Oleic Acid - Production, Uses and Potential Health Effects 2014

BIOCHEMISTRY RESEARCH TRENDS
OLEIC ACID

PRODUCTION, USES AND POTENTIAL
HEALTH EFFECTS

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OLEIC ACID

PRODUCTION, USES AND POTENTIAL
HEALTH EFFECTS

LYNETTE WHELAN
EDITOR

New York

Copyright 2014 by Nova Science Publishers, Inc.

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CONTENTS

Preface vii
Chapter 1 Optimization of the Media Volume, Aeration Rate
and Inoculum Size for Sophorolipid Production
from Candida bombicola ATCC 22214 1
Stephanie Grieb, Fred J. Rispoli and Vishal Shah
Chapter 2 Influence of Oleic Acid on Self-Assembled Liquid
Crystalline Nanostructures 9
Intan Diana Mat Azmi and Anan Yaghmur
Chapter 3 Oleic Acid and Its Potential Health Effects 35
Igor Pravst
Chapter 4 Oleic Acid and Microbial Lipases:
An Efficient Combination 55
Fabiano Jares Contesini, Danielle Branta Lopes,
Elaine Berger Ceresino, Jose Valdo Madeira Junior,
Paula Speranza, Francisco Fbio Cavalcante Barros
and Ricardo Rodrigues de Melo
Chapter 5 Synthesis of Oleic Acid Alkil Esters
via Homogeneous Catalysis 83
Mrcio Jos da Silva and Abiney Lemos Cardoso
Chapter 6 Effects of Temperature on Oleic Acid Percentage
During Grain-Filling in Sunflowers and Other Oil
Crops 99
Rouxlne van der Merwe and Maryke Labuschagne
Index 129

PREFACE

Oleic acid is a monounsaturated fatty acid and natural constituent of a
number of foods, particularly vegetable oils. On the basis of proven beneficial
health effects it is also a possible ingredient in processed functional foods.
However, due to its high energy content it is not recommended to increase the
consumption of any particular fat, but to substitute other lipids with oleic acid.
While there is a well-established consensus that replacing saturated fats in the
diet with oleic acid or other unsaturated fats contributes to the maintenance of
normal blood cholesterol levels, a series of other effects has also been studied,
including the modulation of inflammatory markers, blood pressure, insulin
sensitivity, gastrointestinal functions and even various cancers. This book
discusses oleic acid's health effects, as well as its production, and how it is
used.
Chapter 1 In the current study the influence of aeration rate, inoculum
size and fermentation medium volume on the sophorolipids production from
the yeast Candida bombicola have been studied. Using the data obtained from
a two-level Placket-Burman experimental design, linear and cubic models
were obtained to understand the interaction amongst the ingredients. The cubic
model was used to find the optimal aeration rate, inoculum size and the
fermentation medium volume. The maximum production of SLs is predicted to
be obtained when the medium volume is 10 mL (in 125 mL Erlenmeyer flask),
is inoculated with 5% of the inoculum and incubated at 350 rpm.
Chapter 2 Various studies in the literature suggested a link between the
consumption of olive oil and different food products enriched with oleic acid
(OA) and various positive health effects. The central focus of this research
field is on learning and predicting how OA intake induces these health
benefits. In recent years, there is a growing interest in understanding the
biological role of this monounsaturated cis fatty acid in regulating cell
Lynette Whelan viii
membranes and its effect on biological processes. In this context, it is
interesting to explore the effect of its incorporation on the model membrane
characteristics and properties. These studies are considered as first steps
towards a deeper understanding of the molecular mechanisms underlying OA
beneficial health effects and their association with the biological membrane
properties.
This chapter summarizes recent studies conducted on the influence of OA
and its counterparts (saturated and trans fatty acids) on model lipid
membranes. In particular, the main focus is to present recent investigations on
the structural characterization and also the potential applications of lipidic
non-lamellar self-assembled nanostructures loaded with OA. These lyotropic
liquid crystalline (LLC) phases and microemulsions are attractive as drug
delivery systems. The most investigated LLC phases are the inverted-type
hexagonal (H
2
) and the inverted-type bicontinuous cubic (V
2
) nanostructures.
These unique inverted type self-assembled systems are compatible, digestible,
and bioadhesive matrices that are able to co-exist under equilibrium conditions
with excess water. They display nanostructures closely related to those
observed in biological membranes and posess interesting characteristics such
as the high interfacial area (specific interfacial area up to 400 m
2
/g), the high
solubilization capacities of drugs with different physicochemical properties
(hydrophilic, amphiphilic, and hydrophobic molecules), and the potential of
controlling drug release. In particular, there is an enormous interest in testing
the possibility of utilizing these LLC phases for enhancing the solubilization
of poorly water-soluble drugs, obtaining sustained drug release, and improving
the in vivo performance of various drug substances.
The scope of this chapter also covers recent studies that have attempted to
shed light on the possible fragmentation of these inverted type self-assembled
nanostructures for forming nanoparticlulate formulations attractive for food
and pharmaceutical applications. These nanostructured aqueous dispersions
(mainly cubosomes, hexosomes, and micellar cubosomes) in which the
submicron-sized dispersed particles envelope distinctive well-defined self-
assembled nanostructures can be utilized in different applications owing to
their low viscosity as compared to the corresponding non-dispersed bulk liquid
crystalline phases and their biological relevance.
Chapter 3 Oleic acid is a monounsaturated fatty acid and natural
constituent of a number of foods, particularly vegetable oils. On the basis of
proven beneficial health effects it is also a possible ingredient in processed
functional foods. However, due to its high energy content it is not
recommended to increase the consumption of any particular fat, but to
Preface ix
substitute other lipids with oleic acid. While there is a well-established
consensus that replacing saturated fats in the diet with oleic acid or other
unsaturated fats contributes to the maintenance of normal blood cholesterol
levels, a series of other effects has also been studied, including the modulation
of inflammatory markers, blood pressure, insulin sensitivity, gastrointestinal
functions and even various cancers. Commercial communication of such
effects is only ethical where such effects are relevant to human health and
proven using the highest possible standards, preferably with well-performed,
double-blind, randomised, placebo-controlled human intervention trials. Most
intervention studies investigating the health effects of oleic acid are performed
using vegetable oils which also contain other fatty acids and minor
constituents. This represents a possible confounding factor and makes
interpretations difficult. In this chapter, the health effects of oleic acid are
discussed together with the possibilities of using oleic-acid-related health
claims on foods in commercial communications in the European Union.
Chapter 4 Oleic acid is a monounsaturated fatty acid found in high
concentrations in vegetable oils, presenting a broad number of applications in
many industrial areas, such as food, pharmaceutical, cosmetic, oleochemical
and biodiesel industries. Due to the lipophilicity, unsaturation and acidic
characteristics that this compound presents, oleic acid can be effectively used
in esterification and acidolysis, among other reactions. Recent studies have
used oleic acid as an efficient substrate for synthesis of trimethylolpropane
esters by esterification using lipase from Candida Antarctica, since this polyol
ester is widely applied in hydraulic fluids with several applications. Other
studies used C. antarctica lipase for improving the lipophilicity of bioactive
molecules, such as ferulic acid and L-ascorbic acid by esterification with oleic
acid, which is very interesting, taking into account that it increases the
solubility of these molecules in hydrophobic environments, resulting in higher
biological activities. On the other hand, some studies showed that lipases can
be used to convert oleic acid into epoxies, which are useful intermediates in
organic synthesis due to the high reactivity they present. They are used to
produce plasticizers that increase flexibility, workability or distensibility of
plastics, hence rendering them suitable for several applications. One study
reported biodiesel production by esterification of oleic acid with aliphatic
alcohols using immobilized Candida antarctica lipase, showing high yields of
biodiesel (above 90%) in less than 24 h with ethanol, n-propanol and n-
butanol; whereas with methanol, the enzyme was inactive after ten cycles of
reaction. In addition to the various reactions involving oleic acid as a
promising substrate for various reactions, oleic acid can also be used to induce
Lynette Whelan x
microbial lipase production, as seen in a study using the fungal strain Rhizopus
arrhizus. Therefore, different high-added-value compounds can be obtained
using oleic acid as a cheap and efficient substrate for microbial lipases, which
can be considered as environmentally friendly alternatives for chemical
catalysts. Within this context, this chapter reviews some studies and trends on
the use of oleic acid as an efficient substrate for microbial lipases.
Chapter 5 Recently, due to inevitable exhaustion of the fossil petroleum
reserves, and the environmental impact generated by the green-house effect
gas emission, to develop efficient processes for the production of fuels and
chemicals from the renewable feedstock has been pursued researchers in
worldwide. In this sense, since the oleic acid is a common component of
vegetal oils and animal fatty, it raise as a highly attractive raw material, due to
its high availability and affordability. In general, the oleic acid is present in
different feedstock as a free fatty acid or as glyceryl ester. Several chemicals
of interest for plentiful industries can be obtained via different catalytic
reactions starting from the oleic acid as source, such as alkyl esters or ethers
and epoxide-derivatives. Particularly, alkyl oleate esters are useful as
lubricant, surfactant, emulsifying agent, emollient, fuels additive and
biodiesel. Actually, the main component of biodiesel is in general the methyl
or ethyl oleate, which is manufactured by the alkaline transesterification of
edible or non-edible vegetable oils via a well-established industrial process.
However, the conventional alkaline homogeneous process results in large
generation of effluents and residues of neutralization, in addition the laborious
steps to remove the non-reusable catalyst, being because of these reasons a
non-friendly environment process. In this work, the authors wish the recent
advances achieved in the development of catalytic processes for the production
of alkyl esters of oleic acid via acid catalysis, however, using recyclable
catalysts. They will pay special attention to development of homogeneous
catalysts that can be recovery and reusable without loss of activity in the oleic
acid esterification reactions. These catalysts are solid when pure and soluble in
the reaction being thus recovered after solvent distillation and extraction of
products. Numerous industries in all parts of world have crescent demand by
developing of environmentally friendly technologies for the production of
biodiesel and chemicals, which are especially attractive when are based on
reusable catalysts. Herein, the authors focus the use of two different sorts of
catalysts: the former, Lewis acid such as tin compounds, and the second one,
Brnsted acid catalysts, which are based on Keggin-type heteropolyacids. The
catalysts performance it was assessed in the esterification reactions with short
chain alkyl alcohols (i.e., methyl, ethyl, propyl, isopropyl and butyl alcohols).
Preface xi
A comparison with the traditional catalysts used in these reactions also was
performed. The development of new, efficient, and environmentally benign
catalytic processes that may lead to high value added products, starting of
renewable raw material such as oleic acid, is still an challenge to be overcome.
The authors hope that this work can significantly contribute to improvement of
this important research field.
Chapter 6 Most vegetable oils are obtained from beans or seeds, which
furnish valuable and high quality oil commodities in the world oil market.
Seed oil quality is related to oil percentage and fatty acid composition and
defines the oils value for industry. With emerging new markets and increased
concerns about the health risks of foods, changes in the oil quality of various
crops have been demanded. Plant breeders have been successful in developing
novel oil types in sunflower, soybean, peanut and others with increased
percentages of oleic acid. Genotype is the most important factor that defines
the oil fatty acid composition, but environmental factors, particularly during
the grain-filling period, can widely affect both oil content and oleic acid
percentage. Various environmental factors including temperature (heat and
cold, day/night differences), solar radiation, humidity, day length and moisture
availability (rainfall distribution and intensity, drought or flooding) affect seed
oil percentage and composition. When environmental factors deviate from the
optimal quantity or intensity for the crop plant, stress is caused. Changes in
both oil percentage and fatty acid composition caused by environmental stress
could have a dynamic effect on the quantity and quality of oil that is
extractable by seed processors. Temperature is a major environmental factor
that determines the rate of oil accumulation. Generally warm temperatures
during the entire growing season or a period of heat stress during grain-filling
favors the production of oleic acid, while cooler temperatures favor the
production of linoleic acid in traditional oil crops. However, not all genotypes
are similarly affected by temperature and show strong genotype by
environment interaction. Generally the novel sunflower genotypes with
increased oleic acid contents display more stable oleic to linoleic acid ratios
across different environments than standard types with high linoleic acid
percentages. In novel soybean varieties, the high oleic acid content fluctuates
with temperature differences. In order to improve oil quality in traditional oil
crops, it is necessary to understand the temperature effects on oleic acid
content. In addition, since agricultural and management practices can alter
temperature and other important environmental factors that plants are exposed
to during grain-filling, altered production practices could contribute to
modified oleic acid contents in vegetable oil crops.
In: Oleic Acid ISBN: 978-1-63117-576-3
Editor: Lynette Whelan 2014 Nova Science Publishers, Inc.

Chapter 1

OPTIMIZATION OF THE MEDIA VOLUME,
AERATION RATE AND INOCULUM SIZE
FOR SOPHOROLIPID PRODUCTION FROM
CANDI DA BOMBI COLA ATCC 22214

Stephanie Grieb
1
, Fred J . Rispoli
2
and Vishal Shah
*
1
1
Department of Biology, Dowling College, Oakdale, NY, US
2
Department of Mathematics, Dowling College, Oakdale, NY, US

ABSTRACT

In the current study the influence of aeration rate, inoculum size and
fermentation medium volume on the sophorolipids production from the
yeast Candida bombicola have been studied. Using the data obtained
from a two-level Placket-Burman experimental design, linear and cubic
models were obtained to understand the interaction amongst the
ingredients. The cubic model was used to find the optimal aeration rate,
inoculum size and the fermentation medium volume. The maximum
production of SLs is predicted to be obtained when the medium volume is
10 mL (in 125 mL Erlenmeyer flask), is inoculated with 5% of the
inoculum and incubated at 350 rpm.

*
Corresponding author: Phone: 631-244-3339; Fax: 631-244-1033; Email: ShahV@dowling.
edu.
Stephanie Grieb, Fred J. Rispoli and Vishal Shah 2
Biosurfactants have become increasingly popular in the recent times
owing to their environmental friendly properties. One of the biosurfactants that
is gaining attraction for its biological properties are Sophorolipids (SLs). SLs
are low-molecular weight biosurfactants produced by yeasts such as Candida
bombicola, Yarrowia lipolytica, Candida apicola, and Candida bogoriensis
when grown on carbohydrates and lipophilic substrates. [1] The biological
properties of the compounds include anticancer [2], antibacterial [3],
antifungal [4], antiviral [5] and spermicidal activity. [6] In addition, SLs have
also shown to be an effective septic shock antagonist [7,8] and have been
proposed to have applications in food thickening, herbicide and pesticide
formulations, consumer product manufacturing (e.g. detergents and
cosmetics), and lubricant formulations. [9]
Not many studies have been published to optimize the fermentation
conditions for obtaining maximum SL yields. In our recent study, we
optimized the fermentation medium for the maximum production of SLs using
the yeast Candida bombicola ATCC 22214. [8] Sixteen different media
ingredients were screened and the fermentation medium composed of sucrose,
malt extract, oleic acid, K
2
HPO
4
and CaCl
2
was shown to provide the highest
yield of the glycolipids. However, no physical parameters were optimized in
the earlier study. Using a two-level Placket-Burman design, three physical
process parameters are optimized in the current study to obtain high yields of
SLs under batch fermentation. The process parameters are aeration rate,
medium volume and the age of the inoculum. Aeration rate and medium
volume are critical in determining the amount of oxygen transferred into the
fermentation medium. Oxygen supply is important in the SL fermentation
because the yeast is very sensitive to the oxygen limitation during their
exponential growth phase Guilmanov et al. have carried out a detailed
investigation on the influence of oxygenation on the SL production under fed-
batch conditions using shake-flask method [9]. They reported that higher
levels of oxygenation resulted in increased SL formation and that the oxygen
transfer rate has to be between 50 and 80 mM O
2
/L h
-1
for obtaining high
yields. The study however was carried out using an un-optimized media of
glucose, yeast extract and urea, and also included a step of centrifuging the
cells from the inoculum media before introducing them into the fermentation
media. In our preliminary study, we found that centrifugation of cells before
introducing them to the fermentation media decreases the yield of SL (data not
shown). Thus, the process parameters of media volume and agitation rate were
selected in the current study. As the culture flasks will be of identical size,
cultures of higher medium volumes represent lower oxygenation rate and those
Optimization of the Media Volume, Aeration Rate 3
with smaller volumes represent higher oxygenation. Higher aeration rate
results in higher oxygenation rate, and smaller aeration rates results in lower
oxygenation rate. Inoculum volume was selected as the third parameter
because it is known that the production of SL begins only when the nitrogen in
the fermentation media is depleted. [10] The inoculum size would determine
how many yeast cells are introduced in the fermentation medium and hence
the rate at which the nutrients are utilized.
Candida bombicola ATCC 22214 was used for SL production. The
protocol described in Rispoli et al. [8] was used for Sophorolipid production.
The fermentation was carried out in 125 mL Erlenmeyer flasks and the
fermentation media was composed of sucrose, 125 g/L; oleic acid, 166.67 g/L;
CaCl2, 2.5 g/L; K2HPO4, 1.5 g/L and malt extract 25 g/L. The amount of
fermentation medium in the flask and the volume of inoculum added to the
media were varied as per the experimental design described in Table 1. The
flasks were incubated for 8 days at 30 1.5 C in a rotary shaker. The
extraction and estimation of SLs was carried out following the protocol
described earlier [8] A Plackett-Burman two-level experimental design was
obtained with one block for three independent variables. Fusion Pro version
7.3.20 (S-Matrix Corp., USA) software was used to obtain the design. The
obtained design is shown in Table 1. The statistical analysis of data was
carried out using Statistica release 8 (StatSoft Inc., USA).

Table 1. Experimental design matrix and the obtained yields of
Sophorolipids under each condition

Experiment
Number
Aeration
(rpm)
Media
volume
a
(mL)
Inoculum
(%)
SL Yield
(g/L)
1 50 10 5 26.14
2 50 10 15 23.33
3 50 40 5 9.67
4 50 40 15 7.85
5 200 25 10 15.49
6 350 10 5 87.84
7 350 10 15 74.2
8 350 40 5 15.29
9 350 40 15 15.2
a
The media volume is the final volume in the flask after addition of the inoculum.

As can be seen in Table 1, the media volume in the flask was varied from
1/10 (10 mL) of the total flask volume to 1/3 (40 mL). Similarly, the aeration
was varied from 50 rpm to 350 rpm. Thus, experiment number 6 and 7 which
have a volume of 10 mL and were incubated at 350 rpm receive highest
oxygenation. Whereas experiment number 3 and 4 have the lowest
oxygenation. SL yield indicates that the highest yield was obtained when the
yeast received high amount of oxygen. When one compares the SL yield
obtained in experiment 1 and 3, 6 and 8 it can be concluded that increasing the
media volume decreases the production of SLs. These comparisons were
carried out because between the experiments, the other two variables have
same value. Comparison between experiments 1 and 6, 2 and 7, indicates that
increasing aeration has a positive influence on the yield.

Table 2. Linear and cubic model obtained by analyzing the data described
in Table 1

Variable Linear model Cubic model
R
2
= 0.71 R
2
= 0.94
x
1
50.72 86.30
x
2
-21.53 8.13
x
3
14.74 21.79
x
1
.x
2
- -80.68
x
1
.x
3
- -35.43
x
2
.x
3
- -23.61
x
1
.x
2
.x
3
- 37.16

Both a linear and a cubic model were obtained using regression analysis
(Table 2). The primary effect of each of the variables can be evaluated based
on a liner regression model. Based on the coefficients, aeration has the highest
positive influence on the yield, whereas media volume has a strong negative
influence. The amount of inoculum added also has a positive influence on the
production of SLs. The low fit of the linear model with the experimental data
is an indication that apart from the primary effect for each independent
variable, there is a high degree of interaction that is undetected by the linear
model. The quadratic model result has R
2
value of 0.94. The improvement of
the R
2
value from 0.70 to 0.94 is due to the two-way and three-way interaction
terms incorporated into the cubic model. Interestingly, the cubic model shows
that the primary effects of all the variables (including media volume) are
positive and the observed overall effect for each variable is due to the
interactions with other variables. The model shows that all the two-way
interactions are negative. Confirmation of the interaction can be obtained from
the ternary plot illustrated in Figure 1. Maximum yield is predicted near the
vertex of the aeration and along the inoculum aeration axis. Very low yield
is predicted when the aeration has a lower value (along the inoculum media
volume axis).

Figure 1. Ternary plot of the quadratic model predicting the production of
Sophorolipids under various conditions.
The optimization of the process variables was carried out using Frontline
Solver, optimization software built into Microsoft Excel. The cubic model
described in Table 2 was selected as the objective function. The optimal
solution obtained was aeration of 350 rpm, inoculum volume of 5% and media
volume of 10 mL and the maximum yield predicted is 86.29 g/L under optimal
conditions. The optimal conditions predicted by Solver are similar to those in
experiment 6, and the yield obtained experimentally was 87.84 g/L.
In conclusion, the influence of the aeration, inoculum volume and media
volume have been studied in the current study and the optimal values of the
three obtained to achieve highest SL yield. During the course of study we have
also identified several confounding variables including the amount of cells in
the inoculum and the physiology of the organisms (data not shown). Studies
are now being carried out in our laboratory to investigate how these variables
influence the SL production by Candida bombicola. In addition, it has been
recently shown that the structural composition of SL is highly dependent on
the aeration rate. [12] A regression model that is able to predict the
composition of the SL based on the fermentation conditions is also being
developed.

ACKNOWLEDGMENT

The study was funded by National Science Foundation (Grant # CBET
0828292).

REFERENCES

[1] Gobbert, U., Lang, S. and Wagner, F. (1984) Biotechnol Lett. 6, 225-
230.
[2] Chen, J., Song, X., Zhang, H. and Qu, Y. (2006) Enzyme Microbial
Technol. 39, 501-506.
[3] Shah, V., Badia, D. and Ratsep, P. (2007) Antimicrobial Agents and
Chemotheraphy. 51, 397-400.
[4] Gross, R. and Shah, V. (2004) Antifungal properties of various forms of
sophorolipids. US Patent application No. 20050164955.
[5] Shah, V., Doncel, G. F., Seyoum, T., Eaton, K. M., Zalenskaya, I,
Hagver, R., Azim, A. and Gross, R. (2005) Antimicrobial Agents and
Chemotherapy. 49, 4093-4100.
[6] Bluth, M.H., Kandil, E., Mueller, C. M., Shah, V., Lin, Y. Y., Zhang, H.,
Dresner, L., Lempert, L., Nowakowski, M., Gross, R., Schulze, R. and
Zenilman, M. E. (2006) Crit. Care Med. 34, 188-195.
[7] Solaiman, D. K. Y. (2005) Inform. 16, 408-410.
[8] Rispoli, F. J., Badia, D. and Shah, V. (2010) Biotechnol. Progress, 26,
938-944
[9] Guilmanov, V., Ballistreri, A., Impallomeni, G.. and Gross, R. A. (2002)
Biotechnol. Bioeng, 77, 489-494.
[10] Lien, C-C. (2007) Ph. D. Thesis. Polytechnic University of New York.
2007.
[11] Shah, V., Jurjevic, M. and Badia, D. (2007) Biotechnol. Prog. 23, 512-
515.
[12] Ratsep, P. and Shah, V. (2009) J. Microbiol. Methods. 78, 354-356.


Chapter 2

INFLUENCE OF OLEIC ACID
ON SELF-ASSEMBLED LIQUID
CRYSTALLINE NANOSTRUCTURES

I ntan Diana Mat Azmi and Anan Yaghmur
*

Department of Pharmacy, Faculty of Health and Medical Sciences,
University of Copenhagen, Denmark

ABSTRACT

Various studies in the literature suggested a link between the
consumption of olive oil and different food products enriched with oleic
acid (OA) and various positive health effects. The central focus of this
research field is on learning and predicting how OA intake induces these
health benefits. In recent years, there is a growing interest in
understanding the biological role of this monounsaturated cis fatty acid in
regulating cell membranes and its effect on biological processes. In this
context, it is interesting to explore the effect of its incorporation on the
model membrane characteristics and properties. These studies are
considered as first steps towards a deeper understanding of the molecular
mechanisms underlying OA beneficial health effects and their association
with the biological membrane properties.
This chapter summarizes recent studies conducted on the influence of
OA and its counterparts (saturated and trans fatty acids) on model lipid

*
Corresponding author: Tel.: +45 35 33 65 41, Fax: +45 35336030, e-mail: anan.yaghmur
@sund.ku.dk.
Intan Diana Mat Azmi and Anan Yaghmur 10
membranes. In particular, the main focus is to present recent
investigations on the structural characterization and also the potential
applications of lipidic non-lamellar self-assembled nanostructures loaded
with OA. These lyotropic liquid crystalline (LLC) phases and
microemulsions are attractive as drug delivery systems. The most
investigated LLC phases are the inverted-type hexagonal (H
2
) and the
inverted-type bicontinuous cubic (V
2
) nanostructures. These unique
inverted type self-assembled systems are compatible, digestible, and
bioadhesive matrices that are able to co-exist under equilibrium
conditions with excess water. They display nanostructures closely related
to those observed in biological membranes and posess interesting
characteristics such as the high interfacial area (specific interfacial area
up to 400 m
2
/g), the high solubilization capacities of drugs with
different physicochemical properties (hydrophilic, amphiphilic, and
hydrophobic molecules), and the potential of controlling drug release. In
particular, there is an enormous interest in testing the possibility of
utilizing these LLC phases for enhancing the solubilization of poorly
water-soluble drugs, obtaining sustained drug release, and improving the
in vivo performance of various drug substances.
The scope of this chapter also covers recent studies that have
attempted to shed light on the possible fragmentation of these inverted
type self-assembled nanostructures for forming nanoparticlulate
formulations attractive for food and pharmaceutical applications. These
nanostructured aqueous dispersions (mainly cubosomes, hexosomes, and
micellar cubosomes) in which the submicron-sized dispersed particles
envelope distinctive well-defined self-assembled nanostructures can be
utilized in different applications owing to their low viscosity as compared
to the corresponding non-dispersed bulk liquid crystalline phases and
their biological relevance.

INTRODUCTION

The negative health effects associated with the consumption of food
products containing trans-fatty acids (TFAs) remain a major concern for the
consumers [1]. The overall awareness about the significant role of these fatty
acids in human nutrition has been raised since 1980s [2-4]. These unsaturated
fatty acids contain at least one double bond of trans configuration and are
mainly generated by the process of partial hydrogenation of vegetable oils,
which is used in food manufacturing industry to commercially produce edible
solid fats with an increased shelf life that can substitute animal fats in diet [2,
5-7]. The major concern is that the trans configuration affects not only the
Self-Assembled Liquid Crystalline Nanostructures 11
physicochemical properties of the fatty acids [2, 7] but also it attributes to
multiple negative effects [6-14]. Various epidemiologic and clinical studies
reported on the influence of the TFA intake on increasing the risk of coronary
heart disease [5-8, 15] and cancer [6, 7, 16], increasing the blood low density
to high density lipoprotein (LDL/HDL) ratio [6, 17, 18]. More than one third
of cancer incidence and other chronic diseases such as cardiovascular risk
factors were claimed to be associated with the nutrition-related attitudes [19-
22]. In addition, different studies suggested an important link between the
TFA intake and insulin sensitivity [6], systemic inflammation [6, 23], and
impairing the endothelial function [14]. Diabetes was also reported to be
associated with the TFA dietary that stimulated a greater adipogenic effect [11,
24]. A growing body of evidence on the adverse negative health effects
associated with TFA consumption suggests introducing TFA-free food
products to the market [25-27].
In contrast to trans-fat dietary, the consumption of olive oil, which is rich
in oleic acid (a monounsaturated fatty acid with the natural cis configuration),
is associated with positive health effects [28-30]. In European countries such
as Greece and Italy and in the Middle East the intake of olive oil is high and is
linked in different regions to a relatively reduced blood pressure and a reduced
risk of developing coronary heart disease, a reduced breast cancer, and a low
level of plasma cholesterol [30-32]. The past decade has witnessed a
tremendous interest in understanding why the consumption of oleic acid-rich
diet is important to our health and wellness. It was reported that oleic acid
(OA) reduces a cluster of prevalence metabolic syndrome (MetS) including
obesity, hypertension, impaired fasting glucose (insulin resistance at pre-
diabetic state), blood pressure, high-density lipoprotein cholesterol [HDL-C]
levels, and the risk of coronary heart disease [20, 33-39]. It was also found that
this monounsaturated fatty acid (MUFA) is an active component that
influences the proliferation of immune cells in comparison with other fatty
acids [36, 40] as well as it reduces the risk of ulcerative colitis (UC) disease
[41]. Not only that, OA is used as a penetration enhancer to increase the
permeability of active molecules to the skin [42-44]. Most interestingly, the
role of OA in inhibiting cell proliferation and inducing apoptosis in carcinoma
cells has received great attention [16, 45, 46]. It was suggested therefore to use
OA as an antitumoral agent [29, 40, 47-50]. In an interesting report, it was
found that the combination of OA with the drug trastuzumab leads to the
occurrence of a synergistic cytotoxic effect towards breast cancer [51].
There is a growing research interest on exploring the effect of OA on
biological membrane structures due to the implications of its daily
consumption in vital biological processes related to health and disease, and its
possible use as one of the main components in the formation of soft lipidic
nanoparticlulate formulations attractive for delivering drugs or functional
foods [25, 52-55]. Understanding the effect of this free cis-fatty acid on
regulating cell membranes is considered as first step towards a deeper
understanding of the biological membrane properties and the molecular
mechanisms underlying OA beneficial health effects. In this contribution, the
main attention is to focus on the influence of OA and its counterparts
(saturated and trans fatty acids) on the structural characterization and the
potential pharmaceutical applications of lipidic non-lamellar lyotropic liquid
crystalline (LLC) phases and their corresponding aqueous dispersions
(cubosomes and hexosomes).

I. OLEIC ACID: BIOLOGICAL ACTIVITY
AND PHARMACEUTICAL USES

OA-rich diets are associated with increasing the level of this fatty acid in
human plasma membrane [56, 57]. The health benefits of OA intake has been
subjected to a large number of reports [16, 46, 58], but its specific mechanism
of action remains poorly understood. It was suggested that OA intake
modulates the structure of cell membranes [59-61]. For instance, a recent
interesting study suggested an important role of this monounsaturated cis-fatty
acid in modulating the adrenoreceptor signaling pathway that induces a
reduction in the blood pressure (BP) [62]. This G protein-associated signaling
activity was found in both in vivo (in human) and in cell culture studies, but
apparently not detected in the membrane-free system [62, 63]. In contrast, the
counterparts elaidic (EA, trans C18:1t9) or stearic (SA, C18:0) acids, which
are structurally different than OA at the molecular level, do not induce
significant activity on the adrenoreceptor signaling pathway. This structural
difference between trans- (a rod-like structure) and cis-FA (a boomerang-
shaped structure with prominent kink in the molecular backbone) leads to
important biophysical and biological consequences [64]. It was reported that
the conformational flexibility of OA molecule induces a major structural
alteration of the hydrophobic core of the lipid bilayer and perturb the
membrane structure as compared to the rod-like molecular structure of trans-
FA that leads only to a little disorganization [62].
It was reported that the molecular mechanisms by which OA affects the
biological membrane involve a very specific link between the membrane lipid
structure and the BP regulation [39]. In this context, it was demonstrated that
the penetration of OA molecules into the lipid membrane structure leads to a
marked reduction in the lamellar (L
)-to-hexagonal (H
2
) phase transition as
compared to the trans or saturated FA counterparts [59, 60]. The presence of
this non-lamellar prone lipid in the cell membrane significantly alters the
membrane curvature strain to be more negative [60]. It was assumed that the
transition to hexagonal (H
2
) phase favors the docking of certain peripheral
signaling G protein, which in turn affects the BP [39, 65]. It is also interesting
that the structural analogue of synthetic OA, 2-hyroxyoleic acid (2OHOA) acts
as a potent antitumor drug for glioma by inducing important signaling changes
that end up with cell death [66, 67]. Martnez et al. reported on the propensity
of 2OHOA to organize the lipid membrane into a non-lamellar phase, which
promotes the recruitment of protein kinase C (PKC) to the cell [68]. It was
suggested that the transition to the H
2
phase leads to impair of cell progression
and simultaneously inhibits the growth of the tumor cells [68]. In another
report, the apoptotic activity of OA/protein complexes, known as HAMLET
(Human Alpha-lactalbumin Made LEthal to Tumor cells) was attributed also
to the role of OA in membrane perturbation. As an initial step of killing the
tumor cells, OA alters the membrane and compromises its integrity [64, 69,
70].
Besides the widespread research interests in understanding the role of OA
in regulating biological functions, the use of OA as a main essential
constituent in various drug nanoparticulate formulations including liposomes,
microemulsions, and nanoemulsions has attracted a great attention in the last
two decades [1, 55, 71]. For instance, the utilization of OA-loaded liposomes
(LipoOA) as promising candidates in transdermal applications was suggested
in the literature due to the therapeutic efficacy of these soft drug nanocarriers
in eradicating drug resistance and enhancing its skin penetration [72, 73]. It
was also reported that the association of OA in lipidic nanoparticles (LNPs)
enhances the cellular uptake and hepatic delivery of siRNA and microRNA
[74]. In addition, self-assembled gelatin-OA nanoparticles and OA-loaded
microemulsion were found attractive candidates for improving the
solubilization of poorly water-soluble drugs and controlling their release [71,
75-77].

II. FORMATION OF SELF-ASSEMBLED NANOSTRUCTURES

Surfactant-like lipids adopt either normal (type 1) or inverted (type 2) self-
assembled phases, resulting in either oil-in-water (o/w) phases with convex
curvature lipid/water interface or water-in-oil (w/o) phases with a concave
interface, respectively. The formation of a normal or an inverted self-
assembled nanostructure in water mainly depends on the lipids molecular
shape, as discussed in the seventies by Israelachvili and co-workers [78]. In
this regard, the geometric shape of the lipid can be a useful tool for predicting
the water-lipid interface curvature and also can be helpful in understanding the
phase behavior of binary, ternary, and even multi-component systems [79].
For this purpose, the shape factor or more commonly known in the literature as
the critical packing parameter (CPP) was defined [78] as:

(1)

where v
s
is the effective hydrophobic chain volume, a
0
is the headgroup area,
and l is the hydrophobic chain length. The inverted type phases are favored
when CPP > 1 and therefore are generally formed when adding to water
wedge-shaped lipids with hydrophobic tails having a relatively large volume
(v
s
) as compared to the hydrophobic chain length (l) and the headgroup area
(a
0
). Balanced surfactants with CPP 1 tend to form planar bilayers (the
lamellar phase); whereas normal type liquid crystalline phases and micellar
solutions are displayed in the presence of surfactants having CPP < 1. It is
worth noting that the CPP is affected by different variables including lipid
composition, hydration level, electrostatic interactions, presence of
hydrophilic, hydrophobic and amphiphilic additives, and applied experimental
conditions [79-82].
From applicational point of view, there is a noteworthy difference in the
hydration behavior between the normal and inverted type self-assembled
phases. The normal type phases can be easily destabilized in the presence of
excess water, as the surfactant monomers are dissolved in the aqueous
environment when approaching a concentration lower than its critical micellar
concentration (cmc). In contrast, the inverted type phases are independent of
water content under full hydration conditions and therefore are stable against
water dilution [83]. Thus, these systems have recently gained considerable
CPP
v
s
a
0
l
interest in designing drug and functional food delivery systems due to their
unique properties [84].
Owing to this attractiveness to potential pharmaceutical applications, the
focus in the next sections will exclusively be on describing the formation and
the characterization of inverted type dispersed and non-dispersed phases.

III. INVERTED TYPE LYOTROPIC LIQUID
CRYSTALLINE PHASES

Certain biologically relevant amphiphilic (surfactant-like) lipids including
monoglycerides, glycolipids, and phospholipids have the ability to self-
assemble upon hydration into inverted type lyotropic liquid crystalline (LLC)
phases or micellar systems [79, 85].
This process of self-assembly depends on various parameters including
the chemical structure of the lipid and its composition [86]. It results under
certain experimental conditions on the formation of highly ordered liquid
crystalline phases or micellar solutions consisting of discrete aqueous and
lipidic regions upon direct contact of the surfactant-like lipid with water [87].
These self-assembled systems include lamellar (L
) and non-lamellar (two

and three dimensional bicontinuous and discontinuous nanostructures) phases,
and inverted type micellar solution (L
2
).
Among the inverted type non-lamellar phases, various studies have been
reported on the formation of bicontinuous cubic (V
2
) phases, the hexagonal
(H
2
) phase, and the discontinuous cubic (I
2
) phase of the symmetry Fd3m [81,
88, 89].
The three dimensional (3D) cubic V
2
phases are arranged as single
continuous lipid curved bilayers forming a complex network containing two
non-intersecting water channels [90]. Three different bicontinuous cubic
nanostructures (a family of closely related phases) have been identified in the
literature. They have a primitive (P), a gyroid (G), or a diamond (D) infinite
periodic minimal surface (IPMS) [88, 89].
The minimal surfaces have zero mean curvature and are therefore as
convex as concave at all points. The space groups corresponding to these three
IPMSs are Im3m (the primitive type, C
p
), Ia3d (the gyroid type, C
G
), and
Pn3m (the diamond type, C
D
) respectively [79, 88, 91, 92].
The two-dimensional (2D) reverse hexagonal (H
2
) phase consists of
water-filled cylindrical rods (hydrophilic nanochannels) embedded in a
continuous hydrophobic medium. The discontinuous cubic (I
2
) phase with the
space group Fd3m that was identified in different lipid-based systems consists
of two different quasi-spherical micelles packed in a 3D cubic lattice; whereas
the L
2
phase is a reversed micellar solution with no long-range order
[31, 79, 93].
The non-lamellar liquid crystalline matrices (mainly the inverted-type
hexagonal phase (H
2
) and inverted-type bicontinuous cubic (V
2
)) display
nanostructures closely related to those observed in different biological
membranes and have unique properties such as high interfacial area
(estimation of about 400 m
2
/g of surfactant) [94], capability to solubilize
amphiphilic, hydrophobic, and hydrophilic drugs in their highly ordered self-
assembled interiors, biocompatibility and capability to exist under equilibrium
condition with excess water [95-97].
Monolinolien (MLO) is among the surfactant-like lipids with propensity
to form inverted type non-lamellar phases. The binary MLO-water phase
diagram is shown in Figure 1 [83].
A variety of mesophases is formed depending on the water content and the
investigated temperature. Right of the phase separation line, the mesophases
co-exist with excess water, thus their fully hydrated structures are independent
of water content in the biphasic regions.
It is evident that the bicontinuous cubic phases can solubilize significantly
more water at ambient temperatures in their hydrophilic nanochannels as
compared to those of the H
2
and L
2
phases that are formed at higher
temperatures [83].
The phase behavior of the binary MLO-water system is similar to that of
the well-studied monoglyceride monoolein (MO) [98]. Both amphiphilic lipids
have cis-configuration that introduces a kink in their acyl chain [79]. These
lipids are widely used in food industry as they are specified as GRAS
(generally recognized as safe). They are subject to enzymatic lipolysis in a
wide range of tissues and therefore are considered biocompatible and
biodegradable materials [94].
Figure 1 (right) illustrates the phase behavior in a binary or ternary lipid
system. The self-assembled nanostructure follows the phase sequence of L

V
2
H
2
I
2
L
2
with increasing solubilized oil content and/or
temperature, ranking the inverse phases by increasing values of their mean-
interfacial curvature or CPP value [83, 99]_ENREF_96. The CPP increases
with temperature due to the increased fluctuation of the hydrophobic chains of
the investigated surfactant-like lipid [83].

Figure 1. Left: Phase diagram of the binary MLO-water system. Right: Phase sequence in a binary or ternary lipid system that is
displayed upon increasing temperature and/or solubilizing oil. The phases are the following: (A) a fluid lamellar (L
) phase, (B) three

bicontinuous cubic (V
2
) phases, (C) a H
2
phase, (D) a discontinuous cubic Fd3m phase, (E) and an inverted-type water-in-oil (W/O)
microemulsion system (the L
2
phase) (the figures have been taken with permission from reference [83]).

Figure 2. Left: SAXS patterns taken from MLO-based aqueous dispersions (red lines) and its corresponding fully hydrated non-
dispersed system (black lines) at three different temperatures (the figure was adapted with permission from reference [83]). Right: cryo-
TEM images of four tetradecane-free and tetradecane-loaded MLO-based aqueous dispersions; (a) tetradecane-free cubosomes, (b)
hexosomes, (c) micellar cubosomes, and (d) EMEs (the figures have been taken with permission from references [100, 108]).
This results in a larger effective hydrophobic chain volume (v
s
) with a
simultaneous decrease in the solubilized water content (a decrease of a
0
value
due the dehydration of the hydrophilic headgroups of the lipid). A similar
effect on v
s
and a
0
can be obtained upon the solubilization of hydrophobic
additives at a constant temperature [83, 100-102].

IV. AQUEOUS DISPERSIONS OF LYOTROPIC LIQUID
CRYSTALLINE PHASES AND MICROEMULSIONS

The non-dispersed bulk non-lamellar LLC phases (the V
2
and H
2

nanostructures) are highly viscous. This limits their pharmaceutical
applications as they are difficult to inject and can cause irritation when having
direct contact with epithelial cells [103]. Therefore, an interesting approach in
literature is based on dispersing these LLC phases into low viscous
nanoparticles with retained internal structures [83, 104, 105]. Examples of
these aqueous dispersions are cubosomes with an internal V
2
phase and
hexosomes with an internal H
2
phase [106, 107]. In addition, other aqueous
nanostructured dispersions were reported including micellar cubosomes with
an internal I
2
phase of the symmetry Fd3m, emulsified L
2
system (oil-free L
2

phase), and emulsified microemulsions (EMEs) with an internal W/O
microemulsion system (L
2
). These aqueous dispersions consist of kinetically
stabilized submicron sized particles enveloping internally self-assembled
nanostructures. They have identical unique properties as their corresponding
non-dispersed LLC phases and microemulsions, including high interfacial area
and biological relevance [100].
The most used techniques for characterizing the internal nanostructures of
aqueous dispersions of LLC phases are the small angle X-ray (SAXS) and
neutron (SANS) scattering techniques. Figure 2 (left) shows the typical SAXS
patterns for the fully hydrated non-dispersed V
2
, H
2
, and L
2
bulk phases (black
lines) and their corresponding nanostructured aqueous dispersions (red lines)
[83]. It is evident from the SAXS patterns in Figure 2 (left) that the internal
nanostructures are preserved upon dispersing the bulk phases in excess water,
as the same characteristic X-ray diffraction peaks are observed for the
dispersed and the non-dispersed phases.
As a complementary technique to SAXS, the cryogenic Transmission
Electron Microscopy (cryo-TEM) enables the visualization of the shape of the
dispersed particles and their internal nanostructures. The right side of Figure 2
presents cryo-TEM observations of four MLO-based aqueous dispersions
loaded with tetradecane.

V. EFFECT OF OA AND ITS COUNTERPARTS ON LIPIDIC
SELF-ASSEMBLED NANOSTRUCTURES

Fatty acids (FAs) are abundant components in plasma and other biological
membranes that are present as free or bound to phospholipids or cholesterol
esters [60]. It is crucial to understand how low levels of free fatty acids (FFAs)
affect the membrane structure in order to gain insight into the underlying
mechanisms behind the interaction of OA with biological membranes and its
influence on the associated positive health effects. In spite of the fact that
elaidic acid (EA, C18:1t9: the most abundant fatty acid in TFAs) and its
counterpart oleic acid (OA, C18:1c9) have the same molecular weight, but the
difference in the structure at the molecular level and the associated health
effects with their intake is significant. Funari et al. studied the effect of loading
OA, EA and stearic acid (SA, C18:0) on the structural properties of fully
hydrated phosphatidylethanolamines (PEs) [60]. They found that OA
significantly alters the membrane structure and reduces up to 2023 C of the
lamellar-to-hexagonal transition temperature. Interestingly, the replacement of
OA with its congeners EA and SA does not induce a significant effect on the
structure. Both EA and SA display a very modest effect of about 1-4 C
reduction of the transition temperature. It was suggested that the effect of OA
on the structure is not attributed only to the presence of a double bond at the
position 9 in its backbone or the total carbon atoms, but it is most likely
attributed to the molecular shape as OA has a wedge-shaped molecule with a
kink in the middle of its acyl chain [59-61].
In a recent report, the effect of solubilizing EA and OA on the
nanostructure of fully hydrated monoelaidin (ME, a neutral rod-like
monoacylglycerol with a hydrophobic tail consists of a straight acyl chain
(EA, C18:1t9)) was investigated [31]. It was proposed in the literature to use
ME as a model lipid for investigating the lamellar-to-nonlamellar transitions,
which are of biological relevance and take place in different biological
membranes under certain circumstances [109-112].
Figure 3 shows a rich polymorphism upon the solubilization of OA and
EA in the fully hydrated ME-based system: different inverted-type self-
assembled liquid crystalline phases and microemulsions are displayed [31].
OA shows a greater tendency to perturb the ME bilayers and makes the
membrane curvature more negative and therefore it is more efficient than EA
in inducing the formation of the discontinuous Fd3m and L
2
(inverted-type
microemulsion) phases [31].
The addition of vegetable oils or fatty acids to fully hydrated
monoglycerides such as ME, monoolein (MO) or MLO makes the spontaneous
curvature more negative and therefore induces the formation of highly curved
structures (discontinuous Fd3m and L
2
phases) [31,86,95,100,114].
As a consequence, these hydrophobic guest molecules can be added to
tune the interface curvature for obtaining the desired nanostructure. The
solubilization of the saturated hydrocarbon tetradecane tunes the internal
nanostructure of aqueous dispersions based on MLO (Figure 2) in the classical
sequence described above for the non-dispersed fully hydrated monoglyceride-
based systems (see section IV): a transition from (a) cubosomes, via (b)
hexosomes and (c) micellar cubosomes, to (d) EMEs was reported [100,108].
Similar behavior was also observed when loading OA to MO in the non-
dispersed and dispersed states [113,114].
MO has a different molecular shape than ME due to the cis configuration
present in its hydrophobic tail and therefore it tends at ambient temperatures to
form the bicontinuous cubic Pn3m phase under full hydration conditions;
whereas the fully hydrated rod-like lipid ME adopts a lamellar phase [31,109,
113-115].

Figure 3. Temperature-dependence behavior of the fully hydrated OA-loaded (A) and
EA-loaded (B) ME systems. The experiments for both self-assembled systems were
performed with RWT ratio in the range of 00.6 and were used to construct the partial
phase diagrams. The dashed/dotted curves indicate the approximate phase boundaries
between the different phases. These phase boundaries are tentative (they are not well
characterized) (the figure has been taken with permission from reference [31]).

Figure 4. Representative animal SPECT/CT images showing biodistribution of
subcutaneously administered 99mTc-SpmTrien-hexosomes at different time points.
(A) 99mTc-SpmTrienhexosomes 5 min post-injection; (B) 3 h post-injection of
99mTc-SpmTrien-hexosomes; (C) 6 h post-injection of 99mTc-SpmTrien-hexosomes,
and (D) 99mTc-SpmTrien-hexosomes at 24 h post-injection (the figure has been
adapted with permission from reference [119]).

VI. RADIOLABELING OF OA-LOADED HEXOSOMES
FOR THERANOSTIC APPLICATIONS

The research area of molecular imaging has been rapidly developed due to
the potential of biomedical and pharmaceutical applications and the
advantages of non-invasive visualization of delivering, targeting, detection of
cancer, adjustment of treatment protocols, and so forth [116]. Among different
imaging techniques, the radiotracer imaging based on single-photon emission
computed tomography (SPECT) or positron-emission tomography (PET) is a
useful tool in the detection and treatment of severe disease such as cancer by
the conjugation of radionuclides to nanoparticles and monitoring their uptake
in the whole-body basis [117, 118]. In a recent report, a highly efficient
radiolabelling method based on OA-loaded hexosomes using SpmTrien
(polyamine 1, 12-diamino-3, 6, 9-triazododecane) as a chelating agent was
successfully developed [119]. The
99m
Tc-labeled SpmTrien-hexosomes were
synthesized with good radiolabeling (84%) and high radiochemical purity (>
90%). The interested reader is referred to ref. 119 for further details on the
applied surface chelation method. The
99m
Tc-SpmTrien-hexosomes were
subcutaneously injected to the flank of healthy mice and the in vivo imaging
for the distribution of these radiolabeled nanoparticles was followed by
SPECT in combination with computed tomography (CT). Figure 4 shows
representative SPECT/CT images of the biodistribution and accumulation of
99m
Tc-SpmTrien-hexosomes at different time intervals after administration
[119]. It is interesting that the investigated
99m
Tc-SpmTrien-hexosomes form a
depot in the subcutaneous adipose tissue without any significant accumulation
in other tissues or organs after 24 hrs of injecting the nanostructured aqueous
dispersion [119]. These radiolabeled hexosomes can serve as a promising non-
invasive visualization tool applicable for investigating the in vivo performance
of hexosomal nanocarriers intended for theranostic applications by using
SPECT/CT [119].

CONCLUSION

The last two decades have witnessed an enormous interest in
understanding the role of oleic acid (OA) in modulating the function of various
proteins and the related health-promoting effects as well as the protective
effects against tumoral and hypertensive pathologies. It was the main attention
in the present contribution to summarize recent studies on the role of OA in
regulating biological functions and its use as an essential component in
formulating soft self-assembled drug nanocarriers. In spite of various
published studies to date, the relationship between the molecular interactions
of OA with the plasma membrane and the activation of different intracellular
pathways associated with the health implications is still lacking. It is still of
utmost importance to examine the reasons behind the potential beneficial
effects associated with OA intake.

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Chapter 3

OLEIC ACID AND ITS POTENTIAL
HEALTH EFFECTS

I gor Pravst
*
, Ph.D.
Nutrition Institute, Ljubljana, Slovenia

ABSTRACT

Oleic acid is a monounsaturated fatty acid and natural constituent of
a number of foods, particularly vegetable oils. On the basis of proven
beneficial health effects it is also a possible ingredient in processed
functional foods. However, due to its high energy content it is not
recommended to increase the consumption of any particular fat, but to
substitute other lipids with oleic acid. While there is a well-established
consensus that replacing saturated fats in the diet with oleic acid or other
unsaturated fats contributes to the maintenance of normal blood
cholesterol levels, a series of other effects has also been studied,
including the modulation of inflammatory markers, blood pressure,
insulin sensitivity, gastrointestinal functions and even various cancers.
Commercial communication of such effects is only ethical where such
effects are relevant to human health and proven using the highest possible
standards, preferably with well-performed, double-blind, randomised,
placebo-controlled human intervention trials. Most intervention studies
investigating the health effects of oleic acid are performed using
vegetable oils which also contain other fatty acids and minor constituents.
This represents a possible confounding factor and makes interpretations

*
igor.pravst@nutris.org.
Igor Pravst 36
difficult. In this chapter, the health effects of oleic acid are discussed
together with the possibilities of using oleic-acid-related health claims on
foods in commercial communications in the European Union.

Keywords: Oleic acid, health effects, health claims, scientific substantiation

ABBREVIATIONS

CAD coronary artery disease
CH carbohydrate
CO coconut oil
DHA docosahexaenoic acid
EC European Commission
EFSA European Food Safety Authority
EPA eicosapentaenoic acid
EU European Union
HDL high-density lipoprotein
LDL low density lipoprotein
MCFA medium-chain fatty acid
MUFA monounsaturated fatty acid
OA oleic acid
PO palm olein
PUFA polyunsaturated fatty acid
SA stearic acid
SFA saturated fatty acid
VLDL very-low-density lipoprotein
VOO virgin olive oil

1. INTRODUCTION

While there is no harmonised definition of functional foods, a consensus
on the functional foods concept was reached in the European Union in 1999
when a working definition was established whereby a food can be regarded as
functional if it is satisfactorily demonstrated to beneficially affect one or more
target functions in the body beyond adequate nutritional effects in a way that is
relevant to either an improved state of health and well-being or a reduction of
disease risk (Ashwell, 2002). Functional foods must remain foods and
Oleic Acid and Its Potential Health Effects 37
demonstrate their effects when consumed in daily amounts that can be
normally expected. In practice, examples of functional foods include foods in
which a component has been added or removed, foods in which a component
has been replaced by an alternative component with favourable properties, or
even unmodified natural foods (Ashwell, 2002). Regardless of the various
definitions, the main purpose of functional food should be clear to improve
human health and well-being.
Health claims are a very convenient tool when it comes to marketing
functional foods due to consumers sensitivity to health-related
communications. The use of nutrition and health claims on foods in the
European Union (EU) was harmonised in 2006 by Regulation (EC) No
1924/2006 on nutrition and health claims made on foods (EC, 2006). Only the
use of authorised nutrition and health claims is allowed. All health claims
require specific authorisation by the European Commission (EC) through the
comitology procedure, following the scientific assessment and verification of a
claim by the European Food Safety Authority (EFSA) (Pravst, 2012a).
Oleic acid (OA) is a monounsaturated fatty acid with a number of
potential health effects (Sales-Campos, de Souza, Peghini, da Silva, and
Cardoso, 2013) and can therefore be a constituent of composite functional
foods when it substitutes saturated fats. In addition, foods naturally high in
oleic acid, e.g. olive oil, can be considered as functional food. However, the
high energy content of all oils represents the main limitation on
recommendations to increase the consumption of any particular oil. The
overconsumption of oleic acid should not be recommended but to substitute
other lipids with oleic acid, always aiming to strike a balance between energy
intake and expenditure, preferably at a higher rather than a lower level
(Trichopoulou and Dilis, 2007).

2. POTENTIAL HEALTH EFFECTS OF OLEIC ACID

It is well established that fatty acids are an essential component of human
nutrition (Burr and Burr, 1929); besides being the densest dietary energy
source, they have important structural roles in the human body. Fatty acids that
cannot be synthesised by humans are considered essential and must be
provided by the intake of food (Miles and Calder, 1998).
Oleic acid [C18:1 (n-9)] is a monounsaturated fatty acid. In nature, it is
mainly found in cis form and in this chapter the term oleic acid is reserved
solely for its cis isomer. Humans possess an enzymatic mechanism which
Igor Pravst 38
enables the desaturation of saturated fatty acids; for example, stearic acid can
be converted to oleic acid by enzyme
9
desaturase and oleic acid is therefore
considered to be non-essential to humans. On the contrary, polyunsaturated
linolenic and alpha-linolenic acid are considered essential (Miles and Calder,
1998). Oleic acid is found in significant amounts in olive oil, which contains
approximately 72% oleic acid (Newmark, 1999) and in avocado oil, which has
similar characteristics to olive oil (Oliveira et al., 2013).
Dietary intakes of fats vary among countries significantly. While the main
dietary sources of monounsaturated fatty acids in the USA are whole milk,
peanut butter and various fast foods such as French fries, pizzas and salty
snacks (Nicklas, Hampl, Taylor, Thompson, and Heird, 2004), olive oil
represents the primary source of monounsaturated fatty acids in Mediterranean
countries (Waterman and Lockwood, 2007). Further, substantial differences
can be observed within different population groups. With progress in food
technology and innovation in the development of new functional foods, there
is a trend to modify the fatty acid profile of foods high in saturated fats by
introducing oleic acid (Lopez-Huertas, 2010) and it is expected that processed
foods as a source of OA will become more important in the future.
Non-communicable chronic diseases such as cardiovascular disease and
diabetes are becoming a huge burden on Western societies. Nutrition and other
lifestyle factors are well-recognised, modifiable external factors affecting
these diseases (Suburu et al., 2013). The consumption of fats and of specific
fatty acids can have either a negative or a protective health effect. Saturated
fatty acids have been linked with negative impacts and it is recommended to
keep their intake below 10% of total energy (WHO). Conversely, a number of
studies have investigated the protective effects of monounsaturated (MUFAs)
and polyunsaturated (PUFAs) fatty acids (Tvrzicka, Kremmyda, Stankova, and
Zak, 2011).
In general, Southern European countries record the lowest values of the
accumulated incidence of myocardial infarction (Tunstall-Pedoe et al., 1999)
and many studies have indicated a possible link between adherence to the
Mediterranean diet and a reduced risk of overall mortality, cardiovascular
mortality, cancer incidence and mortality, as well as the incidence of
neurodegenerative diseases (Benetou et al., 2008; De Lorgeril et al., 1999;
Keys, 1980; Scarmeas, Stern, Tang, Mayeux, and Luchsinger, 2006; Sofi,
Cesari, Abbate, Gensini, and Casini, 2008; Trichopoulou, Costacou, Bamia,
and Trichopoulos, 2003). The Mediterranean diet is characterised by: high
consumption of vegetables, legumes, fruit and cereals; regular, but moderate,
wine intake; moderate consumption of fish and white meat; a moderate intake
of dairy products; low consumption of red meat; and relatively high
consumption of fat (up to 40% of total energy intake), mostly from MUFAs
(up to 20% of energy) (Urpi-Sarda et al., 2012). Traditionally, the health
effects of olive oil were chiefly attributed to its high content in oleic acid, but
today we know that these effects must also be ascribed to other constituents of
olive oil, particularly its phenolic fraction (Martin-Pelaez, Isabel Covas, Fito,
Kusar, and Pravst, 2013). In addition to a healthy diet, the candidate protective
factors which may explain this Mediterranean Paradox also include other
lifestyle factors such as regular physical activity and the existing social
cohesion in Southern European countries. Nevertheless, mainly because of its
high OA content olive oil is recognised as an optimal source of fat for the
modulation of cardiovascular risk, gastrointestinal and metabolic functions
(Bermudez et al., 2011). It is not easy to investigate the health effects of OA
alone as interventions usually include other fatty acids and other compounds
that are natural constituents of vegetable oils. These could interfere with the
effect of OA, rendering interpretations difficult. Observed effects are therefore
commonly linked to MUFAs, or to an even wider group of unsaturated fatty
acids.

2.1. Cardiovascular Health

The effects of dietary fats on the risk of coronary artery disease (CAD)
have traditionally been estimated from their effects on LDL cholesterol. A
meta-analysis conducted in 1992 of 27 intervention trials on the impact of
dietary fatty acids on serum lipids and lipoproteins showed a specific
cholesterol-lowering effect of unsaturated fats over and above that of replacing
saturates in the diet with carbohydrates; the effect was more significant with
PUFAs than with MUFAs (Mensink and Katan, 1992). A theoretical model
has shown the most favourable lipoprotein risk profile for coronary heart
disease if saturated fatty acids are replaced by unsaturated fatty acids, with no
decrease in total fat intake (Mensink and Katan, 1992). However, fats also
affect HDL cholesterol and the ratio of total:HDL cholesterol is also
recognised as a specific marker of CAD. The effects of the amount and type of
fat on total:HDL cholesterol and on other lipids were calculated in a meta-
analysis of 60 human intervention trials in 2003 (Mensink, Zock, Kester, and
Katan, 2003). The ratio did not change when carbohydrates replaced SFAs, but
it decreased when cis unsaturated fatty acids replaced SFAs. After 2003, a
series of additional human intervention studies was performed to investigate
Igor Pravst 40
the effects of OA or foods/diets high in OA on blood lipids and other
biomarkers related to cardiovascular health (Table 1).
Tholstrup et al. compared the effects of a diet rich in either medium-chain
fatty acids (MCFAs) or OA on fasting blood lipids, lipoproteins, glucose,
insulin and lipid-transfer protein activities in 17 healthy men using a double-
blind, randomised, crossover design (Tholstrup et al., 2004). It had generally
been claimed that MCFAs do not increase plasma cholesterol, although this
claim had been poorly documented and the objective of the study was
therefore to compare the effects of MCFAs with OA. Volunteers replaced part
of their habitual dietary fat intake with 70 g MCTs (66% 8:0 and 34% 10:0) or
high-OA sunflower oil (89.4% 18:1). Compared with the intake of high-OA
sunflower oil, the MCT intake unfavourably affected the lipid profiles and
resulted in 11% higher plasma total cholesterol, 12% higher LDL cholesterol,
a 12% higher ratio of LDL to HDL cholesterol, 22% higher plasma total
triacylglycerol, and higher plasma glucose. Plasma HDL-cholesterol and
insulin concentrations did not differ significantly between the diets.
Effects of stearic (SA), linoleic (LA) and oleic acids on the serum
lipoprotein profile of healthy subjects were tested on 45 subjects using three
experimental diets (Thijssen and Mensink, 2005). The diets provided 38% of
energy from fat, of which 60% was supplied by the experimental fats. The
dietary compositions of the diets were the same, except for 7% of energy
which was provided by SA, OA or LA. Interestingly, no significant diet-
induced changes in serum lipids and lipoproteins were found and the LDL,
HDL and VLDL particle sizes and lipoprotein subclass distributions also did
not differ significantly between the three diets. It was concluded that with
realistic intakes of stearic, oleic and linoleic acids the differences between
their effects on the serum lipoprotein profile are small.
Mensink further compared the effects of high-PA fat with those of high-
OA fat on the serum lipoprotein profile of 44 healthy subjects (Mensink,
2008). Two experimental diets provided 40% of energy from fat; 15% energy
was supplied by one of two experimental fats, both low trans and with
comparable functional characteristics (semiliquid fat). The high-OA fat had a
more favourable effect on the serum lipoprotein profile than the fat high in
PA: decreased serum LDL cholesterol levels were observed and the total:HDL
cholesterol ratio was also lowered.
While most studies with OA were performed in Western countries, Teng
and co-workers investigated the effects of three vegetable oils on serum
inflammatory markers, lipids and lipoproteins in South East Asia (Teng, Voon,
Cheng, and Nesaretnam, 2010). Using a crossover design, 41 healthy
normolipidemic subjects consumed high-OA palm olein (HOPO diet: 15% of
energy 18:1n-9, 9% of energy 16:0), partially hydrogenated soybean oil
(PHSO diet: 7% of energy 18:1n-9, 10% of energy 18:1 trans) and an
unhydrogenated palm stearin (PST diet: 11% of energy 18:1n-9, 14% of
energy 16:0) diet for 5 weeks. In particular, the PHSO diet as well as the PST
diet significantly increased the total:HDL cholesterol ratio compared to the
HOPO diet (by 23% and 13%, respectively; P < 0.05), indicating it is
preferable to use vegetable oils in their natural state rather than processed ones
(Teng et al., 2010). Also on a South East Asia population, Voon and co-
workers investigated the effects of high-protein Malaysian diets prepared with
palm olein (PO), coconut oil (CO) or virgin olive oil (VOO) on blood lipids,
homocysteine and selected markers of inflammation and cardiovascular
disease in 45 healthy adults using a crossover design (Voon, Ng, Lee, and
Nesaretnam, 2011). While the postprandial total cholesterol and fasting lipid
index for the VOO diet were significantly lower than for the CO diet, no
significant differences were observed in the effects of the three diets on plasma
total homocysteine and other studied inflammatory markers.
The role of dietary fat and specific fatty acids in altering concentrations of
markers of inflammation was also studied by Baer et al. using a crossover
design and 50 subjects (Baer, Judd, Clevidence, and Tracy, 2004); one of five
experimental diets was enriched with OA. No significant differences were
observed in any of the studied markers between the control carbohydrate (CH)
diet and a diet high in OA. On the contrary, higher fibrinogen concentrations
were observed with the diet enriched in SA, and higher C-reactive protein
concentrations with the diet enriched with trans fatty acids (Baer et al., 2004).
Further, Pachco and co-workers investigated the influence of diets high in
either PA or OA on postprandial markers of endothelial activation and
vascular inflammation and observed that a meal high in OA appears to have a
significant postprandial benefit on soluble forms of intercellular adhesion
molecule 1 and vascular cell adhesion molecule 1 in healthy and, more
importantly, in hypertriglyceridemic (normotensive and hypertensive)
subjects.
The potential of OA to affect thrombotic tendency is limited. Thijssen and
co-workers compared the effects of three experimental diets (rich in OA, LA
and SA) using 45 subjects and a crossover design; ex vivo and in vitro platelet
aggregation, and variables of coagulation, fibrinolysis and haematology were
evaluated but very limited differences were observed between the diets
(Thijssen, Hornstra, and Mensink, 2005).
Igor Pravst 42
Other heart-health-related effects of OA were also investigated in animals,
such as hypotensive effects. Observations that virgin olive oil reduces blood
pressure have usually been ascribed to the phenolic fraction and other minor
constituents, although it was demonstrated on rats that OA alone might also be
responsible for such an effect (Teres et al., 2008). It was proposed that a high
OA intake increases OA levels in membranes which regulate the membrane
lipid structure in such a way as to control G-protein-mediated signalling,
causing a reduction in blood pressure.

2.2. Insulin Sensitivity

In general, there is an association between dietary fat intake, the type of
fatty acids consumed and the development of type 2 diabetes (Rocca, LaGreca,
Kalitsky, and Brubaker, 2001; Salmern et al., 2001; Vessby et al., 2001).
Intervention studies show that a decrease in the intake of SFAs and an increase
in the intake of MUFAs improves insulin sensitivity, although it has no effect
on insulin secretion. The favourable effects on insulin sensitivity are only seen
when the total fat intake does not exceed 37% of the energy intake. When the
fat intake is higher, the type of dietary fat only has a small effect. However,
additional benefits of long-chain PUFAs over MUFAs were not observed in
this context (Vessby et al., 2001).
Shah and co-workers studied the effect of fatty acids on postprandial
insulin, glucose and triglyceride responses on 11 type 2 diabetic subjects
(Shah, Adams-Huet, Brinkley, Grundy, and Garg, 2007). They tested meals
rich in OA, PA LA, eicosapentaenoic acid (EPA) and docosahexaenoic acid
(DHA) using a randomised crossover design; serum insulin, glucose and
triglyceride concentrations were measured. It was observed that, in
comparison with PA and LA, OA or EPA and DHA modestly lower the insulin
response in patients with type 2 diabetes without deteriorating the glucose
response (Shah et al., 2007). In addition, a study with healthy subjects
investigated the effect of diets enriched with either PA or OA on markers
related to the risk of type 2 diabetes (Kien, Bunn, Poynter, et al., 2013).
Performed using 18 subjects and a randomised crossover design, the study
provided evidence that the dietary PA:OA ratio impacts the risk of diabetes in
women. The insulin sensitivity and disposition indexes were higher during a
diet rich in OA in women, but not men. The effect of such a diet on the
sensitivity index correlated positively with physical fitness upon enrolment.

2.3. Satiety, Intake of Nutrients and Gastrointestinal Functions

In subjects with longstanding and severe short bowel syndrome,
supplementation of the diet with OA (in soy milk) reduced the utilisation of
energy, yet there was no significant change in the transit time or in the
absorption of fats and protein; neither diarrhoea nor patient body weight was
changed (Compher, Kinosian, Rubesin, Ratcliffe, and Metz, 2009) In geriatric
patients, an intervention with an energy-dense, OA-rich formula also improved
the appetite and utilisation of energy (Faxen-Irving and Cederholm, 2011).
However, no change in general satiety was observed in a healthy population in
a controlled trial in which dietary fat was replaced with LA, GLA or OA
(Kamphuis, Westerterp-Plantenga, and Saris, 2001). Lin and co-workers
investigated the effect of ingesting OA emulsion on gastrointestinal transit in
both healthy subjects and patients with chronic diarrhoea (Lin, Van Citters,
Heimer, and Bonorris, 2001). In the patients, a significant increase in transit
time was observed in a dose-dependent relationship with the intake of OA,
enabling an improved intake of nutrients. A reduction of bowel movements
and stool volume was also observed (Lin et al., 2001).

2.4. Other Functions

Oleic acid, mainly as a constituent of olive oil, has been studied with
regard to a series of other possible health effects in both human and animal
trials. Some reports indicate that OA could help in preventing the progression
of and metastasis in several human cancers and reduce the risk of developing
certain types of cancer, especially colorectal, breast and prostate cancer
(Trichopoulou, Lagiou, Kuper, and Trichopoulos, 2000); various possible
mechanisms were suggested (Carrillo, Cavia, and Alonso-Torre, 2012;
Menendez and Lupu, 2006). Other reports indicate OA anti-inflammatory
effects in autoimmune and chronic inflammatory diseases, although the
findings are sometimes contradictory (Sales-Campos et al., 2013). Olive oil
was experimentally used as a source of OA on mice to treat inflammatory
bowel disease, showing an improvement in the clinical score due to reduced
inflammation, probably due to the endogenic formation of substances with
anti-inflammatory properties (Borniquel, Jansson, Cole, Freeman, and
Lundberg, 2010).

Table 1. Overview of selected human intervention studies investigating the effects of oleic acid on blood lipids,
homocysteine and inflammatory markers (2004-2013)

Design, sample size,
duration
Intervention Response Reference
Double-blind,
randomised, crossover
trial,
17 subjects,
3 weeks
Part of habitual dietary fat intake
replaced with either 70 g of high-OA
sunflower oil (89.4% 18:1) or MCTs
Compared with the intake of high-OA
sunflower oil, MCT intake resulted in 11%
higher plasma total cholesterol, 12% higher
LDL cholesterol, a 12% higher ratio of LDL to
HDL cholesterol and 22% higher plasma total
triacylglycerol. Plasma HDL cholesterol and
insulin concentrations did not differ
significantly between the diets.
(Tholstrup et al.,
2004)
Randomised, crossover
trial,
45 subjects,
5 weeks
Three experimental diets in which 7%
of energy was provided by SA, OA or
LA
No significant diet-induced changes in serum
lipids and lipoproteins
(Thijssen and
Mensink, 2005)
trial,
44 subjects,
3 weeks
Two experimental diets (high-PA and
high-OA) providing 40% of energy
from fat; 15% energy was supplied by
one of two experimental fats
The high-OA diet had a more favourable effect
on the serum lipoprotein profile than a high-PA
diet: decreased serum LDL cholesterol levels
were observed and the total:HDL cholesterol
ratio was also lowered.
(Mensink, 2008)
Single-blind,
randomised, crossover
trial,
41 subjects,
5 weeks
Three experimental diets: high-OA
palm olein (HOPO diet), partially
hydrogenated soybean oil (PHSO diet)
and unhydrogenated palm stearin (PST
diet)
In particular, the PHSO diet as well as the PST
diet significantly increased the total:HDL
cholesterol ratio compared to the HOPO diet (by
23% and 13%, respectively), with the PST diet
having a smaller effect than the PHSO diet).
(Teng et al.,
2010)

Design, sample size,
duration
Intervention Response Reference
trial,
45 subjects,
5 weeks
3 experimental diets with the inclusion
of various fats (30% energy) in high-
protein diets:
PA-rich palm olein (PO), a lauric- and
myristic-acid-rich CO diet, and VOO
rich in OA.
The postprandial total cholesterol and fasting
lipid index for the VOO diet were significantly
lower than for the CO diet. No significant
differences were observed in the effects of the
three diets on plasma total homocysteine and
inflammatory markers.
(Voon et al.,
2011)
trial,
50 subjects,
5 weeks
5 experimental diets, including one
high in OA
No significant differences observed in any of
the studied markers between the control
carbohydrate diet and a diet high in OA
(Baer et al.,
2004)
trial,
45 subjects,
5 weeks
3 experimental diets (38% of energy
as fat) with 7% of energy from SA,
OA or LA
SA is not highly thrombogenic compared with
OA or LA
(Thijssen et al.,
2005)
trial,
two cohorts:
28 hypertriglyceridemic
and 14 healthy subjects,
1 week
Supplementation with either refined
olive oil or high-PA sunflower oil
A positive effect on endothelial activation and
vascular inflammation was observed in the
group consuming refined olive oil
(Pachco et al.,
2008)

Igor Pravst 46
Studies on rats and mice showed that OA may also have some beneficial
effects on the healing of cutaneous wounds. After treatment with OA, surgical
skin wounds on rats healed sooner, had less edema and favoured tissue repair.
The positive effects of oleic acid on wound repair were seen using both an oral
(Rodrigues et al., 2012) or a topical administration (Cardoso et al., 2011).
The Western diet increases the risk of metabolic disease and it was
recently investigated whether lowering the ratio of SFAs to MUFAs in that
diet would affect physical activity and energy expenditure (Kien, Bunn,
Tompkins, et al., 2013). In randomised, double-blind, crossover trials, a three-
week, high-PA diet (similar to the fat composition of the Western diet) was
compared with a high-OA diet similar to the fat composition of the
Mediterranean diet. The replacement of dietary PA with OA was associated
with increased physical activity and resting energy expenditure and less anger.

3. OLEIC ACID AND THE USE OF HEALTH CLAIMS
ON FOODS

Consumers are very sensitive to health-related communications, making
the use of health claims a very convenient tool in the marketing of healthy
foods (Pothoulaki and Chryssochoidis, 2009). While various countries take
different approaches to ensure that health claims do not mislead consumers,
the general approach is proper substantiation by generally accepted scientific
data. The use of health claims in the European Union (EU) was harmonised in
2006 following the acceptance of a challenging and controversial regulation
(Cappuccio and Pravst, 2011; Mariotti, Kalonji, Huneau, and Margaritis, 2010;
Pravst, 2011, 2013). A health claim is defined as any claim that states,
suggests or implies that a relationship exists between a food category, a food
or one of its constituents and health. The regulation requires the authorisation
of all health claims by the European Commission through the comitology
procedure, following the scientific assessment and verification of a claim by
the EFSA (Pravst, 2010). Claims are scientifically substantiated by taking
account of the totality of the available pertinent scientific data and by
weighing up the evidence, in particular whether the effect is relevant to human
health. In this process, well-performed human intervention trials are
particularly important and double-blind, randomised, placebo-controlled trials
are considered the gold standard (Pravst, 2012b).
Currently, in the EU there is only one authorised health claim related to
the health effects of oleic acid (Table 2). Foods high in OA (or other
unsaturated fatty acids) can be labelled with a statement stating that replacing
saturated fats in the diet with unsaturated fats contributes to the maintenance
of normal blood cholesterol levels. In practice, such a claim may be used
where at least 70% of the fatty acids present in the product derive from
unsaturated fat on the condition that unsaturated fat provides more than 20%
of energy of the product. The use of such a claim has a series of regulatory
consequences related to the labelling of mandatory information, including a
mandatory nutrition declaration and a statement indicating the importance of a
varied and balanced diet and a healthy lifestyle (Pravst, 2012a). The claim was
approved on the basis of a well-established consensus that a mixture of SFAs
increases blood total and LDL-cholesterol concentrations relative to mixtures
of MUFAs or PUFAs with a linear dose-response relationship, indicating that
the effects are proportional to the amounts of long-chain SFAs consumed
(EFSA, 2011a, 2011b). During the evaluation it was also noted that there is an
established consensus that the consumption of a mixture of SFAs results in
increased blood HDL-cholesterol concentrations compared with the
consumption of mixtures of MUFAs or PUFAs and that, in comparison with
other fatty acids except for trans fatty acids, SFAs increase the total-to-HDL
cholesterol ratio (EFSA, 2011a, 2011b).

Table 2. List of evaluations of general functional health claims for oleic
acid by the European Food Safety Authority (2014)

Status Health claim wording Conditions of use Ref.
Authorised Replacing saturated fats
in the diet with
unsaturated fats
contributes to the
maintenance of normal
blood cholesterol levels.
Oleic acid is an
unsaturated fat.
The claim may be used
where at least 70% of the
fatty acids present in the
product derive from
unsaturated fat on the
condition that
unsaturated fat provides
more than 20% of energy
of the product.
(EFSA,
2011a,
2011b)
Unauthorised Replacing saturated fats
with oleic acid
contributes to normal
(fasting) blood
concentrations of
triglycerides
Use is not authorised (EFSA,
2011a)
Igor Pravst 48
On the contrary, a cause-and-effect relationship has not been established
between oleic acid replacing SFAs in foods or diets and the maintenance of
normal (fasting) blood concentrations of triglycerides (EFSA, 2011a). It was
noted that when carbohydrates are replaced with fats fasting triglyceride
concentrations are reduced, but there is a lack of evidence that different fatty
acids have different effects. Additionly, there is a series of other possible
health effects of oleic acid, but stronger clinical evidence is needed before
additional health claims may be used in commercial communications.

CONCLUSION

On the basis of a well-established consensus that replacing saturated fats
in the diet with oleic acid contributes to the maintenance of normal blood
cholesterol levels, functional foods high in oleic acid can be promoted by
related health claims in the EU. However, due to its high-energy content it is
not recommended to increase the consumption of any particular fat, but to
substitute other lipids with oleic acid.
A series of other effects has also been studied, including the modulation of
inflammatory markers, blood pressure, insulin sensitivity, gastrointestinal
functions and even various cancers. However, scientific evidence in these
areas is currently not yet strong enough to enable the use of additional health
claims and further well-performed human intervention studies are needed.
These should properly address all possible confounding factors, including the
presence of other fatty acids and minor oil constituents in the experimental
diets.

ACKNOWLEDGMENTS

I gratefully acknowledge the support of iva Koroec in the literature
search and Murray Bales for providing help with the language. The work was
financially supported by the Ministry of Agriculture, Forestry and Food of the
Republic of Slovenia, and the Slovenian Research Agency (Contract 1000-11-
282007, Research project V7-1107).

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Chapter 4

OLEIC ACID AND MICROBIAL LIPASES:
AN EFFICIENT COMBINATION

Fabiano J ares Contesini
, Danielle Branta Lopes,

Elaine Berger Ceresino, J ose Valdo Madeira J unior,
Paula Speranza, Francisco Fbio Cavalcante Barros
and Ricardo Rodrigues de Melo
Laboratory of Biochemistry.Department of Food Science.
State University of Campinas.Campinas SP, Brazil

ABSTRACT

Oleic acid is a monounsaturated fatty acid found in high
concentrations in vegetable oils, presenting a broad number of
applications in many industrial areas, such as food, pharmaceutical,
cosmetic, oleochemical and biodiesel industries. Due to the lipophilicity,
unsaturation and acidic characteristics that this compound presents, oleic
acid can be effectively used in esterification and acidolysis, among other
reactions. Recent studies have used oleic acid as an efficient substrate for
synthesis of trimethylolpropane esters by esterification using lipase from
Candida Antarctica, since this polyol ester is widely applied in hydraulic

Address: Laboratrio de Bioqumica. Departamento de Cincia de Alimentos - FEA,

Universidade Estadual de Campinas. Rua Monteiro Lobato, 80. Cx. Postal 6121. 13083-
862. Campinas-SP, Brasil. Corresponding author. Tel./fax: +55 19 3521 2175 E-mail:
fabiano.contesini@gmail.com.
F. J. Contesini, D. B. Lopes, E. B. Ceresino et al. 56
fluids with several applications. Other studies used C. antarctica lipase
for improving the lipophilicity of bioactive molecules, such as ferulic
acid and L-ascorbic acid by esterification with oleic acid, which is very
interesting, taking into account that it increases the solubility of these
molecules in hydrophobic environments, resulting in higher biological
activities. On the other hand, some studies showed that lipases can be
used to convert oleic acid into epoxies, which are useful intermediates in
organic synthesis due to the high reactivity they present. They are used to
produce plasticizers that increase flexibility, workability or distensibility
of plastics, hence rendering them suitable for several applications. One
study reported biodiesel production by esterification of oleic acid with
aliphatic alcohols using immobilized Candida antarctica lipase, showing
high yields of biodiesel (above 90%) in less than 24 h with ethanol, n-
propanol and n-butanol; whereas with methanol, the enzyme was inactive
after ten cycles of reaction. In addition to the various reactions involving
oleic acid as a promising substrate for various reactions, oleic acid can
also be used to induce microbial lipase production, as seen in a study
using the fungal strain Rhizopus arrhizus. Therefore, different high-
added-value compounds can be obtained using oleic acid as a cheap and
efficient substrate for microbial lipases, which can be considered as
environmentally friendly alternatives for chemical catalysts. Within this
context, this chapter reviews some studies and trends on the use of oleic
acid as an efficient substrate for microbial lipases.

1. INTRODUCTION

Oleic acid has a long 18-carbon chain length with a cis bond located at
carbons 9 and 10 from the methyl end. This monounsaturated fatty acid is the
main component in various vegetable oils such as olive, canola and peanut
(Bermudez et al., 2011; O'Brien, 2009). In olive oil, the percentage of oleic
acid may exceed 80% of the total of fatty acids (Nunes et al., 2011). New
hybrid varieties, such as high-oleic sunflower and high-oleic soybean, are
composed of more than 80-90% of this fatty acid (O'Brien, 2009). Oleic acid,
with its unique double bond, presents relevant industrial properties such as
good fluidity at low temperatures and good oxidative stability (Sverac et al.,
2011). From a biological point of view, evidence from epidemiological studies
suggests that a higher proportion of oleic acid in the diet is linked to a reduced
risk of coronary heart disease and reducing inflammatory responses (Lunn,
2007; Pacheco et al., 2008). Therefore, the widespread availability of oleic
acid, in addition to its industrial and biological properties, justifies the use
thereof for the synthesis of compounds with more potential applications.
Oleic Acid and Microbial Lipases 57
On the other hand, lipase-catalyzed synthesis has become a promising
method due to the advantages of mild conditions, simplified downstream
processing, high region- and stereo- selectivity, low energy consumption and
environmental friendliness over chemical catalysis (Hen et al., 2002). Lipases
differ with regard to their origins and properties. They can be obtained from
microorganisms, plants and animals. Microbial lipases have been the most
attractive because of their thermal stability and different specificities (Eigtved,
1992). These enzymes have great potential in commercial applications, such as
in the production of foods, fine chemicals, detergents, waste water treatment,
chemicals, cosmetics, pharmaceuticals, leather and medical products (Li et al.,
2014; Azudin et al., 2013, Chauhan et al., 2013; Tsuji et al., 2013; Frampton
and Zelisko, 2013; Sun et al., 2013; Brabcova et al., 2013; Saran et al., 2013;
Quian et al., 2013).
Studies indicate that lipase-catalyzed synthesis using oleic acid has shown
promising results. Lipase catalyzed esterification of alkyl oleates for biodiesel
applications has become an alternative method to the traditional production of
biodiesel using base-catalyzed transesterification of oils with methanol (Zhong
et al., 2013). The production of synthetic oleochemical esters by enzymatic
esterification and transesterification reactions using oleic acid as substract is
useful as a diesel additive, plasticizer, water-resisting agent and hydraulic fluid
(Dossat et al., 2002). These oleochemical esters are better alternatives,
environmentally speaking, than lubricants based on mineral oil, exhibiting a
combination of technical performance with favorable ecological properties
(kerman et al., 2011; Schneider, 2006).
In the food area, due to the characteristics of oleic acid mentioned above
and the specificity of lipases by the type and positions of fatty acid on the
glycerol, it is possible to synthesize lipids rich in oleic acid with improved
physicochemical, nutritional and biological properties (Lin and Huey, 2009;
Sellami et al., 2012; Farfn et al., 2013). The lipase-catalyzed reaction
between oleic acid and phytosterols also produces sterol esters with higher
solubility, which contributes to practical applications in food products
(Pan et al., 2012).
In the pharmaceutical area, the reaction of oleic acid catalyzed by lipase is
used for the production of monoolein and carbohydrate fatty acid esters,
compounds with emulsifiers and surfactant properties (Wang et al., 2013;
Abdulmalek et al., 2012). Another important surfactant produced is the fatty
acid ester of vitamin C (Stojanovic et al., 2013). Enzymatic synthesis of
ascorbyloleate is very attractive, as well as surfactants, which have high
antioxidant potential (Viklund; Alander; Hult, 2003). Due to biological
characteristics, production of sterol esters of oleic acid catalyzed by lipase can
also be applied in cosmetic composition for preventing skin aging or
improving skin quality (Chaibakhsh et al., 2012).
Therefore, the objective of this chapter is to highlight the research
conducted in recent years on the application of oleic acid in synthesis reactions
using lipases. The review focuses predominantly on studies for application in
the industries of biodiesel, chemicals, food and pharmaceuticals.

2. BIODIESEL

Recently, the high prices of petroleum-based fuels and predictions that in
the near future the main oil reserves would completely run out have stimulated
the search of alternate energy sources to petroleum-based diesel (Leung et al.,
2010). Furthermore, there are serious environmental issues involving burning
fossil fuel, which are related to the emission of so-called greenhouse gases
(Robles-Medina et al., 2009; Chen et al., 2010; Leung et al., 2010). In view of
these aspects, biodiesel has been categorized as one of the most promising
substitutes for petroleum-based diesel. Biodiesel, a clean renewable fuel, is
nontoxic, biodegradable and contributes to the reduction of greenhouse gas
emission (Meher et al., 2006; Knothe, 2010).
Biodiesel is a mixture of monoalkyl esters of long-chain fatty acids,
preferably methyl and ethyl esters, derived from renewable sources such as
animal fats and vegetable oils (Knothe, 2010). Animal fats can be used in
biodiesel synthesis, however they are not a very promising feedstock since
they contain higher levels of saturated fatty acids, normally present in solid
form at room temperature, which may cause problems in the production
process. Furthermore, the cost is higher than that of vegetable oils (Singh and
Singh, 2010). Thus, vegetable oils, which are renewable in nature and
environmentally friendly, are most frequently used as feedstock (Patil and
Deng, 2009). However, a drawback to producing biodiesel in commercial
scale with edible crude oil is the high cost. In addition, the use of edible crude
oils such as rapeseed, sunflower, soybean and palm oil in biodiesel production
has raised the concern of food versus fuel. Thus, recent biodiesel development
has switched from the use of edible oils to non-edible and waste oils as new
and sustainable raw materials (Du et al., 2004; Chen et al., 2008; Wang et al.,
2008). Nevertheless, most non-edible oils contain high levels of free fatty
acids, may require multiple chemical steps or alternative approaches to
produce biodiesel, increasing the production cost and achieving generally
lower ester yield of biodiesel (Haas, 2005; Patil and Deng, 2009; Sahoo and
Das, 2009).
The modification and adjustment of vegetable oil and animal fat properties
for biodiesel production can be developed using methods of transesterification,
esterification, microemulsification and cracking (Knothe, 2005; Ranganathan
et al., 2008). However, transesterification and esterification reactions have
been the most commonly used methods by the biofuel industry as synthetic
routes (Figure 1) (Robles-Medina et al., 2009).

Figure 1. (1) Transesterification of triacylglycerides and (2) esterification of fatty acids
to biodiesel.
The most common approach for biodiesel synthesis in the world is carried
out by an alkaline-catalyzed transesterification reaction using alkaline catalysts
like potassium hydroxide (KOH), sodium hydroxide (NaOH) and/or the
corresponding sodium or potassium methoxide (Meher et al., 2006; Moser,
2009). These alkali-catalyzed processes have a high reaction rate at a low
temperature, and they are usually more efficient and less corrosive compared
to others (Zheng et al., 2006; Soriano Jr et al., 2009). Despite the advantages
of alkaline catalysts in large scale production of biodiesel, the alkaline-
catalyzed reactions result in soap and emulsion formation when low-quality
raw materials are used (vegetable oil sources with high contents of water and
free fatty acids), decreasing the efficiency of the process and harming the
biodiesel purification and glycerol separation (Meher et al., 2006; Miao et al.,
2009). Therefore, the use of more expensive refined oils is necessary in
alkaline-catalyzed processes (oil should have fatty acid concentrations of less
than 1%), which precludes the use of low quality and cheap oils such as
cooking oil waste (Lam et al., 2010).
The problems caused by high free fatty acid content in low quality oils,
which may cause interference in biodiesel production using alkali catalysts,
could be overcome using an acid-catalyzed process. However, acid catalysis
has shown some inconveniences, such aslower yields than thosereached by
alkali catalysis, need of high temperatures and more corrosiveness than other
processes (Meher et al., 2006; Akoh et al., 2007).
Biodiesel can also be produced from noncatalytictransesterification with
supercritical alcohol. Supercritical alcohol transesterification is a process that
has several advantages, such as reduced reaction time and easy separation and
purification of the products, besides allowing the use of raw material with high
contents of free fatty acids. However, this technique requires relatively severe
operating conditions, therefore requiring special equipment that makes the
costs associated with the process very expensive (Shimoyama et al., 2009; Tan
et al., 2010; Atadashi et al., 2011).
Contrary to these processes, enzyme-catalyzed reactions using lipases
have proven to be an interesting alternative for industrial-scale biodiesel
production in order to reduce production costs. Lipases (triacylglycerol acyl-
hydrolases, E.C. 3.1.1.3.) are powerful tools that can catalyze not only
hydrolysis but also various synthetic reactions including esterification and
transesterification.
Their utilization for biodiesel production is advantageous because these
enzymes have catalytic activity and stability on aqueous-organic interfaces,
and their specificity, regioselectivity and enantioselectivity can be successfully
used for many applications in organic synthesis (Paizs et al., 2004; Bencze et
al., 2010; Brem et al., 2010).
In addition, these enzymes are versatile and robust and show high activity
to catalyze free fatty acids from esterification reactions that involve
condensation of a fatty acid with monoalcohol, forming esters, which is not
possible when using conventional alkaline catalysts (Paizs et al., 2004; Fukuda
et al., 2008; Nielsen et al., 2008; Robles-Medina et al., 2009; Solomons and
C.B. Fryhle, 2009; Bencze et al., 2010; Brem et al., 2010). A further advantage
of the enzymatic process is that less waste and effluents are generated, making
it more environmentally friendly. Furthermore, the amount of energy resources
necessary for management of the process also culminates in lower energy
expenditure, since enzymatic catalysis is conducted at lower temperatures
(Robles-Medina et al., 2009).
In view of the catalytic ability of lipase, the production of biodiesel by
direct esterification of fatty acids catalyzed using this enzyme can be utilized
as an interesting alternative to decrease the operating costs associated with the
conventional process, as well as to overcome the above-mentioned problems.
In addition, the use of fatty acid mixtures is possible in this process, usually
obtained from vegetable oil refinement with lower cost than triacylglycerides
(Vieira et al., 2006).
The animal fats and vegetable oils used in biodiesel synthesis normally
present five main types of fatty acid chainsin their composition: palmitic,
stearic, oleic, linoleic and linolenic.
Among these fatty acids, the oleic acid is a monounsaturated fatty acid
found in significant concentrations in animal fats and vegetable oils (Leung et
al., 2010; Mulalee et al., 2013; Yin et al., 2013).
Recent studies have demonstrated the use of oleic acid as an efficient
substrate for production of biodiesel by esterification using lipase. Mulalee et
al. (2013) studied the production of biodiesel from oleic acid and bioalcohols
(ethanol and butanol) using immobilized lipase (Novozym 435) as biocatalyst
in a batch esterification process. The optimal conditions were 45C, oleic acid
to alcohol molar ratio of 1:2, Novozym 435 loading at 5% based on oleic acid
weight and 250 rpm, in which the free fatty acid conversion at 91.0% was
obtained after 12 hours of the reaction.
Rosset et al. (2013) reported biodiesel production by esterification of oleic
acid with aliphatic alcohols using immobilized Candida antarctica lipase,
showing high yields of biodiesel (above 90%) in less than 24 h with ethanol,
n-propanol and n-butanol; whereas with methanol, the enzyme was inactive
after ten cycles of reaction. In another report, Yin et al. (2013) studied an
efficient bifunctional catalyst lipase/organophosphonic acid-functionalized
silica (SG-T-P-LS) for biodiesel synthesis by esterification of oleic acid with
ethanol. In this system, the process had a conversion ratio reaching 89.94
0.42% under the conditions that the ethanol/acid molar ratio was 1.05:1 and
the SG-T-P-LS to free fatty acid weight ratio was 14.9 wt.% at 28.6 C (Yin et
al., 2013).
Therefore, the use of efficient lipases in the esterification reactions of free
fatty acids has been shown to be a very important tool in developing cleaner
and more economical processes for biodiesel production, as they could reduce
equipment needs and costs (Yin et al., 2013).

Table 1. Types and characteristics of hydroxyl fatty acids from oleic acid by lipase transformation

HFA Compounds Characteristics Function Reference
Ricinoleic acid Flexibility, heat, resistant,
degradable and non-toxic;
Emulsifier, dispersant, lubricant,
polyester polyols, polyurethane
and plasticizer;
Palaskar et al., 2010

-Hydroxy fatty acid Polyester-like property,
degradable;
Plastic manufacture; Kim and Oh, 2013

12-Hydroxystearic acid High melting point,
thermostable, gel state at room
temperature and non-toxic;
Acrylic polymer, rubber, wax,
grease;
Kim and Oh, 2013

10-Hydroxystearic acid High melting point,
thermostable, gel state at room
temperature and non-toxic;
Lubricant; Kim and Oh, 2013

3. CHEMICAL INDUSTRY

Traditionally, chemical catalysts have been used to perform several
reactions. By replacing the chemical catalysts with enzymes, final products
can proceed in a controlled manner. Enzymatic reactions can be used to
upgrade cheap and saturated fats or to add value to commercial fats and oils.
Vegetable oil-based polyurethane, polyester, polyether and polyolefin are the
four most important classes of polymers, many of which have excellent
biocompatibilities and unique properties including shape memory. Many
researchers have investigated lipase-catalyzed reactions as an alternative to
green processes and as a way to improve the physical properties of final
products (Miao et al., 2013).
Different new products with enhanced functions of interest can be
obtained from oleic acid by way of enzymatic reactions. The product is
dependent upon the enzyme used and environmental conditions, such as
aeration and temperature of reaction. Thus, there is an increasing interest in
optimizing the production of these compounds in order to enable their use on a
larger scale.
One of the most important products in chemical fields is the Hydroxy fatty
acids (HFAs). They were originally produced in nature mainly by plant
systems and are known to exhibit special properties such as high viscosity and
reactivity compared to normal fatty acids (Martin-Arjol et al., 2013). Based on
these structural peculiarities, HFAs possess high industrial potential in a wide
range of applications, including resins, waxes, nylons, plastics, lubricants,
cosmetics, and additives in coatings and paintings. Despite poor studies on
microbial enzymes, especially lipases, this type of reaction can synthesize
region-specific hydroxyl fatty acids (Metzger et al., 2006; Gow, 2010;
Biermann et al., 2011; Joo et al., 2012; Kim et al., 2012; Tunaru et al, 2012).
In Table 1, the production of mono-, di-, and tri-hydroxy fatty acids by
microbial lipase transformation is presented.
Most research about lipase in the chemistry industry use the epoxidation
reaction, which is a reaction of a carbon-carbon double bound with active
oxygen, converting the original double bond into a three-membered epoxide
(oxirane) ring (Gamage et al., 2009). The products of this reaction are
generally used for formulation of polyvinyl chloride (PVC), widely used in
plastics. Nowadays, this reaction is catalyzed by chemical compounds
(cations), which increases the cost of the process and the risk of the degrader
environment. Substituting the chemical compounds with lipase improves the
yield of the reaction and results in an environmentally friendly process
(Figure 2).

Figure 2. Enzymatic epoxidation reaction: from oleic acid to epoxystearic acid
(adapted from Roumanet et al., 2013).
The chemo-enzymatic epoxidation of olefins has been studied as an
alternative to the traditional chemical epoxidation method, where peracetic (or
performic) acid is used to oxidize the unsaturated bonds to form epoxide rings.
The peracid is usually formed in situ by hydrogen peroxide, H
2
O
2
, along with
acetic (or formic) acid. The main disadvantage with the chemical method is
the acid-catalyzed side-reaction of ring-opening, resulting in several by-
products. This can be avoided by using the chemo-enzymatic method, which
has been explored initially with reactions in solvents such as toluene, and more
recently in solvent-free reactions with various substrates (Roumanet et al.,
2013; Saithai et al., 2013).
However, enzymatic method has a major problem inactivation of the
enzyme by H
2
O
2
. To minimize this effect, optimization of several factors such
as reactor design, feed distribution of H
2
O
2
, solvent choice, etc. is needed.
Also, for industrial scale, which hinders its implementation, optimization of
the following factors must be achieved: cost of the enzyme in the process,
conditions of enzymatic action and reaction time. Thus, many studies are
being conducted to improve the performance of lipases and thus minimize
their costs in the final product (Roumanet et al., 2013; Saithai et al., 2013).
Roumanet et al. (2013), used oleic acid from sunflower oil fatty acid as a
substrate by lipase epoxidation to produce aliphatic polyesters. The synthesis
of polymeric materials based on monomers from renewable feedstock is a
steadily growing field. The main attractive aspects of these polyesters are the
renewable biomass origin of the diacid and the presence of double bonds on
the polymer backbone (oleic acid derivative), allowing for subsequent
chemical modifications. The epoxidation of these double bonds was
undertaken on the macromolecular chains to induce the photochemical cross-
linking of the epoxide functions. This successful enzymatic transformation
will contribute to a new and large field for biodegradable polymer production
derived from renewable resources.
Corra et al. (2012) studied in oleic acid epoxide production by using
lipase from Burkholderiacepacia as a biocatalyst in the reaction (first time
used to catalyze the epoxidation reaction by using an aqueous solution of
hydrogen peroxide as oxidant). An experimental design adopting surface
response was applied for this purpose. Reactions were performed in shaker
equipment, and different variables were investigated, such as temperature (25
55C), enzyme load (1020 wt% of oleic acid mass), hydrogen peroxide load
(0.10.2%) and reaction time. The lipase showed its best behavior as
biocatalyst after 3 h of reaction at 55C, 10% enzyme load, 0.2% hydrogen
peroxide and applying 150 rpm for stirring. Also, the enzyme concentration
was not significant in the reaction within the range studied. Focusing on the
industrial applicability, this result is considerably relevant as it has direct
implications on operating costs due to the smaller amount of biocatalyst
required. In conclusion, under these conditions, the epoxide yield was
improved from 30% to 88%, and the variables studied had a great impact on
the response of the epoxidation.

4. ASPECTS IN FOOD SCIENCE AND TECHNOLOGY

Oleic acid is a naturally-occurring fatty acid in animal and vegetable oils.
At the present, edible and industrially fatty acids are obtained from seeds of
highly oleaginous plants, and oleic acid naturally occurs in greater quantities
than any other fatty acid. This characteristic makes it an important compound
for the diet and for increasing the lipophilicity of bioactive molecules (Lin and
Huey, 2009; Aki et al., 2005).
The benefits of the intake of oleic acid have been associated with the
decrease of ingestion of saturated fat by changing dietary patterns and the use
of food technology to modify the fatty acid profile of foods naturally rich in
saturated fatty acids in favor of oleic acid. Recently, oleic acid has received
much attention because of its availability and health benefits, which include
the reduction of LDL cholesterol levels in blood. Beneficial effects on risk
factors regarding thrombogenesis, in vitro LDL oxidative susceptibility and
insulin sensitivity have also been reported (Lopez-Huertas, 2010). Besides
health benefits of this fatty acid, various highly oleic sunflower oils containing
from 75% to 90% oleic acid are now on the market, mainly because of their
higher frying stability in relation to conventional sunflower oil (Aladedunye
and Przybylski, 2013; Marmesat et al., 2008). Oleic acid is, at the moment,
attracting attention to its applicability because of the combination of these
features. However, it is necessary to elucidate mechanisms to add or react
oleic acid with other substances in order to obtain high-added-value
compounds.
The control of gene expression of the fatty acid modifying enzymes and
other related enzymes enables oleaginous organisms to produce oils with new
compositions. An increased level of oleic acid has been achieved by DuPont
Pioneer in soybean by genetic modification, decreasing the expression of an
endogenous soybean fatty acid desaturase gene (gm-fad2-1) using gene
silencing (La, 2013).
In addition to genetic manipulation, the use of enzymes from
microorganisms makes possible the conversion of selected substrates into
desirable products with many benefits to the process regarding food
technology. The advantages include selectivity, mild reaction conditions and
little or no unwanted side reactions, which concurs to high product purity.
Little waste material is produced, thus, it is hoped that the use of lipase in
biotransformation of fats and oils increases, expanding the implementation of
environmental friendly processes in food industries (Shimada, 2006).
Enzymatic catalysis can be regio-(1, 3 or 2-position) and fatty acid-
specific and can result in products with better-defined chemical structure and
composition (Ray et al., 2013). This flexibility allows the use of specific
lipases to prepare fully acylated glycerin, such as triolein, as well asmono- or
diglycerides that are used in the formulation of healthier products and as a first
step in the production of structured lipids (Rodrigues and Fernandes-Lafuente,
2010).
The biotransformation enables the conversion of many other types of fats,
as reported in literature. Lipase-catalyzed acidolysis was successfully used to
incorporate oleic acid into palm olein. An increment of 33% oleic acid was
obtained, resulting in high oleic content of 5556% oleic in palm oil products
(Lin and Huey, 2009).
The enzymatic transesterification of palm stearin and olein blends allowed
the production of a zero-trans margarine fat. The study was perfomed by
Sellami et al. (2012) in a solvent free system using a Rhizopusoryzae lipase
immobilized onto CaCO
3
to produce a suitable fat for margarine formulation.
This ecofriendly approach used low value bioresources like palm stearin and
palm olein to obtain a healthier product. The triacylglycerol (TG) profile
showed a decrease in the amounts of saturated TG and increased the lower
melting point TG ratio, especially triolein (OOO).
Lipase-catalyzed modification of milk fat is used commercially to
improve physical, chemical, nutritional, and sensory properties of milk fat, and
to achieve desired properties in the manufacturing of milk products (Kalo and
Kemppinen, 2003). Balco et al. (1998) produced butterfat with higher oleic
acid content and lower lauric, myristic, and palmitic acid contents by a lipase
from Mucorcircinelloides. This exchange widens the melting temperature
range and produces an engineered butterfat with 27% more esterified oleic
acid. Another study performed by Bazmi and Relkin (2009) shows that the
enrichment of milk fat with olein-rich fraction increased the whipping ability
of the emulsions. Lower crystalline fat content contributed to higher air
incorporation into the emulsion,resulting in higher overrun.
Recently, oleic acid has been widely applied in phytosteryl esters(i.e.,
fatty acid (FA) ester forms of phytosterols), which are preferred in food
formulations to overcome the problems of free forms of phytosterols since
they possess very low solubility in edible oils and have a very high melting
point (Contesini et al., 2013). Phytosterols are known to have a
hypocholesterolemic effect, allowing the reduction of low-density lipoprotein
cholesterol in plasma, whereas high-density lipoprotein cholesterol
concentration is not affected by their consumption (Villeneuve et al., 2005).
The lipase-catalyzed esterification reaction between phytosterols and oleic
acid to produce phytosteryl esters of oleic acid were optimized by Kim and
Akoh (2007) using microbial lipases from Candida rugosa. The best reaction
conditions were shown to be simple and mild, which is favorable to industrial
application (i.e., monophasic media of hexane, low temperature 55 C, short
reaction time 24 h, and without the use of water-trapping agents or reduced
pressure system). Temoin (2013) immobilized a lipase from Candida rugosa
on poly(ethylene terephthalate)-grafted glycidyl methacrylate (PET-g-GMA)
fiber. The hydrolysis ofolive oil followed by the esterification of oleic acid
were evaluated, and the immobilized lipase retained65 % of its original
activity at 50 C for 2 h. Moreover, the esterification percent yield of the
immobilized CRL decreased slightly from 29 to 27% after five reuses.
Despite many benefits regarding biotransformation by microbial lipases,
the costs associated with this process are higher than chemical processes.
Nowadays, the use of lipases is limited to the development of fats and oils that
cannot be produced by chemical means. However, research regarding the
increase of the lipophilicity of bioactive molecules is promising, and scale-up
studies simulating industrial processes are necessary to evaluate their
feasibility.

5. PHARMACEUTICAL APPLICATIONS

In the pharmaceutical field, oleic acid is used in several circumstances,
being widely used for the production of monoolein (Wang et al., 2013),
carbohydrate fatty acid esters (Abdulmalek et al., 2012), phytosteryl esters of
fatty acids (Pan et al., 2012) and fatty acid esters of vitamin C (Stojanovic et
al., 2013), which will be mentioned below. This is in addition to other
applications, to a lesser extent, such as dopamine, a phenolic compound
derived from tyrosine and belonging to the catecholamine family that has been
shown to be an effective scavenger of superoxide and hydroxyl radicals
(Sellami et al., 2013) and prodrugs, compounds that suffer biotransformation
before presenting pharmacological effects (Arouri et al., 2013).
Monoacylglycerols (MAGs) are nonionic surfactants with hydrophilic and
hydrophobic parts in the molecules, which have GRAS status (Generally
Recognized as Safe) by the FDA (Food and Drug Administration-USA). They
are precursors to the synthesis of many active lipids and important amphiphilic
emulsifiers, which are widely used in pharmaceuticals, food and cosmetics, for
not presenting ingestion side effects or skin irritations, unlike ionic surfactants.
They are basically formed by fatty acid monoesters and glycerol (Freitas et al.,
2008). In the pharmaceutical industry, MAGs are used as emollients to
plasters, by slowly releasing the medication; whereas in the cosmetic industry,
they are used as texturizing agents and improve the consistency of creams and
lotions (Kaewthong et al., 2005).
Recently, the synthesis of MAGs catalyzed by lipases has been studied
intensively as an alternative to the conventional method, mainly due to the use
of mild reaction conditions, which imply low energy consumption and
selectivity of enzymes lipases that, in an integrated manner, result in best
quality products. Furthermore, the operation of the specificity of these
enzymes enables the synthesis of products, which could not be achieved by the
conventional chemical route. It is noteworthy that from an environmental point
of view, the enzymatic process is technically clean and safe. In this context,
different alternatives have been proposed with regard to the enzymatic
synthesis of MAGs (Freitas et al., 2008). An example of MAG widely used by
the pharmaceutical industry is the monoolein, used as a drug delivery system,
pharmacist charger and emulsifier. It is a mixture of the glycerides of oleic
acid and other fatty acids, consisting mainly of the monooleate (Zeng et al.,
2010). Wang et al. (2013) reported an improved method for the synthesis of
MAGs. Commercial oleic acid was purified, and 1,2-acetonide-3-
oleoylglycerol was synthesized by the esterification of 1,2-acetonide glycerol
with the purified oleic acid through the use of Novozym 435 lipase as catalyst.
The use of 1,2-acetonide glycerol as starting material to synthesize monoolein
was made to avoid the formation of diacyl- and triacylglycerols during
esterification and the cleavage reaction. The cleavage of unpurified 1,2-
acetonide-3-oleoylglycerol in methanol was conducted to obtain 1-monoolein.
The cleavage reaction resulted in the formation of 76.5% 1-monoolein, and
96.2% 1-monoolein was obtained at 72.8% yield after repeated
recrystallization in hexane to remove nonpolar impurities and water washing
to remove glycerol. Bellot et al. (2001) investigated MAG synthesis by a
commercial Rhizomucormiehei lipase (Lipozyme) via direct esterification
between glycerol and oleic acid in organic solvents. With the use of n-hexane
and 2-methyl-2-butanol, they reached a production of 42 mM of monoolein,
and proposed that an increase in solvent polarity using mixtures of both
solvents drastically improves the selectivity toward MAG formation.
Major applications of oleic acid have also been made recently in the
production of carbohydrate fatty acid esters or carbohydrate esters in
biotechnological industries with emulsifier and surfactant properties, due to
their specific properties and wide applications in cosmetics and pharmaceutics,
as well as food and detergent industries (Abdulmalek et al., 2012). These
esters are non-toxic, non-irritant, non-ionic, tasteless and odorless surfactants,
besides being synthesized from cheap and renewable materials. Harmless and
biodegradable, carbohydrate esters have potential as antibacterial, insecticidal
and antitumoral agents (Sabeder, Habulin and Knez, 2006). Besides chemical
synthesis, which produces a variety of carbohydrate esters and leads to low
selectivity, an enzymatic synthesis has been chosen as a significant route to
produce these esters (Kennedy et al., 2006). Abdulmalek et al. (2012)
synthesized sugar fatty acid esters by lipase-catalyzed esterification.
Galactoseoleate ester was specifically produced from galactose and oleic acid
in ionic liquids {1-butyl-3-methylimidazolium tetrafluoroborate [(Bmim)
(BF4)]} with the addition of dimethylsulfoxide (DMSO) as a solubilizing
agent and co-solvent. Lipozyme RM IM (lipase from Rhizomucormiehei
immobilized on macroporous anion exchange resin) was used as biocatalyst.
Different reaction parameters (type of solvent, type of enzyme, amount of
enzyme, reaction time, temperature, stirring rate and substrate molar ratio)
were also studied. A high conversion (87%) was obtained after just 2 hours at
optimal synthesis conditions (1:20 DMSO: (Bmim) (BF4) ratio with 2 %
(w/w) lipase, temperature 60C, stirring rate of 300 rpm and a molar ratio of
galactose to oleic acid of 1:3).
Oleic acid is also employed in the production of important phytosterols.
These substances are sterols derived from plant sources, such as vegetable oils
and cereals and have structure similar to animal tissue sterol, cholesterol.
However, phytosterols are known to have a hypocholesterolemic effect by
lowering total plasma and low-density lipoprotein (LDL) cholesterol levels
without affecting plasma high-density lipoprotein (HDL) cholesterol
concentration (Kim and Akoh, 2007). They are widely applied in food and
cosmetic industries and have recently received a great deal of attention as
nutraceutical additives (Pan et al., 2012). Nevertheless, phytosterols have
limitations in usage for this purpose since they possess very low solubility in
edible oil and have a very high melting point (Kim and Akoh, 2007). For this
reason, there is a growing interest in phytosteryl esters of fatty acids. These
compounds are presently synthesized by chemical esterification and
transesterification. However, the chemical method involves some problems,
such as the formation of side products, which is why the use of enzymes now
has great interest. It has already been reported that phytosteryl esters are
synthesized with different lipases (Vu et al., 2004; King et al., 2001). Pan et al.
(2012) synthesized the phytosteryl esters from oleic acid using different kinds
of immobilized lipase (Novozym 435, TLIM, RMIM and Candida sp. 99-125)
as catalyst. The most effective result was achieved with 1:1 molar ratio of
oleic acid/phytosterol in 10 mL isooctane, employing immobilized Candida
sp. 99-125 lipase, incubated in an orbital shaker (200 rpm) at a temperature of
45C for 24 h, obtaining 93.4% of the sterols. Kim and Akoh (2007) also
performed enzymatic esterification of phytosterols with oleic acid to produce
phytosteryl esters of oleic acid in hexane. Response surface methodology was
used to model the reaction and Candida rugosa lipase was the biocatalyst for
the reaction, achieving 97.2% yield at 55C and 24 h.
Another important application of oleic acid in the pharmaceutical field is
in the production of fatty acid esters of vitamin C. These substances are natural
antioxidants that could be used as adequate substitutes of synthetic ones, such
as BHT (butylatedhidroxytoluene) and BHA (butylatedhidroxyanisole)
(Karmee, 2009; Burham et al., 2009). They present an interesting lipophilicity,
in distinction to vitamin C, and possess high free radical scavenging capacity.
Fatty acid ascorbyl esters are good examples of these substances because they
have an amphiphilic nature, presenting both hydrophobic and hydrophilic
functionality, and therefore can be used as non-ionic surfactants in cosmetics,
pharmaceuticals and other applications. These surfactants are renewable and
eco-friendly, which makes them more suitable for use in comparison to those
obtained from petrochemical resources (Karmee, 2008). Nowadays, efforts are
being made in order to develop an industrial process for enzymatic synthesis
of these compounds, considering numerous advantages over conventional
chemical methods (Karmee, 2011). In recent years, several microbial lipases,
such as lipases from Candida antarctica type B, Thermomyceslanuginosus,
Bacillus stearothermophylus SB1, and Rhizomucormiehei were successfully
used as biocatalysts (Lerin et al., 2012; Reyes-Duarte et al., 2011; Viklund,
Alander and Hult, 2003). Enzymatic synthesis of ascorbyloleate and other
ascorbyl esters of unsaturated fatty acids are very attractive, especially since it
was reported that ascorbyloleate has stronger antioxidative activity in
comparison to others, such asascorbylpalmitate and vitamin C (Viklund,
Alander and Hult, 2003). Stojanovic et al. (2013) synthesized ascorbyloleate
by enzymatic catalysis with immobilized lipase from Candida antarctica and
oleic acid, used as acyl donor. Acetone was applied as a reaction medium.
Effects of experimental factors, like enzyme loading, vitamin C concentration,
initial water content, substrate molar ratio, and temperature, were evaluated
using response surface methodology. The best result was achieved (19.3 mmol
g-1) using 0.2 % (w/v) lipase, 0.135 M of vitamin C, 0.018 % (v/v) water, and
1:8 substrate molar ratio at 60C. Bezbradica et al. (2013) also studied the
production of ascorbyloleate using the same immobilized lipase (from
Candida antarctica) in an acetone medium. They observed that an excess of
ascorbic acid causes an inhibition in the synthesis of this compound. The
kinetic study was performed at optimum experimental factors (temperature,
initial water content, and enzyme concentration), which were determined using
response surface methodology. The greatest result was reached (15.8 mM h-1):
1 % (w/v) immobilized lipase, 0.22 M of ascorbic acid, 1:3 molar ratio, at
60C, and addition of water was not necessary, indicating that the solvent
contains a sufficient amount of water for keeping enzymes in the open
conformation, essential for providing its activity.
After this pharmaceutical approach to the application of oleic acid through
the use of enzymes, performed in this chapter, the great importance of this
substance in this important industrial area can be noticed. In other
applications, such as in the preparation of dopamine derivatives, which are
performed less frequently, the use of oleic acid in production through
bioconversion has become extremely important to open up potential
applications in cosmetic and pharmaceutical industries (Sellami et al., 2013).
Similar to dopamine derivatives, the synthesis of 1,3-diacylglycerols has
attracted considerable attention. Being healthy components of pharmaceutical
intermediates and due to their low content in natural form, the synthesis of this
substance has increased considerably and has become important (Meng et al.,
2014).

CONCLUSION

The use of the combination of oleic acid and lipase is a promising
technology. The chemical, nutritional and functional characteristics of oleic
acid when associated with the specificity of the reactions catalyzed by
enzymes make it possible to use a wide number of molecules derived from this
process in different fields, such as bio combustibles, chemistry, food and
pharmaceuticals. In addition to these features, different high added value
compounds can be obtained using oleic acid as a cheap and efficient substrate
for microbial lipases, which can be considered as environmentally friendly
alternatives to chemical catalysts.
However, despite many benefits regarding biotransformation by microbial
lipases, the costs associated with this process are higher than that of chemical
processes. For this reason, in recent years, a huge effort has been made to
develop this type of technology. Nevertheless, the development of
technologies with the aim of reduction of process costs is still an important
target in the following years. Furthermore, new applications in the fields
highlighted in this chapter present a promising development trend in the
future.

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Chapter 5

SYNTHESIS OF OLEIC ACID ALKIL ESTERS
VIA HOMOGENEOUS CATALYSIS

Mrcio J os da Silva
*
and Abiney Lemos Cardoso
Universidade Federal de Viosa, Chemistry Department, Viosa,
Minas Gerais, Brazil

ABSTRACT

Recently, due to inevitable exhaustion of the fossil petroleum
reserves, and the environmental impact generated by the green-house
effect gas emission, to develop efficient processes for the production of
fuels and chemicals from the renewable feedstock has been pursued
researchers in worldwide. In this sense, since the oleic acid is a common
component of vegetal oils and animal fatty, it raise as a highly attractive
raw material, due to its high availability and affordability. In general, the
oleic acid is present in different feedstock as a free fatty acid or as
glyceryl ester. Several chemicals of interest for plentiful industries can be
obtained via different catalytic reactions starting from the oleic acid as
source, such as alkyl esters or ethers and epoxide-derivatives.
Particularly, alkyl oleate esters are useful as lubricant, surfactant,
emulsifying agent, emollient, fuels additive and biodiesel. Actually, the
main component of biodiesel is in general the methyl or ethyl oleate,
which is manufactured by the alkaline transesterification of edible or non-
edible vegetable oils via a well-established industrial process. However,

*
asilvamj2003@ufv.br.
Mrcio Jos da Silva and Abiney Lemos Cardoso 84
the conventional alkaline homogeneous process results in large
generation of effluents and residues of neutralization, in addition the
laborious steps to remove the non-reusable catalyst, being because of
these reasons a non-friendly environment process. In this work, we wish
the recent advances achieved in the development of catalytic processes
for the production of alkyl esters of oleic acid via acid catalysis, however,
using recyclable catalysts. We will pay special attention to development
of homogeneous catalysts that can be recovery and reusable without loss
of activity in the oleic acid esterification reactions. These catalysts are
solid when pure and soluble in the reaction being thus recovered after
solvent distillation and extraction of products. Numerous industries in all
parts of world have crescent demand by developing of environmentally
friendly technologies for the production of biodiesel and chemicals,
which are especially attractive when are based on reusable catalysts.
Herein, we focus the use of two different sorts of catalysts: the former,
Lewis acid such as tin compounds, and the second one, Brnsted acid
catalysts, which are based on Keggin-type heteropolyacids. The catalysts
performance it was assessed in the esterification reactions with short
chain alkyl alcohols (i.e., methyl, ethyl, propyl, isopropyl and butyl
alcohols). A comparison with the traditional catalysts used in these
reactions also was performed. The development of new, efficient, and
environmentally benign catalytic processes that may lead to high value
added products, starting of renewable raw material such as oleic acid, is
still an challenge to be overcome. The authors hope that this work can
significantly contribute to improvement of this important research field.

Keywords: Oleic acid alkyl esters, biodiesel, heteropolyacids, tin catalysts,
esterification, homogeneous and heterogeneous catalysis

1. INTRODUCTION

Esterification reactions of fatty acids with alcohols are well-known class
of liquid-phase reactions of great industrial interest due to the wide practical
importance of fatty esters ingredients of different commercial products.
Herein, we describes acid-catalyzed oleic acid esterification reactions with
alkyl alcohols without a solvent. The use of alkyl oleate esters has increased in
the past few years and its applications include cosmetics, pharmaceuticals,
food additives, and high- pressure lubricants [1].
Recently, due the inevitable depletion of fossil resources, the production
of fatty acids methyl and ethyl esters, which have similar properties to the
fossil diesel become goal of numerous researches [2]. In general, oleic acid
Synthesis of Oleic Acid Alkil Esters via Homogeneous Catalysis 85
alkyl esters are obtained via oleic acid esterification (Figure 2) or vegetable oil
transesterification reactions (Figure 3), which are commonly catalyzed by
Brnsted or Lewis acids.

Figure 1. Acid-catalyzed oleic acid esterification with alkyl alcohols.

Figure 2. Esterification reaction of fatty acid with alkyl alcohol.
Vegetable oils and animals fatty are the feedstock frequently employed,
however, wastewater and frying oil residues have been also attractive raw
materials because are abundant and inexpensive and their transformation into
esters avoids that they could be disposal into the environment.

Figure 3. Triglycerides transesterification reaction with ethyl alcohol.
The transforming efforts of oleic acid into alkyl esters are justified
because it is frequently present in low joined value greasy materials such as
refined oils residues. Consequently, it allows make biodiesel or surfactants at
OH
O
ROH
acid catalyst
OR
O
+
H
2
O
C
O
R OH
R OH C
O
R R O
H
2
O
CH
2
-COOR'
l
CH-COOR'' + 3C
2
H
5
OH
l
CH
2
-COOR'''
Catalyst
R'COOC
2
H
5
+
R''COOC
2
H
5
+
R'''COOC
2
H
5
CH
2
-OH
l
CH-OH
l
CH
2
-OH
+
more competitive and attractive costs for the market that those of conventional
production process. For this reason, it has calling the attention of several
researchers in all over the world.
The oleic acid esterication is particularly interesting from the perspective
of biodiesel production. Oleic acid is present in different type of vegetable
oils, as well as also in domestic and industrial, therefore, it is required to study
esterification of this fatty acid mainly when ethyl or methyl alcohol are
employed during the process. Oleic acid as well as its alkyl esters are easily
detectable by gas chromatography analysis (Figure 4).

Figure 4. Typical chromatogram of oleic acid esterification with ethyl alcohol.
The vegetable oils esterification reactions containing high amount of fatty
acids when performed in presence of alkaline catalysts are normally more
complex than ones acid-catalyzed. In alkaline solution (i.e. sodium or
potassium hydroxide and methoxyde catalysts), fatty acids may be converted
into organic salts, which compromise the reactions yielding; the soap
formation hamper the organic esters separation from the phase containing
glycerol.
Although lipase-based enzymatic catalysts are an alternative, however, its
high cost besides low stability compromise their performance. Indeed, mineral
acid such as sulfuric acid are the most used catalysts at industrial scale, in spite
of the high corrosiveness and large generation of residues in the neutralization
steps and isolation of products. Consequently, to develop new and efficient
catalysts able to promote the esterification reactions becomes fundamentally
important from viewpoint industrial [3-5].
2. HETEROPOLYACID CATALYSTS

Actually, the large demand for cleaner environment has forcing the
chemical industry to develop less environmental hazardous acid catalysts.
Heteropolyacid catalysts have attracted considerable amount of interest due to
the its easy handling in addition of having higher acidity. They have been
widely used in fine and chemicals synthesis [6-8]. There are so much
information related to heteropolyacid synthesis methods, nevertheless,
however, only the Keggin-structure heteropolyacids (HPAs) are well described
in respect of their physicochemical and catalytic properties [9].
Heteropolyacids are well known to be strong Brnsted acids and their
acidity has been quantitatively characterized and compared with the acidity of
mineral acids such sulfuric acid [10]. Differently of H
2
SO
4
which has only one
totally ionizable proton, heteropolyacids when in aquous solution are
completely dissociated at the first three steps because of the solvent leveling
effect [11]. Several works described the H
3
PW
12
O
40
heteropolyacid as an
efficient super-acid, which has been used either as soluble catalyst in polar
solvents, or in the heterogeneous phase, supported silica or on active coal as
solid matrix.
A mechanism frequently proposed for the Brnsted acid-catalyzed
esterification reactions is based on fatty acid carbonyl activation by proton
generated by the catalyst, followed by alcohol attacks in the carbonylic carbon,
generating a protonate intermediate who after water elimination results in the
ester formation (Figure 5).

O
OH R
H
+
OH
OH R
+
O
OH R
R
`
OH
OR'
H
H
+
- H
2
O
OH
OR R
+
-H
+
O
OR R
..
..
..
..
..
..
.
.
.
.
.
.
..
..
..
..
..
..
..
..
..
..
..
..
..
..
.
.
.
.

Figure 5. General mechanism for the formation of alkyl ester catalyzed by Brnsted
acid.
Nowadays, the majority of the catalysts conventionally used at industrial
scale in the FFA esterification reactions are Brnsted acids [12]. Nevertheless,
features such as high corrosiveness besides large generation of residues and
salts generated during the neutralization steps compromises the use of these
traditional catalysts [13].
Particularly, we have concentrating efforts in the developing alternative
esterification processes based on two recyclable catalysts linked to both
classes of acid catalysts: (i) H
3
PW
12
O
40
heteropolyacid, the highest acid
catalyst of Keggin series [14] and (ii) tin chloride, simple, easily handling,
water tolerant and inexpensive Lewis acid [15].

3. LEWIS ACID CATALYSTS: TIN COMPOUNDS

Lewis acid catalysts are an interesting option for the fatty acid esters
production [16,17]. However, Lewis acid compounds that are traditionally
used in organic synthesis such as BF
3
are more expensive than Brnsted acid
catalysts, and are more hardly manipulated, in addition to be few water
tolerant. Those unfavorable aspects difficult the use of Lewis acid catalysts in
FFA esterification at industrial scale [18].
Recently, we have studied the use of SnCl
2
2H
2
O as catalyst for the oleic
acid esterification pure or added to the soybean oil [19]. That investigation
described the correlation between main parameters of reaction such as catalyst
and oleic acid concentration, as well as the influence of the molar ratio oleic
acid/ethyl alcohol and the temperature upon the ethyl oleate yielding.
Likely vegetable oils, ethyl alcohol is also a renewable raw material that
when employed in the biodiesel production turns the process more beneficial
to environment [20]. However, some acid catalysts fall in FFA esterification
with this alcohol, being efficient only on the methanol reactions [21].

4. MAIN RESULTS OBTAINED IN THE USE
OF HETEROPOLYACID AND TIN CATALYSTS
IN OLEIC ACID ESTERIFICATION REACTIONS

A comparison of H
3
PW
12
O
40
(HPW) and SnCl
2
catalytic activity against
sulfuric acid and p-tolenesulfonic acid (PTSA) in the oleic acid esterification
reactions with ethyl alcohol was made by us and the resulting kinetic curves
are shown on Figure 6.
Reaction conditions: oleic acid (5.0 mmol); H
2
SO
4
(0.0288 mmol); PTSA
(p-toluenesulfoinc acid; 0.0576 mmol); HPW (H
3
PW
12
O
40
; 0.0192 mmol);
SnCl
2
(0.100 mmol); ethyl alcohol (167.0 mmol); temperature (60 C); 5h;
conversion determinate by GC analysis.
0 1 2 3 4 5
0
20
40
60
80
100
(
%
)

e
t
h
y
l

o
l
e
a
t
e

y
i
e
l
d
i
n
g
time (h)
no catalysed
PTSA
H
2
SO
4
SnCl
2
HPW

Figure 6. Kinetic curves of oleic acid esterification reactions with ethyl alcohol.
It is noteworthy that all of the Brnsted acid catalysts were used in the
same hydrogen concentration. It was observed that although high molar ratio
has been employed (i.e. ethyl alcohol: oleic acid molar ratio = 167: 5), only a
low yielding of ethyl oleate (ca. 17%) was reached after 5 hours of reaction in
absence of catalyst.
Conversely, in presence of either Lewis or Brnsted acid catalysts, high
ethyl oleate yielding (ca. 90-95%) were reached (Figure 6). Noticeably, the
initial rate of reactions that were catalyzed by the HPW or H
2
SO
4
, which are
stronger Brnsted acids, were the highest.
Nevertheless, it is important to note that the reaction yields increase
steadily, reach the maximum values (ca. 90%) after a reaction time of ca. 5 h
and stay almost invariable afterwards. Furthermore, observe that the three
Brnsted acid catalysts, in spite of bearing so different structures, displayed
quite similar activities as can be confirmed by the attainment of comparable
ethyl oleate yields at a given reaction time (Figure 6).
Although SnCl
2
being the only Lewis acid studied herein, and
consequently it may display a different catalytic action mechanism, it was also
included in this topic for comparison. Actually, it seems that the SnCl
2
is also
an efficient catalyst, taking in account that high yields in ethyl oleate were
achieved, similarly to those obtained in the Brnsted acid-catalyzed reactions.
0 10 20 30 40 50 60 70 80 90 100 110 120 130
0
1
2
3
4
5
6
7
8
9
10
f
r
e
e

o
l
e
i
c

a
c
i
d

(
m
m
o
l
)
time (min)
SnCl
2
= 0.01 mmol
SnCl
2
= 0.10 mmol
SnCl
2
= 0.20 mmol
SnCl
2
= 0.40 mmol

Figure 7. Effect of the SnCl
2
.2H
2
O concentration on conversion rate of waste cooking
oil (WCO) esterification reactions with ethyl alcohol. Reaction conditions: ethanol
(551.0 mmol, 31.7 mL); WCO (3.26 g., 3.3 mL).
The HPW and SnCl
2
catalytic activity on oleic acid esterification who was
added to the WCO (waste cooking oil) samples was also evaluated. Soybean
oil samples containing oleic acid (ca. 10 mmol) were prepared and the SnCl
2

catalyst activity was then evaluate in the esterification reaction with ethyl
alcohol (Figure 7).
The acidity values of oil samples are around of 10-20 times above those
found into major of the feedstock commonly used for biodiesel production.
The kinetic curves reveals that when used in the concentration equal to 0.40
mmol, a reduction of 95 % on initial oleic acid content it was achieved.
Initially, our intention was to determinate the activation energy of HPW-
catalyzed esterification reaction of oleic acid with ethyl alcohol. However, an
auspicious resulted was found, as displayed in Figure 8. Surprisingly, at
temperature range studied (ca. 25-55 C) high yields were achieved after 4
hours reaction in all of catalytic runs (Figure 8).
This result was motivated us to study the esterification reactions under
room temperature, where we proved that only heteropolyacid was active under
this reaction conditions [22].
0 1 2 3 4 5 6 7 8
0
20
40
60
80
100
c
o
n
v
e
r
s
i
o
n

(
%
)
time (hs)
25
o
C
35
o
C
45
o
C
55
o
C

Figure 8. Effect of temperature on HPW-catalyzed oleic acid esterification with
ethanol.
In general, homogeneous catalytic systems have several disadvantages
owing to the typical problems of separation of the products from the catalyst.
We proposed an efficient method for the oleic acid esterification with ethyl
alcohol, where a homogeneous, notwithstanding recyclable catalyst was
established as an alternative to the traditional supported heterogeneous
catalysts (Figures 9 and 10) [23].
In this homogeneous and recyclable catalytic system, after the reaction
end, hexane is added to the system, the upside comprises the ethyl esters and
the downside containing the aqueous/ethyl alcohol phase. It was possible
because the hexane and aqueous/ethyl alcohol phase are immiscible at room
temperature. Consequently, the catalyst (i.e. HPW or SnCl
2
as convenient)
remains in the polar phase and may be then collected as a solid after remove
via evaporation both alcohol and water (Figure 10).
Recently, we have reported some preliminary results of SnCl
2
-catalyzed
oleic acid esterification reaction with ethyl alcohol esterification with this
recycle procedure [24]. The similar way, the literature has reported a simple
procedure for recycling of HPW catalyst after FFA esterification reactions
[25].
supported- acids solid
catalysts synthesis
catalyst
characterization
catalytic run
solvent/products
liquid-liquid extration
destillation
solvent
products
solvent/products
solid catalyst
catalyst
reuse
step 1
step 2
step 3
catalyst recovery
via filtration
support and
catalyst
supported catalyst
step 4

Figure 9. A simplified block flow diagram of the heterogeneous acid solid-catalyzed
process for the production of biodiesel.
catalytic
run
liquid-liquid
extration
solvent/products
solved catalyst
recycle
step 1
step 2
step 3
solvent and
products
destillation
products
solvent
solvent and
catalyst
catalyst
solvent
destillation

Figure 10. A simplified block flow diagram of the homogeneous acid solid-catalyzed
process for the production of biodiesel [23].
We have successfully applied also the same procedure to the
recovery/reuse of HPW catalyst in glycerol esterification reactions with acetic
acid [26]. Herein, those protocols were used for evaluate the
recovery/reutilization of both catalysts (i.e. HPW and SnCl
2
) from the soybean
oil sample esterification reactions containing high content of oleic acid using
ethyl alcohol under free solvent-conditions (Figure 11).
Figure 11 shows that both catalysts were efficiently recovered and reused
without loss of activity after three successive cycles. Actually, very high
recovery yields of both catalysts were achieved by them extraction from the
reaction medium. In addition, it was observed that the activity of the catalysts
stayed almost unaltered even after three recovery/reutilization cycles.
Furthermore, it was also verified that Sn and W contents in a sample of
biodiesel, prepared via the HPW or SnCl
2
-catalyzed esterification reactions,
were 0.0095 mg of W per g of sample (average of three measurements). This
remarkable result indicates that the content of homogeneous tungstate or tin
species seems to stay at an acceptably low level in the final product.
SnCl2 HPW
0
20
40
60
80
100
1
st
1
st
2
nd
2
nd
3
rd
3
rd
e
t
h
y
l

o
l
e
a
t
e

y
i
e
l
d

(
%
)
catalyst

Figure 11. Ethyl oleate yields obtained for the 1
st
, 2
nd
and 3
rd
consecutive
recovery/reuse on esterification reactions of soybean oil sample (10 % w/w oleic acid)
using the SnCl
2
or HPW as catalysts. Reaction conditions: ethanol (167 mmol), SnCl
2

(0.400mmol); HPW (0.192 mmol); reflux temperature (ca. 78 C); 4 hours reaction.
0 1 2 3 4 5 6 7 8 9
0
10
20
30
40
50
60
70
80
90
100
E
t
h
y
l

o
l
e
a
t
e

y
i
e
l
d
i
n
g

(
%
)
Time (h)
SnO
SnO
2
SnSO
4
SnF
2
SnCl
2
SnOAC

Figure 12. Effect of the tin catalyst nature on the oleic acid esterication with ethyl
alcohol. Reaction conditions: oleic acid (1.0 mmol); ethyl alcohol (155.0 mmol);
catalyst (50 mg); 60C.
The kinetic curves shown in Figure 12 reveal that reactions in the presence
of tin catalysts containing the less labile ligand and with strongest electron
withdrawing character achieved the lowest ester yield (i.e., F
-
and
-
OAc
anions, respectively). On the other hand, the catalyst containing more bulky
halides (i.e., Cl
-
) and consequently the more softness and stable anionic ligand
were the most active. Although not showed herein, the same behaviour was
verified in SnBr
2
-catalyzed oleic acid esterification reactions [27].
The solubility of catalyst was also a key feature in these reactions; for
instance, the solid tin oxides (SnO and SnO
2
) were equally inactive on the
oleic acid esterification with ethyl alcohol, certainly, due to its almost
complete insolubility (Figure 12).
The efficiency of tin bromide catalyst was assessed in oleic acid
esterification with different alcohols and the kinetic curves are displayed in
Figure 13 [27]. It was observed that both steric hindrance as well as the carbon
chain of alcohol affect drastically the final yielding of reactions.

0 2 4 6 8
0
10
20
30
40
50
60
70
80
90
100
C
o
n
v
e
r
s
i
o
n
(
%

)
Time(h )
methanol
ethanol
propan-1-ol
butan-1-ol
propan-2-ol

Figure 13. SnBr
2
-catalyzed oleic acid esterification with different alcohols [27].

a
Reaction conditions: alcohol: oleic acid: SnBr
2
molar ratio equal to 170 : 1 : 0,1;
temperature 60C.
0 2 4 6 8
0
20
40
60
80
100
C
o
n
v
e
r
s
i
o
n

(
%
)
Time (h)
methanol
ethanol
propan-1-ol
propan-2-ol
butan-1-ol

Figure 14. H
3
PMo
12
O
40
-catalyzed oleic acid esterification with different alcohols.

a
Reaction conditions: alcohol: oleic acid: H
3
PMo
12
O
40
molar ratio equal to 170: 1:
0.014; temperature 60C.
0 2 4 6 8
0
20
40
60
80
100
H
3
PMo
12
O
40
0.008 mmol
H
3
PMo
12
O
40
0.014 mmol
C
o
n
v
e
r
s
i
o
n

t
o

e
t
h
y
l

o
l
e
a
t
e
(
%
)
Time (h)

Figure 15. H
3
PMo
12
O
40
-catalyzed esterification of oleic acid with ethyl alcohol.
0 2 4 6 8
0
20
40
60
80
100
C
o
n
v
e
r
s
i
o
n

t
o

m
e
t
h
y
l

o
l
e
a
t
e

(
%
)
Time (h)
H
3
PMo
12
O
40
0.008 mmol
H
3
PMo
12
O
40
0.014 mmol

Figure 16. H
3
PMo
12
O
40
-catalyzed esterification of oleic acid with methyl alcohol.
Recently, our research group performed a similar study using as catalyst
another Keggin catalyst: dodecamolibdophosphoric acid (H
3
PMo
12
O
40
) [28].
Similarly to the verified in the tin catalyzed reactions, alcohols with great
carbon chain and hydroxyl group more hindrance were the lesser reactive than
the ones with short chain and terminal hydroxyl group (Figure 14).
The effect of catalyst concentration was also assessed; we find out that
using 0.008 or 0.014 mmol % of H
3
PMo
12
O
40
catalyst concentration a almost
complete conversion of oleic acid into ethyl oleate was reached after 8 hours
reaction (Figure 15).
A similar result it was verified when methyl alcohol was employed,
nevertheless, after 1 hour reaction, almost oleic acid was converted to methyl
oleate (Figure 16).

CONCLUSION

The use of Lewis and Brnsted acids as catalysts in oleic acid
esterification reactions it was assessed. Although being homogeneous
catalysts, both compounds can be recovered and reused in the reactions via a
simple procedure developed by us. Heteropolyacid catalysts were highly
efficient in the oleic acid esterification reactions with different alcohols. On
the other hand, tin compounds, in special SnCl
2
and SnBr
2,
were the most
effective tin catalysts on these reactions esterification. We hope that the
finding presented in this work may contribute to development of efficient and
environmentally benign process for the synthesis of oleic acid alkyl esters,
which comprise important ingredients for different chemical industries.

ACKNOWLEDGMENTS

The authors thanks FAPEMIG, CAPES and CNPq, by financial support.

REFERENCES

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[7] I.V. Kozhevnikov, Catalysts for Fine Chemicals, 2, (2002).
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[12] S. Zheng, M. Kates, M.A. Dube, D.D. McLean, Biomass Bioenerg., 30,
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[13] M., Di Serio, R. Tesser, M. Dimiccoli, F. Cammarota, M. Nastasi, E. J.
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[14] M. N. Timofeeva, Acid, Appl. Catal. 256, 19, (2003).
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71, 1035, (1994).
[16] M. Di Serio, M. Cozzolino, R. Tesser, P. Patrono, F. Pinzari, B. Bonelli,
E. Santacesaria, Appl. Catal. A. 320, 22, 1, (2007).
[17] P. S. Sreeprasanth, R. Srivastava, D. Srinivas, P. Ratnasamy, Appl.
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[18] E. Lotero, Y. Liu, D.E. Lopez, K. Suwannakaran, D.A. Bruce, J.G.
Goodwin Jr. Ind. Eng. Chem. Res. 44, 5355, (2005).
[19] A. L. Cardoso, S. C. G. Neves, M. J. da Silva, Energies, 1, 79, (2008).
[20] A. Corma, H. Garcia. Chem. Ver. 103, 4307, (2003).
[21] F.R. Abreu, D.G. Lima, E.H. Ham, C. Wolf, P.A.Z. Suarez. J. Mol.
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[22] V. W. de G. Silva, L. O. Laier, M. J. da Silva, Catal Lett 135, 207,
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[23] M. L. da Silva, A. P. Figueiredo, A. L. Cardoso, R. Natalino, M. J. da
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[24] A.L. Cardoso, S.C.G. Neves, M.J. da Silva, Energ. Fuels, 23, 1718,
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3
PMo
12
O
40

Heteropolyacid: A Versatile and Efficient Bifunctional Catalyst for the
Oxidation and Esterification Reactions. In: Anja Li Patel. (Org.).
Environmentally Benign Catalysts for Clean Organic Reactions. Ed.
Mumbai: Springerlink, 170, 2013.


Chapter 6

EFFECTS OF TEMPERATURE ON OLEIC ACID
PERCENTAGE DURING GRAIN-FILLING
IN SUNFLOWERS AND OTHER OIL CROPS

Rouxlne van der Merwe
and Maryke Labuschagne

Department of Plant Sciences, University of the Free State,
Bloemfontein, South Africa

ABSTRACT

Most vegetable oils are obtained from beans or seeds, which furnish
valuable and high quality oil commodities in the world oil market. Seed
oil quality is related to oil percentage and fatty acid composition and
defines the oils value for industry. With emerging new markets and
increased concerns about the health risks of foods, changes in the oil
quality of various crops have been demanded. Plant breeders have been
successful in developing novel oil types in sunflower, soybean, peanut
and others with increased percentages of oleic acid. Genotype is the most
important factor that defines the oil fatty acid composition, but
environmental factors, particularly during the grain-filling period, can
widely affect both oil content and oleic acid percentage. Various
environmental factors including temperature (heat and cold, day/night
differences), solar radiation, humidity, day length and moisture
availability (rainfall distribution and intensity, drought or flooding) affect
seed oil percentage and composition. When environmental factors deviate

Corresponding author: vandermerwer@ufs.ac.za.

Rouxlne van der Merwe and Maryke Labuschagne 100
from the optimal quantity or intensity for the crop plant, stress is caused.
Changes in both oil percentage and fatty acid composition caused by
environmental stress could have a dynamic effect on the quantity and
quality of oil that is extractable by seed processors. Temperature is a
major environmental factor that determines the rate of oil accumulation.
Generally warm temperatures during the entire growing season or a
period of heat stress during grain-filling favors the production of oleic
acid, while cooler temperatures favor the production of linoleic acid in
traditional oil crops. However, not all genotypes are similarly affected by
temperature and show strong genotype by environment interaction.
Generally the novel sunflower genotypes with increased oleic acid
contents display more stable oleic to linoleic acid ratios across different
environments than standard types with high linoleic acid percentages. In
novel soybean varieties, the high oleic acid content fluctuates with
temperature differences. In order to improve oil quality in traditional oil
crops, it is necessary to understand the temperature effects on oleic acid
content. In addition, since agricultural and management practices can
alter temperature and other important environmental factors that plants
are exposed to during grain-filling, altered production practices could
contribute to modified oleic acid contents in vegetable oil crops.

INTRODUCTION

The properties of a vegetable oil are almost exclusively determined by the
fatty acid composition of its lipids, mainly triacylglycerols (TAG), which
constitute the oil. In temperate oil crops, the main fatty acids are the
unsaturated oleic and linoleic acids (Padley et al., 1994).In oilseeds, fatty acids
are synthesized at high rates during a short period and used preferentially for
TAG synthesis. Oleate (18:1), the main product of plastidial de-novo
synthesis, is desaturated to linoleate (18:2) by the oleoyl-phosphatidyl-choline
desaturase (ODS) enzyme (Garcs et al., 1992). Oils with different fatty acid
composition are required depending on their use for human consumption or in
non-food applications. In some cases oils are transformed to modify fatty acid
composition in order to obtain the best properties required for their final use.
In the production of margarine, shortenings and other specialty products, oil
with a high plasticity and stability are required. However, polyunsaturated oils
such as traditional sunflower and soybean oil are often hydrogenated to
improve the oils stability and plasticity. Hydrogenation or chemical hardening
of the oil increases its stability but also produces trans fatty acids which are
harmfully related to heart disease (Ascherio and Willett, 1997). Since
Effects of Temperature on Oleic Acid Percentage 101
consumer and end-user preferences for various oils are changing, breeding
goals to modify fatty acid composition are needed to improve uses in food,
industrial and other products (Wilson, 2004; Fernndez-Martnez et al., 2007).
Plant breeders have been successful in developing a wide range of novel
vegetable oil types that are regarded healthier and that have the necessary
properties for various applications in the food and non-food industry. Selection
for breeding and genetic engineering resulting in elevated oleic acid levels
were reported in many oilseed crops including sunflower (Soldatov, 1976);
safflower (Knowles and Hill, 1964), soybean (Alt et al., 2005), peanut (Jung et
al., 2000), canola (Stoutjesdijk et al., 2000), cotton (Liu et al., 2002) and maize
(Bel et al., 2008). Oils with a high proportion of oleic acid are important
because of their health benefits and increased oxidative stability. A diet in
which fat composition is high in oleic acid is often associated with reduced
cholesterol, arteriosclerosis and heart disease (Wardlaw and Snook, 1990). In
addition, high oleic acid content increases oxidative stability and extends the
utility of the oil at high cooking temperatures (OKeefe et al., 1993; Van der
Merwe, 2010).
To further improve on oil quality a better understanding of the effects of
environment on fatty acid composition is needed. When environmental factors
deviate from the optimal quantity or intensity for the crop plant, stress is
caused. Environmental factors, that may cause any type of stress especially
during the seed maturation stage, influence the proportions of fatty acids in the
oil. Instability of oleic and linoleic acids when plants are grown under
different environments (Van der Merwe et al., 2013) is a major concern. High
temperature stress results in crop yield losses (Wheeler et al., 2000) because of
damage to reproductive organs, acceleration in the rate of plant development
(Gan et al., 2004) and shortened growth period of the reproductive organs
(Angadi et al., 2000). High temperatures during reproductive development
often negatively impact pollen viability and fertilization (Hall, 2004), floral
bud development, grain-filling and seed composition (Rondanini et al., 2003).
When plant organs are exposed to direct sunlight, the temperature of these
organs can exceed air temperature (Lewis and Nobel, 1977). In sunflower,
grain temperatures could be up to 5C warmer than air temperature during the
daylight period on sunny days (Ploschuk and Hall, 1995). Atmospheric
temperatures are expected to increase in the future due to potential climatic
changes (Easterling et al., 1997). This might increase the frequency of
episodes of high temperatures and of temperature stress for crops grown in
warmer climates (Wheeler et al., 2000). Knowing the effect of temperature on
oil fatty acid composition in traditional and modified varieties is useful for
selecting the environment in which to produce oil seeds with the desired oil
composition (Izquierdo et al., 2013). Therefore, producing specific oil, such as
high oleic, requires detailed knowledge of the effects of environmental
conditions, crop management and their interaction with genotype on this trait.
It is well known that seed oil quality closely depends on environmental
conditions and that temperature, especially during flowering to physiological
maturity, is the main environmental factor driving oil chemical composition.
In this chapter, the effects of different temperature regimes, grain-filling stages
and their relationships on oleic acid content in oil crops will be reviewed. The
effect of temperature on the ODS enzyme involved in oleic acid biosynthesis
will be discussed as well as the response of genotypes with genetically altered
fatty acids.

1. ENVIRONMENTAL FACTORS INFLUENCING
OIL QUALITY

The fatty acid composition of lipids is largely species-dependent; however
within a species considerable variation may occur in the proportions of the
individual acids. Some of this variation is due to genotype, but within one
variety the ratio may vary widely in response to environmental conditions.
Genotype by environmental interactions which influence the oil content and
fatty acid profile of oil crops have been addressed in many studies. Apart from
genetic factors, diverse environmental and agronomical factors including
planting location, climate, temperature, rainfall or water regime, sowing date,
intercepted solar radiation, nitrogen availability, soil salinity and crop health
influence seed oil content and composition (Salunkhe et al., 1992; Seiler and
Brothers, 1999; Flagella et al., 2002; Izquierdo et al., 2002; Qadir et al., 2006;
Echarte et al., 2010).The fatty acid composition of sunflower seed varies with
planting location and climatic conditions during the growing season (Kinman
and Earle, 1964; Cummins et al., 1967; Chunfang et al., 1996; Qadir et al.,
2006). In general there exists a correlation between latitude and fatty acid
composition. Sunflower seed grown in the northern hemisphere had higher
linoleic acid content than seed grown in the southern hemisphere. However,
this correlation was only because of the influence of latitude on the climatic
conditions (Lajara et al., 1990). Temperature is mostly the direct consequence
of location and therefore microclimatic conditions may determine acute
differences within the same latitude (Chunfang et al., 1996). According to
Flagella et al. (2002), different planting dates and water regimes cause
different environmental conditions during seed-filling and oil synthesis of
sunflower seed and therefore a possible alteration in oil content and fatty acid
composition of the seed. Different planting dates may cause flowering and
seed development to occur during periods of widely different temperatures,
solar radiation, day length and soil water availability which may contribute to
the variation observed in fatty acid composition. Water stress occurring during
the grain-filling period caused an increase in the oleic/linoleic ratio (Flagella et
al., 2000) with respect to more favorable water regimes, while irrigation
resulted in the reduction of this ratio (Flagella et al., 2002). These authors
claimed that upon irrigation, the plant tissue temperature was lowered and this
might have caused a higher activity of ODS and therefore a lower
oleic/linoleic ratio.
On the other hand, Baldini et al. (2002) hypothesized that water stress
caused accelerated and earlier embryo development and lipid accumulation.
This therefore caused a shorter duration of all enzymatic activities, including
those of ODS and this could reflect on the final fatty acid composition. Both
oleic and linoleic acid concentrations of the oil of cultivated sunflower were
significantly related to total solar radiation and day length (Seiler, 1983). A
positive association between intercepted solar radiation and oleic acid
percentage has been reported for sunflower (Seiler, 1986; Echarte et al., 2010)
and soybean oil (Kane et al., 1997).
Various studies showed that temperature affects both oil content and the
fatty acid profile in oil crops during the period of seed development and
maturation (Seiler, 1983; Seiler and Brothers, 1999; Rondanini et al., 2003,
2006; Qadir et al., 2006). Although the effect of temperature on oil quantity is
variable among different reports, it is generally accepted that increased
temperature during seed development resulted in a reduction in total oil
content (Harris et al., 1978; Rondanini et al., 2003). The cause of the reduction
in oil content could be attributed to various factors including a shortening of
the grain-filling period at high temperatures (Connor and Hall, 1997) and
sowing date. High temperatures during the seed maturation stage have been
found to reduce the rate and duration of oil deposition in the grain (Rondanini
et al., 2006). Unger and Thompson (1982) reported that oil content of seed
maturing late in the season (at lower temperatures) was lower compared to
seed from sunflower planted earlier that matured during warmer weather. The
relative proportions of the major unsaturated fatty acids (oleic and linoleic
acid) are strongly influenced by the environmental temperature during
sunflower seed development (Harris et al., 1978). There is an inverse
relationship between temperature and the degree of unsaturation of the oil
(Qadir et al., 2006). High temperatures during seed development (especially
night temperature) have been found to cause a decrease in the amount of
linoleic acid and a corresponding increase in the amount of oleic acid in the oil
(Izquierdo et al., 2002). Seed maturation during periods of low temperature
gave opposite results.
An increase in the oleic/linoleic acid ratio with increasing temperature
during grain-filling has been widely reported (Lajara et al., 1990; Izquierdo et
al., 2002, 2006; Rondanini et al., 2006).The mechanism involved appeared to
be the direct effect of temperature on the activity of the ODS enzyme that is
responsible for the conversion of oleate to linoleate (Canvin, 1965; Harris et
al., 1978; Silver et al., 1984; Garcs and Mancha, 1991). In addition, there is
genetic variability in the response of oleic acid to temperature (Izquierdo and
Aguirrezbal, 2008).
Several studies have made an effort to clarify the main temperature
regimes or thresholds at which major alteration in oleic acid percentage take
place as well as which temperature (daily mean/minimum, night/night
minimum) better explained the variations in fatty acid composition. The
changes in sensitivity to temperature with the stage of grain development as
well as the genetic variability of the response of oleic acid content to
temperature have been addressed. These matters are discussed in more detail
in the next section of this chapter.

2. MAIN EFFECTS OF TEMPERATURE ON FATTY ACID
COMPOSITION WITH THE EMPHASIS ON OLEIC ACID

Some recent efforts that were made to explain the effect of temperature on
fatty acid composition, especially oleic acid percentage, are summarized in
Table 1. The general conjecture is that the higher the temperature, the higher
the oleic acid percentage and the lower the linoleic acid percentage (Silver et
al., 1984; Kabbaj et al., 1996).
However, under field conditions variations in daily minimum (Harris et
al., 1978; Unger 1986), daily maximum (Seiler, 1983) and daily mean
temperatures (Nagao and Yamazaki, 1983) have been reported to be
responsible for the variation in oil fatty acid composition. In a controlled
environment study, oleic acid percentage was decreased by low night
temperatures (Rochester and Silver, 1983).

Table 1. Effects of high temperature on oleic acid content in different oil crops

Crop Heat treatment Growth stage Major effects References
Soybean
(Glycine max L.)
Cultivar Hodgson 78
Day/night air temperatures:
Season 1
29/20C,
35/20C
Season 2
27/20C
33/20C
Grain-filling High air temperature during grain-filling
proportionately reduced the percentage of
polyunsaturated fatty acids and increased
that of oleic acid;
Oleic acid content increased by 10 and 12
points respectively for the two seasons
Dornbos and
Mullen
(1992)
Rape and mustard
(Brassica napus and B. hirta)
Rape varieties
Norin
Westar
Mustard variety
Bet Dagan
Controlled day/night temperatures:
Low, 17/12C
Medium, 22/17C
High, 27/22C

Pod development and seed
maturation;
10-65 DAA
Temperature affected the biosynthesis and
accumulation of fatty acids in seeds;
With an increase in temperature, oleic acid
content increased significantly with 5 points
in both Norin (from 8.2-13.6%) and Bet
Dagan (from 13.3-18.2%);
Oleic acid content in Westar increased, but
was not significant (65-67%)

Yaniv et al.
(1995)
Sunflower (Helianthus annuus)
Traditional hybrid:
Santiago
HO hybrid:
Olbaril
HO hybrid:
Trisun 870
HT: 30C/26C (day/night: 22 C
degree day)
MT: 24C/20C (day/night: 16 C
degree day)
LT:
18C/14C (day/night: 10C
degree day)
Flowering (F1 stage) to
flowering plus 300 C
degree day
Santiago showed sensitivity to temperature;
Oleic acid content increased with
temperature:
15 points from 33.6% (LT) to 48.7% (MT),
8 points from LT to HT (41.95%);
HO hybrids were insensitive to temperature
changes; Hybrids with oleic acid potentials
above 90% seemed to be less affected by
temperature
Lagravere et
al. (2000)

Table 1. (Continued)

HO lines:
OA (unstable oleic acid
content: 60-85%)
OC (stable oleic acid
content: 80-85%)
Hybrids (HO lines crossed
with traditional lines)
OA x RHA801
OC x RHA 801
OA x PPR3
OC x PPR3
Controlled temperature conditions

Cold treatment: 20C/14C
day/night
Heat treatment: 26C/20C
day/night

Flowering to maturity Oleic acid content of OA, but not OC, was
significantly influenced by temperature;
For OA, oleic acid content varied 27 points
from 45% (Cold) to 72% (Heat);
For OC, oleic acid remained constant (81%
Cold to 81% Heat);
All hybrids, except for OA x PPR3, showed
intermediate oleic acid contents (51-67%),
independent of temperature;
Hybrid OA x PPR3 showed oleic acid
contents close to its oleic parent OA (Heat
72%, Cold 41%)
Tribo-
Blondel et al.
(2000)
Traditional hybrid
Dekasol 3881
Mid-oleic hybrid
Milenio
HO hybrids
Trisol 600
Aromo 10

FC: Daily minimum temperature
range: 13-18C
FC: Night minimum temperature
range: 14-21C (Dekasol 3881
only)

GC: Controlled day/night
temperatures:
26/16C
22/20C
16/26C
Grain filling
H1: 0-200C DAF
H2: 200-400C DAF
H3: 400-600C DAF

Flowering to physiological
maturity

Oleic acid content was higher with higher
night temperatures and this was not related to
the daily minimum temperature;
The traditional hybrid showed the largest
variation while the HO hybrids the lowest
variation in oleic acid;
The effect of temperature is most important
during the early grain filling stage;
In Dekasol 3881, oleic acid content increased
with 21 points (from 38.3-59.2%) with an
increase in night temperature;
In the MO hybrid oleic acid content
remained unchanged at >60% among
different night temperatures;
The HO hybrid Trisol 600 showed changes
in oleic acid content among different night
Izquierdo et
al. (2002)

temperatures
Traditional line:
CAS-6
High stearic acid mutant:
CAS-14


GC experiments
Day/night temperatures:20/10C
25/15C
30/20C
35/25C
Entire cultivation cycle In CAS 6, oleic acid content increased with
34 points (from 22.7-59.6%) with an
increase in the temperature;

In the high stearic acid mutant, CAS-14,
oleic acid content increased with 10 points
(from 26.2-36.5%) with an increase in
temperature; however, oleic acid content was
reduced to 24.1% in the mutant at 35/25C
temperature range
Fernndez-
Moya et al.
(2002)
Traditional inbred line HA89

Control temperature: 25C
Heat treatment:35C and above
24 hours,7 consecutive days
Grain-filling (Rapid TAG
accumulation): 19-26
DAA
Increased oleic acid percentage of 29%
compared to the control (34% oleic acid);
reduced ODS activity with no recovery in the
post-stress period
Rondanini et
al. (2003)
Traditional hybrids:
Paraso 20
Paraso 30
Whole heads enclosed in
controlled temperature chambers

Alternating day/night
temperatures: with a daily mean
grain temperature of 20-40C
Peak grain temperatures varying
between 26-45C

6 / 7 days

Grain-filling

Early grain-filling stage -
HT1: 10-17 DAA / 12-18
DAA

HT2: 18-25 DAA / 18-24
DAA

HT3: 24-30 DAA / 26-33
DAA
High temperatures reduced both the rate and
duration of oil deposition in the grain in
HT1;
4 days of alternating day/night temperatures
resulting in mean daily grain temperatures
>30C can reduce sunflower grain quality
but is strongly dependent on the timing of
exposure and the effects of temperature on
the active growth process;
The unsaturation ratio, oleic:linoleic, showed
significant increases at the HT3 stage (26-33
DAA) when day/night temperatures were
37/32C and above and at the HT2 stage
(from 18-24 DAA) when day/night
temperatures were 37/32C
Rondanini et
al. (2006)


Traditional hybrid:
Dekasol 3881


Day/night temperature regimes:
GC1:28/20C,
25/23C, 20/28C;
GC2: 26/16C, 22/20C, 16/26C;
GC3: 16/16C, 21/21C, 26/26C;
GC4: 26/16C,
22/20C, 26/26C;
GC5: 20/28C for 10 days at
different periods of grain-filling

Control: 25/23C from flowering
to physiological maturity
Grain filling

FC:
Flowering (0C)-200C
DAF,
200-400C DAF,
400-600C DAF

The response of oleic acid content to
temperature was bilinear;
Oleic acid percentage increased significantly
with 13 points when night temperature
increased from 20-23C; However, further
increases in night temperature did not result
in a significant increases in oleic acid
content;
The largest effect of temperature on fatty
acid composition occurred between 100 and
300C DAF and is the most critical period
for fatty acid composition
Izquierdo et
al. (2006)
Traditional hybrids:
ACA884
ACA885
DK4040
VDH 488
MG 2
Mg 50
Paraso 20
HO hybrid:
Trisol 600

Comparative yield trials (CYT)
Night temperature range: 12.5-
21.5C

FC
Night temperature range:
Balcarce: 13.1-14.2C
Croba: 15.5-19.0C
P.R.S. Pea: 19.0-20.7C

GC experiments
Day/night temperatures:
13/12C
22/17C
23/17C
Grain filling:
100-300C DAF (degree
days after flowering)

Changes in oleic acid content in both
traditional and HO hybrids were accounted
by minimum night temperature during the
period 100-300C day, independent of the
experiment

Izquierdo and
Aguirrezbal
(2008)

24/19C
26/22C
(Night temperature range: 18.5-
25.9C; VDH 488 and Trisol 600:
minimum temperature: 11.3C)

GC treatment: 21-33 days (>210
C day: base temperature: 6C)

HO hybrids
MO hybrids

Whole plants

GC:
Alternating day/night
temperatures:
Control 25/18C
Heat treatment 36/24C
10 consecutive days

Grain-filling:

15-25 DAA
Mid- and high oleic genotypes were
differently affected by the temperature;
HO genotype was insensitive to temperature;
Some mid-oleic hybrids showed significant
increases in oleic acid content;
Increases from 9-24 points were observed
Van der
Merwe
(2010)
HO inbred lines:
342mt
R978
Minimum temperature range: 16C
(sowing date 1) - 17C (sowing
date 2)
Maximum temperature range:
28.7C (sowing date 1) 31.5C
(sowing date 2)
Grain-filling
13-35 DAF
High oleic inbred lines with different genetic
backgrounds respond differently to the same
environmental conditions;
R978 showed less sensitivity to high
temperature with no change in oleic acid
content (91%);
342mt was sensitive to high temperatures
and showed a significant increase in oleic
acid content (from 84% to 87%)

Ferfuia et al.
(2012)


Inbred lines:
High stearic: CAS-3,
ADV-2504, ADV-3512;
High stearic-high oleic
(HSHO):ADV-3807,
ADV-2803, ADV-3816
(Soldatov, 1976 OLD
mutations);
Traditional: HA89B
HO (RHA345)


GC experiments:
Day/night temperatures:
16/16C
26/16C
26/26C
32/26C

Grain-filling

5 DAF-physiological
maturity
Temperature during grain-filling modified
oil oleic acid composition in all inbred lines;
Genotypic differences were observed;
An increase in temperature (16/16C-
26/26C) resulted in significant increase in
oleic acid concentration;
The high stearic lines showed the largest
increase in oleic acid (19 points), followed
by the traditional line (8 points); The HO and
HSHO showed the smallest variation (6
points each);
The largest increase in temperature was
observed between treatments with different
night temperatures
Izquierdo et
al. (2013)
DAA: days after anthesis; DAF: day after flowering; FC: field experiments; GC: growth chamber; HO: high oleic; HT: high
temperature; MT: middle temperature; LT: low temperature; MO: mid-oleic; ODS: oleoyl- phosphatidyl-choline desaturase; TAG:
triacylglycerol.

The question arose whether daily minimum or night temperature better
explained the variations in fatty acid composition. A more recent study by
Izquierdo et al. (2002) addressed this question and they reported that night
temperature, and not daily minimum temperature was the best temperature
predictor for oleic acid content. Increases in minimum night temperature from
11-23C resulted in a strong increment of oleic acid concentration in the
traditional hybrid (Dekasol 3881), but further increases in night temperature
did not increase the concentration of this fatty acid.
Previous reports showed that constant high temperatures and short periods
of extremely high temperatures have different effects on dry matter
accumulation and grain quality (Wardlaw and Wrigley, 1994; Wardlaw et al.,
2002) and this provoked the need to study the direct effects of high
temperatures on grain growth. Rondanini et al. (2003) investigated the direct
effect of brief periods of high temperature on grain growth and oil quality at
different phases of grain-filling in sunflower. When the capitulate of
traditional sunflower plants (HA89) were exposed to continuous extreme
temperatures of 35C for seven consecutive days during the period of rapid
triacylglycerol accumulation, the final fatty acid composition was strongly
modified in the post-stress period. This high temperature stress showed
detrimental effects on oil deposition patterns and oil quality traits. Oleic acid
increased significantly with 29% from 34% for the control (25C) to 63% for
the heat treatment. However, the number of days required for detectable
effects varied for different grain-filling stages.
Rondanini et al. (2006) realized that a more precise definition of the
thermal conditions that evoke reductions in sunflower oil quality could require
the use of alternating day/night temperatures. This temperature regime would
be more representative of that operating under natural conditions. In addition,
the number of days required for heat stress exposure to produce an effect
needed to be explored. In their study, Rondanini et al. (2006) showed the oil
fatty acid composition, as reflected in the oleic/linoleic acid ratio, was altered
after four days exposure to alternating day/night high temperature stress when
the exposure overlapped with the termination of oil deposition. This ratio
showed significant increases when day/night temperatures were 37/32C and
above and when heat stress exposure overlapped with the cessation of storage
lipid deposition. Rondanini et al. (2006) reported that a mean grain
temperature of 35C and above together with a night temperature of 32C and
above were required for strong modifications in the oleic/linoleic ratio.
With the conclusion made that night temperature was the best predictor
for oleic acid content and not daily minimum temperature, Izquierdo et al.
(2006) recognized the need to determine which night temperature variable
(mean, maximum or minimum) was the best oil quality predictor for modeling
purposes. In their attempt to develop a model that would estimate fatty acid
composition as a function of temperature during specific periods within the
grain-filling stage, Izquierdo et al. (2006) discovered that the oleic acid
content of the traditional sunflower hybrid Dekasol 3881 responded linearly to
minimum night temperature up to 22.6C. Increases in minimum night
temperature from 11-23C resulted in a strong increment of oleic acid
concentration in the traditional hybrid, but further increases in night
temperature did not increase the concentration of this fatty acid. Oleic acid
concentration showed a level of saturation at high temperatures and this
response helped to explain why traditional hybrids in high-temperature
environments do not attain oleic acid percentages of above 60%. Their report
concluded that night minimum temperature during a specific grain-filling
stage, 100-300 degree day after flowering (DDAF) could adequately predict
oleic acid content.
In order to understand the effect of the day-night cycle in fatty acid
biosynthesis in developing seeds, Pleite et al. (2008) examined the
liposynthetic metabolism of developing sunflower seeds in vivo during the
day-night period. By using radioactive precursors, these authors were able to
identify variations in the metabolism of fatty acids during the daynight period
in sunflower seeds. Oleic and linoleic acid content was found to oscillate with
the light-dark cycles with oleic acid increasing during the day and decreasing
during the night, at the expense of linoleic acid synthesis. However, under
constant light conditions this alternating behaviour disappeared and oleic acid
percentage increased. It appeared that under low temperature conditions,
during the night, the ODS activity increased and oleic acid was desaturated.
However, during the day, oleic acid increased and this was due to the
inactivation of the ODS enzyme. This confirmed that the ODS enzyme is
regulated by night temperature and that an increase in night temperature could
decrease the activity of ODS and therefore result in an increase in oleic acid
percentage.
A few studies were conducted to compare the response of fatty acid
composition, especially oleic acid content, among different genotypes
including traditional, high oleic (HO) (Lagravere et al., 2000; Rondanini et al.
2006; Izquierdo and Aguirrezbal, 2008; Ferfuia et al., 2012) and mid-oleic
(Tribo-Blondel et al., 2000; Van der Merwe, 2010) sunflower genotypes,
traditional, HO, high stearic (HSHL) (Fernndez-Moya et al., 2002) and high
stearic-high oleic (HSHO) sunflower genotypes (Izquierdo et al., 2013).
The effects of temperature changes on oleic acid content among various
genotypes are discussed in subdivision 6 of this chapter.

3. TEMPERATURE CHANGES AT DIFFERENT PERIODS
IN GRAIN-FILLING HAVE DIFFERENT EFFECTS ON FINAL
FATTY ACID COMPOSITION

The stage of rapid oil accumulation commences with the development of
the embryo about 150 day-degrees after pollination and oil content reaches a
maximum value just prior to physiological maturity of the seed (Harris et al.,
1978). The final oil composition is highly sensitive to the timing of heat stress.
Attempts have been made to predict fatty acid composition using temperatures
at different periods of seed development or grain-filling. Initially a model that
predicts linoleic acid concentration based on temperature from flowering to
physiological maturity was established by Harris et al. (1978). They reported
that in order for the linoleic acid content in sunflower oil to be 64% or more,
mean minimum (night) temperatures of 16C or less are required during the
grain-filling phase of crop development. From field and controlled
environment survey data they indicated that the percentage linoleic acid
decreased with an increase in mean minimum temperature from flowering to
maturity, while the percentage of oleic acid increased at an equivalent rate.
They suggested that the close negative relationship between temperature and
linoleic acid percentage was due to the adaptation of the desaturase enzymes to
function best in cool temperature conditions. Nagao and Yamazaki (1984)
reported that linoleic acid concentration was best estimated by temperature
from 12 days after flowering (DAF) to physiological maturity (34 DAF). The
period 24-34 DAF was most sensitive to temperature since oleic and linoleic
acid contents were significantly correlated to temperature during this maturity
stage. They suggested that the average temperature 10 days before harvesting
might be a useful index of the fatty acid composition of harvested sunflower
seeds. These results were consistent with the findings of Sobrino et al. (2003).
Their data indicated that changes in oleic acid level was more correlated to the
stage of physiological maturity before harvesting (30 DAF) than to the stage of
achene development (2 DAF).
In more recent studies, the effects of temperature on oil quality at different
stages in the grain-filling period were investigated. Izquierdo et al. (2002)
studied the effect of night temperature during three different periods of grain-
filling on fatty acid composition of sunflower plants. These included (1) the
period when the amount of oil accumulated in the achene is low - flowering to
200 DDAF (0-200 DDAF), (2) the first half of the rapid oil accumulation
phase (200-400 DDAF) and (3) the last half of the rapid oil accumulation
phase (400-600 DDAF). They reported that the period from flowering to 400
DDAF showed a stronger effect of temperature on oleic acid content than
during the later grain-filling period. According to Izquierdo et al. (2002) it
appeared that a moderate increase of the night temperature only during the
early stage of grain-filling affected the final fatty acid composition of
sunflower oil, by increasing the oleic acid percentage at physiological
maturity. However, during this early stage of grain growth the amount of oil
accumulated in the achene is low. Therefore they suggested a memory effect
of an early temperature treatment on the fatty acid desaturation mechanism.
Rondanini et al. (2003) imposed high temperatures during three different
grain-filling periods; 12-19, 19-27 and 27-34 days after anthesis (DAA) to
identify the stage that is most sensitive to temperature stress. According to
these authors the grain-filling period from 19-26 DAA was the most sensitive
to temperature since exposure to heat stress (35C) during this period
strongly modified the fatty composition of sunflower seed oil. The period 19-
26 DAA corresponded to the main rapid oil accumulation phase. When plants
were exposed to heat stress temperatures at an earlier grain-filling period (12-
19 DAA) the fatty acid composition profile showed post-stress recovery at
maturity, with values similar to the control. These findings were consistent
with the observations of Fernndez-Moya (2001) who found that the final fatty
acid composition was determined by temperature during the main oil
accumulation stage (15 DAA), but was opposed to the findings of Izquierdo et
al. (2002).
In contrast to reports indicating that the early grain-filling stage was more
sensitive to temperature, Rondanini et al. (2006) reported that a later stage in
the grain-filling period was more sensitive. They exposed sunflower
capitulums to high alternating day/night temperatures in three main separate
phases during grain-filling: (1) early - 10(12) to 17(18) DAA, (2) middle -18
to (24)25 and (3) late - 24(26) to 30(33) DAA. It was observed that the
oleic/linoleic acid ratio showed significant increases at two grain-filling stages,
middle grain-filling (18-24 DAA) and late grain-filling (26-33 DAA), but only
when day/night temperatures were 37/32C or above. It was determined that
the oil fatty acid composition, as reflected in the oleic acid/linoleic acid ratio,
was significantly altered after exposure to alternating day/night high
temperature stress when the exposure to heat stress (37/32C) overlapped with
the termination of oil deposition.
The apparently conflicting findings of Rondanini et al. (2003, 2006) and
Izquierdo et al. (2002) regarding the most sensitive period of grain-filling,
(early or during the main oil accumulation phase) needed further research.
However, Rondanini et al. (2006) suggested that several aspects of oil quality
responses in the various experiments may derive from the cultivars that were
used. Sunflower genotypes show differences in the growth patterns of their
grain components as well as oil accumulation (Mantese et al., 2006). In some
hybrids, oil deposition ceases 3-5 days before physiological maturity, while in
others it occurs closer to physiological maturity. This difference could impact
oil quality as the oleic/linoleic acid ratio may respond to different grain-filling
stages. Therefore, the critical period of termination of oil deposition is
determined by either the genotype of the hybrid used in the study or by the
effect of temperature stress on this stage (Rondanini et al., 2006). When the oil
deposition period is shortened by stress (at 35C), the same response in
altering the oleic/linoleic ratio would be obtained at an earlier grain-filling
stage such as from 18 DAA compared to from 24 DAA (a week earlier).
Further understanding of the response of oleic acid content to temperature
during grain-filling has been obtained by Izquierdo et al. (2006). They
identified the period 100-300 DDAF as the most critical for fatty acid
determination in the traditional sunflower hybrid Dekasol 3881. The minimum
night temperature during this period was identified the best predictor of oleic
acid content in Dekasol 3881.These authors suggested that the period 100-300
DDAF was the most critical for fatty acid composition and by knowing the
temperature during this period, it is feasible to predict fatty acid composition
before crop harvest.

4. RELATIONSHIP TYPE BETWEEN OLEIC ACID
CONCENTRATION AND TEMPERATURE

In order to predict the type of oil that will be produced in a particular area,
knowledge of the effects of temperature on the oleic acid content is necessary.
According to Izquierdo et al. (2006), studies on the type of response of oleic
acid content to temperature have been vague and are limited. Linear
relationships between oleic acid content and temperature were established for
ranges of daily mean temperature between 15 to 27C (Harris et al., 1978;
Goyne et al., 1979; Silver et al., 1984). A report by Trmolires et al. (1982)
indicated a curvilinear relationship between oleic acid content and mean
temperature, with a maximum value at roughly 27C. Results for these reports
showed that there is an optimum temperature for maximum oleic acid content.
In order to better explain and predict the percentage of oleic acid in
sunflower oil, Sobrino et al. (2003) developed models in terms of geographic
or climatic conditions. The oleic acid content was modeled according to
longitude, latitude, altitude and temperature recorded during achene
development and at physiological maturity. Linear regressions were fitted
using the different variables. Through stepwise regression, it was established
that the best results were obtained using the temperature model and the
variables mean minimum development temperature, and mean minimum and
maximum maturation temperatures. Of the three variables, the mean minimum
maturation temperature provided the closest estimate of the percentage oleic
acid. This model was statistically validated and can be used to estimate oleic
acid content based on local temperatures.
Izquierdo et al. (2006) recognized the need to develop a more precise
model to estimate fatty acid composition as a function of temperature during a
specific stage of grain-filling. They identified the period 100-300 DDAF as the
most critical for fatty acid determination in the traditional hybrid Dekasol
3881. The minimum night temperature during this period was identified the
best predictor of oleic acid content in Dekasol 3881. The work done by
Izquierdo et al. (2002) with traditional sunflower hybrids indicated a bilinear
response between oleic acid content and night temperature. Increases in
minimum night temperature from 11-23C resulted in a strong increase of
oleic acid content in the traditional sunflower hybrid (Dekasol 3881), but then
further increases in night temperature did not increase the concentration of this
fatty acid. Oleic acid concentration has saturated at high temperatures and this
response helped to explain why traditional hybrids in high-temperature
environments do not attain oleic acid percentages of above 60%. This
deduction confirmed the existence of an optimum temperature for oleic acid
content. Izquierdo et al. (2006) used the model established for Dekasol 3881 to
predict the fatty acid composition of other traditional hybrids, but it showed
reduced capacity for prediction. This suggested that traditional sunflower
hybrids show genetic variability in the response of fatty acid composition to
temperature. This finding corresponded to the results of Roche et al., (2006)
who also proposed variability in the response of fatty acid composition to
temperature among traditional sunflower hybrids.
In a subsequent study by Izquierdo and Aguirrezbal, (2008) the
concentration of oleic acid in sunflower oil demonstrated a sigmoidal response
to minimum night temperature from 100 to 300C DAF among both traditional
and HO sunflower hybrids. This relationship showed a zone of high response
to temperature and two zones of low response. The two zones of low response
represented the minimum and maximum concentrations of oleic acid at low
and high temperatures respectively. These authors suggested that the response
of oleic acid content to temperature is not linear and the increase in oleic acid
content took place within a range of temperatures. Above and below this
temperature range, the oleic acid content remains relatively constant, which
suggested that the activity of ODS stabilizes at low and high temperatures.
Sobrino et al. (2003) also suggested the existence of a temperature range over
which the efficiency of oleic acid formation is optimum. Oleic acid production
is diminished below and above temperatures that could be classified as critical.
A report by Pereyra-Injuro and Aguirrezbal (2007) was the first to
establish and validate a simple model which could be used to estimate both
yield and oil quality aspects of sunflower under non-limiting conditions. This
model used published empirical relationships to estimate yield and its
components (grain weight and number), grain oil percentage and fatty acid
percentages based on estimates of intercepted radiation, leaf are index and
phenology. Their model was adequate for analyzing yield-oil quality
interactions and its variability among different locations, years, sowing dates
and to evaluate the impact of climate change (Carbone et al., 2003). Results of
Pereyra-Injuro and Aguirrezbal (2007) suggested that with production at low
latitudes, sunflower oil with high quality and oxidative stability could
compensate for relatively low yield, while at higher latitudes, high-linoleic
acid oil production should be compatible with high yield potentials. Their
model could facilitate the selection of the best location, sowing date and
sowing density for the production of a specific sunflower oil with specific
characteristics.

5. EFFECTS OF TEMPERATURE
ON OLEOYL-PHOSHATIDYL-CHOLINE
DESATURASE (ODS) AND ITS ACTIVITY

The ODS enzyme is highly regulated by temperature in sunflower seed
(Garcs et al., 1992) and according to Garca-Daz et al. (2002) different
mechanisms might be involved in the control of the microsomal ODS activity.
This enzyme shows rapid and partial inhibition at high temperatures, but this
inhibition is reversible. Sarmiento et al. (1998) reported that ODS activity was
stimulated by low temperatures and repressed by high temperatures and that
recovery of its activity could be restored once the temperature is reduced
again. The activity of ODS was strongly reduced by heat stress temperatures
of 35C and above in traditional sunflower (Rondanini et al., 2003). However,
the timing of the heat stress period during grain-filling was also important.
Although ODS activity was inactivated by extreme temperatures, its activity
did recover in the post-stress period, but only when the heat stress occurred
during the early grain-filling stage (12-19 DAA). It was reported that the
activity of the ODS enzyme was greatest at early grain-filling (Garcs and
Mancha, 1991; Garcs et al., 1992; Kabbaj et al., 1996). When heat stress
occurred near the end of oil synthesis (19-26 DAA), the activity of ODS did
not recover in the post-stress period and this caused permanent changes in oil
composition at maturity. Rondanini et al. (2006) also observed that the
oleic/linoleic acid ratio was permanently altered only when exposure to heat
stress overlapped with the cessation of deposition of storage lipids.
Izquierdo et al. (2002) claimed that that night temperature was associated
with variation in oil fatty acid composition and not daily mean, maximum or
minimum temperature. They reported that an increase in night temperature
resulted in an increase in oleic acid percentage. This finding was further
supported by the results of Pleite et al. (2008). These authors showed that
under normal growing conditions during the middle of the night, oleic acid
percentage decreased. This decrease was attributed to an increase in the rate of
oleic acid desaturation or an increase in ODS activity during the dark period.
Therefore, during the night, when the temperature is lower than during the
day, the ODS enzyme is active and oleic acid is desaturated at a high rate. This
indicated the influence of night temperature on the desaturation activity in
sunflower seeds. The data presented by Pleite et al. (2008) confirmed the
nocturnal desaturation of oleic acid and the influence that the night
temperature during this period exerts on the activity. Knowing that ODS is
temperature regulated, an increase in night temperature would result in
inhibition of the ODS enzyme and therefore an increase in oleic acid
percentage as observed by Izquierdo et al. (2002).
In addition, this could also help explain why Rondanini et al. (2006) only
observed significant increases in oleic acid content when mean daily grain
temperatures were above 30C. From their data, significant increases in the
oleic/linoleic acid ratio were observed at day/night temperatures of 37/32C,
39/33C, 42/32C and 45/38C. At each of these regimes the night temperature
was extremely high (above 30C) which could have resulted in reduced
activity of the ODS enzyme. Sarmiento et al. (1998) demonstrated that a high
temperature of 30C repressed the activity of ODS.
It was shown that oleic acid desaturation is modulated by the activation of
the enzyme synthesis at low temperatures (Garcs et al., 1992) and/or by the
partial inactivation of the existing enzyme at high temperatures (Sarmiento et
al., 1998). However, Izquierdo and Aguirrezbal (2008) indicated that the total
activity of the enzyme stabilizes at low and high temperatures. These authors
suggested that the response of oleic acid content to temperature is not linear
and the increase in oleic acid content took place within a range of
temperatures. Above and below this temperature range, the oleic acid content
remains relatively constant which suggested that the activity of ODS stabilizes
at low and high temperatures.
Not only ODS but also the stearoyl-acyl carrier protein desaturase (SAD)
enzyme, which is responsible for the desaturation of stearate (18:0) to oleate
(18:1) could be regulated by temperature. Fernndez-Moya et al. (2002)
observed an increase in oleic acid content with an increase in temperature in
both the traditional and high stearic acid mutant sunflower lines. However, a
sudden reduction in oleic acid content was obtained in the high stearic acid
mutant, with a further increase in temperature to 35/25C. Fernndez-Moya et
al. (2002) suggested that this response was probably caused by a reduced SAD
activity at the higher temperature, therefore reducing the amount of oleic acid
produced in the fatty acid biosynthesis pathway. A report by Cheesbrough
(1990) indicated that SAD activity in soybean seed was low at a high
temperature (35C), while its activity was higher at a lower temperature
(20C). From their data it appeared that SAD responded to changes in growth
temperature by altering the level of active enzyme present in the tissue.

6. GENOTYPIC DIFFERENCES PLAY A ROLE

Genotypes with high environmental stability of their fatty acid
composition are preferred so that they can be sown in a wide range
environmental conditions while maintaining oil quality. Although most of the
registered sunflower hybrids are traditional, HO hybrids are available and
planted for specific end use markets. Several researchers reported that oleic
acid content showed a great stability in different environments in HO
sunflower genotypes, even if genetic differences were present (Fick, 1984;
Salera and Baldini, 1998). Salunkhe et al. (1992) reported that in HO
sunflower seed, fatty acid composition was not affected by climatic conditions.
Additionally, in HO mutants the oleic and linoleic acid contents were less
influenced by temperature than standard genotypes (Flagella et al., 2000).
Lagravere et al. (2000) found that the HO hybrids they studied were
insensitive to temperature conditions. In contrast, Champolivier and Merrien
(1996) suggested that temperature had an effect on oleic acid content in HO
sunflower hybrids.
Tatini (1995) showed that an increase in temperature from 10-20C during
seed-filling produced an increment from 45-80% of oleic acid content in a HO
sunflower genotype. Some HO hybrids showed sensitivity to temperature,
however, their response to temperature was lower than that of traditional
hybrids (Izquierdo et al., 2002). Hybrids such as the HO hybrids that contained
a higher concentration of oleic acid at lower temperatures were those which
showed less variation in oleic acid content (Izquierdo and Aguirrezbal, 2008).
Consequently differences between these reports could be related to differences
in the genetic backgrounds of the genotypes.
Oleic hybrids can be characterized as high or low oleic acid potential
hybrids and the largest part of total variation in oleic acid percentage could be
due to differences in potential acid percentages of the genotypes (Izquierdo et
al., 2002). Lagravere et al. (2000) suggested that hybrids with low oleic acid
potentials could be more sensitive to environmental conditions such as
temperature, while hybrids with a higher oleic acid content genetic potential
were insensitive to temperature conditions. Van der Merwe (2010) also
observed that oleic sunflower hybrids containing lower oleic acid potentials
(25-50% oleic acid) were more sensitive to temperature during grain-filling
and showed significant increases in oleic acid percentage in response to an
increase in temperature. The oleic acid percentage of the HO hybrid with
higher oleic acid content (75%) did not change with the increase in
temperature. Yaniv et al. (1995) also observed that when comparing the
responses of the rape variety Westar (containing a high oleic acid content of
65%) with the rape variety Norin (with a low oleic acid content of 11%)
under three different temperature regimes, the variety with the higher oleic
acid content (Westar) showed stability for oleic acid content with changes in
temperature. Therefore, genotypes with higher oleic acid potentials show
better stability to temperature (Lagravere et al., 2000; Izquierdo et al., 2002).
Later Izquierdo et al. (2013) gave an explanation on the variation in oleic
acid percentage among different genotypes. The quantitative magnitude of the
variation in oleic acid content among different genotypes is associated with the
presence or absence of the HO mutation. The HO mutation increases the
stability of oil fatty acid composition. Therefore, the oleic/linoleic ratio was in
general more susceptible to temperature in the traditional genotypes than in the
HO genotype with the HO mutation (Tribo-Blondel et al., 2000; Izquierdo et
al., 2002). In traditional genotypes, the variations in the oleic/linoleic ratio are
explained by the direct effects of temperature on the ODS enzyme (Garcs and
Mancha, 1991; Garcs et al., 1992). An increase in temperature reduces the
total activity of this enzyme and the oleic acid is restored in the TAG instead
of being desaturated to linoleic acid. In genotypes containing the HO mutation,
the activity of the ODS enzyme is already reduced by the mutation. However,
these genotypes are also affected by temperature, but to a lesser extent. This
might be attributed to the lower amount of ODS enzyme in the oil seeds
(Garcs et al., 1992) or reduced activity of this enzyme during the stage of
active lipid biosynthesis.

CONCLUSION

Oil crops grown in temperate climates have to face wide fluctuations of
seasonal and diurnal temperatures. High temperature stress has become a
major concern for crop production worldwide because it largely affects the
growth, development and productivity of plants. Plants respond to changes in
environmental temperature by altering their fatty acid composition in the seed
oil. The effects of episodes of heat stress on fatty acid composition can be
particularly intense, especially during the production of specialty oil types
where a specific fatty acid composition is desired. The impact of temperature
on oleic acid percentage (and oil quality) cannot be solely predicted from the
absolute temperature, but it is reflected by the combination of the magnitude
and duration of the high temperature period and coincidence with the grain
developmental stage. Oil quality may be capable of post-stress recovery
provided sufficient time elapses between the end of exposure of the high
temperature period and the end of oil deposition. Genotypes differ in the
sensitivity of their oil properties to the environment, especially temperature.
Genotypes with high environmental stability of their oil fatty acid composition
are generally preferred since they can be grown in a wide range of
environmental conditions while maintaining oil quality. The sensitivity of
genotypes to temperature has been exploited both from a theoretical
perspective, such as enzyme inactivation by temperature changes under natural
conditions, as well as from an applied standpoint, since it is possible to
produce oil of different characteristics at different environments (latitudes or
sowing dates). Simple empirical models, which use available variables of
previously established effects and that can predict oil quality, have been
established. These models can aid in crop management in selecting the best
location or sowing date in order to obtain a specific oil quality with a high
yield.

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INDEX

A
Abraham, 31
acetic acid, 92
acetone, 71, 80
acid esterification, x, 84, 85, 86, 88, 89, 90,
91, 94, 95, 96
acidic, ix, 55
acidity, 87, 90
acidolysis, ix, 55, 66, 73, 79
activation energy, 90
active oxygen, 63
adaptation, 113, 124, 127
additives, 14, 16, 63, 70
adipose, 23
adipose tissue, 23
adjustment, 22, 59
adults, 29, 41, 51, 54
aeration rate, vii, 1, 2, 6
age, 2
air temperature, 101, 105
alcohols, ix, x, 56, 61, 79, 84, 85, 94, 95, 96,
97
alkyl esters, x, 76, 83, 84, 85, 86, 97
alters, 13, 20
anger, 46
anticancer activity, 30
anticancer drug, 30
anti-inflammatory drugs, 79
antioxidant, 28, 57, 80
antioxidative activity, 71
antitumor, 13, 28, 30
apoptosis, 11
appetite, 43
aqueous dispersions, viii, 10, 12, 18, 19, 21,
31, 33
arteriosclerosis, 101
ascorbic acid, ix, 56, 71, 74
Asia, 41
assessment, 37, 46, 80
atherosclerosis, 24, 53
attitudes, 11
awareness, 10
B
base, 57, 108
Beijing, 123, 125
beneficial effect, 23, 46
benefits, vii, 9, 12, 42, 65, 66, 67, 72, 101
benign, xi, 84, 97
bioavailability, 75
biocatalysts, 71, 75
biocompatibility, 16
bioconversion, 72
biodegradable materials, 16
biodegradation, 79
biodiesel, ix, x, 55, 57, 58, 59, 60, 61, 73,
74, 75, 76, 77, 78, 79, 81, 82, 83, 84, 85,
86, 88, 90, 92
Index 130
biofuel, 59
biological activities, ix, 29, 56
biological consequences, 12
biological processes, viii, 9, 12
biomarkers, 40, 53
biomass, 65, 76
biomaterials, 78
biosynthesis, 102, 105, 112, 119, 121, 126
biotechnological applications, 76
blends, 66, 80
blood, vii, ix, 11, 12, 25, 27, 35, 40, 41, 42,
44, 47, 48, 50, 53, 66
blood pressure, vii, ix, 11, 12, 27, 35, 42,
48, 53
body weight, 43
bonds, 64, 65
bowel, 43, 49
Brazil, 55, 83
breast cancer, 11, 26, 28
breeding, 101, 127
breeding goal, 101
Butcher, 31, 34
by-products, 64
C
CAD, 36, 39
calcium, 31
cancer, 11, 22, 24, 25, 28, 38, 43, 49, 51
cancer cells, 28
candidates, 13
carbohydrate(s), 2, 36, 39, 41, 45, 48, 51,
57, 68, 69
carbon, 20, 56, 63, 76, 87, 94, 96
carbon atoms, 20
carbon dioxide, 76
carcinoma, 11
cardiovascular disease(s), 24, 25, 38, 41
cardiovascular risk, 11, 39
case study, 26
catalysis, x, 57, 60, 61, 66, 71, 74, 77, 84
catalyst, x, 61, 69, 70, 81, 84, 87, 88, 89, 90,
91, 92, 93, 94, 96
catalytic activity, 60, 88, 90
catalytic properties, 87
catalytic system, 91
cation, 29
C-C, 6, 74
cell culture, 12
cell death, 13
cell differentiation, 30
cell membranes, viii, 9, 12
chemical(s), x, 15, 30, 51, 56, 57, 58, 63,
64, 65, 66, 67, 68, 69, 70, 71, 72, 77, 83,
87, 97, 100, 102, 125
chemical characteristics, 30
chemical industry, 87
children, 51
China, 123
chitosan, 28
cholesterol, vii, ix, 11, 20, 25, 35, 39, 40,
41, 44, 47, 48, 50, 51, 66, 67, 70, 81, 101
cholesterol lowering agents, 81
choline, 100, 110
chromatography, 86
chromatography analysis, 86
chronic diseases, 11, 26, 38, 54
classes, 63, 88
cleavage, 69
climate(s), 101, 102, 117, 121, 122
climate change, 117
climatic factors, 123
clinical application, 54
coal, 87
coatings, 63
coconut oil, 36, 41
colon, 28
colon carcinogenesis, 28
commercial, ix, 36, 48, 57, 58, 63, 69, 78,
84
communication, ix, 35
complications, 49
composition, xi, 6, 14, 15, 29, 32, 46, 50,
58, 61, 66, 73, 99, 100, 101, 102, 103,
104, 108, 110, 111, 112, 113, 114, 115,
116, 118, 119, 121, 122, 123, 124, 125,
127, 128
compounds, x, 2, 39, 56, 57, 63, 66, 68, 70,
71, 72, 76, 84, 88, 96
computed tomography, 22
Index 131
condensation, 31, 60, 78
configuration, 10, 11, 16, 21, 33
confounding variables, 5
conjugation, 23
consensus, vii, ix, 35, 36, 47, 48
constituents, ix, 35, 39, 42, 46, 48
consumers, 10, 37, 46
consumption, vii, viii, 9, 10, 11, 12, 26, 35,
37, 38, 47, 48, 67, 100
controlled trials, 25, 46, 51
controversial, 46
conversion rate, 90
cooking, 60, 77, 90, 101, 125
copolymer, 79
coronary artery disease, 36, 39
coronary heart disease, 11, 24, 25, 29, 39,
50, 53, 56
correlation, 88, 102
cosmetic(s), ix, 2, 55, 57, 58, 63, 68, 69, 70,
71, 72, 84
cost, 58, 61, 63, 64, 86
cotton, 101
CPP, 14, 16
critical period, 108, 115
crop(s), xi, 99, 100, 101, 102, 105, 113, 115,
121, 122, 126, 128
crop production, 121
crude oil, 58
crystalline, viii, 10, 12, 14, 15, 20, 28, 31,
32, 33, 34, 67
crystallization, 81
CT, 22, 23, 34, 73
cultivars, 115, 125
cultivation, 107
culture, 2
cycles, ix, 56, 61, 92, 112
CYT, 108
D
deduction, 116
deficiency, 49
dehydration, 16
Delta, 127
Denmark, 9
deposition, 103, 107, 111, 115, 118, 121
derivatives, x, 71, 76, 80, 83
detectable, 86, 111
detection, 22
detergents, 2, 57
diabetes, 38, 42
diet, vii, ix, 10, 11, 25, 26, 28, 29, 35, 38,
39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,
52, 53, 56, 65, 77, 101
dietary fat, 39, 40, 41, 42, 43, 44, 50, 51
dimethylsulfoxide, 69
diseases, 38
dispersion, 23
disposition, 42
distillation, x, 84
distribution, xi, 23, 64, 99
diversification, 77
diversity, 24
docosahexaenoic acid, 36, 42
donors, 79
dopamine, 68, 71, 80
dose-response relationship, 47
double bonds, 65
drought, xi, 99, 123, 124
drug delivery, viii, 10, 27, 28, 31, 32, 33, 69
drug release, viii, 10
drug resistance, 13
drugs, viii, 10, 12, 13, 16, 30, 74
dry matter, 111
E
East Asia, 40
economics, 75
edema, 46
editors, 75
effluents, x, 60, 84
eicosapentaenoic acid, 36, 42
election, 101
electron, 94
e-mail, 9
emission, x, 22, 58, 83
emulsions, 67, 73
encapsulation, 31
Index 132
energy, vii, viii, 35, 37, 39, 40, 41, 42, 43,
44, 45, 46, 47, 48, 50, 57, 58, 60, 68, 125
energy consumption, 57, 68
energy expenditure, 46, 50, 61
engineering, 73
England, 29
environment(s), ix, x, xi, 14, 29, 56, 63, 81,
84, 85, 87, 88, 100, 101, 104, 112, 113,
116, 119, 121, 122, 124, 127, 128
environmental conditions, 63, 102, 109,
119, 120, 121, 126
environmental factors, xi, 99, 101, 127
environmental impact, x, 83
environmental issues, 58
environmental stress, xi, 100
enzyme(s), ix, 38, 56, 57, 60, 61, 63, 64, 65,
66, 68, 70, 71, 72, 73, 76, 100, 102, 104,
112, 113, 117, 118, 119, 121
EPA, 36, 42, 50, 77
epidemiologic, 11
epithelial cells, 19
equilibrium, viii, 10, 16, 33
equipment, 60, 61, 65
ester, ix, x, 55, 57, 59, 67, 69, 72, 74, 76,
80, 81, 83, 87, 94
esterification, ix, x, 55, 57, 59, 60, 61, 67,
69, 70, 73, 77, 79, 81, 82, 84, 86, 87, 88,
90, 91, 92, 93, 95, 96, 97
ethanol, ix, 56, 61, 80, 81, 90, 91, 93
ethers, x, 83
ethyl alcohol, 85, 86, 88, 89, 90, 91, 92, 93,
94, 95
etiology, 27
EU, 36, 37, 46, 47, 48, 52
Europe, 52
European Commission, 36, 37, 46
European Parliament, 50
European Union, ix, 36, 37, 46
evaporation, 91
evidence, 11, 24, 25, 28, 42, 46, 48, 56
exclusion, 49
experimental condition, 14, 15
experimental design, vii, 1, 3, 65, 74
exposure, 107, 111, 114, 118, 121, 126
extinction, 32
extraction, x, 3, 51, 84, 92
F
fast food, 38
fasting, 11, 40, 41, 45, 47, 48, 50, 54
fasting glucose, 11
fat, vii, viii, 11, 26, 35, 39, 40, 42, 44, 45,
46, 47, 48, 49, 50, 51, 52, 54, 59, 66, 67,
80, 101
fat intake, 26, 39, 42, 52
FDA, 68
feedstock(s), x, 58, 64, 75, 78, 83, 85, 90
fermentation, vii, 1, 2, 3, 6
fertilization, 101
fiber, 67
fibrinogen, 41
fibrinolysis, 41
fibroblasts, 27
films, 28
financial, 97
financial support, 97
fish, 38
flank, 23
flexibility, ix, 12, 56, 66
flooding, xi, 99
flour, 128
fluctuations, 121
fluid, 17, 57
food, vii, viii, ix, 2, 9, 10, 16, 27, 31, 36, 37,
38, 46, 49, 55, 57, 58, 65, 66, 67, 68, 69,
70, 72, 84, 100, 101
food additive(s), 84
Food and Drug Administration, 68
food industry, 16, 101
food products, vii, 9, 10, 31, 57
formation, 2, 12, 14, 15, 20, 21, 34, 43, 59,
69, 70, 86, 87, 117
formula, 43, 50
France, 124, 125, 127
free fatty acid, x, 20, 58, 59, 60, 61, 77, 81,
83
fructose, 52, 79
fruits, 51
Index 133
functional food, vii, viii, 12, 14, 35, 36, 37,
38, 48, 49, 80
G
gel, 62
gene expression, 66
gene silencing, 66, 125
genes, 125
genetic background, 109, 120
genetic engineering, 101
genetic factors, 102
genetics, 125
genome, 122
genotype(s), xi, 76, 100, 102, 109, 112, 115,
119, 120, 121, 127
glioma, 13, 30
glucagon, 52
glucose, 2, 40, 42, 53
glycerin, 66
glycerol, 57, 59, 68, 69, 80, 86, 92
glycol, 24
GRAS, 16, 68
Greece, 11
greenhouse, 58
greenhouse gas(s), 58
growth, 2, 13, 101, 107, 110, 111, 114, 115,
119, 121, 122, 123, 124, 126
growth temperature, 119, 123
H
harvesting, 113
healing, 46
health, vii, viii, xi, 9, 10, 11, 12, 20, 23, 24,
25, 26, 27, 28, 29, 35, 36, 37, 38, 39, 40,
42, 43, 46, 47, 48, 50, 51, 52, 53, 65, 99,
101, 102
health effects, vii, viii, 9, 10, 11, 12, 20, 24,
35, 36, 37, 39, 43, 47, 48
health risks, xi, 99
health status, 52
heart disease, 11, 100, 101
hemisphere, 102
herbicide, 2
heterogeneous catalysis, 84
hexane, 67, 69, 70, 91
high density lipoprotein, 11
homocysteine, 41, 44, 45, 54
homogeneous catalyst, x, 84, 96
human, ix, 10, 12, 28, 35, 37, 39, 43, 44, 46,
48, 49, 51, 53, 100
human body, 37
human health, ix, 35, 37, 46
human subjects, 53
humidity, xi, 99
hybrid, 56, 105, 106, 108, 111, 112, 115,
116, 120, 124
hydraulic fluids, ix, 56, 80
hydrogen, 64, 65, 89
hydrogen peroxide, 64, 65
hydrogenation, 10
hydrolysis, 60, 67
hydroxide, 59, 86
hydroxyl, 62, 63, 68, 96
hypertension, 11
hypotensive, 42
hypothesis, 51
I
ID, 50
images, 18, 22, 23
immobilization, 81
immune function, 51
immune response, 49
immune system, 27
impurities, 69
in vitro, 27, 30, 41, 66
in vivo, viii, 10, 12, 23, 27, 112
incidence, 11, 38, 49
independent variable, 3, 4
individuals, 26
industry(s), ix, x, xi, 10, 55, 58, 59, 63, 66,
68, 69, 70, 72, 80, 83, 97, 99
inflammation, 11, 25, 28, 41, 43, 45, 49
inflammatory bowel disease, 43, 49
inflammatory disease, 43
Index 134
inflammatory markers, vii, ix, 35, 40, 44,
45, 48, 53, 54
inflammatory responses, 56
ingestion, 27, 65, 68
ingredients, vii, 1, 2, 84, 97
inhibition, 71, 118
inoculum, vii, 1, 2, 3, 4, 5
insulin, vii, ix, 11, 27, 35, 40, 42, 44, 48, 50,
52, 53, 54, 66
insulin resistance, 11
insulin sensitivity, vii, ix, 11, 35, 42, 48, 50,
54, 66
integrity, 13, 30
intercellular adhesion molecule, 41
interface, 14, 21
interference, 60
intervention, ix, 35, 39, 43, 44, 46, 48, 50,
77
Iran, 125
irrigation, 103, 124
isolation, 86
isomers, 24, 73
Italy, 11
K
kinetic curves, 88, 90, 94
KOH, 59
L
Langerhans cells, 27
Latin America, 81
LDL, 11, 25, 36, 39, 40, 44, 47, 50, 66, 70
lead, xi, 84
learning, vii, 9
Lewis acids, 85
ligand, 94
light, viii, 10, 112, 127
light conditions, 112
linear model, 4
linoleic acid, xi, 24, 40, 50, 52, 53, 73, 100,
101, 102, 103, 104, 111, 112, 113, 114,
115, 117, 118, 120, 121
linoleic acid ratios, xi, 100
lipases, ix, 56, 57, 58, 60, 61, 63, 64, 66, 67,
68, 70, 71, 72, 77, 78
lipids, vii, ix, 13, 14, 15, 16, 25, 26, 27, 31,
32, 35, 37, 39, 40, 44, 48, 51, 53, 57, 66,
68, 75, 80, 100, 102, 118, 126, 127, 128
lipolysis, 16
lipophilicity, ix, 55, 65, 68, 70
lipoproteins, 39, 40, 44, 51, 53
liposomes, 13, 30, 33, 73
liquid crystals, 32
liquids, 69, 72
longevity, 53
low temperatures, 34, 56, 118, 119
low-density lipoprotein, 25, 36, 67, 70
LTC, 80
lubricants, 57, 63, 75, 80, 84
M
macromolecular chains, 65
magnitude, 120, 121
majority, 87
malt extract, 2, 3
management, xi, 60, 100, 102, 122
manipulation, 66
manufacturing, 2, 10, 67
marketing, 37, 46
mass, 65
materials, 31, 69, 85
matrix, 3, 32
MB, 75
measurements, 92
meat, 38
media, 2, 3, 4, 5, 67, 77
medical, 34, 57
medication, 68
Mediterranean, 25, 26, 28, 29, 38, 46, 49,
51, 52, 53
Mediterranean countries, 38
mellitus, 26
melting, 62, 67, 70
melting temperature, 67
membranes, viii, 10, 15, 20, 29, 30, 42
memory, 63, 114
Index 135
meta-analysis, 24, 26, 39, 51
metabolic syndrome, 11, 26
metabolism, 29, 52, 53, 112
metastasis, 43
methanol, ix, 56, 57, 61, 69, 80, 81, 88
methodology, 70, 71, 76, 80, 81
methyl methacrylate, 79
mice, 23, 28, 31, 43, 46
microbial lipases, x, 56, 67, 71, 72
microemulsion, 13, 17, 19, 20
microorganisms, 57, 66
microRNA, 13, 30
Microsoft, 5
Middle East, 11
migration, 81
Missouri, 76
models, vii, 1, 116, 122
modifications, 65, 111
moisture, xi, 99
molecular dynamics, 29
molecular structure, 12
molecular weight, 2, 20
molecules, viii, ix, 10, 11, 13, 21, 56, 65,
68, 72
monomers, 14, 64
monounsaturated fatty acids, 38, 50
mortality, 38, 53
multi-component systems, 14
multivariate analysis, 50
mutagenesis, 127
mutant, 107, 119, 122, 123
mutation(s), 110, 121
myocardial infarction, 38, 49
N
nanoparticles, 13, 19, 23, 28, 30, 31, 34
nanostructures, v, viii, 9, 10, 13, 15, 19, 20,
26, 31
National Academy of Sciences, 27, 30, 81
natural food, 37
negative effects, 11
negative relation, 113
Netherlands, 75, 127
neurodegenerative diseases, 38
neutral, 20
New England, 24, 25, 26
nitrogen, 3, 102
nonequilibrium, 32
nonionic surfactants, 68
nutraceutical, 70
nutrient(s), 3, 31, 43, 50
nutrition, 10, 25, 26, 29, 37, 47, 50, 51, 54
nylons, 63
O
obesity, 11
ODS, 100, 102, 103, 104, 107, 110, 112,
117, 118, 119, 121
oil market, xi, 99
oil production, 117
oil samples, 90
oilseed, 101
olefins, 64
olive oil, vii, 9, 11, 25, 26, 27, 28, 36, 37,
38, 39, 41, 42, 43, 45, 49, 51, 53, 54, 56,
73, 76, 78
omega-3, 50, 77
operating costs, 61, 65
operations, 80
optimization, 5, 64, 74, 76, 77, 79, 80
organic solvents, 69, 77
organize, 13
organs, 23, 101
oxygen, 2, 4, 124
P
paclitaxel, 28, 31
Pakistan, 126
palm oil, 58, 66
pathways, 23
peanut, xi, 26, 38, 56, 99, 101, 125, 126
peptide, 52
permeability, 11
permeation, 24
permission, 17, 18, 21, 22
peroxide, 65
Index 136
pesticide, 2
PET, 22, 67
petroleum, x, 58, 83
pharmaceutical(s), viii, ix, 10, 12, 14, 19,
22, 55, 57, 58, 68, 69, 70, 71, 72, 75, 84
pharmaceutics, 69
pharmacokinetics, 28, 30
pharmacology, 29, 30
phase boundaries, 21
phase diagram, 16, 21, 33
phosphatidylethanolamine, 29
phospholipids, 15, 20, 27
phosphorus, 52
physical activity, 39, 46, 50
physical fitness, 42
physical properties, 63
physicochemical properties, viii, 10, 11
physiology, 5, 123
phytosterols, 57, 67, 70
placebo, ix, 35, 46
plants, xi, 57, 65, 100, 101, 109, 111, 114,
121
plasma membrane, 12, 23
plasticity, 100
plasticizer, 57, 62
plastics, ix, 56, 63
platelet aggregation, 41
PM, 75, 78
polar, 87, 91
polarity, 69
pollen, 101
pollination, 113
poly(ethylene terephthalate), 67
polyamides, 75
polyamine, 23
polyesters, 64, 75, 79
polyether, 63
polymer(s), 33, 62, 63, 65, 78
polymeric materials, 64
polymorphism, 20, 32
polyphenols, 51
polyunsaturated fat, 36, 50, 105
polyunsaturated fatty acids, 50, 105
polyurethane, 62, 63, 78
polyvinyl chloride, 63
population, 38, 41, 43, 53
population group, 38
positron, 22
potassium, 59, 86
pregnancy, 25
preparation, 71, 78
prevention, 25, 26, 28, 29, 54
prodrugs, 68, 73, 79
production costs, 60
project, 48
proliferation, 11
prostate cancer, 25, 43
protective factors, 39
protein kinase C, 13, 30
proteins, 23, 29
psoriasis, 28, 30
PST, 41, 44
public health, 51, 52
purification, 59, 60, 73
purity, 23, 66
PVC, 63
R
race, 74
radiation, xi, 99, 102, 103, 117, 123
radicals, 68
rainfall, xi, 99, 102
rape, 120, 127, 128
raw materials, 58, 59, 74, 85
reaction medium, 71, 92
reaction rate, 59
reaction time, 60, 64, 65, 67, 70, 89
reactions, ix, x, 55, 57, 58, 59, 60, 61, 63,
64, 66, 72, 76, 78, 81, 83, 84, 85, 86, 87,
88, 89, 90, 91, 92, 93, 94, 96
reactivity, ix, 56, 63
receptors, 29, 81
recommendations, 37
recovery, x, 84, 92, 93, 107, 114, 118, 121
recrystallization, 69
recycling, 91
regioselectivity, 60
regression, 4, 6, 116
regression analysis, 4
Index 137
regression model, 4, 6
relevance, viii, 10, 19, 20, 34
renewable fuel, 58
repair, 46, 49
reproductive organs, 101
researchers, x, 63, 83, 86, 119
reserves, x, 58, 83
residues, x, 84, 85, 86, 87
resins, 63
resolution, 73, 74, 78
resources, 60, 65, 71, 77, 78, 84
response, 42, 65, 71, 76, 80, 81, 102, 104,
108, 112, 115, 116, 117, 119, 120, 123,
124, 126
reusability, 80
Rhizopus, x, 56
rings, 64
risk(s), 11, 24, 26, 36, 38, 39, 42, 43, 46, 49,
51, 52, 56, 63, 66
risk factors, 49, 66
risk profile, 39
rods, 15
room temperature, 58, 62, 90, 91
routes, 59
rubber, 62
S
salinity, 102
salts, 86, 87
SANS, 19
saturated fat, vii, ix, 35, 36, 37, 38, 39, 47,
48, 50, 58, 63, 65
saturated fatty acids, 38, 39, 50, 58, 65
saturation, 112
SAXS, 18, 19
scattering, 19
science, 29, 52, 73
scope, viii, 10
secretion, 42, 52
seed, xi, 99, 101, 102, 103, 105, 113, 114,
117, 119, 120, 121, 122, 123, 124, 125,
127, 128
selectivity, 57, 66, 68, 69, 73
self-assembly, 15, 31
sensitivity, 37, 42, 104, 105, 109, 120, 121
septic shock, 2
serum, 27, 39, 40, 42, 44, 51, 53, 128
shape, 14, 19, 20, 21, 63
shelf life, 10
shock, 128
showing, ix, 22, 43, 56, 61
side effects, 68
signaling pathway, 12
signalling, 42
silica, 61, 81, 87
simulation, 126
Singapore, 26
siRNA, 13, 30
skin, 11, 13, 27, 28, 46, 49, 58, 68
sodium, 34, 59, 86
sodium hydroxide, 59
software, 3, 5
solid matrix, 87
solubility, ix, 30, 56, 57, 67, 70, 94
solution, 5, 15, 65, 86, 87
solvents, 64, 69, 87
sophorolipids, vii, 1, 6
South Africa, 99, 128
sowing, 102, 103, 109, 117, 122, 124
soybean, xi, 41, 44, 56, 58, 66, 75, 76, 79,
88, 92, 93, 99, 100, 101, 103, 119, 122,
123, 125
SP, 55, 80
species, 92, 102, 122, 127
Sri Lanka, 75
SS, 76
stability, 56, 60, 66, 73, 86, 100, 101, 117,
119, 120, 121, 126
state(s), 11, 21, 36, 41, 46, 62, 77
statin, 25
sterols, 70
storage, 111, 118
stress, xi, 100, 101, 103, 107, 111, 113, 114,
115, 118, 121, 122, 124, 126, 128
structural transformations, 34
structure, 12, 15, 20, 29, 30, 32, 33, 42, 66,
70, 76, 87, 125
structuring, 31
substitutes, 37, 58, 70
Index 138
substrate(s), ix, 2, 55, 61, 64, 66, 70, 71, 72
sucrose, 2, 3
sulfuric acid, 86, 87, 88
Sun, 57, 77, 80
sunflower, xi, 40, 44, 45, 56, 58, 64, 66, 73,
77, 79, 80, 99, 100, 101, 102, 103, 107,
111, 112, 113, 114, 115, 116, 117, 118,
119, 120, 122, 123, 124, 125, 126, 127,
128
supplementation, 29, 43
suppression, 127
surfactant(s), x, 14, 15, 16, 57, 68, 69, 71,
74, 76, 83, 85
survival, 53
susceptibility, 66
symmetry, 15, 19
syndrome, 43, 49
synthesis, ix, 31, 55, 56, 57, 58, 59, 60, 61,
64, 68, 69, 71, 72, 73, 74, 75, 76, 77, 79,
80, 81, 87, 88, 97, 100, 103, 112, 118,
119, 126, 127
T
target, 36, 72
techniques, 19, 22
technology(s), x, 38, 65, 66, 72, 73, 75, 80,
81, 84, 123, 126
TEM, 18, 19
testing, viii, 10
thermal stability, 57
three-way interaction, 4
time use, 65
tin, x, 84, 88, 92, 93, 94, 96, 97
tin oxide, 94
tissue, 46, 70, 103, 119
TNF, 27
TNF-alpha, 27
toluene, 64
total cholesterol, 40, 41, 44, 45
total energy, 38, 39
traditions, 28, 51, 53
traits, 111, 125, 128
transesterification, x, 57, 59, 60, 66, 70, 77,
78, 80, 83, 85
transformation(s), 62, 63, 65, 74, 75, 85
transition temperature, 20
Transmission Electron Microscopy, 19
transport, 29
treatment, 22, 26, 28, 30, 46, 50, 51, 57, 81,
105, 106, 107, 108, 109, 110, 111, 114
triacylglycerides, 59, 61
trial, 43, 44, 45
triglycerides, 47, 48, 50, 79
tumor, 13, 28, 30
tumor cells, 13
turnover, 127
type 2 diabetes, 26, 42, 52
tyrosine, 68
U
ulcerative colitis, 11, 27
underlying mechanisms, 20
United, 27, 78
United States, 27, 78
urea, 2
USA, 3, 38, 53, 81, 123
USSR, 127
uterus, 81
V
vapor, 80
variables, 4, 5, 6, 14, 41, 65, 116, 122
variations, 31, 104, 111, 112, 121
varieties, xi, 56, 100, 101, 105, 125, 127
vascular cell adhesion molecule, 41
vegetable oil(s), vii, viii, ix, x, xi, 10, 21,
35, 39, 40, 53, 55, 56, 58, 59, 61, 65, 70,
75, 79, 83, 85, 86, 88, 99, 100, 101
vegetables, 38, 75
vegetal oils, x, 83
viscosity, viii, 10, 63
visualization, 19, 22
vitamin C, 57, 68, 70, 74
vitamin E, 32, 33
VLDL, 36, 40
Volunteers, 40
Index 139
W
waste, 57, 58, 60, 66, 77, 82, 90
waste water, 57
wastewater, 81, 85
water, viii, 10, 13, 14, 15, 16, 17, 19, 30, 32,
33, 34, 57, 59, 67, 69, 71, 81, 87, 88, 91,
102, 103, 122, 124, 125
WCO, 90
weight gain, 25
weight ratio, 61
well-being, 36
wellness, 11
Western countries, 40
WHO, 38, 53, 54
Wisconsin, 123
workers, 14, 40, 41, 42, 43
worldwide, x, 83, 121
wound healing, 49, 52
X
X-ray diffraction, 19, 29
Y
yeast, vii, 1, 2, 4, 81
yield, 2, 4, 5, 59, 64, 65, 67, 69, 70, 76, 94,
101, 108, 117, 122, 124, 126, 128
young adults, 50

Oleic Acid - Production, Uses and Potential Health Effects 2014

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Oleic Acid - Production, Uses and Potential Health Effects 2014

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BIOCHEMISTRY RESEARCH TRENDS

) and non-lamellar (two

) phase, (B) three

, Danielle Branta Lopes,

Address: Laboratrio de Bioqumica. Departamento de Cincia de Alimentos - FEA,

and Maryke Labuschagne

Corresponding author: vandermerwer@ufs.ac.za.

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