17.0 ± 0.6'b 14.7 ± o.o'b 17.9±0.1. 6.4 ± 0.4' 3.0 ± 0.3abo 4.6 ± 0.9abo 1.9 ± 0.4b
16.6 ± 0.1b 16.4 ± 0.2bod 14.4 ± o.ob' 17.7 ± 0.2· 6.8 ± 0.9' 3.0 ± 0.2abo 4.8 ± 0.3abo 2.4 ± 0.4bo
16.2 ± 0.2' 16.2 ± 0.4ode 14.2 ± 0.2bod 17.4±0.1. 6.3 ± 0.2' 3.1 ± 0.3'b 5.2 ± 0.2bode 2.9±0.1bo
15.8 ± o.od 15.9 ± 0.3def 13.6 ± 0.3'd 17.7 ± 0.3· 7.0 ± o.o'b 2.8 ± O.Obcd 5.0 ± o.o'bcd 3.2 ± 0.2bo
15.0 ± 0.3· 15.4 ± 0.519 13.4 ± 1.3de 18.1 ± 0.1· 7.8 ± 0.1bo 2.7 ± 0.2'd 5.4 ± 0.9ode 4.2 ± 0.4'd
12.3 ± 0.29 13.1 ±0.4h 10.4 ± 0.91
15.4±1.59 8.1 ± 0.8' 2.5 ± 0.3d 6.8 ± 1.31
8.8 ± 3.11
o.o ± o.o" 3.8 ± o.o' o.o ± o.o' 12.2 ± 0.3h 16.4 ± 0.2· 0.0 ± O.Oh 8.7 ± 0.29 17.7±0.2'
14.0 ± 0.2' 15.6 ± o.o•'g 12.6 ± 0.2· 24.3 ± 0.4b 11.5 ± 0.1d 1.3 ± 0.2· 5.5 ± 0.6ode 3.4 ± 0.2bo
10.8 ± 0.1h 15.6 ± 0.1efg 9.9 ± 0.11
25.3 ± 0.1' 15.7 ± o.o· 1.1 ± o.o·' 6.1 ± 0.1 del 5.2 ± 0.1d
7.8 ± o.o' 15.0 ± 0.09 7.4 ± 0.1 9 25.5 ± o.o' 20.4 ± 0.2' 0.8 ± 0.119 6.0 ± 0.1def 6.8 ± 0.1·
5.6 ± 0.2' 13.4 ± o.2" 5.3 ± o.oh 23.6 ± 0.4bo 24.7 ± 0.1 9 0.5 ± 0.1 9 6.2 ± 0.3·' 9.6 ± 0.119
3.8 ± 0.1' 12.0 ± 0.6' 4.0 ± 0.1' 23.1 ± 0.6' 31.6 ± 0.9h 0.0 ± O.Oh 4.7 ± o.o'b' 10.8 ± 0.29
2.4 ± o.o' 9.9 ± 0.1' 2.5 ± 0.41 20.3 ± 0.2d 36.4 ± 0.4' 0.0 ± O.Oh 4.7 ± 0.3'bc 13.5 ± 0.2"
1.1 ± O.Om 7.2 ± 0.1' 1.2 ± o.o' 16.4 ± o.o' 40.5 ± 0.41 0.0 ± O.Oh 4.1 ± 0.2'b 16.2 ± 0.1'
Values followed by the same letters within the same column are not significantly different (p<0.05)
Abbreviation: P = palmitic, 0 = oleic, L = linoleic acids
ppp
o.o ± o.o'
1.2±0.1abo
1.8 ± 0.1bod
2.6 ± 0.3de
3.7 ± 0.1·'
5.4 ± 0.1 g
6.3 ± 2.49h
12.5 ± 0.3'
41.3 ± 0.3'
o.o ± o.o'
o.o ± o.o'
o.9 ± o.o'b
1.9 ± o.obod
2.4 ± 0.4cde
4.1 ± 0.11
7.0 ± 0.2h
59
After interesterification, the amount of LLO, PLL and LOO decreased
significantly whereas that of PPP and 000 decreased slightly when compared to the
physical blends. On the contrary, the amount of PLO and PLP and POP decreased
significantly while the quantity of POO remained approximately the same. Exchanging
the position of fatty acids in TAG molecule allowed more formation of some TAG species
with the expense of the others and this influenced the crystallization and melting
behavior of the interesterified fats as will be demonstrated later.
4.3 Fatty acid composition
Fatty acid (FA) compositions (in % area) of hPS, RBO and hPS/RBO blends
before and after interesterification with Lipozyme RM IM at 6 h are shown in Table 15.
The chain lengths of FAs present in hPS and RBO ranged from C12 to C24, indicating
that the physical blends and the interesterified fats produced from hPS/RBO has a wide
range of FAs. The major FAs in hPS, RBO and hPS/RBO blends before and after
interesterification were palmitic (C16, 18.55-79.90%), oleic (C18:1, 7.13-44.81%) and
linoleic acids (C18:2, 1.45-32.14%), the quantities of which depended on the hPS/RBO
proportions of the blends. As the amount of hPS in the blend increased, the quantity of
palmitic acid in the blends increase and this coincided with the decrease in oleic and
linoleic acids in the blend.
When compared the results between unreacted and reacted blends, it was
found that the difference between the FA compositions of the unreacted and reacted
blends with the same hPS/RBO ratio was not significant (p<0.05). This was because the
enzymatic interesterification is a process that just alters the position of fatty acid on
glycerol backbone without adding other fatty acid or triacylglycerol in a process (Allen,
1996; Noor lida et al., 2007). However, splitting off and reconnect the FAs at position sn-
1 and sn-3 may be occurred incompletely
60
Table 15 Fatty acid compostion of hPS/RBO blends before and after interesterification with Lipozyme RM IM
hPS/RBO Fatty acid composition (Area %)
wt!wt C12:0 C14:0 C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 C20:0 C22:0 C24:0
Lauric acid Myristic acid Palmitic acid Palmitoleic acid Stearic acid Oleic acid Linoleic acid Linolenic acid Arachidic acid Behenic acid Lignoceric acid
Unreacted
0: 100 o.o2 ± o.oo" 0.21 ± 0.018
18.55 ± 0.318
0.10±0.01° 1.71±0.018
44.81 ± 0.138
32.14 ± 0.308
o.59 ± o.oo" 0.59 ± 0.018
0.17 ± o.oo" 0.27 ± 0.028
20:80 o.o4 ± o.oob 0.42 ± o.oob 30.49 ± 0.41 b 0.12±0.01ab 2.97 ± 0.77bcde 37.07 ± 1.28b 24.21 ± 0.31b 0.51 ± 0.00° 0.60 ± 0.018 o.16 ± o.oo•b 0.24 ± o.oo•b
30: 70 0.04 ± O.OObbc 0.49 ± 0.00° 38.28 ± 0.34" 0.12 ± 0.01 b 2.72 ± 0.02bc 32.35 ± 0.00° 19.40±0.11d 0.46 ± o.ood o.58 ± o.oo•b 0.16 ± o.oobc 0.23 ± o.oobc
40:60 0.05 ± o.oode 0.56 ± 0.03d 44.14 ± 0.741
0.10 ± 0.00° 3.31 ± o.oocde 28.18 ± 0.14d 16.45 ± 0.12" 0.39 ± 0.011
0.52 ± 0.00° o.14 ± o.ood 0.20 ± o.oocd
50:50 0.05 ± o.oocd 0.60 ± 0.01" 51.05 ± 1.06h o.o7 ± o.oo" 3.32 ± 0.22cde 24.52 ± 0.07" 13.89 ± 0.551 0.30 ± 0.01h 0.46 ± o.ood 0.12 ± o.oo" 0.17 ± o.oodel
60:40 0.05 ± o.oodel 0.67 ± 0.031
56.07 ± 0.09; o.o6 ± o.oo"1
4.17 ± 0.469h 20.66 ± 0.091
10.93 ± 0.029 0.25 ± 0.00; 0.42 ± o.oo" 0.10 ± 0.001
o.14 ± o.0019
70: 30 o.o6 ± o.ooh 0.77±0.01h 62.70 ± 0.06j o.o5 ± o.0019 4.00 ± 0.0219h 17.04 ± 0.099 8.48 ± 0.02h 0.20 ± o.ooi o.38 ± o.oo1
o.o9 ± o.009 0.12 ± o.oo9h
80:20 o.o6 ± o.ooh 0.83 ± 0.01; 68.21 ± 0.06k o.o4 ± o.ooh 4.23 ± 0.049h 13.74 ± 0.10h 6.00 ± 0.03; 0.13 ± o.ook 0.34 ± o.oo9 o.o7 ± o.ooh o.o9 ± o.ooh
100: 0 o.o7 ± o.od 0.99 ± o.ooi 79.90 ± 0.191
o.o2 ± o.oo' 4.55 ± o.ol 7.13±0.08; 1.45 ± 0.01j 0.01 ± 0.011
o.26 ± o.ooh 0.04 ± 0.00; 0.04 ± 0.00;
Reacted
20:80 0.04 ± o.oobc 0.42 ± 0.01b 31.76 ± 0.23° 0.13 ± 0.01" 2.35 ± o.o5b 36.65 ± o.o5b 22.41 ± 0.04° 0.53 ± 0.02b 0.60 ± 0.03" 0.17±0.01" 0.26 ± 0.018
30: 70 0.05 ± o.oocd 0.49 ± 0.01° 37.20 ± 1.05d 0.13 ± o.oo" 2.86 ± 0.41 bed 31.93 ± 0.01° 19.51 ± 0.20d 0.47 ± 0.01 d 0.56 ± o.oob 0.15±0.00° 0.23 ± o.oobc
40:60 o.o5 ± o.od9 0.59 ± o.oode 43.85 ± 0.791
0.12 ± o.oob 3.37 ± 0.43cdel 28.26 ± 0.68d 16.89 ± 0.44" 0.41 ± 0.00" 0.52 ± 0.01° 0.14 ± o.ood 0.21 ± 0.01 cd
50:50 o.o5 ± o.oo9h 0.66 ± 0.001
49.95 ± 0.299 0.10±0.00° 3.42 ± 0.03del 24.43 ± o.o8" 14.16 ± 0.081
o.34 ± o.oo9 0.48 ± o.ood 0.12 ± o.oo" 0.18 ± 0.01de
60:40 o.o5 ± o.od9h 0.71 ± 0.039 56.14 ± 0.19; o.o8 ± o.ood 4.14±0.51 9h 20.73 ± o.od 11 .15 ± 0.309 0.26 ± 0.01; 0.42 ± o.oo" 0.10 ± o.oo1
0.15 ± 0.01°19
70: 30 o.o5 ± o.oo9h o.75 ± o.ooh 62.94 ± o.55j o.o5 ± o.oo9h 3.64 ± 0.12"19
17.20 ± 0.289 8.61 ± 0.21 h 0.19 ± o.ooi 0.36 ± 0.029 0.08 ± 0.01 9 0.16 ± 0.06"1
80:20 o.o6 ± o.ooh 0.82 ± 0.03; 67.94 ± 0.23k o.o4 ± o.ooh 4.13±0.01 9h 14.12 ± o.o5h 6.28 ± 0.05; 0.14 ± o.ook 0.34 ± o.oo9 o.o7 ± o.ooh 0.10±0.00h
Values followed by the same letters within the same column are not significantly different (p<0.05)
Values are means± standard deviation.
61
4.4 Slip melting point
Slip melting point (SMP) of a fat is the temperature at which a column of fat in an
open capillary tube softens or becomes sufficiently fluid to slip or run up the tube when it
is subjected to a controlled heating. Table 16 shows SMP of hPS/RBO blends before
and after interesterification with Lipozyme RM IM at 60°C for 6 h. Prior to the enzymatic
interesterification reaction, an SMP of 100% hPS was recorded at 60.3°C. Addition of
RBO at increasing quantity gradually reduced the SMP of the physical blends. The SMP
of unreaction blends with 20-80% of RBO ranged from 47.1 to 58.6°C.
Table 16 Slip melting point of the hPS/RBO blends before and after interesterification
with Lipozyme RM IM
hPS/RBO (wt/wt)
20:80
30:70
40:60
50:50
60:40
70:30
80:20
100:0
Slip melting point (°C)
Unreacted Reacted
47.1 ± 0.3 30.4 ± 1.6
51.1±0.2 36.6 ± 0.2
54.3 ± 0.2 39.1 ± 0.5
56.1 ± 0.2 43.8 ± 0.1
57.1±0.1 48.8 ± 0.2
58.0 ± 0.1 51.3 ± 0.2
58.6 ± 0.2 53.6 ± 0.1
60.3 ± 0.3
Values within the same column and row are significantly different (p<0.05)
Values are means ±standard deviation.
The SMP of the interesterified blend was lowest for blend 20:80 (30.4°C). As the
quantity of RBO in the unreacted blend increased, SMP of the interesterified blends
decreased with blend 80:20 showing the highest SMP (53.6°C). After enzymatic
interesterification, SMP of the interesterified blends were lower than their unreacted
blends at the same amount of RBO. As the quantity of RBO in the unreacted blend
62
increased, the drop in the SMP when the unreacted blend was transformed into the
interesterified blend decreased. The blend with the highest quantity of RBO (blend
20:80) exhibited the biggest reduction in SMP (16.rC) whereas the blend with the
lowest quantity of RBO (blend 80:20) showed the smallest SMP reduction (5°C).
4.5 Solid fat contents
The solid fat content (SFC) is the percentage of lipid that is solid at selected
temperatures from about 1 0-40°C. Since SFC influences physical properties such as
spreadability, hardness, mouthfeel and stability, the SFC values are required to
characterize the properties of plastic fat and are considered one of the qualitative
parameter of the texture of margarine (Lee et al., 2008). Three measurement
temperatures related to the spreadability, product stability, and texture and mouthfeel
are refrigeration, room, and body temperature, respectively (Ribeiro et al., 2009).
100
90
80
70
~ 'E 60 J!! c 8 50
~ :E 40 0 (/)
30
20
10
0
15 20 25 30 35
Temperature (°C)
Figure 11 SFC profile of hPS/RBO blends before interesterification
40
~100:0
-80:20
~70:30
--e--60 :40
-50:50
--.!r- 40 : 60
--+-30: 70
~20:80
The SFC profile as a function of temperature of hPS and the unreacted hPS/RBO
blends is showed in Figure 11 (SFC values are given in Appendix I, Table 18). hPS
63
exhibits high SFC at all temperatures from 15-40oc and only a modest SFC reduction
was observed when the temperature increased. The unreacted blends showed 24.42-
80.52% SFC at 15°C, which then decreased to 11.39-62.15% at 40°C. At any
temperature, the SFC increased as the quantity of hPS in the blends increased. This
was, however, not unexpected due to the high content of high-melting TAGs in hPS such
as PPP and POP (see Table 14).
SFC profiles of hPS/RBO blends after interesterification are presented in Figure
12 (SFC values are provides in Appendix I, Table 19). The enzymatic interesterification
reaction significantly changed the characteristic of the SFC profiles of all fat blends.
Below 20 oc. the SFC of interesterified blends 80:20 and 70:30 were higher than those of
their physical blends whereas the SFC of the rest of the interesterified blends was either
the same as (blend 60:40) or below those of their physical blends. However, at 20 oc
and above, the SFC of all interesterified blends decreased significantly to well below
their physical blends. At 35°C, the average drop in SFC from before to after
interesterification for all blends was around 20%. The changes in SFC after
interesterification were due to the exchange of fatty acids in the TAG species in the
blends, leading to changes in the relative quantity of the TAG components of the
interesterified lipids. The SFC increase at low temperature of the interesterified blends
was probably due to an increase in the content high-melting TAGs (e.g. PLP and POP)
and a decrease in low-melting TAGs (e.g. LLO, PLL and LOO) (Kurashige et'al., 1993)
(see Table 14). The decrease in SFC at higher temperatures following interesterification
was presumably due to the decrease in trisaturated TAG (PPP).
The SFC of margarine at 35°C should be below 10 % in order to show complete
melting in the mouth without leaving a waxy coating on the palate (Kristott et al., 2003).
Based on this criteria, only the interesterified fats of 20:80, 30:70 and 40:60 blends (with
the SFC of 0.47 %, 4.14% and 9.15 %, respectively, at 35°C) fit the requirement.
100 ··r ----------- ----··-·---··-·---···-·---------------------- --- ·····--····-·,.····---------- -- -·· . i
90 ~ 80
70 I
60 I i
50 4
40
30
20
10
0
15 20 25 30 35
Temperature (°C)
Figure 12 SFC profile of hPS/RBO blends after interesterification
60
50
::.!1 40 0
c 2 <::
30 0 (.)
.!§ :2 0 20 (/)
10
0
15.0 20.0 25.0 30.0 35.0 40.0
Temperature (°C)
40
hPS/RBO
-80:20
-70:30
--e-60 :40
-50:50
---e.-40: 60
~30:70
--e--20:80
hPS/RBO
____.__ 80 : 20
~70:30
~60:40
~50:50
-~- 40:60
--+--30 :70
----&- 20 : 80
--Commercial1
-- Commercial 2
--Commercial 3
Commercial 4
Commercial 6
64
Figure 13 SFC profile of the interesterified hPS/RBO blends compared with commercial
margarine fats
65
The SFC profiles of all interesterified blends are compared with the fats
extracted from the five commercial margarines selected from the market in Thailand as
presented in Figure 13 (SFC values of commercial margarine fats are given in Appendix
I, Table 20). It can be clearly seen that only the interesterified fats of the 40:60 blend
exhibited the SFC curve that fit will within the range of the SFC curves of all commercial
margarine fats, indicating the blend's high potential as a suitable trans-free margarine
fat. In addition, the figure shows that the SFC profile of the interesterified blend 40:60
was closest to the SFC curves of commercial margarines fats #1 and #4, which were
from the two most well-known margarines in Thailand. Therefore, the studies in the
following sections would focus on comparing different characteristics of the fat blends
with those of only commercial margarine fats #1 and #4.
4.6 Crystallization and melting profile
The crystallization thermograms of RBO, hPS and the physical blends are shown
in Figure 14. hPS showed one sharp crystallization peak with the highest crystallization
temperature at 38.36aC whereas RBO exhibited one broad crystallization peak at
-6.19°C. The crystallization of the physical blends showed two peaks representing the
crystallization of high-melting (II) and low-melting (I) fractions. As the amount of hPS in
the unreacted blends increased, both DSC peaks moved gradually to the high
temperature area.
The melting thermograms of RBO, hPS and the unreacted blends are shown in
Figure 15. hPS had a single sharp melting peak at 60.8°C and RBO displayed two broad
and overlapping melting peaks at -14.87 and -8.98°C. The exothermic thermograms of
unreacted blends showed mixture of the melting peaks of both hPS and RBO. As the
content of hPS in the blends increased, the peak representing the melting of the high
melting fraction (from hPS) of the blends moved slowly to the high temperature area and
the two broad and overlapping peaks relating to the melting of the low-melting TAGs
(from RBO) diminished gradually and finally disappeared in blend 80:20. The melting
end temperature (Tend), which is the temperature where a fat sample melts completely,
66
depends on the amount and type of the TAG molecular species and also on the type of
FAs. Tend of RBO was the lowest at 8.4 oc whilst Tend of hPS was the highest at 64.46°C.
For the unreacted blends, Tend increased from 52.94°C for blend 20:80 to 60.75 oc for
blend 80:20. All of melting temperatures are showed in Appendix I, Table 24-26.
70 l--"
i R1ce bran oil
60 i Hard palm steann
(a) Unreacted (20:80)
I 50 (b) Unreacted (30.70)
(c) Unreacted (40·60) 0. ::l 40 (d) Unreacted (50 50) 0 ., " w
(e) Unreacted (6040) .. 30 0
1 u:
(f) Unreacted (70:30) iii .. J: 20
(gl Unreacted (80·20)
10 I
II 0
-30 -20 -10 0 10 20 30 40 50 60 70
Temperature ('C)
Figure 14 DSC crystallization thermograms of hPS, RBO and hPS/RBO blends before
interesterification
60
50
I 40
0. ::l 0 ..,
30 " w ;o 0 u: iii 20 ., J:
10
0
RICe bran oil
(a) Unreacted (20:80)
~----------------~<b~l~Um~e~a~cted~(~~7~0) __________ ~~-----
------------------~(c~)=Un~re=act~ed~(4~0~:60~)---------~~---
~----------------~(d~)U~n~re=&~ted~(50~:50~)---------~ (e)Unreacted(60:40) ~
~----------------~~~U-n_rea_c-ted~(?-0:-~~)----------~:.:.·
(g) Unreacted (80:20) __/ L
-50 -40 -30 -20 -10 0 10 20 30 40 50 60 70
Temperature ('C)
Figure 15 DSC melting thermograms of hPS, RBO and hPS/RBO blends before
interesterification
67
The crystallization thermograms of the interesterified fats together with two
commercial margarine fats are provided in Figure 16. The thermograms could be
divided into two areas. The first area is the high-temperature side of the thermogram
which represents the crystallization of high-melting TAGs (peak Ill) and the second area
is the lower-temperature side of the diagram which showed the crystallization of the
lower-melting TAGs (peaks I and II). As the quantity of hPS in the blends increased,
peaks II and Ill moved slowly to the high temperature side but peak I gradually moved
toward lower temperature. All of crystallization temperature was showed in Appendix I,
Table 21-23). Before interesterification each physical blend had its own melting and
crystallization characteristic, presenting higher and lower melting TAGs. However, with
interesterification by lipase-catalysis, rearrangements of fatty acids within or between
TAGs occurred and led to the production of new TAG molecules, and in tum, their
crystallization and melting behaviors were changed (Lee et al., 2008). It can be seen
from Figure 16 that the crystallization thermogram of blend 40:60 was most similar to the
exothermic thermograms of the two fats extracted from the commercial margarines.
Commercial 4
18 -....___/ v ...
16 j Commercial 1
~ ~ ~ 14
Q, 12 ::J 0
""' 10 I: w 30 0 8 :(e) Reacted (6040i u:: '&; ., 6 J:
~
(fl Reacted (70. 30\
i (gl Reacted (8020) 4
2
Ill 0
-60 -50 -40 -30 -20 -10 0 10 20 30 40 50 60 70
Temperature (•c)
Figure 16 DSC crystallization thermograms of hPS, RBO and hPS/RBO blends after
interesterification compared with commercial margarine fats
68
The melting properties of fats can be changed by enzymatic interesterification.
Melting characteristics of margarine fat are important for flavor release and consumer
acceptance. Fat and oil do not have a distinct melting point but rather a melting range
because of the different FAs present (Pande et al., 2013). The DSC melting
thermograms of the interesterified fats and two commercial margarine fats are provided
in Figure 17. The melting thermograms of all samples showed broad and overlapping
peaks. Blends 40:60 and 50:50 displayed the melting thermograms which were most
similar to commercial margarines. Tend of blend 20:80 was the lowest at 36.84oC and Tend
for the blends increased when the hPS content in the blends increased. As a
consequence, Tend of blend 80:20 was the highest (56.58°C). When Tend of the unreacted
and interesterified blends with the same hPS:RBO ratio are compared, for example
blend 40:60, T me of the interesterified blend (42.56°C) was lower than the unreacted
blend (56.6rC). This was possibly because the interesterified fat contained less content
of trisaturated TAG (PPP) (see Table 14).
45 I~ ~
4{) j ___,.-.. 35 ---....._..
I 30 lal Reacted (20:80J --I
, Commercial 41 Commercial 1 1
c. :::1 (b) Reacted (30:70! ---0 25 ... t:: (c) Reacted 140 601 w
20 ..____.. .. 1 0 (d) Reacted (50:50) u:: j ~
iii 15 (e) Reacted (6040) I .. I :I: I 10 ~; In Reacted 170:30)
' !
-50 -40 -30 -20 -10 0 10 20 30 40 50 60 70
Temperature (•C)
Figure 17 DSC melting thermograms of hPS/RBO blends after interesterification
compared with commercial margaine fats
69
When both the crystallization and melting characteristics of the interesterified
blends were considered, it was found that the interesterified 40:60 blend had both the
DSC crystallization and DSC melting profile most similar to those of the commercial
margarine fats. This was in consistence with the results from the SFC studies which
demonstrated that blend 40:60 had the SFC profile most similar to that of commercial
margarine fats (see Figure 13). (DSC melting thermograms of more commercial
margarine fats are given in Appendix I, Figure 26). Consequently, the interesterified
40:60 blend appeared to be suitable for use as a trans-free margarine fat.
4. 7 Polymorphism
Polymorphic forms of fat crystals are most important criteria for the funtional
properties of margarines and greatly influenced the physical properties and processing
of the final product (Reddy and Jayarani, 2001; and Ribeiro et al., 2009). The main
polymorphs of fat crystals are a. W and 13 polymorphic forms. Each polymorph has
different characteristics with a form characterised as unstable with the lowest melting
point and short spacing at 4.15 A; W form, metastable, intermediate melting point and
two strong short spacings at 3.80 A and 4.20 A and three minor short spacings at 4.27
A, 3.97 A and 3.71 A. This type of crystals is small, about 5-7 J...Lm in length, hence
contributing to a shiny surface, smooth texture and spreadability of margarines
(Miskandar et al., 2002a, ldris et al., 1996). 13 form, very stable, highest melting point
and short spacing at 4.60 A, is unfavorable in margarine fats due to its association with
grainy texture (D'souza et al., 1990; Solis-Fuentes et al., 2005). 13 crystals are initially
small but they grow into large needle-like agglomerates about 20-30 J...Lm, leading to
sandy and hard texture (Miskandar et al., 2002a, Liu et al., 2010, deMan and deMan,
2002, Marangoni and Rousseau, 1995).
70
20000 13 (4.6 A)
18000
16000
14000
12000
(j) 0. ..s .?;- 10000 ·u; c:: .2! ..!:
8000
6000
4000
2000
0
14 16 18 20 22 24 26 28 30
2-theta (deg)
Figure 18 X-ray diffraction spectra of hPS/RBO blends before and after interesterification
and commercial margarine fats stored at 4°C 24 h
The polymorphic forms of the physical blends and the interesterified fats were
crystallized at 4 and 25°C, and their polymorphisms were determined with the short
spacings by X-ray diffraction spectra as shown in Figure 18 for 4 oc and in Figure 19 for
25°C. The physical blends had a mixture of both 13' and 13 polymorphic forms when
stored at 4 °C. It was also noticed that an increase in 13' crystal occurred with an increase
in the amount of hPS in the blends. After interesterification, the diffracted intensity of 13
form (at 4.60 A) of the interesterified fats noticeably decreased and the peak
representing 13' structure (at 3.8 A) became predominant over 13 polymorph especially in
71
blends which contained higher amount of hPS. The commercial margarine fats exhibites
mainly W polymorph when stored at 4°C.
20000
J3' (4.2 Al
18000 J3 (46Al
16000
14000
12000
(j) a. .3. z. 10000 ·u; c: 2 E
8000
6000
4000
2000
0
14 16 18 20 22 24 26 28 30
2-theta (deg)
Figure 19 X-ray diffraction spectra of hPS/RBO blends before and after interesterification
and commercial margarine fats stored at 25oC 24 h
When the storage temperature was changed from 4 °C to 25oC and the storage
duration was further extended for 24 hours, the polymorphic structure of both the
unreacted blends and the interesterified fats had transformed further from 13' to 13 form
with an increase in the diffracted intensity of 13 polymorph (at 4.60 A) and a decrease in
the diffracted intensity of 13' polymorph (at 3.80 A and 4.20 A) (Figure 19). The
apparently more rapid polymorphic transformation at 25oC of storage could have been
72
linked to the fact that both the unreacted blends and the interesterified fats had no trans
fatty acid that would normally give stability to the fats and keeping the fats at high
temperature will make a conversion of crystal polymorph from one from to another (in
this case is 13' to 13) occur more quickly than general which would be undesirable in
margarines. Therefore, trans-free margarines that are made from trans-free fats should
be stored at a temperature lower than room temperature.
The content of 13' and 13 structures in all the samples was estimated by the
relative intensity of the short spacings of 13' form at 4.2 A and that of 13 form at 4.6 A
following a method described by Kim et al. (2008) and the results are shown in Table
17. Both of the commercial margarine fats exhibited higher content of 13' than 13 at both
storage temperatures. The unreacted and interesterified blends had more 13' than 13
polymorphs when stored at 4°C, except the physical 80:20 blend. At 25°C, most of the
blends, except the physical 20:80 blend and the interesterified blends 40:60 and 60:40,
showed more 13' than 13. The interesterified blend 40:60 had equal content of 13' and 13
polymorphs. As mentioned before, the predominant presence of 13' over another
polymorph (especially 13) is necessary for imparting desirable textural properties to
margarines. However, the margarine fats with equal content of 13' and 13 polymorphs,
although not ideal, is still acceptable as long as the fats shows other good qualities, e.g.
good crystallization and melting characteristics, etc.
Table 17 Crystal polymorphic forms of hPS/RBO blends before and after
interesterification, commercial margarine fats and hPS at 4 oc and 25°C
Level of 13· and 13 form
hPS/RBO Unreated Reacted Unreacted Reacted
(wt!wt) 40C 4°C 25oC 25°C
20-80 13· > 13 13· > 13 13' = 13 13' > 13
30-70 13' > 13 13' > 13 13' > 13 13' > 13
40-60 13' > 13 13' > 13 13' > 13 13' = 13
50-50 13' > 13 13' > 13 13· > 13 13' > 13
60-40 13' > 13 13' > 13 13' > 13 13' = 13
70-30 13' > 13 13· > 13 13' > 13 13' > 13
80-20 13· = 13 13' » 13 13' > 13 13' > 13
Commercial 1 13' > 13 13' > 13
Commercial 4 13' > 13 13· > 13
hPS 13' > 13
73
*Estimated using the equation ~/ ~· = (intensity of short spacing at 4.6 A)/(intensity of short spacing at
4.2 A) as follows: ~· = ~ (1.2 <: ~/ ~'> 0.8); ~· > ~ (0.8 <: ~/ ~· > 0.4); and ~· » ~ (0.4 <: ~/ ~· > 0).
4.8 Crystal microstructure
The microstructures of hPS, the physical blends, the interesterified fats and the
commercial margarine fats obtained by PLM are shown in Figure 20. The crystal
morphologies between the substrate (hPS) and produced fat were obviously different.
hPS had densely packed crystals of well-defined Maltese cross shape at 4 oc and had
similar crystal microstructure previously obseNed in fully hydrogenated oil, as the
process makes fat harder which is the same as fractionation (Lee et al., 2008). The
physical blend containing hPS between 60-80% showed a similar structure to hPS
whereas the physical blends containing hPS between 20-50% showed closely packed
large rod-like spherulic crystals that were obseNed in palm stearin by Adhikari et al.
(201 Oa). The physical blends with higher amount of hPS tended to form more closely
74
and more densely packed crystals . The interesterified fats displayed different crystal
microstructures from that of their physical blends, exhibiting needle-shaped with certain
degree of aggregation . The crystal size was smaller with higher crystal number when
compared with the physical blends. Moreover, a higher amount of hPS in the physical
blends led to the formation of smaller size crystals in the interesterified fats.
75
Figure 20 Crystal microstructure of fat blend at different ratio of hPS/RBO blends before
and after enzymatic interesterification and commercial margarine fats obtained after
static crystallization at 4 oc and then left at 25°C for 24 h using PLM
When crystallized and stored at a higher temperature (25°C), the interesterified
fats had fewer crystals than when they were kept at 4 oc because an increase in
temperature partly melted fat crystals leading to a lower amount of solid fat and a lower
ability of the fat to hold the liquid oil inside the crystal networks. The interesterified fats
which kept at 25oC showed compact small needle-like crystal formation compared to
76
physical blends and hPS. In comparison, the interesterified hPS/RBO blend of 40:60 has
crystal morphology similar to commercial margarine fats.
Fats containing the small compacted crystal are desired for use in bakery since
these crystals could surround and stabilize air bubbles produced during the creaming
stage and to give a fine and smooth texture to bakery products (Lee et al., 2008). As
mentioned earlier in unit 3, the criteria for selection of the best interesterified fat to
produce trans-free margarine fat are that the interesterified blend should have physical
properties and crystallization and melting behavior as closest to the commercial
margarine fats, possess a tendency to crystallize in 13' form, and have small crystals and
with needle-shaped crystals. Considering all these requirements, it was concluded that
the interesterified blend 40:60 is the best sample that has the highest potential to use for
as a trans-free margarine fat.
Chapter 5
Conclusion
The interesterified blends of hard palm stearin and rice bran oil (hPS/RBO) in
weight ratios of 20:80, 30:70, 40:60, 50:50, 60:40, 70:30 and 80:20 were prepared by
enzymatic interesterification using immobilized Mucor miehei lipase (Lipozyme RM IM)
at 60°C and mixing speed was 300 rpm. The result of TAG composition changes during
enzymatic interesterification shown that the rearrangement of fatty acids on TAG
molecule mostly occurred and had reached equilibrium after 6 h of reaction time.
Therefore, a reaction time of 6 h was chosen for scale-up experiments to ensure the
highest conversion of all fatty acids into newT AG.
Physical properties and crystallization and melting behavior of the interesterified
blends were investigated by different techniques and compared with margarine fats
extracted from the commercial margarines. The Lipozyme RM 1M-catalyzed
interesterification significantly modified the physical properties of the hPS/RBO blends.
The SMP and SFC results of all the interesterified blends had lower than their unreacted
blends. Mostly 13' crystal form and small needle-shaped crystal were found in the
interesterified fats, which is a desirable property for margarines, it provides smooth
texture to the products. In conclusion, the interesterified blend of hPS/RBO 40:60 had
SFC profile, crystallization and melting behavior, polymorphism and crystal
microstructure most similar to commercial margarine fats. Therefore, the interesterified
blend of hPS/RBO 40:60 is suitable for use as a margarine fats without trans-fatty acid.
77
78
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Appendix
30
25
20
I'll
~ 15 ';it.
10
5
0
Appendix I
Laboratory data
0 2 4 6 8 10 12 14 16 18 20 22 24
Reaction time (h)
----tr- LLO
~PLL
~LOO
-PLO
~PLP
........... ooo -+-POO
"""'*""""POP
""""'*""" ppp
Figure 21 Evolution of the relative proportions ofT AG species of hPS/RBO blends (30:70)
30
25
20
10
5
0 T-~-~~~~~~~~;;~~;;J 0 2 4 6 8 10 12 14 16 18 20 22 24
Reaction time (h)
----tr- LLO
~PLL
~LOO
-PLO
-e-PLP
......,..ooo
-+-POO
~POP
""""'*""" ppp
Figure 22 Evolution of the relative proportions ofT AG species of hPS/RBO blends (50: 50)
97
35
30
25
(ll 20
~ <F. 15
10
5
0
0 2 4 6 8 10 12 14 16 18 20 22 24
Reaction time (h)
~LLO
~PLL
~LOO
-PLO
~PLP
-..-ooo
--+-POO
~POP
~ppp
Figure 23 Evolution of the relative proportions of TAG species of hPS/RBO blends (60:40)
40
35
30
25 (ll
~ 20
15
10
5
0
0 2 4 6 8 10 12 14 16 18 20 22 24
Reaction time (h)
~LLO
~PLL
~LOO
-PLO
~PLP
-..-ooo -..-Poo ~POP
~ppp
Figure 24 Evolution of the relative proportions ofT AG species of hPS/RBO blends (70:30)
98
99
45
40
35 ~LLO
~PLL
30 ~LOO
C1l 25 -PLO !!:? <(
-e-PLP <ft. 20
15 --.-ooo ~POO
10 ~POP
5 """"*""" pp p
0
0 2 4 6 8 10 12 14 16 18 20 22 24
Reaction time (h)
Figure 25 Evolution of the relative proportions ofT AG species of hPS/RBO blends (80:20)
Table 18 Solid fat content of hPS, RBO and hPS/RBO blends before interesterification
Temperature 0: 100 20:80 30:70 40:60 50:50 60:40 70:30 80:20 100:0
(OC)
15.0 0.20 ± 0.28 24.42 ± 0.61 34.69 ± 0.13 44.61 ± 0.25 53.62 ± 0.35 62.40 ± 0.04 71.80 ± 0.18 80.52 ± 0.08 95.93 ± 0.01
17.5 0.10 ± 0.16 22.84 ± 0.16 33.20 ± 0.05 43.05 ± 0.09 52.43 ± 0.20 61.70±0.11 70.74 ± 0.18 79.26 ± 0.03 95.65 ± 0.01
20.0 0.23 ± 0.01 21.68 ± 0.10 31.75±0.18 41.47 ± 0.10 50.88 ± 0.06 60.09 ± 0.14 69.18 ± 0.08 77.96 ± 0.04 95.47 ± 0.01
22.5 0.12 ± 0.05 20.26 ± 0.19 30.20 ± 0.12 39.63 ± 0.11 48.97 ± 0.23 58.25 ± 0.18 67.60 ± 0.11 76.51 ± 0.01 95.06 ± 0.01
25.0 0.23 ± 0.16 19.06 ± 0.11 28.81 ± 0.04 37.87 ± 0.01 47.54 ± 0.37 56.59 ± 0.02 66.18 ± 0.09 75.09 ± 0.08 94.33 ± 0.01
27.5 0.07 ± 0.13 17.71 ± 0.26 26.84 ± 0.09 35.97 ± 0.11 45.22 ± 0.11 54.63 ± 0.04 64.15 ± 0.10 73.32 ± 0.01 93.30 ± 0.04
30.0 0.05 ± 0.21 16.55 ± 0.34 25.48 ± 0.18 34.23 ± 0.29 43.58 ± 0.16 52.81 ± 0.13 62.13 ± 0.16 71.57 ± 0.04 91.97 ± 0.01
32.5 0.02 ± 0.20 15.21 ± 0.54 23.76 ± 0.21 32.36 ± 0.11 41.14±0.06 50.83 ± 0.01 60.43 ± 0.02 69.99 ± 0.00 90.48 ± 0.06
35.0 0.06 ± D.01 13.97 ± 0.07 21.81 ± 0.33 30.45 ± 0.01 39.11 ±0.14 48.20 ± 0.24 57.68 ± 0.49 67.73 ± 0.37 88.65 ± 0.17
37.5 0.00 ± 0.40 12.57 ± 0.40 20.24 ± 0.25 28.51 ± 0.11 36.81 ± 0.28 46.09 ± 0.08 55.51 ± 0.15 65.08 ± 0.54 86.07 ± 0.12
40.0 0.00 ± 0.11 11.39 ± 0.29 18.55 ± 0.04 26.06 ± 0.32 34.43 ± 0.02 43.36 ± 0.57 52.70 ± 0.02 62.15 ± 0.16 83.57 ± 0.08
Values are means ± standard deviation.
100
Table 19 Solid fat content of hPS/RBO blends after interesterification at 6 h
Temperature ('C) 20:80 30:70 40:60 50:50 60:40 70:30 80:20
15.0 12.36 ± 0.97 23.13 ± 0.02 36.79 ± 0.21 49.97 ± 0.37 62.07 ± 0.11 76.03 ± 0.70 86.00 ± 0.35
17.5 10.78 ± 0.83 19.23 ± 0.28 31.42 ± 0.38 45.10 ± 0.90 56.93 ± 0.11 71.97±0.74 83.22 ± 0.40
20.0 8.91 ± 0.44 15.40 ± 0.01 26.62 ± 0.42 39.28 ± 0.79 51.73±0.08 67.22 ± 0.91 79.73 ± 0.38
22.5 7.38 ± 0.52 13.19 ± 0.13 22.81 ± 0.00 34.44 ± 0.74 47.11 ± 0.02 62.17 ± 0.62 75.79 ± 0.32
25.0 5.94 ± 0.71 11.17 ± 0.17 19.28 ± 0.14 29.71 ± 0.65 41.91 ± 0.43 56.90 ± 0.81 71.21 ± 0.29
27.5 4.67 ± 0.81 9.15 ± 0.02 16.35 ± 0.03 25.84 ± 0.47 37.08 ± 0.16 51.68 ± 0.59 66.09 ± 0.28
30.0 3.30 ± 0.27 7.59 ± 0.06 13.96 ± 0.43 22.62 ± 0.20 32.79 ± 0.42 46.78 ± 0.35 61.08 ± 0.00
32.5 2.02 ± 0.07 5.57 ± 0.04 11.43 ± 0.18 19.05 ± 0.44 29.17 ± 0.84 41.91 ± 0.43 55.87 ± 0.06
35.0 0.47 ± 0.60 4.14 ± 0.11 9.15 ± 0.47 16.48 ± 0.13 25.11 ± 0.86 36.90 ± 0.53 50.68 ± 0.08
37.5 0.20 ± 0.00 2.40 ± 0.04 7.31 ± 0.08 14.21 ± 0.08 22.46 ± 0.19 32.07 ± 0.08 45.39 ± 0.06
40.0 0.21 ± 0.30 1.11 ± 0.06 4.74 ± 0.90 11.18 ± 0.16 19.77 ± 0.21 29.41 ± 0.49 40.58 ± 0.13
Values are means ±standard deviation.
Table 20 Solid fat content of commercial margarine fats
Temperature ('C) Commercial 1 Commercial 2 Commercial 3 Commercial 4 Commercial 5 Commercial 6
15.0 37.76 ± 0.31 36.92 ± 0.98 39.11 ± 0.23 42.03 ± 0.44 45.83 ± 0.33 30.33 ± 0.42
17.5 29.72 ± 0.25 28.40 ± 0.21 31.14 ± 0.02 33.58 ± 0.69 40.37 ± 0.54 26.62 ± 0.11
20.0 24.81 ± 0.17 22.13±0.23 24.56 ± 0.01 27.09 ± 0.28 33.05 ± 0.04 21.68 ± 0.13
22.5 21.42 ± 0.21 17.41 ± 0.02 19.77±0.11 22.70 ± 0.09 25.99 ± 0.18 17.09 ± 0.25
25.0 18.47±0.26 14.39 ± 0.01 16.33 ± 0.06 19.09 ± 0.16 20.76 ± 0.06 13.33±0.11
27.5 16.33±0.11 11.64 ± 0.07 13.26 ± 0.07 16.71 ± 0.20 17.09±0.13 9.92 ± 0.11
30.0 14.15 ± 0.11 9.35 ± 0.08 10.74 ± 0.08 14.46 ± 0.01 14.04 ± 0.16 7.09 ± 0.00
32.5 12.50 ± 0.12 7.43 ± 0.26 8.43 ± 0.02 12.27 ± 0.13 11.57 ± 0.11 4.70±0.13
35.0 10.85 ± 0.05 5.35 ± 0.23 6.11 ± 0.33 10.47 ± 0.06 8.88 ± 0.54 2.74 ± 0.27
37.5 8.99 ± 0.16 3.54 ± 0.10 4.44 ± 0.36 8.59 ± 0.13 6.92 ± 0.42 0.80 ± 0.11
40.0 7.31 ± 0.07 1.96 ± 0.01 3.41±0.18 6.57 ± 0.13 6.04 ± 0.27 0.15±0.27
Values are means ±standard deviation.
101
Table 21 Crystallization temperature of hPS/RBO blends before interesterification
hPS/RBO (wt/wt) Tonset T peak1 T peak2 Tend enthalpy (J/g)
0: 100 -4.42±0.10 -6.19 ± 0.11 -24.82 ± 0.08 -7.13±0.11
20:80 28.36 ± 0.04 24.18±0.01 -9.80 ± 0.01 -23.67 ± 0.23 -27.91 ± 0.14
30:70 29.91 ± 0.01 27.66 ± 0.04 16.81 ± 0.18 5.42 ± 0.13 -41.29 ± 0.00
40:60 33.29 ± 0.15 30.43 ± 0.05 20.08 ± 0.00 8.16 ± 0.02 -57.33 ± 0.52
50:50 34.83 ± 1.03 32.15±0.01 22.32 ± 0.65 10.52 ± 0.14 -73.27 ± 0.29
60:40 35.81 ± 0.26 33.62 ± 0.02 24.71 ± 0.89 13.96±0.18 -86.30 ± 0.12
70:30 36.93 ± 0.08 34.99 ± 0.06 25.22 ± 0.47 16.26 ± 0.25 -96.69 ± 0.48
80:20 38.68 ± 0.13 36.09 ± 0.06 27.84 ± 1.10 13.17 ± 0.05 -408.11±0.16
100: 0 41.22 ± 0.15 38.36 ± 0.16 10.08±0.10 -122.80 ± 0.20
Values are means ± standard deviation.
Table 22 Crystallization temperature of hPS/RBO blends after interesterification at 6 h
hPS/RBO (wt!wt) Tonset T peak1 Tpeak2 T peak3 Tend enthalpy (J/g)
20:80 24.89 ± 0.57 17.81±2.11 2.40 ± 0.02 -4.09 ± 0.01 -22.56 ± 0.45 -25.38 ± 1.62
30:70 27.04 ± 0.01 23.93 ± 0.31 4.14±0.18 -4.40 ± 0.16 -23.68 ± 0.29 -38.09 ± 1.05
40:60 29.76±0.16 28.09 ± 0.16 5.70 ± 0.09 -4.87 ± 0.07 -23.68 ± 0.34 -48.76 ± 0.33
50:50 31.87 ± 0.00 30.52 ± 0.17 6.91 ± 0.09 -5.31 ± 0.09 -24.02 ± 0.30 -55.66 ± 0.11
60:40 34.61 ± 0.23 32.37 ± 0.90 7.49 ± 0.23 -5.22 ± 0.08 -22.37 ± 0.20 -52.32 ± 2.89
70:30 37.18 ± 0.23 33.85 ± 0.19 7.61 ± 0.01 -5.50 ± 0.03 -23.32 ± 0.23 -63.42 ± 0.96
80:20 38.67 ± 0.16 36.15 ± 0.13 8.24±0.12 -5.40 ± 0.23 -11.27 ± 0.21 -72.67 ± 0.91
Values are means± standard deviation.
Table 23 Crystallization temperature of commercial margarine fats
Commercial margarine Tonset T peak1 T peak2 Tend enthalpy (J/g)
Commercial 1 27.74 ± 0.08 24.62 ± 0.06 -2.73 ± 0.04 -31.09±0.15 -57.53 ± 0.06
Commercial 2 24.55 ± 0.45 18.40 ± 0.04 -7.71 ± 0.08 -28.34 ± 0.18 -40.70 ± 0.01
Commercial 3 21.69 ± 0.28 18.31 ± 0.08 -8.27 ± 0.06 -29.23 ± 0.15 -58.68 ± 0.03
Commercial 4 26.27 ± 0.06 22.73 ± 0.06 -7.51 ± 0.03 -28.42 ± 0.02 -47.06 ± 0.06
Commercial 5 24.72 ± 0.21 19.55 ± 0.11 2.08 ± 0.05 -13.70 ± 0.06 -49.17 ± 0.06
Commercial 6 19.78 ± 0.05 14.75±0.12 -41.19 ± 0.08 -48.47 ± 0.06 -58.39 ± 0.21
Values are means ±standard deviation.
102
Table 24 Melting temperature of hPS/RBO blends before interesterification
hPS/RBO (wt/wt) Tonset T peakl T peak2 Tend enthalpy (J/g)
0: 100 -37.57 ± 0.08 -14.87 ± 0.16 -8.98 ± 0.01 8.40 ± 0.01 56.79 ± 0.08
20:80 -27.18±0.03 -16.51 ± 0.18 50.49 ± 0.21 52.94 ± 0.66 123.60 ± 0.40
30:70 -27.25±0.11 -18.83 ± 0.00 53.13 ± 0.04 56.10 ± 0.00 109.96 ± 1.40
40:60 -27.33 ± 0.03 -20.54 ± 0.82 55.83 ± 2.70 56.67 ± 0.42 115.16±1.99
50:50 -27.15 ± 0.01 -20.96 ± 0.04 56.26 ± 0.03 58.05 ± 0.31 129.37 ± 1.10
60:40 -27.95 ± 1.12 -22.56 ± 0.33 57.01 ± 0.46 58.97 ± 0.21 140.05 ± 0.93
70:30 -27.16±0.01 -22.26 ± 0.25 58.02 ± 0.19 60.19 ± 0.06 154.97 ± 1.40
80:20 -27.52 ± 0.07 -20.63 ± 0.01 58.84 ± 0.01 60.75 ± 0.01 168.99 ± 0.31
100: 0 44.27 ± 0.08 60.80 ± 0.01 64.46 ± 0.06 161.27±0.42
Values are means± standard deviation.
Table 25 Melting temperature of hPS/RBO blends after interesterification at 6 h
hPS/RBO Tonset T peakl T peak2 T peak3 T peak4 Tend enthalpy (J/g)
(wt/wt)
20:80 -26.12 ± 1.89 2.05 ± 0.21 30.56 ± 0.88 36.84 ± 0.54 50.83 ± 4.32
30:70 -23.47 ± 0.45 5.00 ± 0.23 36.45 ± 0.54 39.67 ± 0.10 75.32 ± 0.58
40:60 -23.53 ± 0.54 1.64 ± 0.01 7.49 ± 0.09 39.83 ± 0.15 42.56 ± 0.08 101.45±4.73
50:50 -23.30 ± 0.23 3.99 ± 0.13 9.61 ± 0.06 42.13 ± 0.11 45.24 ± 0.11 83.89 ± 1.65
60:40 -23.17 ± 0.43 5.88 ± 0.40 10.43 ± 0.12 43.73 ± 0.21 49.40 ± 0.71 51.86 ± 0.04 116.64 ± 0.16
70:30 -22.81 ± 0.47 6.60 ± 0.07 14.04 ± 0.64 44.88±0.15 52.15±0.11 56.10±0.00 123.07 ± 0.58
80:20 -17.70 ± 0.10 8.47 ± 0.09 15.31 ± 0.23 46.36 ± 0.33 54.25 ± 0.28 56.58 ± 0.10 129.60 ± 1.97
Values are means ± standard deviation.
Table 26 Melting temperature of commercial margarine fats
Commercial margarine Tonset T peakl T peak2 Tend enthalpy (J/g)
Commercial 1 -13.55 ± 0.42 7.52 ± 0.05 42.75 ± 0.23 47.71 ± 0.05 97.09 ± 0.80
Commercial 2 -14.36±0.13 11.45 ± 0.16 43.28 ± 0.10 96.85 ± 0.18
Commercial 3 -15.48 ± 0.15 6.41 ± 0.06 18.55±0.14 42.26 ± 0.23 96.59 ± 0.13
Commercial 4 -16.11 ± 0.14 6.29 ± 0.03 20.78 ± 0.05 47.69 ± 0.06 108.55 ± 0.15
Commercial 5 -22.54 ± 0.06 1.34 ± 0.06 22.27±0.16 43.57 ± 0.08 117.69 ± 0.58
Commercial 6 -25.91 ± 0.08 -17.39 ± 0.23 17.56 ± 0.08 40.18 ± 0.07 107.12 ± 0.20
Values are means ± standard deviation.
45 ~
4{) - Commercial 1
I 35 Commercial 2
-----------------~ c. ::::l 30 ~
0 'C c w
Commercial 3
----------~ CommerCial 4
lO 25 0
u:: Oi
20 .. :I:
-----------~ '' V Commercial 5
----------------~------------~ Commercial 6
15
-20 0 20 40 60
Temperature (•c)
L--------------------~-------~~---c_om_m_e_rc_ia_l_1 ___ !
Commercial 2
L-------------------~~----~,, _____ c_om __ me_r_c_ia_l3 __ __
L---------------~ ~~---C-om __ m_erc_,_al_4 __ _
, -----------~~--------------~--------~--,----~C~o~m~m~e~rc~ia~I~S---15 -;..
i
10 l~---~ -50 -30 -10 10 30 50 70
Temperature (•c)
Figure 26 DSC crystallization and melting thermograms of extracted commercial
margarine fats
103
104
Appendix II
Preparation of fatty acid methyl esters (FAMEs); Boron trifluoride method
1. Weight sample in a flask about 0.2000 g then add NaOH/MtOH 6 ml and boiling
chip 5-6 beads
2. Place a flask on a hot plate 1 oooc and connect to condenser
3. Reflux 10 min until fat drop disappear
4. Add BF 3 7 ml through the condenser and heat continuously 2 min
5. Add n-heptane 5 ml through the condenser and heat continuously 1 min
6. Leave the flask away from hot plate to cool down solution and condense
7. Add saturated salt (NaCI) about 15 ml
8. Use glass stopper plug a flask and then vigorous shaking for 15 second while the
solution was warm
9. Pour into test tube and pipette upper clear solution into new beaker
10. Add a pinch of Na2S04 anhydrous to eliminate water
11. Filter the solution by syringe filter 0.45 i-Jm into vial and got sample for GC analysis
Source: AOAC official method 969.33 (1997)
105
Appendix Ill
Free Fatty Acid Removing
1. The lnteresterified fat 0.85 g was solved in hexane 5 ml
2. Ethanol 95% (v/v) was added 3 ml
3. Phenolphthalein indicator was added 3-4 drops
4. The solution was titrated by 0.5N Potassium hydroxide in ethanol 20% (w/v) until
pink color appear in solution and separate into two layers
5. The solution was heated at 45°C for 3 min
6. The top layer of solution was separated into new beaker and evaporated hexane out
7. Got interesterified fat without free fatty acid
Source: modified from Alim et al. (2008)
Appendix IV
Determination of solid fat content (SFC)
106
1. The sample was melted at sooc until fat melt completely to destroy crystal structure
2. NMR tubes filled with fat about 4 em
3. The sample in NMR tubes were melted at sooc and held for 10 minutes
4. Temperature of all samples is maintained at ooc for 60 minutes
5. Each sample is held at required temperature for measurement (15, 17.5, 20, 22.5,
25, 27.5, 30, 32.5, 35, 37.5, 40°C) for 30 minutes
6. Every sample is placed into sensor and measurement process is started
7. Plot graph between SFC value (%) and temperature
Source: AOCS Method Cd 16-93 (1997)
Appendix V
Study of crystallization and melting behavior of fat
by differential scanning calorimeter (DSC)
1. Weight sample in liquid aluminum pan for DSC 3-S mg and hermetically sealed
2. Put sample pan and reference pan (blank) into DSC
3. Set temperature program as follow below:
- The samples were heated from 20oc to sooc at a rate of 30°C/min and
held at this temperature for 10 min to destroy the memory effect
- Then cooled from sooc to -60oC at a rate of SOC/min and hold at this
temperature for 30 min
- Finally the sample was heated to sooc at a rate of soC/min
107
4. The crystallization and melting profiles were generated during the cooling and
heating, respectively. The profiles were analyzed by the software provided with the
DSC (Pyris software, Perkin-Elmer, Shelton, CT, USA). The crystallization onset
(T c0), melting completion temperatures (T Mc) and enthalpy (Jig) were obtained from
the peaks located at the highest temperature of each fat sample. T co and T Mc were
considered to be the temperatures at which the crystallization began and the
melting ended, respectively.
Source: AOCS Method Cj 1-94 (1998)
Appendix VI
Determination of slip melting point (SMP)
1. The sample was melted at sooc until fat melt completely
2. Capillary tubes filled with fat about 10 mm
3. The samples were chilled in a temperature-controlled cabinet at 10 ± 1 oc for 16
hours before being immersed in a cold water in water bath
4. Capillary tubes were attached with thermometer and were immersed in a
temperature-controlled water bath
5. The water was stirrer and heated slowly by gradually adjust temperature
6. The temperature was recorded when the fat begins to rise in the tube due to
hydrostatic pressure
7. The temperature at the movement was taken as the SMP
Source: AOCS Method Cc 3-25 (1997)
108
109
Appendix VII
Study of polymorphism by XRD
1. The samples were melted at sooc for 10 min and poured into rectangular plastic
mould (20 mm x 25 mm x 3 mm) and then left to crystallize at 4°C and 25oC for 24 h
2. The samples were analyzed using scans were performed in wide angle x-ray
scattering (WAXS) from 3°28 to 35°28 with a scan speed and a step width of
4°28/min and 0.01°28, respectively
Source: Kim et al. (2008)
110
Appendix VII
Study crystal microstructure by PLM
1. The sample was melted at soac until fat melt completely to destroy crystal structure
2. 20 f.JL of each molten sample was placed on a preheated glass slide and then a
preheated cover slip was placed over the sample
3. The samples were transferred to and stored in a temperature-controlled cabinet.
The temperature of which was maintained at 4 ± OZC for 24 hours and 25 ± 0.2°C
for 24 hours
4. Microstructure at 4°C was observed using the specimen, which was kept at 4°C for
24 hours
5. Microstructure at room temperature (25°C) was observed using the specimen,
which was kept at 4°C for 24 hours and then left at 25aC for 24 hours
6. The fat crystal microstructure in the gray scale photographs was taken by 40x lens.
Source: modified from Kim et al. (2008)
Appendix IX
Commercial margarine fat extraction
1. The commercial margarine was melted at soac until fat melt completely
2. The top fat layer was decanted into a separatory funnel
3. Washed three times with the same volume of warm water
111
4. Filtered margarine fat through an anhydrous sodium sulpate layer with a Whatman
filter paper no.1 under vacuum
Source: Pande et al. (2013)
112
Appendix X
Pictures of enzymatic interesterification process
II ·~qw
A l B
f
l
Picture A and B: Small-scale of enzymatic interesterification
Picture C: Scale-up of enzymatic interesterification
Picture D: Separation of enzyme and molecular sieves from produced margarine fat by
vacuum filtration
Appendix XI
Pictures of commercial margarine fat extraction process
Picture A: Melt commercial margarine at sooc until fat melt completely
Picture B: Decant the top fat layer into a separatory funnel and wash with warm water
Picture C: Filter margarine fat under vacuum filtration
113
Name (Thai)
(English)
Bibliography
.o:::::tt. IS'" Clo" e"" I -=II
\..n'I~I'J~t.J~'lfln.J"ll 'fl'fl'Wfl:::L'flt.l~
MISS PIMWALAN ORNLA-IED
114
Home address 1511 Moo 2 Tambon Huayploo Nakhon Chaisi Nakhon Pathom 73120
Education
2009 High school I Photisarn pittayakorn school
Major: Science and Mathmetics
2012 Bachelor degree (second class honor) I Silpakorn university
Major: Food Technology (B.Sc.)
Minor: Food Processing
2013 Studying in Master degree I Silpakorn university
Major: Food Technology (M.Sc.)
Research
2013 Poster presentation in topic "Characterization of Physicochemical
and thermal properties and crystallization behavior of rambutan
seed fat" in the national conference as concept 'Sustainable
Technology Development' for engineering and industrial
technology 2013 on 2-3 December 2013 at faculty of engineering
and industrial technology sanamchandra palace campus
Silpakorn university, Nakhon Pathom
2014 Poster presentation in topic "Production of trans-free margarine
fat by enzymatic interesterification of rice bran oil and
fractionated palm stearin" in 1st Asian Conference on Oleo
Science (ACOS2014) Organized by Japan Oil Chemists' Society
on 8-10 September 2014 at Royton Sapporo, Japan