Studies on the preparation of structured lipids from rice bran oil

Rice bran oil (RBO) is the byproduct of rice milling and it is consumed widely in
Asia. It is produced from the bran and polish of rice, which are by-products of the rice
milling industry. India is the second largest producer of rice in the world and has high
potential to produce RBO. It is used for both edible and industrial applications. RBO is
unconventional oil with its fatty acid composition very close to that of groundnut oil and
has high content of unsaponifiable matter. There has been much interest in RBO in
recent years because of its nutraceuticals like tocopherols, tocotrienols, and γ-oryzanol,
which have been found to have several health beneficial effects. Structured lipids are
triacylglycerols (TAGs) that have been modified to incorporate new fatty acids or have
been restructured to change the position of fatty acids to produce novel/new TAGs.
Structured lipids can be produced to alter the physical characteristics, or improve the
nutritional quality of fats and oils. RBO contains approximately 38 % oleic acid, 34 %
linoleic acid and 18.6 % palmitic acid and it lacks n-3 PUFA. The physical properties of
these fatty acids do not confer any special properties for its use in food applications. It is
interesting to note that stearic acid content of RBO is very low. Though stearic acid is a
saturated fatty acid, studies have been shown non-atherogenic nature of stearic acid.
Saturated fatty acids are known to impart various physico-chemical and thermal stability
properties to oils and fats. Cocoa butter which is rich in stearic acid is currently the fat of
choice in the confectionery industry. Because of its demand for confectionery and
desirable physical and nutritional quality, cocoa butter substitutes and cocoa butter
equivalents have recently become more common. It is of interest to see of whether
structured lipid with RBO containing n-3 fatty acids such as α - linolenic acid,
eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which are having
several health benefits, can be synthesized. Therefore, the objective of this study was to
modify the RBO to produce structured lipids containing stearic acid and n-3 fatty acids
so that utilization of RBO can be increased and value added products can be prepared
from RBO.
Objectives and plan of work
The major objectives of the present investigation are to prepare structured lipids
from RBO rich in stearic acid, α- linolenic acid and EPA+DHA by enzyme catalyzed
reaction using immobilized lipase from Rhizomucor miehei. The structured lipids
enriched with stearic acid and n-3 PUFA were evaluated for their physico-chemical and
hypocholesterolemic and anti-aggregation effects respectively and accordingly the
following work plan has been envisaged.
1. Standardization of different reaction conditions like incubation time, temperature,
enzyme concentration and substrates molar ratio on the incorporation of stearic
acid into RBO. Purification of reaction product and studies on physico-chemical
studies.
2. Optimization different reaction conditions like incubation time, temperature,
enzyme concentration and substrates molar ratio using response surface
methodology on the incorporation of n-3 PUFA into RBO. Synthesis of large
scale structured lipids rich in n-3 PUFA and their purification.
3. Evaluation of structured lipids rich in n-3 PUFA for their hypocholesterolemic
effects and hepatic antioxidant enzymes and platelet aggregation in rats fed n-3
PUFA rich structured lipids.
Chapter I:
Introduction
This chapter deals with the general introduction of fats and oils, lipid
classification, different types of fatty acids and their physiological effects, biological
significance and health benefits of n-6 and n-3 fatty acids, metabolic pathway of n-6 and
n-3 fatty acid synthesis. This chapter also includes the effects of dietary lipids on health,
health benefits of n-3 PUFA on cardiovascular system, enzymes regulating calcium ion
channels, in the vascular retina, haemostatic system, endothelial function and nervous
system. A description on the edible oils used in India, dietary recommendation of
different fatty acids and need for the balance of fatty acids in the diet is given. Uses and
importance of structured lipids, importance of immobilized enzymes in modification of
fats and oils are described in this chapter.
Chapter II:
Materials and methods
In this chapter brief protocols of all procedures carried out throughout the
investigation have been given with appropriate references. Described here are
standardization of different reaction conditions for the synthesis of structured lipids
enriched in stearic acid, purification of newly synthesized TAG by thin layer
chromatography, quantitation of triglycerides, analysis of fatty acids by gas
chromatography and fatty acids at sn-2 position of triglyceride and differential scanning
calorimetric studies of structured lipids rich in stearic acid. Analytical methods for minor
constituents like γ- oryzanol, tocopherols and tocotrienols of native and modified RBO
were also given. This chapter describes the preparation of free fatty acids by the
hydrolysis of linseed and cod liver oil and preparation n-3 fatty acid concentrate from
linseed oil by urea inclusion method. Optimization of reaction conditions like incubation
time, temperature, enzyme concentration and substrates molar ratio by using response
surface methodology for the acidolysis reaction were also described. Scale up of the
enzyme catalyzed acidolysis and large scale preparation of structured lipids rich in n- 3
PUFA and preparation of blended oil from RBO with linseed oil and cod liver oil and
nutritional evaluation of these structured lipids were enumerated.
Chapter III:
Synthesis of structured lipids with RBO enriched in stearic acid
This chapter begins with the brief introduction of RBO, structured lipids,
importance of structured lipids for the improvement of physical properties of fats and oils
and importance and effect of stearic acid on cholesterol level. Enzymatic acidolysis
reactions were carried out in 50 mL stoppered conical flasks under inert atmosphere.
Different reaction conditions like incubation time, temperature, substrates molar ratio and
enzyme concentration on the incorporation of stearic acid into RBO were studied. The
effect of reaction time on the incorporation of stearic acid into RBO was studied from 0 to
48 h keeping other parameters constant. The effect of reaction temperature on the
incorporation of SA into RBO was studied from 25 to 60 0C keeping other parameters
constant. The effect of molar ratio of substrates on incorporation of SA into RBO was
studied from 1:1 to 1:10. The effect of enzyme concentration on incorporation of SA into
RBO was studied from 1 to 10% of the weights of both the substrates. Stearic acid was
successfully incorporated (48.5 %) into RBO under optimal conditions. After
incorporation of stearic acid into RBO, fatty acid profile and sn-2 positional analysis was
carried out in modified and original RBO using pancreatic lipase catalyzed reaction.
Analysis of oryzanol, tocopherols and tocotrienols were carried out in native and
modified RBO. The type of the triacylglycerols in modified and native rice bran oil were
analyzed by HPLC. After incorporation of stearic acid into RBO, physical properties of
native and modified RBO were studied by differential scanning calorimeter and physical
properties of modified RBO were compared with vanaspati and cocoa butter. The
chapter was concluded by discussion of the results obtained.
Chapter IV:
Enrichment of RBO with n-3 PUFA by enzymatic acidolysis: optimization of
parameters by RSM
This chapter begins with the brief introduction about the importance of n-3 PUFA
in human health and need of balance between n-6 and n-3 PUFA. The importance of
response surface methodology (RSM) for optimizing different reaction conditions for
incorporation of n-3 PUFA into the RBO was elaborated. Fatty acid concentrate from
linseed oil (LSO) rich in α- linolenic acid (ALA) and free fatty acids from cod liver oil
(CLO) were prepared and further used for acidolysis reaction to enrich RBO with ALA
and EPA+DHA respectively. Two structured lipids (SL) were obtained with RBO, one
was rich in ALA and other rich in EPA+DHA. RSM was used to optimize the reaction
conditions for lipase-catalyzed incorporation of n-3 PUFA into RBO. A CCRD with four
variables was used to study the response pattern. Purification of the reaction product
was done with TLC and column chromatography. Pancreatic lipase hydrolysis was used
to determine the fatty acids at sn-2 position in triacylglycerol. The optimum incorporation
of ALA (n-3 PUFA) into RBO was about 49 % when incubation time and temperature
were 11.5 h and 43.750C respectively and substrates molar ratio and enzyme
concentration (%) was maintained at 7.75. The optimum incorporation of EPA+DHA into
RBO was about 10 % at 37 h and 32.5 0C incubation time and temperature respectively,
where as enzyme concentration (%) and substrates molar ratio was 7.75. The fatty acid
composition of substrates and reaction products were analyzed by gas chromatography.
Large scale synthesis of these SL rich in PUFA was done. The chapter ends with the
discussion.
Chapter V:
Effects of SL and blends from RBO enriched with n-3 PUFA on liver and serum
lipids in rats
This chapter focused on the effect of SL with RBO rich in n-3 PUFA on the lipid
profile in rats. This chapter begins with a brief mention on the role of dietary fatty acids in
health and disease prevention in humans and cardiovascular protective effects of n-3
PUFA. This chapter deals with the hypocholesterolemic effect of RBO rich in n-3 PUFA.
Different experimental diets were prepared by including SL with ALA and EPA+DHA and
blended oils (RBO and LSO and RBO and CLO) having similar fatty acid composition
was fed to rats. Growth and food intake were monitored and food efficiency (FER) was
calculated. The serum lipid parameters like total cholesterol, triglycerides, phospholipids,
low density and high density cholesterol were estimated. Liver lipid parameters like total
cholesterol, triglycerides and phospholipids were estimated. Fatty acid analysis was
done for serum, liver, heart, eye, brain and adipose tissues. The chapter was concluded
with the discussion.
Chapter VI:
Antioxidant activity and platelet aggregation in rats fed SL and blends from RBO
enriched with n-3 PUFA
This chapter begins with a brief introduction about the importance of n-3
PUFA in prevention of inflammatory diseases, including cancer and coronary heart
diseases. Effect of RBO rich in n-3 PUFA on platelet aggregation of rats was studied.
Antioxidant enzymes like catalase, superoxide dismutase, glutathione reductase and
glutathione transferase activity in liver homogenate and serum of rats fed structured
lipids enriched with n-3 fatty acid was studied. Lipid peroxides in liver and serum were
done. Total Na+, K+ ATPase activity in RBC membrane of rats fed SL rich in PUFA was
also studied. Structured lipids enriched with n-3 fatty acid were studied for their inhibitory
effects on platelet aggregation of rats. The chapter concludes with discussion.
The summary and conclusions of the present findings were given and the thesis
concludes with the bibliography arranged alphabetically in sequential order