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Study of Field Pea Accessions for
Development of an Oilseed Pea
Ehsan Khodapanahi
Department of Bioresource Engineering
McGill University
Montreal, Quebec, Canada
A thesis submitted to McGill University in partial fulfillment of the
requirements of the degree of Master of Science
© Ehsan Khodapanahi, 2011
I
Abstract
The global interest in vegetable oil is due to greater environmental
concerns and increasing demand for renewable sources of energy in recent
decades. In order to meet the growing demand for vegetable oil, oilseed
production has increased globally, and needs to be further extended. In warm
temperate regions of Canada, protein and vegetable oil are primarily produced by
soybean, which is replaced by canola (Brassica napus) and field pea (Pisum
sativum) in less temperate regions of western Canada. The objective of this
research was to examine a variety of field pea accessions for the total lipid
content in the seeds to create a comparable dual purpose (protein and oil) crop
for western Canada. The research was initiated by validation of lipid extraction
methods, and multiplication of 174 acquired pea accessions in 2009 and 2010 at
McGill University (Quebec, Canada). Lipid extraction was carried out by the
validated method (the butanol extraction procedure) presented in chapter 2 and
applied to the seeds of pea accessions which were grown to maturity as
presented in chapter 3. Lipid content ranged from 0.3 % to 6.3 % with the
accession (p<0.0001), the year (p=0.0002) and the interaction of accession by
year (p <0.0001) being significant factors on the total lipid production in pea
seeds. Among the plant characteristics, which were investigated in the research,
seed surface type (wrinkled as compared to smooth) had a significant effect (p=
II
0.001) on the total lipid production in the seeds. The data can contribute to the
selective breeding of field pea accessions for specific traits suitable for lipid
production
Résumé
L’intérêt global dans l’huile végétal est due en partie aux problémes
environnementaux et en partie à la hausse de la demande pour des ressources
d’énergie renouvables ces dernières décennies. Afin de pouvoir répondre à
cette hausse de besoin pour l’huile végétal, il y a eu lieu une hausse globale de
la production de l’huile de graines, une hausse qui continue d’augmenter. Dans
les régions tempérées chauds du Canada, les protéines et les huiles végétales
sont produites surtout par les graines de soja. Dans les régions moins tempérées
du Canada, les graines de soja sont remplacées par le canola et les pois.
L’objectif de cette recherche a été d’examiner une variété de pois desquelles
nous avons extracté les lipides afin de définir une variété destinée à produire une
récolte à l’Ouest du Canada. Cette récolte de pois visant à: extraire les protéines
et l’huile. La recherche a été initiée par la validation des méthodes d’extraction
de lipides, et par la multiplication de 174 accessions de pois en 2009 et 2010 à
l’Université de McGill (Québec, Canada). L’extraction de lipides a été effectuée
par la méthode validée (la procédure d’extraction par le butanol) présenté au
III
chapitre 2 et a été appliquée aux graines de pois d’accessions qui ont été
produites jusqu'à maturité maturité (chapitre 3). La quantité de lipides variait de
0.3% à 6.3% selon l’accession (p<0.0001), l’année (p=0.0002) et l’intéraction
d’accession par année (p <0.0001). Pour les autres charactérisques des plantes
étudiées dans cette recherche, le type de surface des graines (ridée ou lisse) a
eu un effet important (p= 0.001) sur la production totale de lipide dans les
graines. Ces données peuvent contribuer à facilité la sélection des pois
d’accession: en faveur de ceux qui ont un meilleur potentiel pour la production
de d'huile végétale.
IV
Acknowledgements
I would like to acknowledge the valuable advice and guidance of my
supervisor Dr. Mark Lefsrud and of my committee member Dr. Valérie Orsat,
without whose dedication, knowledge and assistance I would not have finished
this degree. I would also like to thank my other committee member, Dr.
Jaswinder Singh from the Plant Science Department and our research advisor
Dr. Tom D. Warkentin from the Crop Development Centre, University of
Saskatchewan.
Funding and in-kind for this project was provided by our industry partners,
Lefsrud Seed and Processors Ltd. and Agrocenter Belcan. Further funding came
from Natural Sciences and Engineering Research Council (NSERC) as
Collaborative Research and Development (CRD) grants and Consortium de
Recherche et Innovations en Bioprocédés Industriels au Québec (CRIBIQ).
I want to extend a large thank you to Dr. Grant Clark, Dr. Vijaya Raghavan
and Dr. Michael Ngadi for granting us the permission to use their laboratories
and assets.
Special thanks to Raghav Narayanapurapu for his distinguished work, to
Jamshid Rahimi for devoting his time and support, to former students, Jenna
Senecal-Smith and Aliya Bekmurzayeva, whose data has contributed to the
research, and all of my lab mates.
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Finally, I want to thank my dearest wife, Maryam, for her ongoing support
and encouragement, and my parents, who always make their unseen presence
known and felt.
Authorship and Manuscript
The contributions of the authors are:
1. Reviewing the literature for common extraction procedures on oilseeds,
executing laboratory experiments, performing calculations, data analysis and
writing of manuscripts (student).
2. Supervising the research and reviewing of manuscripts (supervisor)
3. Advising throughout the research and reviewing of manuscripts (advisors)
This thesis is written in paper-based format. The authorship of preparing
papers is as follow: E. Khodapanahi; M. Lefsrud; V. Orsat; J. Singh; T. D.
Warkentin.
VI
Table of Contents
Abstract .............................................................................................................................. I
Résumé ............................................................................................................................. II
Acknowledgements .......................................................................................................... IV
Authorship and Manuscript ................................................................................................ V
List of acronyms ................................................................................................................ 1
Chapter 1: Literature Review ............................................................................................. 2
1.1 Introduction .................................................................................................................. 2
1.2 What are lipids? ........................................................................................................... 3
1.3 Lipid value ................................................................................................................... 4
1.3.1 Food, health and medicinal applications .............................................................. 5
1.3.2 Oleochemical Industry .......................................................................................... 6
1.3.2.1 Surfactants .................................................................................................... 7
1.3.2.2 Cosmetic and personal care products ........................................................... 8
1.3.2.3 Lubricants.................................................................................................... 10
1.3.2.4 Biofuel ......................................................................................................... 11
1.4 Lipid Analysis ............................................................................................................. 13
1.4.1 Determination of Total Lipid Concentration ........................................................ 13
1.4.1.1 Solvents Extraction ..................................................................................... 14
1.4.1.2 Lipid extraction history ................................................................................ 17
1.4.2 Determination of Lipid Composition.................................................................... 18
1.4.2.1 Bulk properties methods ............................................................................. 19
1.4.2.2 Chromatographic methods .......................................................................... 19
1.4.2.3 Spectrometric Methods ............................................................................... 20
1.4.2.4 Enzymatic methods ..................................................................................... 20
1.5 Natural lipid sources .................................................................................................. 21
1.5.1 Canola ................................................................................................................ 22
1.5.1.1 Extraction methods ..................................................................................... 23
1.5.1.2 Fatty acid composition ................................................................................ 24
1.5.2 Soybean ............................................................................................................. 26
1.5.2.1 Extraction methods ..................................................................................... 27
1.5.2.2 Fatty acid composition ................................................................................ 29
1.6 Field pea .................................................................................................................... 30
1.6.1 Lipid content and fatty acid composition............................................................. 31
1.7 Conclusion ................................................................................................................. 34
1.8 References ................................................................................................................ 36
VII
Connecting Statement ..................................................................................................... 47
Chapter 2: Method validation for lipid extraction .............................................................. 48
2.1 Abstract ..................................................................................................................... 48
2.2 Introduction ................................................................................................................ 49
2.3 Experimental .............................................................................................................. 53
2.3.1 Sampling ............................................................................................................ 53
2.3.2 Chemicals .......................................................................................................... 54
2.3.3 Instrumentation .................................................................................................. 54
2.3.4 Methods used for gravimetric determination of total lipid content ....................... 55
2.3.4.1 Butanol extraction method .......................................................................... 55
2.3.4.2 Hexane/Isopropanol .................................................................................... 56
2.3.4.3 Chloroform/methanol ................................................................................... 57
2.3.4.4 Soxhlet extraction ........................................................................................ 57
2.3.4.5 Modified Bligh and Dyer .............................................................................. 58
2.3.4.6 Microwave extraction .................................................................................. 59
2.3.5 Statistical analysis .............................................................................................. 60
2.4 Results....................................................................................................................... 60
2.5 Discussion ................................................................................................................. 63
2.6 References ................................................................................................................ 66
Connection Statement ..................................................................................................... 70
Chapter 3: Lipid content variation in field pea (Pisum sativum) accessions .................... 71
3.1 Abstract ..................................................................................................................... 71
3.2 Introduction ................................................................................................................ 72
3.3 Experimental .............................................................................................................. 78
3.3.1 Sampling ............................................................................................................ 78
3.3.2 Chemicals .......................................................................................................... 80
3.3.3 Instrumentation .................................................................................................. 80
3.3.4 Methods used for Gravimetric determination of total lipid content ...................... 80
3.3.4.1 Butanol extraction method .......................................................................... 80
3.3.5 Statistical analysis .............................................................................................. 81
3.4 Results and Discussion ............................................................................................. 82
3.5 Conclusion ................................................................................................................. 85
3.6 References ................................................................................................................ 86
Chapter 4 ......................................................................................................................... 96
4.1 General conclusion .................................................................................................... 96
4.2 Future research ......................................................................................................... 97
VIII
List of Tables
Table 1.1: Fatty acid composition of genetically modified canola oil ................................ 26
Table 1.2: Fatty Acid composition of Soybean oil ............................................................ 29
Table 1.3: Total oil content and composition in different crops ........................................ 33
Table 2.1: Solvents polarity index .................................................................................... 51
Table 2.2: The extractable lipid content variation by extraction method .......................... 61
Table 2.3: Analysis of variance, sample type and extraction method .............................. 61
Table 3.1: World Population, 1950-2050 ......................................................................... 72
Table 3.2: World total oilseed crop production................................................................. 73
Table 3.3: World total oilseed crop production................................................................. 74
Table 3.4: Analyse of variance, accession and year ........................................................ 83
Table 3.5: Least square means for analysed pea characteristics .................................... 83
Table 3.6: Lipid variation in pea accessions. ................................................................... 89
List of Figures
Figure 3.1: Pea growing area in Canada ......................................................................... 76
1
List of acronyms
DM
ESA
EU
FAME
FFA
GC
HEAR
HPLC
IPA
LCT
LEAR
MC
MCT
MEAR
PI
PE
PUFA
SFC
SFE
TAG
TLC
UV
USDA
dry mass
essential fatty acids
European Union
fatty acid methyl ester
free fatty acid
gas chromatography
high erucic acid rapeseed
high performance liquid chromatography
Isopropanol
low chain triglycerides
low erucic acid rapeseed
moisture content
medium erucic acid rapeseed
medium erucic acid rapeseed
polarity index
petroleum ether
polyunsaturated fatty acid
supercritical fluid chromatography
supercritical fluid extraction
triacylglycerol
thin layer chromatography
ultraviolet
United States Department of Agriculture
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Chapter 1: Literature Review
1.1 Introduction
Vegetable oils and fats are used in a vast spectrum of industries, from the
production of edible oil to the non-food applications, such as lubricants,
surfactants, emulsifiers and biodiesels (Gunstone, Harwood et al. 1994;
Simanzhenkov and Idem 2003). The energy crisis in the 1970s, coupled with the
fast diminishing energy reserves and a greater environmental awareness
aroused strong interest in renewable energy sources (Wengenmayr and Bührke
2008). There are several advantages involved in replacing petrochemical
products by vegetable oils. Vegetable oils are biodegraded more quickly and
disappear from the environment faster when used. Biofuels do not add to the
total carbon dioxide in the atmosphere, which is one of the greenhouse gases
responsible for the climate change. Biofuels liberate the carbon dioxide which
was trapped only months earlier as compared with carbon dioxide that was
trapped in fossil fuel millennia earlier (Gunstone, Harwood et al. 1994).
Pea seeds are primarily produced for protein and starch (Sosulski, Hoover
et al. 1985; Sosulski and McCurdy 1987; Small 1997); but, there is research that
supports the idea of lipid production from field pea (Letzelter, Wilson et al. 1995;
Bastianelli, Grosjean et al. 1998). This novel lipid source can be used in various
industries, such as biofuel (Kemp 2006).
3
This thesis will review the importance of lipid, applications in various
industries and common methods in the processing and analysis of canola and
soybean, which are the most common oilseeds in Canada (FAOSTAT 2009). The
study will review previous research on field pea, to develop a convenient method
to quantify total lipid content in field pea seeds.
1.2 What are lipids?
The term lipid includes oil and fat which are often used interchangeably by
food scientists. Oils and fats are defined as liquid and solid, respectively at
ambient temperature (24 °C) (Gunstone, Harwood et al. 1994; Haas 2005). Lipid
is a diverse group of biological substances primarily made up of non-polar
compounds such as triglycerides, diglycerides, monoglycerides and sterols, as
well as more polar compounds such as free fatty acids, phospholipids and
sphingolipids (Gunstone, Harwood et al. 1994; Gurr, Harwood et al. 2002; Vance
and Vance 2002). Lipids can be in a free form, or covalently bound to
carbohydrates and proteins to form glycolipids and lipoproteins. Edible oils and
fats are mainly esters of fatty acids and glycerol (Gurr, Harwood et al. 2002).
Fatty acids are aliphatic chains with a methyl group at one end and a
carboxylic acid at the other (Petersson Grawé 2003). Fatty acids differ in chain
length as well as the number and position of double bonds or substitution groups.
4
In theory, fatty acid chains can be of any length, but most natural fatty acids have
4 to 22 carbons, among which the most common number is 18 (Gunstone,
Harwood et al. 1994). From the health perspective, fatty acids are divided into
two groups of essential and non-essential, depending upon the human or animal
body’s ability to bio-synthesize the compounds (Insel, Ross et al. 2010).
Essential fatty acids are polyunsaturated (PUFA), which are necessary for a
good health and cannot be naturally synthesized in the body (Gunstone,
Harwood et al. 1994).
1.3 Lipid value
As a cell component in all living organisms, lipids contribute to cell
structure, act as an energy storage, and are responsible for a range of biological
processes (Gurr, Harwood et al. 2002). Vegetable oil is mainly used for human
consumption (80%), followed by oleochemical industry (14%) and animal feed
(6%) (Gunstone, Harwood et al. 1994). A growing demand for vegetable oil and
fat is predicted almost everywhere in the world. The increase would be mainly
toward edible uses in East Asia and Latin America, and biofuel in Europe and
North America. In order to meet the growing demand of vegetable oil, the
production has been increasing in last few decades and is expected to increase
by 30% by 2015 (Vollmann and Rajcan 2009).
5
Having numerous applications in a variety of industries has made the lipid
composition and efficient extraction an important subject of research. In general,
lipid industrial uses can be split into two categories: food, health and medicinal
applications, and oleochemical applications.
1.3.1 Food, health and medicinal applications
Lipids play important roles in food quality by contributing to attributes such
as texture, flavour, nutrition and caloric density (Gunstone 2004b). The
importance of lipids in food, health and medicinal products can be viewed from
different perspectives:
• A source of energy by supplying 25 to 30 % of calories in a normal diet
(Jones 1974).
• A source of essential fatty acids (ESA) that cannot be bio-synthesized in
the human body (Rosdahl and Kowalski 2008).
• A carrier of important minor components, such as soluble vitamins and
phytosterols (Smith and Charter 2009).
• A factor that contributes to the flavour and texture of foods (Brown 2007).
Lipid content quantification is a basic requirement in testing food products
(Ruiz-Jiménez and Luque de Castro 2004). Saturated and unsaturated fatty
acids and cholesterol content must be analyzed in order to reveal the caloric
6
value and nutritional quality of foods. The measurement of quality factors, such
as degree of unsaturation, saponification value, refractive index, free fatty acid
(FFA) content, solid fat index and oxidative stability are needed to determine the
market value and potential application of bio-lipids (Akoh and Min 2002). The
degree of unsaturation of fatty acids in vegetable oils can be a determinant factor
to propose the best usage of the oil on the basis of PUFAs nutritional value
versus their negative contribution to oxidative instability and oil degradation
during frying (Johnson, White et al. 2008).
The application of lipids and fatty acids in drug delivery and water-
insoluble drugs in clinical development is mainly due to the solubility properties of
lipid materials. Vegetable oil from oil crops such as soybean, safflower and
coconut are used to obtain LCT and MCT (low and medium chain triglycerides,
respectively) required in water insoluble drug formulations, and oleic acid is used
in parenteral and oral lipid-based drug formulations (Liu 2008).
1.3.2 Oleochemical Industry
The manufacturing of fatty acids, soaps, methyl esters, alcohols, amines,
and glycerols are the main uses of oils and fats in oleochemical industry
(Gunstone, Harwood et al. 1994). Over the past ten years since 2000, the
production of oleochemicals has raised one-third, from 5.76 to 7.75 million
7
tonnes (Gunstone 2009). The proportion of the vegetable oil commodities used
for industrial purposes in European Union (EU-15) for 2003/04 was reported by
U.S. Department of Agriculture (USDA) as rapeseed oil (39%), palm kernel oil
(29%), coconut oil (17%), palm oil (10%), soybean oil (7%) and sunflower oil
(6%), with olive oil, groundnut oil, and cottonseed oil at 1% or less. Generally, in
oleochemical industries vegetable oil is used to produce four following classes of
lipid based materials: surfactants, cosmetics and personal care products,
lubricants and biofuels (Gunstone and Hamilton 2001).
1.3.2.1 Surfactants
Surfactants are surface-active molecules for lowering the surface tension
of a liquid, allowing easier spreading, and decreasing the interfacial tension
between two liquids (Goodwin 2004). This characteristic is derived from the
amphiphilic nature of surfactants, which means one end of the molecule (the
alkyl chain) is lipophilic (hydrophobic), and the other end (usually the polar head
group) is lipophobic (hydrophilic) (Baran and Maibach 2005). The balance
between the two forces is an important property of surfactant molecules.
Surfactants are widely used in industrial processes, such as emulsification for
emulsion polymerization, foaming for food processing, detergency for household
8
and industrial cleaning, wetting and phase dispersion for cosmetics and
solubilisation for agrochemicals (Lin 1996).
Surfactants are made from petroleum and natural feedstock (Kjellin and
Johansson 2010). The environmental and economic advantages of plant-based
materials have highlighted their importance during the time of high-priced oil and
gas (Gunstone, Harwood et al. 1994). The higher environmental awareness and
strict legislations have made the environmental compatibility of surfactants an
important factor for various uses (Maier and Soberón-Chávez 2000). Numerous
research projects in the last few decades have attempted to find alternatives to
petrochemically produced surfactants. Recently, bio-surfactants have gained
attention as natural and promising products due to several advantages over
petrochemical surfactants, such as lower toxicity, biodegradable nature and
ecological acceptability (Zhou and Kosaric 1995).
1.3.2.2 Cosmetic and personal care products
Lipids have beneficial properties in cosmetic formulations that can be
reviewed from two aspects; First, the physicochemical properties and second,
bioactivity of lipids. Physicochemical properties of lipids help the consistency of
cosmetic products while lipids bioactivity refers to the components such as
essential fatty acids, tocopherols and phytosterols that contribute to the skin
9
health by acting as an antioxidant and anti-inflammatory (Gunstone, Harwood et
al. 1994). Some of the bioactive components such as linoleic acid, linolenic acid
and arachidonic acid, which are grouped under vitamin F, are essential for the
skin health, and their shortage can cause cutaneous (skin related) problems
(Servel, Claire et al. 1994). As a cosmetic product ingredient, lipids may function
as a moisturiser, an emulsifier, a texturiser or a skin-feel improver (Gunstone,
Harwood et al. 1994).
Bio-materials were reported as renewable sources of compounds required
in the cosmetic industry. Marine microalgae was reported as a possible source of
PUFAs for cosmetic products (Servel, Claire et al. 1994). Lipid content from
different oilseed crops, such as palm (Mattsson, Cederberg et al. 2000), canola
(de Morais, dos Santos et al. 2006) and soybean (Wu and Wang 2003) can be
used as emulsifiers in cosmetic products. Squalene, which is a natural organic
compound, 2, 6, 10, 15, 19, 23-hexamethyl-2, 6, 10, 14, 18, 22-tetracosahexaene
(Budin, Breene et al. 1996), acts as a biosynthetic precursor to all steroids as
well as having a photo protective property (He, Cai et al. 2001). Squalene is
mainly extracted from marine animals, such as shark and whale liver oils (Budin,
Breene et al. 1996). Recently, it was shown that grain amaranth (Amaranthus
species) has a great potential to replace the current sources of squalene (He, Cai
et al. 2001; Sun, Wiesenborn et al. 1995).
10
1.3.2.3 Lubricants
A lubricant is a substance that improves the smoothness of movements by
reducing surface friction (Walker and Wood 2009). The majority of manufactured
lubricants are used in motor oils (about 70%) for automotive engines, or in
hydraulic fluids (approximately 10%). There are four types of lubricants namely:
liquid lubricants, grease, solids and gas lubricants (Gunstone, Harwood et al.
1994). Lubricant functions are to:
• reduce, or control friction between metal parts to save energy (Asthana
and Asthana 2002),
• reduce wear, or prevent weld of metal surfaces (Larsen 1949)
• clean metal surfaces of dirt or sludge to prevent scratching or scoring
(Gunstone, Harwood et al. 1994),
• clean metal surfaces of water and acids to prevent corrosion and
overheating (Ferry 1988).
Vegetable oil can be used as a liquid lubricant directly (Liao 2009), or as a
grease with additives (Adhvaryu, Erhan et al. 2004). Rapeseed oil, soybean oil,
sunflower oil, palm oil and castor oil have found applications as lubricants in
olechemical industry for years (Mang and Dresel 2007). Bio-lubricant’s
performance and stability is limited at lower temperature as compared with
mineral oils (Erhan, Sharma et al. 2008), which is mainly due to the high
11
unsaturation level of fatty acids in bio-lubricant. This is partially solved in the
genetically modified oilseeds in which the PUFA content was lowered. The
oxidative stability of high-oleic oils is three to six times greater than a normal
vegetable oil (Rudnick 2006).
Vegetable oils and other lipid derivatives have been increasingly used as
bio-lubricants over the last decade, but still contribute less than 2% of all base-
oils used in the market (Gunstone, Harwood et al. 1994).
1.3.2.4 Biofuel
Animal fat and vegetable oil have been used as alternative resources for
biodiesel (usually methyl esters derived from oils and fats) and bioethanol.
Sudden price spike of fossil fuel during the 70s and a lack of power to control the
market motivated western countries to look for alternative resources of power.
The environmental advantage of green fuel sources has reinforced this trend
during the last few decades (Demirbas 2002).
Petroleum and vegetable based diesel fuels have different chemical
structures (Gurr, Harwood et al. 2002). Diesel fuel contains only carbon and
hydrogen atoms arranged in a straight chain or branched chain structures along
with aromatic configurations. In contrast, biodiesel structure is based on
12
triglycerides, which contains up to three fatty acids linked to a glycerine molecule
with ester linkages (Demirbas 2002).
For biodiesel production, crude oils are degummed (removal of
phospholipids) and neutralised (removal of free acids) while bleaching and
deodorisation is not required. In general, oils and fats (triacylglycerol) are
converted to methyl esters by reacting with methanol (transesterification) in the
presence of an acidic, basic, or enzymatic catalyst (Gunstone, Harwood et al.
1994). Animal fat and vegetable oil are composed of triacylglycerol (TAG) as the
main component. This implies that the production of biodiesel is potentially
possible from all extractable bio-lipids (Kemp 2006). Biodiesels have to meet
certain criteria that have been accepted by most vehicle companies. It is
desirable to avoid saturated esters content, which are solidified at ambient
temperature, or polyunsaturated esters (especially those with more than two
double bonds) which lead to undesirable oxidations and may cause problems
during storage of the fuel or at the moment of use (Gunstone, Harwood et al.
2007). Biodiesel produced from animal or vegetable oil is used as an additive to
petroleum fuel since its production cost is still higher than traditional fuel
(Gevorkian 2007).
13
1.4 Lipid Analysis
Lipids are mainly characterized by fatty acid composition (Gunstone
2004b). As a result of a distinctive composition in different bio-materials and also
analysis objectives, numerous methods have been developed through the years
for fatty acid profiling. The best method for the extraction and characterization of
lipid content in a sample can be determined by comparing the results of different
methods on the same sample (Barthet, Chornick et al. 2002; Moreau, Powell et
al. 2003).
1.4.1 Determination of Total Lipid Concentration
The total lipid content can be quantified by extraction or non-extraction
methods. In non-extraction methods, such as density measurement, dielectric
method, near-infrared spectroscopy, low-resolution nuclear magnetic resonance
spectroscopy, ultrasonic method and X-Ray absorption, the total lipid
concentration is determined from a measured physical or chemical property of a
sample (Akoh and Min 2002). In extraction methods, the lipid content is
separated from other compounds of the cell. Various lipid extraction methods
have been developed for lipid analysis in the laboratory and for vegetable oil
production on an industrial scale (Gunstone and Padley 1997).
14
Natural lipids interact with other cellular components via van der Waals
interaction (such as lipid-proteins interaction), electrostatic and hydrogen bonding
(such as in lipoproteins) and covalent bonding (with lipids, carbohydrates and
proteins). In order to isolate lipids from a complex cellular matrix, different
chemical and physical treatments must be administered (Akoh and Min 2002).
The common property of lipids used in lipid extraction methods is water
insolubility (Rahman 2008). Extraction methods are divided into two categories:
solvents and non-solvent methods. Solvent methods, which are mostly used for
plant tissues, oilseeds, and marine samples, extract the lipid content with one or
a mixture of organic solvents, whereas in non-solvent methods no solvent is
used, and lipid content are quantified after digestion by chemical reagents, such
as by alcohol. Non-solvent methods are commonly used in dairy product analysis
(Gunstone 2004a; Wrolstad 2005). In extraction methods, it is always desirable
to maximize the oil yield with minimal chemical and physical damage to the
extract components, and the remaining materials which may have other uses, for
instance in animal feeding (Gunstone 2004a; Francis 2000).
1.4.1.1 Solvents Extraction
Lipids have a range of hydrophobicity, which is caused by a molecular
variation in their structure. TAG and sterols are non-polar whereas free fatty
15
acids (FFAs), phospholipids and sphinglolipids are slightly polar (Gunstone,
Harwood et al. 1994; Gurr, Harwood et al. 2002; Vance and Vance 2002). Polar
lipids are more soluble in polar solvents while non-polar lipids can be better
dissolved in non-polar solvents. For a more efficient lipid extraction, the polarity
of a selected solvent should be in agreement with the overall polarity of the lipid
molecules (Akoh and Min 2002).
The ability of lipids to bind to other molecules and the capability of
different solvent mixtures to solubilize lipid classes has led to the concept of the
total lipid content and extractable lipid fraction. Solvents used for lipid extraction
should have a high solubility for all lipid compounds in a sample, and be
sufficiently polar to remove bound lipids from their binding sites with cell
membranes, lipoproteins and glycolipids (Smedes 1999). A mixture of non-polar
and polar solvents is suggested for more exhaustive extraction results (Schäfer
1998). In addition to polarity, boiling point, which has to be low enough for easy
evaporation, low flammability and health and environmental concerns should be
effectively brought into consideration for lipid solvent selection (Burton and
Guerra 1974).
In general, the following steps are involved in lipid extraction from plant
samples (Akoh and Min 2002):
• Drying, size reduction or hydrolysis.
16
• Homogenization of sample in the presence of solvent.
• Separation of liquid and solid phase.
• Removal of non-lipid contamination and solvent.
• Drying the extract.
Hexane, petroleum ether and diethyl ether are the most common solvents
for oilseeds, for both low or high fat sources, considering that the moisture
content (MC) of sample does not exceed 10% (Wrolstad 2005). Although
hexane is known as the most common lipid solvent in the industry (Starck 1991),
its environmental disadvantages and safety concerns has prompted researchers
to look for alternative solvents (Springett 2001). Moreau et al. (2003) has studied
four organic solvents, hexane, methylene chloride (also known as
dichloromethane), isopropanol (IPA), and ethanol, to determine the polar and
non-polar lipid content of corn and oat using accelerated solvent extraction. The
extraction efficiency was investigated in two different temperatures, 40 and 100
°C. Their results showed that the highest amount of TAG was extracted by using
methylene chloride at 100 °C while hexane and IPA at both temperatures were
the second most efficient solvents.
17
1.4.1.2 Lipid extraction history
Modern lipid extraction has begun to develop as per findings of Chevreul
(1823) and his studies that showed the dissolution of lipid materials in various
solvents in the 18th century. Franz Von Soxhlet (1879) introduced the first
automated solvent extraction apparatus by using diethyl ether as a solvent. The
innovation used hot solvent passing through the sample, which improved the
extraction efficiency. However, this method was not industrialized until 1946
(Temelli 1992). In 1914, a mixture of ethanol/ether was used for extraction by
Bloor (1914). A remarkable improvement was brought to extraction techniques by
Folch et al. (1957), who developed a method to extract polar lipids from animal
tissue. Bligh and Dyer (1959) and Sheppard (1963) have developed
methodologies that are still common in lipid analysis, using a mixture of
chloroform:methanol and ethanol:diethyl ether, respectively.
A relatively new method of supercritical fluid extraction (SFE) was developed in
the 1980s to replace organic solvent with CO2. In this method, CO2 becomes a
supercritical fluid by controlling pressure and temperature. A supercritical fluid
can penetrate into a sample as a gas, and dissolve and carry lipids as a liquid
(Schwartzberg and Rao 1990).
To improve the lipid solubility in solvents, a microwave digester was first
reported by Ganzler et al. (1986) to heat a mixture of solvent and sample. They
18
compared Soxhlet and shake flask method with microwave, and found that
microwave gives a better result for more polar compounds.
The ultrasound-assisted extraction method prior to the gravimetric
determination of total lipid content is one of the most recent advancements in
lipid extraction (Castro and García 2002). In this method, ultrasound is used to
assist in the release of the lipid content into the solvent. The ultrasound-assisted
extraction was reported by Ruiz-Jiménez and Luque de Castro (2004) to give a
similar result to Soxhlet extraction with hexane.
1.4.2 Determination of Lipid Composition
Fatty acids are the main component of lipid content in plants (Gunstone,
Harwood et al. 1994). In order to evaluate the extracted oil, individual classes of
fatty acids must be separated for further analysis. It is not always easy to quantify
lipid content of cereal grain and other hard seed crops because a part of the lipid
content is in the impermeable cells and starch granules, and is not accessible by
solvents under normal condition. Moreover, quantification of around 20 natural
fatty acids is not easily achieved by a single method (Morrison, Tan et al. 1980).
It is important that samples are prepared in a pure form and free of extraneous
matter. Moisture should be at minimum to avoid any interference with the
analysis. It may be necessary to add antioxidants with no exposure to light and
19
heat to avoid oxidation in unsaturated fatty acids (Simanzhenkov and Idem
2003).
Lipids analysis can be divided into four groups, namely: bulk properties
methods, chromatographic methods, spectrometric methods and enzymatic
methods (Akoh and Min 2002).
1.4.2.1 Bulk properties methods
Methods to determine the degree of unsaturation, FFA content, oxidative
stability, refractive index, saponification value, etc. are based on gravimetrical or
volumetrical measurements, or a combination of the two. The combined methods
are commonly used to evaluate food quality through measuring oil and fat
characteristics (Akoh and Min 2002; Gunstone, Harwood et al. 1994).
1.4.2.2 Chromatographic methods
The ultimate goal in the chromatographic method is to separate the lipid
content of a sample based on the polarity of the components. Liquid-liquid
extraction (partitioning), liquid-solid column chromatography (adsorption) and ion
exchange chromatography are the three traditional fractioning methods (Kuksis
1987).
20
Widely used chromatographic techniques for lipid characterization are (Akoh and
Min 2002):
• Gas chromatography (GC)
• High performance liquid chromatography (HPLC)
• Supercritical fluid chromatography (SFC)
• Thin layer chromatography (TLC)
1.4.2.3 Spectrometric Methods
UV-visible spectroscopy, infrared absorption spectroscopy, nuclear
magnetic resonance spectroscopy and mass spectrometry are the four
techniques prevalently used to identify and quantify lipid components (Akoh and
Min 2002).
1.4.2.4 Enzymatic methods
Lipid extraction with enzymes, such as lipase (Higgins 1984) has gained
enormous attention as an environmentally cleaner alternative technique for oil
extraction. The benefits of this method are: elimination of solvent usage, less
capital investment and energy usage, simultaneous recovery of protein and oil,
elimination of degumming process and capability of removing toxin and anti-
21
nutritional components from the extracts, such as removal of phytic acid from
high protein meal (Caragay 1983; Rosenthal, Pyle et al. 1996).
1.5 Natural lipid sources
Oilseed production has considerably increased in the last 25 years
(Gunstone, Harwood et al. 2007). This was achieved by increasing the yield per
unit as well as increasing the cultivation area (Vollmann and Rajcan 2009).
Throughout this period, genetic engineering and breeding has contributed to
improve the lipid content in oilseed crops. Improving yield and quality of lipid
content in major crops has been studied in different research. Yield increase was
gained by refining agricultural practices while desired fatty acid compositions
have been produced by growing transgenic plants (Gunstone, Harwood et al.
2007).
There are fourteen vegetable oil commodities, which can be divided into
three groups based on the type of crops (Gunstone 2002):
• Vegetable oil extracted from annual plants, such as canola (Brassica
napus L.), sunflower (Helianthus anuus) and flax (Linum usitatissimum).
• Vegetable oil derived from trees, such as coconut (Cocos nucifera) and
olive (Olea europaea).
22
• Vegetable oil extracted as by-products in crops such as cotton (Genus
gossipium) and corn (Zea mays).
For the purposed of this study, the top two Canadian oilseeds, canola and
soybean (FAOSTAT 2008), were selected for further review on common lipid
extraction methods, total lipid content and lipid characteristics.
1.5.1 Canola
For thousands of year Brassica seed was used to feed animals, operate
home-based oil lamps, and the high erucic acid rapeseed (HEAR) oil was used
as a lubricant (Shahidi 1990). Erucic acid is considered an antinutritional and
toxic component for human consumption, which is largely found in the Cruciferae
family, such as rapeseeds (Brassica napus and Brassica campestris) and
mustard seeds (Brassica hirta and Brassica juncea) (Concon 1988; Guil,
Rodríguez-Garcí et al. 1997). In the 1960s, through a revolutionary research
program, the HEAR oil was altered to low erucic acid rapeseed (LEAR). The
novel crop named canola (Canadian oil low acid) since it was bred in Canada
(Downey and Craig 1964), and it became a valuable source of edible oil in the
food industry (Gunstone 2004b; Shahidi 1990). China, Canada and India are the
top three canola producers while it is a common crop in some European
countries (FAOSTAT 2009). Canola seeds are rich in oil content, and the oil
23
extraction residue, which has high protein content, is used for animal feed
(Shahidi 1999). A higher oil and protein as well as lower fiber content, makes the
yellow color seeds a more desirable product compared to black color seeds
(Vollmann and Rajcan 2009).
1.5.1.1 Extraction methods
Lipid extraction method in the canola industry can be mechanical, solvent,
or a combination of the two, which is determined by the production scale (Boer
and Ella 2000). The most common extraction solvent used in canola oil
production is n-hexane due to its availability, high solubility and low boiling point
(easy removal) (Unger 1990). There are required pre-treatment steps in canola
extraction process, such as dehulling, flaking and thermal treatment that ease the
extraction process and improve its efficiency. The optimal pre-treatment before
hexane extraction was described by Sosulski et al. (1988) as flaking, autoclaving
adjustment to 30% seed MC, 12% (g/g) of enzyme concentration and incubation
for 12 h at 50 °C, followed by drying to 4% MC.
In laboratory-scale, various lipid solvents were tested on canola, such as
chloroform and methanol (Zaderimowski and Sosulski 1978), hexane (McKillican
1966), tetrachloroethylene (Evans, Rothnie et al. 1987), petroleum benzene
(Matthäus and Brühl 2001) and 2-propanol (Kanth Rao and Arnold 1957). In
24
1988, supercritical carbon dioxide extraction was studied on canola seeds by
Fattori et al. (1988) at different temperatures, ranging from 25 to 90 °C and
pressure from 10 to 36 MPa. They also studied five different pre-treatments such
as crushing, chopping, flaking, cooking, and pressure in order to physically
rupture seeds. They reported positive correlation between canola oil solubility
and pressure at all temperatures with flaking and cooking being the most efficient
pre-treatments, which can produce a similar result to hexane extraction.
Enzymatic and hydrothermal treatment (Do and Sabatini 2010) and
aqueous extended-surfactant based method (Szydlowska-Czerniak, Karlovits et
al. 2010) for vegetable oil extraction have been recently shown to have a
potential to replace the traditional solvent extraction methods on canola and
other oilseeds.
1.5.1.2 Fatty acid composition
Traditional rapeseed oil contains about 20 to 45% erucic acid (cis-22:1(n-
9)) (Potts, Males et al. 2001). In the early 1970s, maximum allowed erucic acid
(EA) content in edible oil was set at 5% in Canada, which was lowered to 2% in
the early 1980s. To meet the food industry criteria in terms of EA content while
supplying oleochemical industry with high EA content product, two types of
canola varieties have been developed: low erucic acid rapeseed (LEAR) with ≤ 2
25
% EA, to be used in foods, and high erucic acid rapeseed (HEAR), with up to
55% of EA, to respond to the olechemical industry needs (Gunstone 2004b).
The crude oil extracted from low erucic acid rapeseed (LEAR) and
medium erucic acid rapeseed (MEAR) was quantified by Zaderimowski and
Sosulski (1978) at 43%. In this study, they fractioned lipid content to polar and
non-polar, and calculated a ratio of 22:1 for non-polar to polar lipids, with TAG
being the main component of non-polar fraction (92 %). The polar fraction was
primarily composed of phospholipids, 3.2 to 3.6 %, and 1% glycolipid. They used
GLC analysis to measure different fatty acids, and found 40% oleic, 20% linoleic,
9% linolenic, 12% eicosanoic and 15% erucic acids in MEAR, and 60% oleic acid
and 2 % of 20- and 22-carbon fatty acids in LEAR. A similar experiment,
conducted by McKillican (1966), showed a slight difference of 2% more TAG and
less polar lipids.
Typically, canola oil contains palmitic (4%), stearic (2%), oleic (62%),
linoleic (22%) and linolenic (10%) acids, and has less saturated acids than any
other commodity oil. It contains large amounts of fatty acids with chain lengths
above 18 carbons (Gunstone 2004b). Fatty acid composition was reported by
Evans et al. (1987) as oleic acid (18: 1), linoleic acid (18: 2) and low level of
eicosenoic (20: 1) and erucic (22: 1) acid. Table 1.1 shows fatty acid composition
of canola oil in genetically modified varieties (O'Brien 1998).
26
Table 1.1: Fatty acid composition of genetically modified canola oil
Fatty
acid,%
Canola Low
Linolenic
Canola
High
Oleic
Canola
High
Lauric
Canola
C-12:0
C-14:1
C-16:0
C-16:1
C-18:0
C-18:1
C-18:2
C-18:3
C-20:0
C-20:1
C-20:2
C-22:0
C-22:1
C-24:0
C-24:1
Lauric
Myristic
Palmitic
Palmitoleic
Stearic
Oleic
Linoleic
Linolenic
Arachidic
Gadoleic
Eicosadienoic
Behenic
Erucic
Lignoceric
Nervonic
0
0.1
4.2
0.3
2.3
62.5
19.2
7.9
0.7
1.3
0.1
0.3
0.3
0
0.2
0
0.1
3.8
0.3
2.4
64.1
23.8
2.1
0.7
1.2
0.1
0.3
0.3
0
0.2
0
0.1
3.0
0.3
2.0
73.7
14.4
2.9
0.7
1.4
0.1
0.3
0.1
0.2
0.2
37.0
4.4
3.2
0.3
1.3
31.5
13.1
6.7
0.5
1.0
0.1
0.3
0.2
0
0.1
Data source: O'Brien, R. D. (1998). Fats and oils: formulating and processing for application
1.5.2 Soybean
Soybean (Glycine max) is the second oilseed crop in Canada in terms of
seeded area (Statistics Canada 2011). World production of soybean has almost
quadruple over the past three decades (FAOSTAT 2009). During the past five
27
decades, the USA has been leading the world in soybean production,
representing 33% of the total production, followed by Brazil (27%), Argentina with
(21%), China (7.2%), India (4.4%), Paraguay (1.8%) and Canada (1.3%)
(FAOSTAT 2009). Early breeding has converted soybean ancestor with low oil
content, high protein, small, black hard seeds to yellow seeds with 20% oil
content and 40% protein content (Vollmann and Rajcan 2009). High quality
protein can be used for animal feed or human processed food (Gunstone 2004a).
Approximately 29% of the world vegetable oil production is from soybean
(Johnson, White et al. 2008). Oil content in soybean seeds is positively
correlated with yield, but it usually causes loss of protein (Chung, Babka et al.
2003). A desired variety of soybean usually contains a balanced amount of
protein and lipid.
1.5.2.1 Extraction methods
Hard screw and hydraulic pressing had been the two extraction methods
predominantly used in the industry until the 1950s, when direct solvent became
the preferable method (Nakamura and Hieronymus 1965). Today, solvent
extraction is solely the most common method for oil extraction from soybean
(Adesehinwa 2008). Similar to other oil crops, some pre-treatment steps are
needed before extraction. Seed should be dried to approximately 13 % MC,
28
cleaned and dehulled, but oil content is as high as to require pre-pressing before
solvent usage (Johnson, White et al. 2008). Although different solvents’
efficiency, such as ethanol, isopropanol, acetone, iso-hexane, heptane and
trichloroethylene (Johnson and Lusas 1983) have been investigated on soybean,
the solvent of choice is hexane (Johnson, White et al. 2008; Moscardi 2004). The
incorporation of enzymatic treatment without a significant alteration of the
conventional process is a growing domain in soybean oil extraction. In a study by
Domínguez et al. (1995), a mixed activity consisting of cellulase, hemi-cellulase
was studied in hexane extraction on soybean. During the study, the particle size
and MC was found to be the common difficulty in any enzymatic treatment on the
industrial scale. An increase of 5% or 8 -10% was demonstrated in extractable oil
if the treatment was administrated prior or simultaneous to the extraction. They
reported that the digestibility of the meal was improved by 3 % after the
treatment, and contained a higher concentration of free fatty acids and
phosphorous. The application of high-intensity ultrasound during extraction was
evaluated by Li et al. (2004) with the application of hexane, isopropanol and
hexane/isopropanol (3:2) as a solvent. A higher yield was achieved in ultrasound
assisted extraction with a mixed solvent. But, gas chromatography (GC) analyses
did not show a significant difference in fatty acid composition among the
extracted oil obtained from the individual solvents or their mixture.
29
1.5.2.2 Fatty acid composition
Soybean oil is classified as semi-drying oil, and is rich in polyunsaturated,
linoleic, and linolenic fatty acid (O'Brien 1998). More than 50 % of the dry mass
(DM) of soybean seeds is composed of protein and oil. Mature seeds usually
contain around 40% protein, 20% oil, 17% cellulose and hemicellulose, 7%
sugars, 5% crude fiber and 6% ash on a dry-mass basis (Rubel, Rinne et al.
1972). Triglycerides in soybean oil are characterized by a total absence of any
saturated fatty acids in the sn-2 position, high proportion of linoleic fatty acid in
the sn-2 position and random distribution of oleic and linolenic fatty acids on all
glycerol positions (O'Brien 1998).
In terms of fatty acid composition, soybean oil is characterized by linoleic
(53%), oleic (23%), palmitic (11%), linolenic (8%) and stearic acids (4%)
(Gunstone 2004a). A typical range of variation of fatty acids in soybean oil is
shown on Table 1.2.
Table 1.2: Fatty Acid composition of Soybean oil
Fatty acid composition,
%
Typical Range
C-14:0 Myristic
C-16:0 Palmitic
C-16:1 Palmitoleic
C-17:0 Margaric
0.1
10.6
0.1
0.1
<0.2
8.0 to 13.3
<0.2
-
30
C-18:0 Stearic
C-18:1 Oleic
C-18:2 Linoleic
C-18:3 Linolenic
C-20:0 Arachidic
C-20:1 Gadoleic
C-22:0 Behenic
C-22:1 Erucic
C-24:0 Lignoceric
4.0
23.3
53.7
7.6
0.3
-
0.3
-
-
2.4 to 5.4
17.7 to 26.1
49.8 to 57.1
5.5 to 9.5
0.1 to 0.6
<0.3
0.3 to 0.7
<0.3
<0.4
Data source: O'Brien, R. D. (1998). Fats and oils: formulating and processing for applications
1.6 Field pea
Field pea is a legume crop grown to eradicate protein malnutrition in the
cereal-based diet of people in the Mediterranean region (Osman, Ibrahim et al.
1990). Protein, starch, dietary fiber, low molecular weight carbohydrates, ash and
crude fat are the main components in the seeds (Daveby, Abrahamsson et al.
1993). In Canada, field pea is grown in the western regions of the country, and
the production is mostly used for domestic protein consumption. Similar to what
has been reported in soybean (Chung, Babka et al. 2003), protein content in field
pea seeds shows a negative correlation with lipid content, total yield and starch
content (Daveby, Abrahamsson et al. 1993; Al-Karaki and Ereifej 1997).
31
1.6.1 Lipid content and fatty acid composition
There is limited published research on pea lipid content, mainly due to a
relatively small fraction of lipid deposit in the seed, compared to protein and
starch. Sessa and Rackis (1977) measured the crude oil at 2.57 % in a study to
identify lipid-derived flavours of underblanched pea seeds. Research by Welch,
and Wynne Griffiths (1984) reported a variation range of 1.4 to 2.8 % in lipid
content, measured in 18 pea cultivars. Coxon and Wright (1985) reported that
field pea seeds typically contain 3% of oil content. They screened the lipid
content of a variety of pea genotypes to quantify crude lipid and FAMEs with a
gravimetrical method and GC analysis. The analyses were performed by
chloroform:methanol (2:1 V:V) for crude oil measurement and water-saturated n-
butanol for the FAME extraction. They calculated approximately 2.5 % FAMEs
and 4 % crude oil, and described that the difference between the two contents is
because the FAME extraction method is only reliable when all lipid content
consists of triglyceride. Daveby et al. (1993) made a comparison between
carbohydrate, protein, crude fat and other seed components in four stages of
plant development. In their study, crude fat was extracted from three Swedish
pea varieties with diethyl ether in a Tecator Soxtec after acid hydrolyses. The
study showed a range of 1.9 to 2.6 % in oil content in mature plants’ seeds. It
was reported by the study that the lipid content is reduced through plant
32
maturation. A relatively higher upper limit of 4.7 % (Bastianelli, Grosjean et al.
1998) and 9.7 % (Letzelter, Wilson et al. 1995) was reported by other studies on
field pea seeds. Al Karaki and Ereifej (1997) studied chemical composition of
seeds grown in arid and semi-arid areas, and showed an inverse relation
between yield, lipid and starch content with protein and three types of sugar
content (glucose, fructose and sucrose). In terms of fat production, the study
reported lipid content of twenty field pea genotypes ranging from 6.4 to the
maximum of 22.9 Kg/ha. The highest fat production was observed among the
peas grown in semi-arid location. Data presented by Nikolopoulou et al. (2007)
confirms the climate conditions and soil characteristics of the cultivation area
during the growing season to have significant effect on the total lipid content and
its composition. A higher lipid production, at least by 1.2 %, was observed in
2003 production compared with 2004 in the same growing location, which was
due to a lower average temperature in 2003.
Murcia and Rincón (1992) characterized the fatty acid composition in field
pea seeds, and divided seed to four groups according to their size: super fine
(SF, from 4.7 to 7.5 mm), very fine (VF, from 7.6 to 8.2 mm), fine (FN, from 8.3 to
8.8 mm) and middle (MD, from 8.9 to 10.2 mm). They extracted crude oil and
FAME by water based n-butanol, and the results showed the most commonly
found fatty acids in fresh peas are linoleic acid in small and medium (SF, VF, FN)
33
and palmitic acid in larger (MD) seed accessions. Linolenic acid was the rarest
fatty acid in all sizes. The study reported that lipid accumulation ends when pea
seeds are still quite small (in terms of diameter), and when the seeds grow in
size, lipid composition changes toward saturation of fatty acids. The lipid content
in pea seeds was reported by Ryan et al. (2007) to contain phytosterol.
Phytosterol is an unsaponifiable lipid fraction in food, which has a wide spectrum
of biological effects, such as anti-inflammatory, anti-oxidative and
anticarcinogenic activities as well as restraining the intestinal absorption of
cholesterol. Total phytosterol content found in pea seeds was 242 mg/100 g on a
dry-mass basis. They employed the hexane/isopropanol (3:2 v/v) for lipid
extraction and GLC for fatty acid analysis. Oil content in pea seeds was
estimated at 1.5%, with more than half being composed of PUFA (Table 1.3).
Table 1.3: Total oil content and composition in different crops
Data source: Ryan, E., K. Galvin, et al. (2007).
34
Field pea seed oil is mainly composed of 61% triglycerides (more than half
being 18:1) and 49% phospholipids (Sessa and Rackis 1977). This composition
was slightly different in another research, where the lipid content was reported to
have a distribution of phospholipids from 52.2 to 61.3% and triacyglycerids from
31.2 to 40.3% (Yoshida, Tomiyama et al. 2007). In terms of fatty acid content,
palmitic (16:0), stearic (18:0), oleic (18:1), linoleic (18:2) and linolenic (18: 3)
were reported to compose more than 99 % of the total lipid content in field pea
seeds (Coxon and Wright 1985).
1.7 Conclusion
The global growing demand of vegetable oil is due to the higher
consumption of edible oil, the novel industrial applications and a global interest to
find alternatives to petrochemical products. To balance the demand and supply
of vegetable oil commodities, oilseed production has been increasing by different
approaches, such as extending cultivation area of oil crops, breeding and genetic
modification.
Field pea is a valuable source of protein and starch, but lipid content in
their seeds has been usually considered an undesirable component that
deteriorates food properties. A new value can be added to pea seeds by
developing lipid content, which has always been underestimated. A convenient
35
method to quantify lipid content in pea seeds can be found by a comparison of
common extraction methodologies on field pea. Determination of fatty acid
composition is needed to propose the product’s usage in the industry.
Modification of this composition by genetics and breeding is a useful method to
acquire nutritious food and environmentally-friendly materials for the
oleochemical industries.
36
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Connecting Statement
Diversity among various lipid extraction methods emphasizes the
importance of analytical method selection in research. Lipid extraction methods
vary in extraction efficiency, which is due to the physical or chemical compatibility
on different substances. In the context of this research, a variety of lipid
extraction methods were examined on canola and soybean, and validated by
comparing the results to previous research. Furthermore, a comparison between
the results of the selected methods on field pea led to determine the most
convenient method for screening lipid content in pea accessions.
48
Chapter 2: Method validation for lipid extraction, a comparative study
of seven analytical procedures on field pea (Pisum sativum), canola
(Brassica napus) and soybean (Glycine max)
2.1 Abstract
A lipid extraction method is selected based on the solvents’ chemical
compatibility to solubilize the maximum lipid content in a sample. In order to
select the most efficient extraction method on field pea (Pisum sativum) seeds,
seven extraction methods were evaluated by comparing the results on canola
(Brassica napus) and soybean (Glycine max). The results of the selected
methods on field pea ranged from 0.66 % to 2.0 % of the dry mass (DM).
Analysis of variance (ANOVA) found the difference in sample type (p< 0.0001)
and extraction method (p=0.0114) to be statistically significant. The most
effective method on pea was the Bligh & Dyer with 2.0 % of yield, compared to
the Soxhlet (with hexane or petroleum ether), as the least effective method,
averaged 0.8 % in yield, and the butanol and the hexane/isopropanol methods
with a medium result between 1.5 to 1.7 %. Our experiments on field pea showed
that a binary solvent system of hexane/isopropanol gives a relatively higher result
than the single solvent hexane. According to the experiments, the butanol and
the hexane/isopropanol methods are the most convenient, fast screening
methods to be employed in the oilseed pea project. The objective of this paper
was to investigate the variation of total lipid content (including fat and oil) in pea
49
seeds in a broad selection of accessions including bred cultivars and wild
accessions.
2.2 Introduction
Lipid quantification methods are routinely chosen according to the
research purpose and the nature of the samples. Lipid quantification methods are
generally classified into three groups known as solvent, non-solvent and
instrumental methods (Ghatak 2011). In the solvent methods, lipids are isolated
from cellular milieus by being dissolved in lipid solvents, such as hexane,
petroleum ether and chloroform (Akoh and Min 2008). The mixture of solvent and
lipid has to be separated from the remaining pellet before quantification, and can
be measured directly or indirectly. In direct measurement, the oil content is
weighed after the solvent is evaporated from the mixture of solvent and dissolved
lipid. In indirect measurements, the oil content is quantified by calculating the
mass difference between the initial sample and the remaining pellet after it is
completely dried (Luthria 2004). Solvent methods are extensively used for plant
tissues, oilseeds and marine organisms (Wrolstad 2005; Gunstone 2004). In non-
solvents methods, the lipid content is measured by using non-organic reagents
such as alcohols. In these methods, lipid content is measured after the sample is
digested and the bound lipids are released. Non-solvent methods are common
50
extraction methodologies in dairy product analyses (Wrolstad 2005; Gunstone
2004). With the instrumental methods, the concentration of specific types of lipids
are estimated by measuring their physical property in the sample, such as the
mass and charge employed in chromatography and spectrometry (Ghatak 2011).
Lipids have non-polar groups in their structure, which cause all lipids to be
insoluble in water (hydrophobicity or amphiphilicity) (Rahman 2008). Since
hydrophobicity is a relative quantity (DeVido, Dorsey et al. 1998), lipids are
classified into two groups of non-polar and slightly polar (Gunstone, Harwood et
al. 1994; Gurr, Harwood et al. 2002; Vance and Vance 2002), depending on the
molecular constituents. Organic solvents, which have a high capability of
dissolving lipid materials, can be similarly sub-grouped by polarity index (PI)
comparison (Table 2.1). The more the polarity of a solvent is in agreement with a
solute, the more efficient the chemical is in extracting the targeted lipid material
(Akoh and Min 2002).
51
Table 2.1: Solvents polarity index
Non-polar solvent Polarity index
Hexane 0
Cyclohexane 0.2
Petroleum ether 0.1
Polar solvent Polarity index
n-butanol 4
Chloroform 4.1
ethul ether 2.8
Methanol 5
isopropanol 3.9
Data source: Online data base, Solvent Polarity and Miscibility by Byers (2003).
There are various organic solvents used for lipid extraction. Since the
1930s, when the preferred solvent of almost all oilseed mills was paraffin
hydrocarbon (Goss 1946), researchers have been continuously updating the list
of functional lipid solvents by testing organic chemicals on oilseeds. Among the
studied solvents, hexane became the most common choice of producers due to
its efficiency, minimum extraction of non-oil materials, and easy separation from
the extracted crude oil (Akoh 2006; Johnson and Lusas 1983). There are other
solvents, such as petroleum ether, diethyl ether, methanol, ethanol, propanol,
isopropanol, butanol and acetone, which have been shown to have great
advantages in extracting the lipid content of food samples. However, methods
involving these solvents are mainly applied in laboratories since they are not
52
economically feasible in the oilseed industry, resulting in the production of toxic
meal and other safety issues, or do not meet the criteria of bio-renewability and
environmental friendliness (Wan and Wakelyn 1997). In addition to organic
solvents, solvent extraction methods differ by the extraction apparatuses. There
are several automated and semi-automated extraction apparatuses that offer
more accurate and efficient lipid extraction by reducing errors via less human
involvement and optimizing extraction condition. There has been considerable
development in designing precise extraction apparatuses, such as Soxhlet or
supercritical fluid extraction (SFE) systems. However, they are not easily
accessible because of the cost of equipments and need for highly trained
operators.
There has been a limited amount of research on lipid content in field pea
seeds, since starch and protein content have always been considered as the
main values of the crop (Sosulski, Hoover et al. 1985; Sosulski and McCurdy
1987; Small 1997). As a result, there is a lack of data in the literature about the
extraction procedures that produce reliable results for lipid quantification in field
pea. The choice of extraction method needs to be evaluated by conducting a
comparison of approved methods in oilseeds (Barthet, Chornick et al. 2002;
Moreau, Powell et al. 2003).
53
The research presented in this paper will first, evaluate the result of
different extraction methodologies on current oilseeds to validate the
experimental conditions; and second, determine the most expedient extraction
procedure on field pea. The first objective was carried out by testing a selection
of extraction methods on canola and soybean, for which there is agreement on
the range of lipid content in the literature (Singh and Hymowitz 1999; Taylor, Eller
et al. 1997). The results were compared with the published research to confirm
the experiment conditions and evaluate methods’ efficiency. The second
objective of the research was achieved by comparing the results of the lipid
extraction methods on field pea seeds to determine the most efficient
procedures. The results were used to select the most convenient method to
apply in the screening stage of different field peas, where approximately 170
accessions were evaluated for the total lipid content in their seeds.
2.3 Experimental
2.3.1 Sampling
Seed of field pea (Pisum sativum L., cv. Cutlass) and canola (Brassica
napus L., cv. Roper) were obtained from plants grown in 2009 on the Lefsrud
farm (Viking, Alberta, Canada). Seeds of soybean (Glycine max, cv. Champion),
were obtained from plants grown in 2009 at the Belcan Center (Saint-Marthe,
54
Quebec, Canada). The seeds were dried by placing the pods in paper bags and
left in the oven at 60 °C for 48 h. After drying, seeds were ground by a Black and
Decker coffee grinder (CBG100S, Richmond Hill, Ontario, Canada) for 1-2 min,
until fine powder was obtained as described by Hoover et al. (1988), but the size
of particles was not measured.
2.3.2 Chemicals
1-butanol (Certified ACS), hexanes (Certified ACS), 2-propanol (Certified
ACS Plus), methanol (Certified ACS), chloroform (Approx. 0.75% ethanol as
preservative/Certified ACS), cyclohexane (Certified ACS), petroleum ether
(Certified ACS), were purchased from Fisher Chemical (Ottawa, Ontario,
Canada).
2.3.3 Instrumentation
50 ml plastic centrifuge tubes, plastic pipette (15 ml) and glass pipette (15
ml) were acquired from Fisher Scientific. Test tubes were weighed by an
analytical balance (APX-153). Other instruments used in our experiments, such
as tube rotator (VWR, H005302, Mississauga, Ontario, Canada), Fischer
centrifuge, Fisher vortex mixer (Standard 120V), nitrogen evaporator (NEVAP-
55
111, Berlin, MA, USA), microwave reactor (CEM, Ottawa, Ontario, Canada) and
Soxhlet extractor (VELP scientifica, SER-148 Italy) were accessed at McGill
University.
2.3.4 Methods used for gravimetric determination of total lipid content
2.3.4.1 Butanol extraction method
The summary of the butanol extraction is listed below, as described by
Murcia and Rincón (1992). Two grams of ground sample was added to screw-
capped centrifugal plastic tubes of known-mass in triplicate. A second tube with
the same amount of sample was prepared as a control tube to measure moisture
content. 20 ml of n-butanol was added to the test tubes and was placed in the
tube rotator for 30 min, followed by 10 min of centrifuge at 3000 rpm. The two
separated phases consist of solid material in the lower layer, and a mixture of
solvent and dissolved lipid in the top layer. The top layer was decanted off into a
waste container with special attention to avoid sample loss. The experiment was
continued by adding fresh solvent, and the extraction steps were repeated twice.
The test tubes were placed in the nitrogen evaporator for up to 30 min at 70 °C
until the remaining solvent was completely evaporated. The test tubes along with
the control were placed in the oven for 24 h at 95 °C, and were covered with
caps after removal from the oven. The final mass of the tubes was recorded after
56
leaving them in a lab-made drierite box to allow them to reach room temperature.
The difference between initial and final sample mass of the control tube, which
represents the moisture loss during the drying period, was subtracted from the
difference of test tube mass to calculate the oil content percentage of the
samples.
2.3.4.2 Hexane/Isopropanol
The hexane method was a modified version as described by Ryan et al.
(2007). Two grams of ground sample was weighed into three test tubes. Six ml of
solvent (hexane/Isopropanol 3:2, v:v) was added to the tubes and placed in the
tube rotator for one h. After 10 min of centrifuge, the solvent layer was
transferred into a second tube of known mass. The remaining pellet was washed
twice with four ml of fresh solvent. Each wash was followed by a transfer of the
solvent into the solvent tube after a 30 s of vortex and 10 min of centrifuge at
3000 rpm. Contrary to the butanol extraction, the oil content was quantified by
direct measurement of lipid left in the solvent tube after the solvent was
evaporated under nitrogen stream at 60 °C for 3 h.
57
2.3.4.3 Chloroform/methanol
The chloroform/methanol method is a modification of the Bligh and Dyer
(1959) method which was developed for dry samples, as described by
Manirakiza et al. (2001). In the first extraction, eight ml of methanol and four ml of
chloroform were added to 2 g of ground sample in the test tubes. Tubes were
vortexed for two min, and another four ml of chloroform was added to the
sample. Tubes were shaken vigorously by hand for two min. 7.2 ml of distilled
water was added to each tube, and vortexed for two min. After 10 min of
centrifuge at 3000 rpm, the lower layer was transferred into an empty weighed
tube (solvent tube) by a Pasteur pipette or a syringe.
The second extraction was started by adding eight ml of methanol in chloroform
(10% v/v) to the test tubes. The tubes were vortexed for two min, and centrifuged
for 10 min. The upper layer was decanted off into the solvent tube. The solvent
was evaporated off under nitrogen stream at 104 °C for 3 h. Total lipid content
was calculated directly from the mass of the lipid recovered in the solvent tubes.
2.3.4.4 Soxhlet extraction
The Soxhlet extraction method was performed as described in the
apparatus manual. A 5 g sample was added to a cellulose thimble in triplicate.
58
Soxhlet apparatus was assembled with the thimbles and a solvent (petroleum
ether or hexane). The Soxhlet extraction with petroleum ether solvent was
performed on the ground sample with 30 min of immersion, 45 min of washing
and 15 min of recovery at 130 °C.
The Soxhlet extraction with hexane solvent was performed on the ground
sample with 45 min of immersion, 45 min of washing and 15 min of recovery at
180 °C. The lipid content of sample was directly measured by the mass of lipid
recovered in the Soxhlet extraction beaker.
2.3.4.5 Modified Bligh and Dyer
The modified Bligh and Dyer is similar to the Blight & Dyer (Bligh and
Dyer 1959), modified by replacing chloroform and methanol with propanol and
cyclohexane as described by Manirakiza et al. (2001). The first extraction cycle
was performed by adding water:propan-2-ol: cyclohexane (11: 8: 10) to the test
tubes, and vortexed for two min. 7.2 ml of distilled water was added to the tubes
and vortexed for two min followed by 10 min of centrifuge at 3000 rpm. Three
layers formed in the test tubes: water, the solvent layer and the solid material,
from top to bottom. The solvent layer (in the middle) was transferred into a
second tube of known mass by a Pasteur pipette or a syringe.
59
The second extraction was carried out by adding eight ml of propan-2-ol:
cyclohexane (10% v/v) to the test tubes. The mixture was vortexed for two min,
and centrifuged for 10 min. The cyclohexane phase was transferred into the first
extract and the solvent was evaporated by placing the tubes in the nitrogen
evaporator for 1 h at 104 °C. The total lipid content was directly quantified from
the mass of recovered lipid in the solvent tube.
2.3.4.6 Microwave extraction
The microwave extraction method was performed by adding 5 g of sample
and 25 ml of hexane into a glass vessel. The vessel was fitted in the microwave
digester and run for 2 min at 2.45 GHz frequency and 200 W power. After the
vessel cooled, the content was decanted off into a second vessel. The extraction
steps were repeated twice and the content of both vessels were filtered through
an assembled vacuum filter system. The mixture of solvent and lipid was
transferred into a regular centrifuge tube of known mass. The supernatant was
dried under nitrogen stream for 3 h at 60 °C, and the total lipid content was
directly measured by mass. The experiment was run in triplicate for each sample.
60
2.3.5 Statistical analysis
Statistical analyses of data were performed using SAS 9.2, Version
6.1.7601 for Windows operating system (SAS Institute Inc., Toronto, ON,
Canada). The effect of sample types and extraction methods were analyzed as
fixed effects in a mixed ANOVA model (Multi-way Classification), and calculated
F-ratios were compared with the tabulated F-value at P, 0.05 to determine the
significance of the terms in the model.
2.4 Results
The results (Table 2.2) show an overall range of recovered lipid from 0.66
% on field pea seeds to 46.2 % on canola seeds. Analysis of variance (ANOVA)
of the result found the difference in sample type (p<0.0001) and extraction
method (p=0.0114) to be statistically significant (Table 2.3).
61
Table 2.2: The extractable lipid content variation by extraction
method, %
Method Field pea (Pisum
sativum L., cv.
Cutlass)
Soybean
(Glycine max, cv.
Champion)
Canola (Brassica
napus L., cv.
Roper)
1- Butanol 1.22 ± 0.21 13.9 ± 1.9 41.8 ± 3.2
2- Hexan/isopropanol 1.6 ± 0.04 15.8 ± 0.4 34.8 ± 0.3
3- Bligh & Dyer 2.0 ± 0.02 15.8 ± 0.2 41.0 ± 0.8
4-Soxhlet (PE) 0.7 ± 0.03 13.3 ± 0.2 40.5 ± 0.3
5-Soxhlet(Hexane) 0.9 ± 0.05 16.6 ± 0.3 46.0 ± 0.2
Table 2.3: Analysis of variance, sample type and
lipid extraction method
Degree of
freedom
Sums of
squares
Mean
square F - ratio
Tabulated
F value Pr > F
Mean 1 16328.3 16328.3 3230.7 4.09
Sample 2 12063.4 6031.7 1193.4 3.24 <0.0001
Method 4 75.8 19.0 3.8 2.61 0.0114
Residual 38 192.1 5.1
62
Our experiments showed the total lipid content for canola ranges from
34.4 to 46.2 % and for soybean from 12.0 to 17.2 % of the DM, depending on the
extraction method. Among the selected methods, Soxhlet with hexane showed
the highest extraction efficiency for both oilseeds. The least efficient methods
were the hexane/isopropanol and the butanol methods for canola and soybean,
respectively. According to the extraction result on canola seeds, the extraction
methods can be divided into three groups of: hexane/isopropanol as low, Blight &
Dyer, Soxhlet with petroleum ether as medium and butanol, Soxhlet with hexane
as high efficiency methods. Similar analysis on soybean revealed only two
groups of Soxhlet (with hexane), Bligh & Dyer and the hexane/Isopropanol
methods characterized with medium and butanol, Soxhlet (petroleum ether) with
limited capability in the lipid extraction.
Due to the nature of the sample (low lipid deposition), extracted lipid from
field pea seeds was considerably lower than canola and soybean, ranging from
0.66 to 2.0 % of the DM. The most effective method was the Bligh & Dyer with
2.0 % of yield, compared to the Soxhlet (with hexane or petroleum ether), which
is shown to be the least effective method, averaged 0.8 % in yield. The butanol
and the hexane/isopropanol methods gave a medium result between 1.5 to 1.7
%.
63
The results of microwave and modified Blight & Dyer are not reported in
the study since the results were extremely low and inconsistent for the three
types of seed.
2.5 Discussion
The total lipid content ranges from 13% to 22% in different soybean
varieties (Singh and Hymowitz 1999), with an average of 20% (Johnson, White et
al. 2008). This value is doubled in canola, which stores approximately 40% of its
mass as lipid content in seeds during plant maturation (Taylor, Eller et al. 1997).
However, some varieties of canola can extend the oil composition in their seeds
by up to 50% (Assadi, Janmohammadi et al. 2011). Field pea seeds were
reported by previous studies to contain 1 to 4 % of lipid content (Sessa and
Rackis 1977; Ryan, Galvin et al. 2007). The lipid extraction results for canola
averaged 40.8 ± 3.9 %, 15.1 ±1.6 % for soybean, and 1.3 ± 0.5 % for field pea.
The selected methodologies were confirmed to yield a result within the reported
range of lipid content by previous research on the three crops (Ryan, Galvin et al.
2007; Sessa and Rackis 1977; Singh and Hymowitz 1999; Taylor, Eller et al.
1997).
The statistically significant difference between the methods for each
sample was due to the variation in the solvents chemical compatibility to
64
solubilize various lipid molecules (Dobush, Ankney et al. 1985). An observed
orange color in Bligh & Dyer extracts compared with yellow color from the other
methods refers to chloroform capability of extracting carotenoids
(Taungbodhitham, Jones et al. 1998). The larger amount of lipid extracted by
Soxhlet extractor with hexane in contrast with petroleum ether, was due to a
better solubility of non-polar lipids in hexane (Wrolstad 2005). This indicates a
larger fraction of lipid content in the seeds being non-polar, which is supported by
previous studies on canola (Zaderimowski and Sosulski 1978), soybean
(Salunkhe 1992) and field pea (Sessa and Rackis 1977).
Our experiments on field pea showed that a binary solvent system of
hexane/isopropanol gives a relatively higher result than a single solvent of
hexane used in the Soxhlet method (Schäfer 1998). However, the result was
opposite on canola. The higher level of recovered oil from canola seeds by
hexane in Soxhlet extractor was due to a greater lipid solubility in a hot solvent
(Schäfer 1998). But, the lower result of the same method on pea may be a
device limitation for a minimum lipid content required for extraction.
Microwave extraction and modified Blight & Dyer did not result in uniform
results. During microwave extraction, the lipid content was underestimated,
which was due to parts of the lipid content immobilized on the sides of the flask,
making it impossible to include their mass in the final calculation of the extracted
65
oil. A similar difficulty was experienced in the modified Blight & Dyer procedure.
After the last centrifuge, there were three definite layers from which the middle
one contained the lipid content. Failure to separate the whole layer from the
surrounded media by a Pasteur pipette or a syringe caused a consistent
dissimilarity in the results of this method. In fact, this issue occurred in the
butanol and hexane/isopropanol as well, but a solution was discovered to solve
the problem. It was experienced that by carefully drawing off the upper level of
the fluid with a slow and gradual inclination of the tube, the amount of solid
material transferred into a new tube was minimized. As an additional approach in
the hexane/isopropanol method, the tubes were re-centrifuged to purify the liquid
from the solid, and the amount of solid material was negligible in the solvent.
In conclusion, our experiments shows that among the selected methods, the
butanol and the hexane/isopropanol methods are the most convenient, fast
screening methods to be employed in the oilseed pea project.
66
2.6 References
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biotechnology. New York: Marcel Dekker.
Akoh, C.C. 2006. Handbook of functional lipids: CRC Press.
Akoh, C.C., and D.B. Min. 2008. Food lipids: chemistry, nutrition, and
biotechnology: CRC Press.
Assadi, E., H. Janmohammadi, A. Taghizadeh, and S. Alijani. 2011. "Nutrient
composition of different varieties of full-fat canola seed and nitrogen-
corrected true metabolizable energy of full-fat canola seed with or without
enzyme addition and thermal processing." The Journal of Applied Poultry
Research no. 20 (1):95.
Barthet, V.J., T. Chornick, and J. K. Daun. 2002. "Comparison of Methods to
Measure the Oil Contents in Oilseeds." Journal of Oleo Science no. 51
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Bligh, E.G., and W.J. Dyer. 1959. "A rapid method of total lipid extraction and
purification." Canadian Journal of Biochemistry and Physiology no. 37
(8):911-917.
Byers, J. A. . 2003. Solvent Polarity and Miscibility.
DeVido, D. R., J. G. Dorsey, H. S. Chan, and K. A. Dill. 1998. "Oil/Water
Partitioning Has a Different Thermodynamic Signature When the Oil
Solvent Chains Are Aligned Than When They Are Amorphous." The
Journal of Physical Chemistry B no. 102 (37):7272-7279.
Dobush, G.R., C.D. Ankney, and D.G. Krementz. 1985. "The effect of apparatus,
extraction time, and solvent type on lipid extractions of snow geese."
Canadian Journal of Zoology no. 63 (8):1917-1920.
Ghatak, K.L. 2011. Techniques and Methods in Biology: PHI Learning Private
Ltd.
Goss, W. 1946. "Solvent extraction of oilseeds." Journal of the American Oil
Chemists' Society no. 23 (11):348-354.
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Gunstone, F.D. 2004. Rapeseed and canola oil : production, processing,
properties and uses: Blackwell Publication ; CRC Press.
Gunstone, F.D., J.L. Harwood, and F.B. Padley. 1994. The Lipid handbook:
Chapman and Hall.
Gurr, M.I., J.L. Harwood, and K.N. Frayn. 2002. Lipid biochemistry. Malden,
Mass.: Blackwell Science.
Hoover, R., L. Cloutier, S. Dalton, and F. W. Sosulski. 1988. "Lipid Composition
of Field Pea (Pisum sativum cv. Trapper) Seed and Starch." Starch -
Stärke no. 40 (9):336-342.
Johnson, L., and E. Lusas. 1983. "Comparison of alternative solvents for oils
extraction." Journal of the American Oil Chemists' Society no. 60 (2):229-
242.
Johnson, L.A., P.J. White, and R. Galloway. 2008. Soybeans : chemistry,
production, processing, and utilization: AOCS Press.
Luthria, D. L. 2004. Oil extraction and analysis critical issues and comparative
studies: AOCS Press.
Manirakiza, P., A. Covaci, and P. Schepens. 2001. "Comparative Study on Total
Lipid Determination using Soxhlet, Roese-Gottlieb, Bligh & Dyer, and
Modified Bligh & Dyer Extraction Methods." Journal of Food Composition
and Analysis no. 14 (1):93-100.
Moreau, R., M. Powell, and V. Singh. 2003. "Pressurized liquid extraction of polar
and nonpolar lipids in corn and oats with hexane, methylene chloride,
isopropanol, and ethanol." Journal of the American Oil Chemists' Society
no. 80 (11):1063-1067.
Murcia, M.A., and F. Rincón. 1992. "Size as source of variance in lipid
composition of pea." Food Chemistry no. 44 (1):29-35.
Rahman, A. 2008. Studies in Natural Products Chemistry: Elsevier.
Ryan, E., K. Galvin, T. O’Connor, A. Maguire, and N. O’Brien. 2007. "Phytosterol,
Squalene, Tocopherol Content and Fatty Acid Profile of Selected Seeds,
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Grains, and Legumes." Plant Foods for Human Nutrition (Formerly
Qualitas Plantarum) no. 62 (3):85-91.
Salunkhe, D.K. 1992. World oilseeds: chemistry, technology, and utilization: Van
Nostrand Reinhold.
Schäfer, K. 1998. "Accelerated solvent extraction of lipids for determining the
fatty acid composition of biological material." Analytica Chimica Acta no.
358 (1):69-77.
Sessa, D., and J. Rackis. 1977. "Lipid-Derived flavors of legume protein
products." Journal of the American Oil Chemists' Society no. 54 (10):468-
473.
Singh, R.J., and T. Hymowitz. 1999. "Soybean genetic resources and crop
improvement." Genome no. 42 (4):605-616.
Small, D.B.M.E. 1997. Vegetables of Canada: NRC Research Press.
Sosulski, F.W., R. Hoover, R.T. Tyler, E.D. Murray, and S.D. Arntfield. 1985.
"Differential Scanning Calorimetry of Air Classified Starch and Protein
Fractions from Eight Legume Species." Starch Stärke no. 37 (8):257-262.
Sosulski, F.W., and A.R. McCurdy. 1987. "Functionality of Flours, Protein
Fractions and Isolates from Field Peas and Faba Bean." Journal of Food
Science no. 52 (4):1010-1014.
Taungbodhitham, A. K., G. P. Jones, M. L. Wahlqvist, and D. R. Briggs. 1998.
"Evaluation of extraction method for the analysis of carotenoids in fruits
and vegetables." Food Chemistry no. 63 (4):577-584.
Taylor, S.L., F.J. Eller, and J.W. King. 1997. "A comparison of oil and fat content
in oilseeds and ground beef--using supercritical fluid extraction and related
analytical techniques." Food Research International no. 30 (5):365-370.
Vance, D.E., and J.E. Vance. 2002. Biochemistry of lipids, lipoproteins, and
membranes: Elsevier.
Wan, Peter J., and P.J. Wakelyn. 1997. Technology and solvents for extracting
oilseeds and nonpetroleum oils: AOCS Press.
Wrolstad, R. E. 2005. Handbook of food analytical chemistry: John Wiley & Sons.
69
Zaderimowski, R., and F. Sosulski. 1978. "Composition of total lipids in
rapeseed." Journal of the American Oil Chemists' Society no. 55 (12):870-
872.
70
Connection Statement
Lipid content has not been developed as a trait in field pea cultivars, and
the possibility of having a lipid deposit in pea seeds is yet to be explored. In order
to investigate the possibility of developing a novel oilseed pea, a broad range of
wild accessions and cultivars were acquired and grown in McGill University,
Quebec, Canada. By this research, the variation of lipid content in the 134 field
pea accessions was measured by the butanol extraction, which was validated in
the previous chapter. The interaction between lipid content and physical
characteristics of field pea accessions was statistically analyzed to find the
controlling factors on the total lipid content. This information will help breeders to
progress the trait through selective breeding.
71
Chapter 3: Lipid content variation in field pea (Pisum sativum)
accessions
3.1 Abstract
The greater need for calorie supply in the human diet along with
increasing interest to replace petrochemical products has increased the demand
for vegetable oil in the second half of the 20th century. To meet this growing
demand, oilseed production has increased globally by different approaches. With
this research, 134 field pea accessions, grown in 2009 and 2010 at McGill
University, Canada, were evaluated for the lipid content of their seeds. The data
will be used by pea breeders to investigate the possibility of developing a dual-
purpose oilseed pea for western Canada, which can add to the total vegetable oil
production in Canada. The results collected from 134 grown accessions, ranged
from 0.3 % to 6.3 %. Analysis of variance (ANOVA) revealed a significant
difference between accessions (p<0.0001), the growing years (p=0.0002) and
the interaction between the two factors (p<0.0001). The analysis of variance
revealed a significant difference between wrinkled and smooth surfaced seeds
(p= 0.001), but seed color, flower color, plant height and mass of 100 seeds had
no effect on the total lipid production in pea seeds. Wrinkled seeds were shown
to contain more lipid content as compared with smooth and medium surfaced
seeds.
72
3.2 Introduction
The term vegetable oil commonly refers to the lipid materials derived from
oilseed crops, which can be either liquid (oil) or solid (fat) at room temperature
(Gunstone, Harwood et al. 1994). Vegetable oils are used in a broad range of
applications in both the food and the oleochemical industries.
Increasing the production of agricultural commodities has been considered
as a means of combating the world hunger in the last three decades (Byron
1982). The world population has almost tripled from 2.5 billion in 1950 to 6.9
billion in 2010, and is expected to reach 8 billion by 2025 (Table 3.1).
Table 3.1: World Population, 1950-2050
73
Edible vegetable oil is one of the main sources of energy in the human
diet supplying approximately 30% of daily calories (Dosti and Kidushim-Allen
1991). The greater need for calorie supply by growing population along with
increasing attention to replace petrochemical products has caused a greater
demand for vegetable oil in the second half of the 20th century (Green 1991).
To meet the fast growing demand for vegetable oil, oilseed production has
increased globally, from 56 million tons in 1990 to 88 million tons in 2000
(Demirbaş 2008), and was reported to reach 130 million tons in 2009 (Table 3.2).
Table 3.2: World total oilseed crop production
Crop Production (MT)
Palm
Soybean
Rapeseed
Sunflower seed
Cottonseed
Peanut
Palm Kernel
Coconut
Olive
Total
41.7
35.7
19.9
10.8
4.8
4.9
5.3
3.5
3
129.5
Source: USDA, Oil Crops by Vollmann et al. (2009)
74
The increase in vegetable oil production is achieved by a combination of
approaches according to the agronomic potentials of a district, such as available
agricultural land to increase oilseed cultivation area (OECD 2008). An approach
being evaluated by this paper is to develop a dual-purpose crop in Canada for
lipid content and a second product (protein and starch). An example of a dual-
purpose crop is soybean, which is produced for both lipid and protein from the
seeds (Singh 2010).
The major oilseed crops in Canada are canola and soybean (FAOSTAT
2009). The total production of canola in 2008 was approximately 12.5 million
tons, and soybean production was 3.3 million tones (Table 3.3).
Table 3.3: World total oilseed crop production
Rank Commodity Production (Int
$1000)
Production (MT)
1
2
3
4
5
6
7
8
9
10
11
Wheat
Rapeseed
Indigenous pig meat
Indigenous cattle meat
Cow milk, whole, fresh
Indigenous chicken meat
Potatoes
Peas, dry
Soybeans
Maize
Hen eggs, in shell
4462759
3442243
2874331
2647437
2164751
1166420
656272
653547
598918
451757
328160
28611100
12642900
2838425
1280019
8140000
1000000
4724460
3571300
3335900
10592000
419013
75
12
13
14
15
16
17
18
19
20
Lentils
Linseed
Tomatoes
Indigenous turkey meat
Blueberries
Mushrooms and truffles
Apples
Beans, dry
Tobacco, unmanufactured
225685
194577
182450
171596
150773
144110
122602
112334
80221
1043200
861100
770059
157000
95516
86946
426858
266200
44000
Source: USDA, Oil Crops by Vollmann et al. (2009)
Canola is predominantly grown in western Canada due to the crop cold
tolerance while soybean is mainly cultivated in more tempered regions in the
eastern part of country. Soybean seeds contain about 40 % of edible protein,
suitable for human consumption or animal feed, as well as 20 % of oil content
(Vollmann and Rajcan 2009). In addition to seed composition, soybean has other
advantages, such as lower need for nitrogen fertilizer in the soil. Soybean is not
well established in western Canada since it requires a temperature of 24 to 32 °C
(Winch 2007) for optimum growth while the average temperature in western
Canada normally does not rise above 20 °C during the growing season. Low
temperatures have a negative effect on soybean production and no great
success has yet been achieved in developing a cold-tolerate soybean for western
Canada (Schmid and Keller 1980; Gass, Schori et al. 1996).
76
Field pea, which is a cold-tolerate legume crop is the most common
legume grown in western provinces of Alberta and Saskatchewan and Manitoba
(Figure 3.1).
Figure 3.1: Pea growing area in Canada
Source: The Siliceous Group, (2010)
Field peas (also known as dry peas), belongs to the family of cool season
legume crops, commonly referred to as pulses. Pea is the eighth most important
crop in Canada with total production of 3.5 million tons in 2008 (Table 3.3). Field
pea acreage in Canada has increased since 1985, rising from 74,400 ha to
1,261,000 ha in 2007 (PRRP et al. 2008), and has made Canada the world
77
leader in pea production. There are economic and environmental advantages of
field pea to be developed as a novel oilseed crop for western Canada:
Like other plants from Fabiaceae (Leguminosae) family, field pea forms a
symbiotic relationship with rhizobia bacteria and fixes atmosphere
nitrogen, and therefore, reduces the amount of nitrogen fertilizer required
for the cropping system.
The crop is well adapted to the cold climate of western regions of
Canada.
The crop is grown throughout western Canada, and it requires a shorter
time for acceptance by farmers.
Field pea seeds contain crude protein, 22% to 25% and carbohydrate,
50% to 60% (Coyne, Grusak et al. 2005; Daveby, Abrahamsson et al.
1993; Nikolopoulou, Grigorakis et al. 2007).
Field pea and soybean belong to the same family; the genomic
knowledge of soybean can be used to develop a novel oilseed pea.
There is limited research on the variation of lipid content in field pea
accessions. Field pea has been traditionally grown and evaluated for protein and
carbohydrate content (Small 1997; Sosulski and McCurdy 1987; Sosulski,
Hoover et al. 1985). Past research conducted on total lipid content of field pea
78
were mostly interested in the effect of lipid content in relation with the quality of
foods made from pea seeds. Sessa and Rackis (1977) who have reported 2.57
% of crude oil in field pea in their research, were primarily interested in lipid-
derived flavors in the seeds. Lipid content in field pea seeds was estimated at 1.5
% by Ryan et al (2007) in a research to investigate phytosterols (an
unsaponifiable lipid in foods with preferred biological effects, such as anti-
inflammatory, anti-oxidative, and anticarcinogenic) in pea seeds. Letzelter et al
(1995) reported total fat in pea ranging from 1.7 to 9.7 % of the dry mass (DM).
Bastianelli et al. (1998) arranged field peas into different categories and
evaluated feeding value of the seeds according to chemical composition, and
reported the lipid content in field pea seeds could range up to 35%.
The objective of this paper is to investigate the variation of total lipid content
(including fat and oil) in pea seeds in a broad selection of accessions including
bred cultivars and wild accessions.
3.3 Experimental
3.3.1 Sampling
Seeds of 174 field pea accessions (Pisum sativum L.) were acquired from
the Plant Gene Resources of Canada (Saskatoon, SK) and the pea collection of
the U.S. Department of Agriculture (Pullman, WA). Seeds were grown in two
79
following years, 2009 and 2010 at a field site, 25 by 40 m plot with loamy clay soil
condition, located at Macdonald Campus of McGill University, Ste-Anne-de-
Bellevue, Quebec, Canada (Lat: 45 24' 29'' Long: -73 56' 10''). The plot was tilled
twice before planting in each year. In 2009 seeds were planted on May 20th, and
harvested on Aug 30th. In 2010 seeds were planted on May 2nd, and harvested
on Aug 30th. Weeds were controlled by hand and a small gas rototiller during the
growing season. Peas were harvested when the pods were brown and dry. Each
pod was harvested by holding the steam at the joint of the flower and holding the
pod firmly in the other hand and pulling. The pods were placed in paper bags and
left in the oven at 60 °C for 48 hours to dry. Pods were emptied, and seeds were
ground by a Black and Decker coffee grinder (CBG100S, Richmond Hill, Ontario,
Canada) for 1-2 min until a fine powder was obtained as described by Hoover et
al. (1988), but the size of particles was not measured.
Plant characteristics, such as flower color, seed color and seed surface
type were visually compared and documented, and plant height was manually
measured and averaged among grown plants of the same accession. Seed
density was measured by weighting 100 seeds, or adjusting the mass if less than
100 seeds were acquired from the grown pea accession.
80
3.3.2 Chemicals
1-butanol (Certified ACS) was purchased from Fisher Scientific (Ottawa, Ontario,
Canada).
3.3.3 Instrumentation
50 ml plastic centrifuge tubes, plastic pipettes (15 ml) and glass pipettes
(15 ml) were acquired from Fisher Scientific. Test tubes were weighed by an
analytical balance MK (APX-153, Buckinghamshire, United Kingdom). Other
instruments used in our experiments, such as tube rotator VWR (H005302,
Mississauga, Ontario, Canada), Fisher centrifuge, Fisher scientific vortex mixers
(standard 120V), nitrogen evaporator (NEVAP-111, Berlin, MA, USA) were
accessed at McGill University.
3.3.4 Methods used for Gravimetric determination of total lipid
content
3.3.4.1 Butanol extraction method
The butanol extraction is listed below, as described by Murcia and Rincón
(1992). This method was confirmed to be convenient for the purpose of this
research (Chapter 2). Two g of pea flour was added to screw-capped centrifugal
81
plastic test tubes of known-mass in duplicate. A third tube with the same amount
of sample was prepared as a control tube to measure moisture content. 20 ml of
n-butanol was added to the test tubes and were placed in the tube rotator for 30
min, followed by 10 min of centrifuge at 3000 rpm. The two separated phases
consist of solid material in the lower layer and a mixture of solvent and dissolved
lipid in the top layer. The top layers were decanted off into a waste container with
special attention to avoid sample loss. The experiment was continued by adding
fresh solvent, and repeating the extraction steps twice. The two test tubes were
placed in the nitrogen evaporator for up to 30 min at 70 °C until the remaining
solvent was completely evaporated. The test tubes along with the control were
placed in the oven for 24 h at 95 °C. Tubes were covered with caps after removal
from the oven. The final mass of the tubes was recorded after leaving them in a
lab-made drierite box to allow them to reach room temperature. The difference
between initial and final sample mass of the control tube, which represents the
moisture loss during the drying period, was subtracted from the difference of test
tube mass to calculate the oil content percentage of the samples.
3.3.5 Statistical analysis
Statistical analyses of data were performed using SAS 9.2, Version
6.1.7601 for Windows operating system (SAS Institute Inc., Toronto, ON,
82
Canada). The effect of accession, growing year, the interaction between
accession and year, flower color, seed color and seed surface type were fitted in
a mixed Multi-way Classification model with mass of 100 seeds and plant height
as regression factors. The calculated F-ratios were compared with the tabulated
F-value at P=0.05 to determine the significance of the terms in the model. The
least square mean of significant factors were compared using Bonferroni
comparison method.
3.4 Results and Discussion
From the 174 acquired accessions, the lipid extraction results were
collected from the 134 accessions, which were grown to maturity and sufficient
seeds were produced for the experiment. Statistical analysis revealed a
significant difference between accessions (p<0.0001), the growing years
(p=0.0002) and the interaction between the two factors (p <0.0001) (Table 3.4).
83
Table 3.4: Analysis of variance, accession and year
The total lipid content in pea seeds grown in 2010 (2.6 ± 0.1) was
estimated greater than in 2009 (2.4 ± 0.1) (Table 3.5).
Table 3.5: Least square means for analysed pea characteristics
Pea
characteristic
Estimate Standard error
Year
2009 2.4 0.1
2010 2.6 0.1
Flower color
White 2.5 0.1
Color 2.5 0.1
Seed surface
Degree of
freedom
Sums of
squares
Mean
square F-ratio
Tabulated
F-value Pr > F
Accession 89 88.1 1.0 8.7 1.36 <0.0001
year 1 1.8 1.8 15.2 3.90 0.0002
Accession
by year
69 57.8 0.8 7.3 1.39 <0.0001
Residual 147 16.9 0.12
84
Wrinkled
Smooth
Medium
2.8
2.4
2.3
0.1
0.1
0.1
Seed color
Grey
Green
White
Yellow
Red
Brown
Black
Mixed
2.8
2.5
2.3
2.4
2.7
2.3
2.3
2.6
0.1
0.1
0.2
0.1
0.3
0.2
0.3
0.3
The majority of flowered pea accessions (58%) possess colored flower as
compared to white flower. The mature plants ranged in height from 30 to 155 cm
with the average of 105 cm. A variety of seed color was observed in the
accession, such as yellow, green, gray, black and red, but a dominant proportion
of seeds were in a spectrum, from yellow to green. The three types of seed
surface, smooth, medium or wrinkled were equally distributed among the
accessions. The result of our extraction methodology showed the average of lipid
content in grown field pea accessions ranges from 0.3 % (accession 112340 in
2009) to 6.3 % (accession 29569 in 2009) (Table 3.6).
It has been reported that lipid content is dependent on plant accession,
seed size (Murcia and Rincón 1992) and seed surface (Kosson, Czuchajowska
85
et al. 1994), but no research has investigated the effect of seed color, flower
color, plant height or seed density on total lipid content in field pea seeds. The
analysis of variance revealed a significant difference between the growing years
(p=0.02) and different classes of seed surface (p= 0.001) but seed color, flower
color, plant height and mass of 100 seeds had no effect on the total lipid
production in pea seeds. There was a significant difference between wrinkled
seeds and smooth seeds (p=0.0007) as well as wrinkled seeds and medium
seeds (p=0.0008), but the difference between medium and smooth seed was not
statistically significant. Wrinkled seeds were found to have a greater lipid deposit
(2.8 ± 0.1) as compared to medium (2.3 ± 0.1) and smooth seeds (2.4 ± 0.1).
3.5 Conclusion
According to literature, lipid content in field pea seeds usually ranges from
1 to 4 % (Coxon and Wright 1985; Daveby, Abrahamsson et al. 1993; Welch and
Wynne Griffiths 1984). The results of the butanol extraction on the selected
accessions were within the expectation of other research. A relatively high lipid
content was previously reported in pea seeds by Letzelter et al. (1995) and
Bastianelli et al. (1998) at 9.7 % and 35%, respectively. However, no accession
was found to exceed 8 % of lipid content in seeds in our experiment condition.
86
3.6 References
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"Feeding value of pea (Pisum sativum L.). 1. Chemical composition of
different categories of pea." Animal Science -Glasgow no. 67 (3):609-620.
Byron, W.J. 1982. The Causes of world hunger: Paulist Press.
Coxon, D.T., and D.J. Wright. 1985. "Analysis of pea lipid content by gas
chromatographic and microgravimetric methods. genotype variation in lipid
content and fatty acid composition." Journal of the Science of Food and
Agriculture no. 36 (9):847-856.
Coyne, C.J., M.A. Grusak, L. Razai, and B.K. Baik. 2005. "Variation for pea seed
protein concentration in the USDA Pisum core collection." Pisum Genet
no. 37:5-9.
Daveby, Y.D., M. Abrahamsson, and P. Åman. 1993. "Changes in chemical
composition during development of three different types of peas." Journal
of the Science of Food and Agriculture no. 63 (1):21-28.
Demirbaş, A. 2008. Biodiesel: a realistic fuel alternative for diesel engines:
Springer.
Dosti, R., and D. Kidushim-Allen. 1991. Light style: the low fat, low cholesterol,
low salt way to good food and good health: HarperSanFrancisco.
FAOSTAT. 2009. "Agriculture Organization of the United Nations." Statistical
Database.
Gass, T., A. Schori, A. Fossati, A. Soldati, and P. Stamp. 1996. "Cold tolerance
of soybean (Glycine max L. Merr.) during the reproductive phase."
European Journal of Agronomy no. 5 (1-2):71-88.
Green, A.E.S. 1991. Solid fuel conversion for the transportation sector: presented
at the 1991 International Joint Power Generation Conference, October 6-
10, 1991, San Diego, California: American Society of Mechanical
Engineers.
Gunstone, F.D., J.L. Harwood, and F.B. Padley. 1994. The Lipid handbook:
Chapman and Hall.
87
Hoover, R., L. Cloutier, S. Dalton, and F. W. Sosulski. 1988. "Lipid Composition
of Field Pea (Pisum sativum cv. Trapper) Seed and Starch." Starch -
Stärke no. 40 (9):336-342.
Kosson, R., Z. Czuchajowska, and Y. Pomeranz. 1994. "Smooth and wrinkled
peas. 2. Distribution of protein, lipid, and fatty acids in seed and milling
fractions." Journal of Agricultural and Food Chemistry no. 42 (1):96-99.
Letzelter, N.S., R.H. Wilson, A.D. Jones, and G. Sinnaeve. 1995. "Quantitative
determination of the composition of individual pea seeds by fourier
transform infrared photoacoustic spectroscopy." Journal of the Science of
Food and Agriculture no. 67 (2):239-245.
Murcia, M.A., and F. Rincón. 1992. "Size as source of variance in lipid
composition of pea." Food Chemistry no. 44 (1):29-35.
Nikolopoulou, D., K. Grigorakis, M. Stasini, MN Alexis, and K. Iliadis. 2007.
"Differences in chemical composition of field pea (Pisum sativum)
cultivars: Effects of cultivation area and year." Food Chemistry no. 103
(3):847-852.
OECD. 2008. OECD Economic Outlook, Volume 2008 Issue 2: OECD
Publishing.
PRRP (Pesticide Risk Reduction Program), PMC (Pest Management Centre),
and AAFC (Agriculture and Agri-Food Canada). 2008. Crop Profile for
Field Pea in Canada.
Ryan, E., K. Galvin, T. O’Connor, A. Maguire, and N. O’Brien. 2007. "Phytosterol,
Squalene, Tocopherol Content and Fatty Acid Profile of Selected Seeds,
Grains, and Legumes." Plant Foods for Human Nutrition (Formerly
Qualitas Plantarum) no. 62 (3):85-91.
Schmid, J., and ER Keller. 1980. "The behavior of three cold-tolerant and a
standard soybean variety in relation to the level and the duration of a cold
stress." Canadian Journal of Plant Science no. 60 (3):821-829.
88
Sessa, D., and J. Rackis. 1977. "Lipid-Derived flavors of legume protein
products." Journal of the American Oil Chemists' Society no. 54 (10):468-
473.
Singh, G. 2010. The Soybean: Botany, Production and Uses: CABI.
Small, D.B.M.E. 1997. Vegetables of Canada: NRC Research Press.
Sosulski, F.W., R. Hoover, R.T. Tyler, E.D. Murray, and S.D. Arntfield. 1985.
"Differential Scanning Calorimetry of Air Classified Starch and Protein
Fractions from Eight Legume Species." Starch Stärke no. 37 (8):257-262.
Sosulski, F.W., and A.R. McCurdy. 1987. "Functionality of Flours, Protein
Fractions and Isolates from Field Peas and Faba Bean." Journal of Food
Science no. 52 (4):1010-1014.
The Siliceous Group. Canada's pea growing region [Image] 2010.
Vollmann, J., and I. Rajcan. 2009. Oil Crops: Springer.
Welch, R. W., and D. Wynne Griffiths. 1984. "Variation in the oil content and fatty
acid composition of field beans (Vicia faba) and peas (Pisum spp.)."
Journal of the Science of Food and Agriculture no. 35 (12):1282-1289.
Winch, T. 2007. Growing Food: A Guide to Food Production: Springer.
89
Table 3.6: Lipid variation in pea accessions.
Peas were grown in St. Anne de Bellevue, QC Macdonald Campus of McGill University.
Plant ID accession
number
country of
origin
Lipid content 2009 Lipid content 2010
plant characteristics
flower
color height
seed
color
seed
surface
100
seed
weight 1 2 Ave 1 2 Ave
1 ILCA - - 1.3 1.2 1.2 2.7 2.4 2.6 Color 100 grey medium 18.0
2 Wando - USA,SC 2.5 2.3 2.4 3.5 3.3 3.4 White 70 green wrinkled 23.8
3 22722 PI 343990 Turkey 3.3 3.1 3.2 2.8 2.8 2.8 - 100 grey wrinkled 16.4
4 Austrian Winter Pea PI 517922 US, Idaho - - - - - - Color 75 - - -
5 ILCA 3005 PI 505062 Greece 1.2 1.7 1.5 3.2 3.2 3.2 Color 120 green medium 18.6
6 227313 - Iran 2.6 1.2 1.9 2.7 1.9 2.3 Color 0 red smooth 16.8
7 Dual - - 2.4 2.2 2.3 3.7 3.3 3.5 Color 110 white wrinkled 17.6
8 ILCA 5006 PI 505063 Afghanistan 1.8 - 1.8 - - - Color 105 - - -
9 ILCA 5032 PI 505074 Yugoslavia 1.6 1.6 1.6 1.6 1.6 1.6 Color 130 green smooth 7.2
10 ILCA 5072 PI 505108 Greece - - - 2.1 2.1 2.1 Color 85 - - -
11 ILCA5075 PI 505111 Syria 0.8 0.9 0.9 - - - Color 135 green wrinkled 30.9
12 Galena - - 2.7 2.5 2.6 2.2 2.2 2.2 White 70 - - -
13 ILCA 5052 PI 505092 Cyprus 3.1 2.0 2.5 1.5 2.2 1.8 White 115 - - -
14 Chinese Snow Pea PI 279933 USA, N.Y. 1.8 2.1 1.9 1.9 2.2 2.1 Color 120 - - -
15 Green Small Pea PI 471211 India 2.1 2.3 2.2 - - - White 125 green smooth 15.0
16 22719/343988 PI 343988 Turkey 1.8 1.9 1.9 1.8 1.9 1.9 Color 100 - - -
17 Oleggon Sugar II - USA 2.0 3.0 2.5 2.5 2.1 2.3 White 75 green smooth 26.0
90
18 Super Sugar Snap - USA - - - - - - No growth - - -
19 Maple Pea NZ PI 236494 US, Iowa 2.1 1.9 2.0 - - - Color 115 - - -
20 YI PI 391630 China 2.3 2.5 2.3 2.3 2.7 2.5 White 125 yellow smooth 7.0
21 22718 PI 343987 Turkey 2.5 2.3 2.4 - - - White 60 green smooth 22.4
22 ILCA 5094 PI 505127 Albania 2.1 1.9 2.0 - - - Color 130 - - -
23 Marx 609 - - 2.1 1.8 1.9 - - - Color 75 - - -
24 ILCA 5115 PI 505144 Spain 1.4 1.9 1.7 1.8 1.9 1.9 White 130 green medium 23.3
25 AWP 517923 PI 517923 Canada 2.1 3.6 2.9 - - - Color 45 - - -
26 Lincoln - USA - - - - - - No growth - - -
27 Red Small Pea PI 471293 India 2.3 1.8 2.1 2.6 2.2 2.4 Color 115 green medium 17.2
28 AA38 PI 269762 UK 3.2 2.3 2.7 - - - Color 150 green wrinkled 19.7
29 ILCA 5089 PI 505122 Albania 3.3 3.2 3.3 1.9 2.5 2.2 Color 110 grey medium 7.8
30 Big Pea PI 262189 Costa Rica 3.3 2.1 2.7 2.3 1.9 2.1 White 120 yellow smooth 30.5
31 AWP 517926 - - - - - 2.3 - 2.3 - 60 black smooth 8.6
32 G 611 764 - - 2.3 2.8 2.3 2.2 1.7 2.0 Color 130 green medium 12.6
33 Dull White Pea PI 471312 India 1.8 2.2 1.8 2.3 1.9 2.1 - 115 - - -
34 ILCA 5117 PI 505146 Iran 3.8 3.1 3.5 3.2 3.2 3.2 Color 110 red medium 25.3
35 Frosty - USA 2.8 - 2.8 3.6 3.3 3.5 White 70 yellow medium 22.6
36 ILCA 5073 PI 505109 Afghanistan - - - - - - Color - - - -
37 AA134 PI 269818 UK - - - - - - No growth - - -
38 Wando - - 2.5 2.1 2.3 2.8 2.5 2.6 White 95 white wrinkled 25.0
39 Super Sugar Snap - USA 2.1 2.0 2.1 2.3 2.2 2.2 White 100 green wrinkled 20.3
40 Dakota (Early Dwarf) - - 3.8 3.2 3.5 - - - - - - - -
41 Oregon Sugar Snap II - - 1.5 2.2 1.9 2.1 2.7 2.4 White 65 yellow medium 19.1
42 Lincoln (mid-season) - - 2.5 1.6 2.1 - - - White 85 yellow wrinkled 26.6
43 Frosty - - - - - 3.3 3.1 3.2 White 75 - - -
44 Sugar Sprint (mid- - - - - - - - - White - - - -
91
season)
45 Dual (early-season) - - 3.2 3.1 3.2 - - - White 85 - - -
46 Dual (early-season) - - - - - - - - No growth - - -
47 Galena (mid-season) - - 2.8 2.5 2.6 2.4 2.3 2.3 White 55 green smooth 15.0
48 Thomas Lacton
(early) - - 1.7 1.6 1.6 3.9 3.4 3.6 White - yellow medium 17.8
49 112373 - - 2.3 2.4 2.3 2.6 2.8 2.7 White 100 yellow smooth 26.7
50 112369 - - - - - 4.4 4.9 4.6 White 130 - - -
51 112367 - - 1.3 - 1.3 1.7 2.3 2.0 Color - grey medium 9.9
52 112365 - - 2.3 1.9 2.1 2.5 2.5 2.5 Color 130 brown medium 11.0
53 112363 - - 3.0 2.5 2.8 1.9 2.3 - Color 135 green medium 9.1
54 112355 - - 2.7 2.9 2.8 2.3 2.6 2.5 White 120 yellow smooth 23.0
55 112358 - - - - - 2.1 1.9 2.0 White 55 yellow smooth 12.1
56 112356 - - 3.1 - 3.1 3.1 2.9 3.0 White - green wrinkled 19.2
57 112408 - - 2.0 1.9 1.9 - - - White 65 green smooth 30.3
58 112406 - - 2.4 2.6 2.5 2.0 2.3 2.2 White 130 - - -
59 112405 - - 2.0 2.1 2.0 2.2 1.5 1.9 White 110 yellow smooth 23.1
60 112394 - - 2.0 - 2.0 - - - White 130 yellow smooth 41.5
61 112393 - - - - - 2.3 2.0 2.2 White 115 yellow smooth 22.3
62 112385 - - - - - 2.4 2.5 2.5 White 65 green medium 18.6
63 31655 - - 2.3 - 2.3 2.8 3.4 3.1 White 75 yellow wrinkled 19.6
64 31656 - - - - - 4.3 5.7 5.0 White - yellow wrinkled 19.1
65 31657 - - 0.8 0.4 0.6 2.5 1.9 2.2 Color 130 - - -
66 31659 - - - - - - - - White 110 yellow wrinkled 14.5
67 31660 - - - - - 2.2 1.9 2.0 White - - - -
68 31653 - - 2.5 1.4 1.9 2.9 2.2 2.6 Color 70 grey medium 28.6
69 33551 - - 2.5 - 2.5 2.5 2.7 2.6 White 135 yellow smooth 16.6
92
70 33555 - - - - - 2.1 1.8 2.0 White 125 yellow smooth 31.5
71 35748 - - 2.3 2.3 2.3 3.1 3.1 3.1 White 110 yellow smooth 22.2
72 35751 - - 2.2 2.1 2.2 3.6 3.3 3.4 White 80 green wrinkled 23.2
73 36164 - - 2.2 2.8 2.5 2.3 2.0 2.2 Color 120 brown medium 21.4
74 40608 - - - - - 2.4 2.3 2.3 Color - brown smooth 24.0
75 40609 - - 2.2 2.5 2.3 1.5 1.6 1.6 Color 125 green medium 17.7
76 41188 - - 1.7 1.9 1.8 1.9 1.6 1.8 White 135 yellow smooth 15.4
77 42818 - - 2.6 2.3 2.4 1.9 1.9 1.9 White 120 green wrinkled 27.0
78 36165 - - 1.4 - 1.4 1.8 1.9 1.8 White 155 yellow smooth 21.9
79 42819 - - 2.5 2.6 2.5 - - - White 120 - - -
80 43015 - - 1.9 2.7 2.3 3.3 3.4 3.4 White 140 green medium 12.2
81 45762 - - 2.0 2.9 2.5 2.9 3.2 3.1 White 90 grey medium 14.5
82 45763 - - 2.3 2.9 2.6 3.4 2.6 3.0 White 80 yellow wrinkled 19.9
83 43016 - - 3.7 3.2 3.5 3.9 3.6 3.8 White 85 green wrinkled 22.8
84 45760 - - 3.6 3.1 3.4 3.8 - 3.8 White 80 mix wrinkled 22.9
85 45761 - - - - - 3.2 2.8 3.0 White - yellow wrinkled 27.8
86 112311 - - 2.0 2.2 2.1 2.4 2.1 2.2 Color 130 green medium 21.0
87 112313 - - - - - - - - White - - - -
88 112316 - - 1.9 1.4 1.9 2.3 2.4 2.3 Color 100 mix medium 10.1
89 112320 - - - - - - - - White 65 - - -
90 46700 - - 1.4 1.2 1.4 1.2 2.2 1.7 White 120 yellow smooth 20.5
91 46702 - - 3.1 3.2 3.1 2.9 2.8 2.8 White 85 yellow medium 17.0
92 46716 - - 2.7 2.6 2.7 2.4 2.0 2.2 White 105 yellow smooth 23.7
93 46718 - - 2.4 2.5 2.5 - - - Color 120 - - -
94 51215 - - - - - - - - White 50 - - -
95 112302 - - - - - 1.3 1.4 1.4 - 30 green medium 23.8
96 76 - - - - - 2.4 2.1 2.3 Color 35 - - -
93
97 112306 - - 2.0 1.5 1.7 2.5 2.0 2.2 Color 65 green medium 21.1
98 112310 - - 2.7 2.6 2.6 - - - Color 125 - - -
99 112322 - - - - - 3.4 4.0 3.7 Color 70 green wrinkled 22.5
100 112324 - - 2.6 2.5 2.5 - - - White 125 - - -
101 112329 - - - - - 2.4 2.0 2.2 White 120 - - -
102 112330 - - 1.9 0.7 1.9 - - - White - - - -
103 112337 - - 2.2 2.8 2.5 2.9 2.9 2.9 White 115 mix smooth 20.8
104 112338 - - - - - 2.9 3.2 3.1 Color 90 green smooth 9.4
105 112340 - - 0.5 0.2 0.4 2.0 2.5 2.2 Color - red medium 14.1
106 112343 - - 3.3 2.3 2.8 2.8 2.1 2.5 Color - brown medium 15.6
107 112344 - - 2.3 2.1 2.2 3.6 2.9 3.3 - 125 green medium 21.6
108 112347 - - - - - 2.2 1.9 2.0 Color 135 red wrinkled 8.6
109 112349 - - - - - 3.0 3.1 3.1 - - green smooth 18.4
110 112350 - - - - - - - - Color 75 - - -
111 112351 - - 1.8 1.5 1.8 - - - White 90 - - -
112 29434 - - 2.6 2.0 2.3 - - - White 130 - - -
113 299448 - - - - - 2.4 2.6 2.5 White - green smooth 25.5
114 29540 - - - - - 2.3 3.1 2.7 White 70 yellow smooth 30.8
115 29453 - - - - - 2.4 1.8 2.1 White 130 green smooth 20.4
116 29482 - - 2.1 2.3 2.2 1.8 2.1 2.0 White 135 yellow smooth 34.4
117 29486 - - - - - 4.4 3.9 4.1 Color 125 grey medium 7.1
118 29497 - - 2.1 1.5 2.1 1.7 2.2 2.0 White 125 white smooth 18.9
119 29500 - - - - - 2.5 2.0 2.3 White 40 - - -
120 29501 - - 1.6 2.6 2.1 - - - White 135 - - -
121 29508 - - 1.8 1.7 1.8 - - - White 100 - - -
122 29514 - - 2.7 3.3 3.0 - - - Color 130 - - -
123 29525 - - 2.7 2.5 2.6 3.1 2.3 2.7 Color 110 grey medium 14.7
94
124 29526 - - 2.9 3.2 3.1 - - - White 120 - - -
125 29527 - - - - - 2.2 2.1 2.2 - - yellow smooth 17.4
126 29531 - - 3.4 2.3 2.8 3.2 3.5 3.4 White - - - -
127 29534 - - 2.1 2.0 2.1 2.6 1.6 2.1 White 130 white smooth 21.2
128 29535 - - 2.9 2.6 2.8 2.5 2.5 2.5 Color 110 green smooth 14.9
129 29542 - - 2.3 2.5 2.4 3.5 2.6 3.1 Color 120 green smooth 9.5
130 29546 - - 3.2 3.0 3.1 2.6 3.2 2.9 Color 135 green medium 5.8
131 29547 - - 3.1 2.2 2.6 2.0 2.3 2.2 White 155 white smooth 13.4
132 29548 - - 1.7 2.9 2.3 2.8 - 2.8 White 130 yellow smooth 10.4
133 29555 - - 1.5 - 1.5 2.5 2.2 2.3 Color - - - -
134 29562 - - 0.2 - 0.2 3.2 3.5 3.3 Color 100 grey smooth 9.9
135 29559 - - 1.8 1.8 1.8 - - - White 135 - - -
136 29560 - - - - - - - - Color - green smooth 20.9
137 29577 - - 3.1 3.0 3.0 - - - Color 100
138 29578 - - 2.2 2.5 2.3 2.4 1.8 2.1 Color 125 green medium 9.2
139 29579 - - 4.4 3.6 4.0 3.5 3.2 3.4 Color 125 grey smooth 12.3
140 29588 - - 2.4 1.9 2.2 2.5 2.5 2.5 Color 135 grey medium 15.5
141 29590 - - 3.2 2.7 3.0 2.7 3.1 2.9 White 125 green smooth 14.5
142 31649 - - - - - - - - White 65 - - -
143 29563 - - 1.6 1.8 1.7 2.0 - 2.0 Color - grey medium 4.7
144 29564 - - 1.8 1.7 1.8 - - - Color 100 brown smooth 11.8
145 29565 - - - - - 2.0 1.2 1.6 - 125 green medium 12.9
146 29566 - - 2.3 1.9 2.1 2.7 2.3 2.5 White - grey medium 7.3
147 29569 - - 7.0 5.6 6.3 0.7 1.2 1.0 Color 100 green wrinkled 28.0
148 29567 - - 1.9 1.9 1.9 2.4 2.5 2.5 Color - green wrinkled 26.2
149 29572 - - 2.3 2.2 2.3 - - - Color - - - -
150 29575 - - 2.6 2.3 2.5 2.9 - 2.9 Color 130 black medium 14.3
95
151 29595 - - 2.3 2.5 2.4 3.0 2.9 2.9 White 120 green smooth 11.8
152 29596 - - 1.2 1.6 1.4 2.1 - 2.1 White 125 black smooth 11.6
153 29600 - - 2.5 - 2.5 2.5 2.8 2.7 White - yellow wrinkled 12.5
154 29602 - - 2.6 3.7 3.1 2.9 - 2.9 Color 125 green medium 15.8
155 29606 - - 2.4 2.2 2.3 - - - White 120 - - -
156 29608 - - 2.3 2.6 2.4 3.1 - 3.1 White 135 yellow wrinkled 19.8
157 29610 - - 3.1 2.9 3.0 2.0 - 2.0 White 125 green smooth 18.9
158 29612 - - 3.9 4.3 4.1 - - - - - - - -
159 29613 - - 0.8 - 0.8 - - - Color 105 - - -
160 29638 - - 1.2 - 1.2 - - - White 70 - - -
161 31210 - - 0.8 1.1 1.0 3.7 3.6 3.6 Color 75 grey medium 28.6
162 Reward - Denmark 0.4 0.7 0.6 1.6 1.0 1.3 White 80 yellow smooth 25.0
163 Canstar - Canada 2.2 2.2 2.2 3.0 2.8 2.9 White 70 yellow smooth 22.3
164 Agaggiz - Canada 1.5 - 1.5 2.8 2.1 2.4 White 75 yellow medium 20.7
165 Stella - Canada 0.9 - 0.9 2.3 2.5 2.4 White 80 yellow smooth 21.1
166 Thunderbird - Canada 1.6 1.7 1.7 2.1 2.3 2.2 White 100 yellow smooth 23.1
167 Mendel - Canada 3.9 2.4 3.1 - - - White - - - -
168 22713 PI 343985 Turkey 0.3 1.5 0.9 - - - Color - - - -
169 ILCA 5041 PI 505082 Ethiopia 2.8 2.6 2.7 - - - Color 110 - - -
170 ILCA 5077 PI 505112 Greece 3.1 1.9 2.5 - - - Color 130 - - -
182 34393 - - 0.8 - 0.8 - - - - - green medium 17.2
191 505082 - - 1.7 1.5 1.6 - - - - - - - -
197 505112 - - 1.7 1.7 1.7 2.5 2.0 2.2 - - green medium 17.5
198 101-unknown - - 4.9 6.7 5.8 - - - - - - - -
Some of the values are missing in the table, which is due to various reasons, such as the seeds did not germinate, not
enough seeds were produced for the experiment, limited number of seeds for proper replication.
96
Chapter 4
4.1 General conclusion
Global concerns about stable energy supplies as well as environmental
attributes has renewed interest in vegetable oil, and stressed existing oil crop
production. Field pea is a valuable legume crop in western Canada,
predominantly grown for protein and starch, but lipid content in the seeds has not
been assessed for vegetable oil production. To develop a novel dual-purpose
oilseed that mirrors soybean in western Canada, lipid content variation was
examined in wild and domesticated peas. Value can be added to the existing
crop by improving lipid storage in the seeds to the extent that pea oil production
becomes economically feasible in Canadian market. A selection of extraction
methods were evaluated in a comparative study among which the butanol and
the hexane/isopropanol extraction methods are reported as the most efficient
methods on field pea. Lipid content ranged from 0.3 to 6.3 % in the 134 pea
accession grown in McGill University, Quebec, Canada. It is reported that seed
surface type and color have significant effect on the total lipid content on the
seeds while flower color, plant height and 100 seeds weight do not have a
significant impact on it. Lipid content in pea seeds need to be further researched
for the fatty acid composition and market evaluation. Oilseed pea can be
97
developed to become a source of edible vegetable oil, or to supply recently
arisen market demands, such as biofuel.
4.2 Future research
1. Among the examined accessions, 30 accessions with the highest and the
lowest lipid content were selected to be seeded in the summer 2011. The seeds
from the grown plants will be evaluated for the lipid content by the second
recommended lipid extraction procedure, the hexane/isopropanol method. The
accession with the highest lipid content will be crossed into the accession with
the lowest lipid content by breeders in the Plant Science Department, McGill
University, Canada. The objective of the breeding is to develop an oilseed pea
with a minimum of 10% lipid content in the seeds while protein content is not
significantly influenced. The novel oilseed crop can be employed to mirror
soybean protein and oil production in western Canada.
2. In order to investigate the variation of lipid content among the selected
accessions and crossed plants, the extracted lipid content will be characterized
by mass spectrometry (MS) and high-performance liquid chromatography
(HPLC).
98
3. The ultimate goal of the project is to evaluate the lipid production for
market usage, and modify the content by genetic engineering to supply the
growing industrial need for vegetable oil in Canada, such as biofuel production.
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