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THE PRODUCTION OF FOOD-GRADE PROTEIN ISOLATES FROM YELLOW MUSTARD SEED BY
SOLVENT EXTRACTION AM) MEMBRANE PROCESSING TECHNIQUES
Haoqun Luo
Under the supervision of
Prof. L. L. Diosady
A Thesis Submitted in Conformity with the Requirements for the Degree of Master of Applied Science in the
Graduate Department of Chemical Engineering and Applied Chemistry at the University of Toronto
O Copyright by Haoqun Luo I998
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THE PRODUCTION OF FOOD-GRADE PROTEIN ISOLATES FROM YELLOW MUSTARD SEED BY SOLVENT EXTRACTION
AND MEMBRANE PROCESSING TECHNIQUIES
by Haoqun Luo
A thesis submitted in conformity with the requirements for the degree of Master of Applied Science in the Graduate Department of Chemical Engineering and Applied Chemistry at the
University of Toronto 1998
ABSTRACT
A processing scheme developed for the production of canola protein isolates was
modified for yellow mustard. The optimized process consists of alkaline extraction of
protein from MeOH/NH3/H20 treated hexane-defatted yellow mustard meal, isoelectric
precipitation, ultrafiltration and diafiltration of the remaining solution and freeze-drying.
The process was reproducible and yielded two protein isolates and a meal residue with
protein recoveries of 7 1% and 2 1 % respectively based on the treated defatted meal. The
precipitated protein isolate had 98% protein and a bland taste and the soluble protein
isolate contained 86% protein with slightly astringent taste. Both isolates were free of
glucosinolates, low in phytates and very light in color. They are suitable for human
consumption. The meal residue contained approximately 20% protein and was fkee of
glucosinolates. It can be used as animal feed, or a low-grade food ingredient. The process
developed here promises to be economical due to the high protein recovery.
ACKNOWLEGEMENTS
I would like to express my deepest appreciation to Professor L. L. Diosady for
giving me the opportunity to undertake this research project and providing invaluable
advice and guidance throughout the period of my study. I would like to thank Hemam
Laue Spice Company Ltd. for providing mustard seed and giving feedback on the quality
of the products. I am also very grateful to my colleagues in the Food Engineering Group
of the University of Toronto, especially Lei Xu for his kind help and assistance. I am
deeply indebted to my wonderfd husband Zuqiang Zhao who constantly encouraged and
supported me, and my parents who provided the care for my son. Without their
understanding, help and support, this piece of research work would not have been
possible.
TABLE OF CONTENTS
ABSTRACT
ACKNOWLEDGEMENTS
TABLE OF CONTENTS
LIST OF TABLES
LIST OF FIGURES
1. INTRODUCTION
2. LITERATURE REVIEW
2.1 Mustard seed and its utilization
2.2 Yellow mustard mucilage
2.3 Glucosinolates
2.4 Phytic acid
2.5 Application of solvent extraction technique
2.5.1 Introduction
2.5.2 Two-phase solvent extraction process
2.6 Preparation of protein isolates
2.6.1 Introduction
2.6.2 Dissolution of protein
2.6.3 Precipitation of protein
2.6.4 Removal of phytic acid
2.7 Application of membrane processing technique
i
. . i1
... 111
vi
vii
1
4
4
8
11
14
17
17
19
22
22
23
25
26
28
2.7.1 Introduction
2.7.2 Ultrafiltration
2.7.3 Diafiltration
2.7.4 Production of vegetable protein isolates using
membrane processing technique
3. MATERIALS AND EXPERIMENTAL METHODS
3.1 Materials
3.2 Membrane processing unit
3.3 Experimental methods
3.3. I Solvent treatment of ground yellow mustard seed
3.3.2 Alkaline extraction of protein fkom yellow mustard meal
3.3.3 Isoelectric precipitation of yellow mustard protein
3.3.4 Preparation of yellow mustard protein isolates
3.4 Chemical analyses
4. RESULTS AND DISCUSSION
4.1 Production of CH30HNH3/H20 treated and hexane defatted
yellow mustard meal
4.2 Protein extractability of CH30WNH3/H20 treated and hexane
defatted yellow mustard meal
4.3 Precipitability of yellow mustard
4.4 Production of yellow mustard protein isolates
4.4.1 Recovery o f protein products
4.4.2 Removal of phytic acid
4.4.3 Removal of glucosinolates
4.4.4 Cost evaluation
5. CONCLUSIONS
6. RECOMMENDATIONS
7. REFERENCES
8. APPENDICES
8.1 Protein analysis (The Kjeldahl Method)
8.2 Phytic acid analysis
8.3 Glucosinolate analysis
8.4 Preparation of myrosinase
LIST OF TABLES
TABLE 1.
TABLE 2.
TABLE 3.
TABLE 4.
TABLE 5.
TABLE 6.
TABLE 7.
TABLE 8.
TABLE 9.
TABLE 10.
TABLE 1 1.
TABLE 12.
Canadian mustard seed production
Fatty acid compositions of mustard oil
The composition of ground yeiIow mustard seed
The effect of the CH30WM13/H20 treatment on the composition of yellow mustard meal
Mass balance of treatment and defatting
Nitrogen balance of treatment and defatting
Phytic acid balance of treatment and defatting
The effect of ah l i ne extraction time on the re COT rery of protein in extract
The effect of meal residue washing time on the recovery of protein in both combined extract and meal residue
Nitrogen extractability of CH30WNH3/H20 treated and hexane defatted yellow mustard meal at different pH values
Effect of pH on precipitation of yellow mustard protein
Mass balance among products in the production of yellow mustard protein isolates
Protein content of product (N% x 6.25)
TABLE 14. Nitrogen balance among products in the production of yellow mustard protein isolates
TABLE 15. Total protein recovery in isolates
TABLE 16. Phytic acid content of product
TABLE 17. Phytic acid balance among products in the production of yellow mustard protein isolates
TABLE 1 8. Glucosinolate content of products
vi
LIST OF FIGURES
FIGURE 1.
FIGURE 2.
FIGURE 3.
FlGURE 4.
FIGURE 5.
FIGURE 6.
FIGURE 7.
FIGURE 8.
FIGURE 9.
Canadian exports of mustard seed, mustard flour and prepared mustard
Flavor release reactions of yellow, black and Indian mustard
The structures of phytic acid
Schematic representation of a membrane filtration process
Schematic diagram of a batch ultrafiltration
Schematic diagram of a diafiltration process
Flow diagram of single phase solvent treatment for yellow mustard seed
Flow diagram for production of yellow mustard seed protein isolates
Protein recovery in extract at different extraction time
FIGURE 10. Effect of pH on nitrogen extractability o f CH30WNH3/H20 treated and hexane defatted yellow mustard meal
FIGURE 1 1. Effects of isoelectric precipitation at different pH on yellow mustard protein recovery from alkaline extract and on protein content of PPI
FIGURE 12. Cost evaluation of yellow mustard protein isolates based on the mass balance of the whole process
vii
1. INTRODUCTION
Since ancient times, mustard seed has been used as a condiment, a source of
edible oil and medicine (Shankaranarayana et al., 1 972). The original use of mustard was
to mask the taste of degraded perishables. Later, it was added into various food products
as a preservative against the action of yeast and molds and used as a counterirritant to
relieve pain. Today, mustard is the largest volume spice in international trade, accounting
for over 160,000 t o ~ e per year with Canada as one of the major contributors
(Anonymous, 1996).
Mustard seed contains 29-36% oil and 23-30% protein. After the removal of oil,
the meal has about 40% protein with a fairly well-balanced amino acid composition
(Peterson and Johnson, 1978). It has excellent nutritional value as a protein source for rats
(Goering et al., 1960) and is compared favorably with casein by rat bioassay (Niazi et al.,
7 989). It is superior to most oilseed meals due to its richness in lysine, an essential amino
acid. Thus, mustard protein could be used in food fortification such as a supplement to
cereals, soft drinks and other food products lacking lysine.
However, the usellness of mustard seed as a source of food protein is limited by
the presence of undesirable components such as glucosinolates, phytates, and phenolic
compounds. Glucosinolates are the principal antinutrients found in mustard and rapeseed.
Their enzymatic hydrolysis products interfere with the function of thyroid glands and
adversely affect growth (Fenwick et al., 1983; Langer and Greer, 1977). When consumed
by humans in small amounts as part of the normal diet, the enzymatically released
products are desired. But when consumed in larger amounts by animals as part of their
1
feed or by humans as fortified foods, the products may reduce palatability or be toxic.
Phytates are strong chelating agents which reduce the bioavailability of several metals,
especially zinc and iron, and cause mineral deficiency (Rutkowski and Kozlowska, 1979).
Phenoiic compounds have a bitter taste and contribute to the dark colour of protein
products due to their oxidation to quinones which then react with protein (Blouin et al.,
1982). All these toxic and antinutritional factors must be removed or reduced to a certain
level in order to incorporate mustard protein into food products.
With the development of new plant breeding technology, the glucosinolate content
of rapeseed varieties has been lowered significantly. Even twenty years ago, canola, the
Canadian rapeseed variety, contained only 30 p o l glucosinolates per gram of meal.
Nevertheless, this level is still too high for inclusion of canola meal into food products.
Mustard seed has even higher glucosinolate content than the unimproved rapeseed. Thus,
detoxification of mustard meal is indispensable. Extensive work on the detoxification of
rapeseed meal and the production of food grade protein products £iom rapeseed meal has
been reported (Rutkowski, 1970; Afzalpurkar et al., 1974; Rutkowski and Kozlowska,
1979; Maheshwari et al., 1981; Blaicher et al., 1983; Serraino and Thompson, 1984).
However, commercial application of these methods is not feasible due to high processing
costs, high loss of proteins, incomplete removal of the glucosinolates and poor functional
properties of the resultant products.
Since 1984, a new process for the removal of glucosinolates from canola, midas
rapeseed and mustard seed has been developed (Rubin et al., 1984; Naczk et al., 1986;
Diosady et al., 1987; Shahidi et al., 1988). In this process, a combined alkanol-arnmonia-
waterhexme treatment was used to produce an improved meal and simultaneously to 2
extract the oil. The meal thus produced was light in colour and bland in flavour. Its
glucosinolate content was below the detection limit of the analytical method employed,
and the oil extracted by the process was edible. Later, a membrane-based process was
developed to produce high quality protein isolates from canola meal treated by the two
phase solvent system (Diosady et al., 1989a; Tzeng et al., 1990). It consists of alkaline
extraction, calcium chloride treatment, isoelectric precipitation, ultrafiltration and
diafiltration. These steps complement one another to yield three products with excellent
protein recovery. Recently, after some modifications, the two-phase solvent treatment and
the membrane processing were tested on high glucosinolate containing Chinese rapeseed
(Liu, 1992; Xu and Diosady, 1994). It was proven that these processes were adaptable and
had good potential for commercial application.
The objective of this study was to modify the existing solvent extraction and
membrane processes, and to optimize process conditions for yellow mustard seed
processing. The ultimate goal is to produce food-grade protein isolates that are free of
gIucosinolates, high in protein content, low in phytate, light in appearance and bland in
taste.
2. LITERATURE REVIEW
2.1 Mustard seed and its utilization
Mustard belongs to the genus Brassicn of Cruciferae family. It is an erect, much
branched, annual herbaceous plant, chiefly grown in China, India, Canada, France,
Pakistan, United States, United Kingdom and other European countries (Peterson and
Johnson, 1978). According to the most recent statistics published by Statistics Canada,
mustard seed was ranked fourth in production among all major oilseeds in Canada
following canola, soybean and flaxseed (Anonymous, 1998). In 1997, the total area
planted with mustard seed in Canada increased by 22.2% to 292,200 hectares, a
significant increase from previous year. The yields of mustard seed declined 15.9% to 833
kilograms per hectare, and the mustard production increased 5.4% to 243,3 00 tonnes
(Table 1). In 1995, Canada exported 167,647 tonnes of mustard seed and 10,287 tomes
of mustard flour and prepared mustard (Figure 1) with a total value of 81 million dollars
(Anonymous, 1996).
There are three kinds of mustard seeds available around the world, yellow/white
mustard seeds (sinupis alba L or Brassica aZba Boissier), black/brown mustard seeds
(Brassica nigra L. Koch), and Indian mustard seeds (Brassica juncea L Cosson). The
seeds of yellow/white mustard are roughly globular in shape, minutely pitted,
mucilaginous when soaked in water and possess a pale straw or yellowish colour
(Peterson and Johnson, 1978). They are usually larger in size than other types of mustard
seeds and odourless even when crushed in the presence of water. The taste is pungent. 4
TABLE 1. Canadian mustard seed production
Year
Area (hectares)
(Statistics were adapted from CANSIM, Statistics Canada Website)
Yield (kgha)
Production (tomes)
1995
267,000
936
244,300
1996
239,100
1997
292,200
99 1
230,800
833
243,3 00
s e e d M f our and prepared mustard
1988 1989 1990 1991 1992 1993 1994 1995
eB seed .flour and prepared mustard
(Source: TIERS, Statistics Canada, 1996)
FIGURE 1. Canadian exports of mustard seed, mustard flour and prepared mustard
Black mustard seeds are small, roughly spherical, minutely pitted and dark brown or
black in colour. The dried seeds are odourless, but when crushed in the presence of water
they quickly produce a strongly irritating and pungent odour. The taste is initially bitter,
but almost immediately intensely pungent. Indian mustard seeds are similar to black
mustard seeds, but are Iarger and browner.
The composition of mustard seed varies slightly between crop years and regions
of cultivation. Generally, white/yellow mustard seeds contain 2430% fixed oil, about
25% protein, mucilage in the seed-coat, and about 1.5% glucosinolates (as p-
hydroxybenzyl isothiocyanate), but no starch (Reineccius, 1994). Black/brown mustard
seeds contain 24-40% oil, about 30% protein, some mucilage and 0.5-1% glucosinolates
(as ally1 isothiocyanate).
Mustard seed has been well known for its condiment and medical usage since
ancient times. Carbonized seed of Brassica juncea was unearthed in China and in
Pakistan and dated to approximately 4000 B. C. and 2300 to 1750 B. C. respectively
(Prakash, 1980). Mustard was mentioned in Chinese manuscripts of the Chou Dynasty
which was about 1 122 to 247 B. C. (Li, 1980). The use of mustard as a condiment and a
remedy for scorpion stings was recorded around 530 B. C. and 400 B. C., respectively.
The cultivation of mustard was introduced into Europe and this condiment was carried
wound in 1497 by Vasco da Garna (Nieuwhof, 1969). A study of the evidence from the
herbal books of the 16th and 17th centuries described the domestication of Brassica crops
in Europe during that time (Van Marrewijk and Toxopeus, 1979).
At the present time, mustard products can be found in the diets of people in many
parts of the world. The oil of mustard is a pale yellow liquid and is used for edible
7
purposes. It is usually obtained by pressing and is subsequently refined for use in the
manufacture of salad oils, margarine, and shortenings. The oil contains unsaturated fatty
acids such as erucic, oleic, linoleic and linolenic and saturated fatty acids such as behenic,
stearic, myristic, md lignoceric (Peterson and Johnson, 1978). Typical fatty acid
compositions of mustard oil were given by Vorobev (1966) and Viswanarhan and Meera
Bai (1961) in Table 2. Mustard seed also contains natural antioxidants such as
tocopherols which protect the oil from rancidity.
Other commercial products of mustard include mustard flour, ground mustard and
prepared mustard. Mustard flour is a fine powder, generally made tiom the interior
portion of the yellow and brown mustard seeds. It is an essential ingredient in
mayonnaise, salad dressings, sauces, and related products. Ground mustard is a powder
obtained by grinding whole yellow mustard seeds. It is widely used in the meat industry
as an emulsifier, water binder, and inexpensive bulking agent. It is also used in
seasonings for frankfurters, bologna, salami and lunch loaf, and in salad dressings,
pickled products and condiments. Prepared mustard is a smooth paste consisting of
ground mustard, salt, vinegar, spices or other condiments (Cui, 1997).
2.2 Yellow mustard mucilage
The water binding and emulsifying properties of ground mustard are largely
attributed to the presence of mucilage in yellow mustard seed (Weber et al., 1974).
Although all mustard seeds contain mucilage, only the mucilage of yellow mustard is 8
TABLE 2. Fatty acid composition of mustard oil
Yellow mustard (B. alba) oil (%I
Brown mustard (B. juncea) oil (%)
(Sources: Vorobev, 1966; Viswanathan and Meera Bai, 196 1)
significant due to its high yield and hctionality. Yellow mustard mucilage was first
reported more than 60 years ago (Bailey and Norris, 1932), and the yield from four yellow
mustard cuitivars grown at four different locations in Western Canada showed a range
from 0.34% to 2.05% (Woods and Downey, 1980). Since the mucilage is deposited
mainly in the epidermal layer of the seed coat (Vaughan, 1970; Siddiqui et al., 1986), it is
relatively easy to extract. However, the yield of the mucilage differs significantly
depending on the method used for extraction.
The latest procedure used to extract mucilage fkom yellow mustard seeds resulted
in the highest recovery (Cui et al., 1993a). The method includes mixing yellow mustard
seeds with water, stirring at room temperature for a period of time, and centrifbging to
separate the seeds from the liquid solution. The mucilage can be precipitated by pouring
the aqueous extract into three volumes of 95% ethanol, and washing three times with
ethanol. It can then be freeze-dried or vacuum-dried. The dried mucilage is a white,
cottome-like product with a yield of about 5% of the dry seed.
Yellow mustard mucilage can be separated into a water-soluble fraction (55.6%)
and a water-insoluble fraction (38.8%) (Cui et al., 1993a). The water-soluble fraction can
then be separated into a CTAF3-precipitated fraction (52.0%) and a CTAB-soluble
fraction (34.0%) by precipitation with 5% CTAB (hexadecyltrimethylaznmonium
bromide) (Cui et al., 1993 b). Further isolation and analysis of the two CTAB hctions
were also carried out (Cui et al., 1994a; 1995 and 1996).
The chemical compositions of yellow mustard mucilage have been studied by a
number of researchers (Vose, 1974; Theander et al., 1977; Siddiqui et al., 1986; Cui et al.,
1993a). Yellow mustard mucilage is a complex mixture of polysaccharides containing six
10
neutral sugars and two uronic acids. The most recent study suggests that crude mucilage
contains 80.4% carbohydrates, 4.4% protein and 15.0% ash. Of the monosaccharides,
glucose (23.5%) is the predominant neutral sugar followed by galactose (1 3.8%),
mannose (6.1 %), rhamnose (3.2%), arabinose (3 -0%) and xylose (1 3%) together with
14.7% galacturonic acid and 4-methyl-glucuronic acid (Cui et al., 1993a).
The rheological and functional properties of yellow mustard mucilage have also
been studied extensively (Cui et al, 1994b; Gerhards and Walker? 1997). Aqueous
solutions of mustard mucilage exhibit rheological properties similar to xanthan gum such
as shear thinning flow behavior at concentrations above 0.3%, weak-gel properties and
interacting synergistically with galactomannans (Weber et al, 1974; Cui et al., 1995; Cui
and Eskin, 1996). At a low concentration, K 0S%, the mucilage exhibits interfacial
activity as it substantially reduces the surface tension of water and the interfacial tension
between vegetable oil and water (Weber et al, 1974; Cui et al., 1993a). The mucilage has
also been shown to have great emulsion capacity and stability when it is incorporated into
an oil and water system. The ability of the mucilage to form stable emulsion appears to
depend on the lowering of the interfacial tension rather than on an increase in the bulk
viscosity (Weber et al, 1974).
2.3 Glucosinolates
Glucosinolates are the major toxic compounds in mustard seed due to their
hydrolysis products which can cause thyroid enlargement in animals. The structures of 11
glucosinolates and their breakdown products have been studied for more than three
centuries. Detailed reviews of this historical development have been presented by Kjaer
(1960) and Challenger (1959). In general, the structure of glucosinolates contains a P-
thioglucose group, a side chain R and a sulfonated oxirne moiety (Fenwick et al., 1983).
Up to now, more than 100 glucosinolates have been discovered in plants (Dietz and
Harris, 1990), and dmost all of them posses the same basic skeletonne, differing onIy in
the nature of the side chain (Underhill, 1980). The glucosinolates of mustard seeds are
sinapine p-hydroxybenzyl glucosinolate (sinalbin) in yellow mustard seeds (Kjaer and
Rubinstein, 1954) and potassium ally1 glucosinolate (sinigrin) in black and Indian
mustard seeds (Kjaer et al., 1953).
Mustard seeds are well known for their pungent flavour. However, dry mustard
seeds do not possess any characteristic odour. They produce a pungent flavour only when
they are broken down in the presence of water (Fenwick et al., 1983). The pungent
flavour associated with these seeds is due to sharp tasting isothiocyanates, the compounds
which are goitrogenic, affecting both growth and reproduction of experimental animals
(Hill, 1979). The organic isothiocyanates are not present as such in mustard but are
released as a result of enzymatic hydrolysis of their parent glucosinolates (Peterson and
Johnson, 1978). The flavour release reactions of yellow, bIack and Indian mustard are
represented in Figure 2. Myrosinase, the enzyme responsible for the reactions is a
naturally occurring substance present in mustard seed. Yellow mustard is especially rich
in myrosinase and therefore used for the preparation of this enzyme (Holley and Jones,
1984). The reactions are also dependent on pH and temperature (Fenwick et al., 1983).
Yellow mustard:
Black and Indian mustard:
FIGURE 2. Flavour release reactions of yellow, black and Indian mustard
The enzymatic hydrolyses of glucosinolates present in many plants yield a range
of products: isothiocyanates, thiocyanate ion, oxazolidinethiones, nitriles, sulfate and
glucose (McGregor et al., 1983). These products have been used as the basis of analytical
measurements. There are many different analytical methods developed for detecting both
intact glucosinolates (Persson, 1974; Hiltunen et al., 1980; Heaney and Fenwick, 1980;
Olsen and Sorensen, 1980; Helboe et al., 1980) and their breakdown products (Wetter,
1957; Appelqvist and Josefsson, 1967; Josefsson, 1968; Langer and Gschwendtova,
1969; VanEtten et al., 1974; Wetter and Youngs, 1976; Maheshwari et al., 1979; Radwan
and Lu, 1986; DeClercq and Dam, 1989; Kershaw and Johnstonnee, 1990; Zolondek,
1996). However, the methods involving measurement of the hydrolysis products are of
particular interest, because it is the hydrolysis products and not the glucosinolates which
are responsible for the flavours as well as most of the biological effects (Fenwick et al.,
1983). An extensive review of glucosinolate analysis has been given by McGregor et d.
(1983) and the principles of glucosinolate analysis hzve been summarized by Dietz and
Harris (1990).
The removal of glucosinolates f7om plant seeds has been a subject of research for
many years. Detailed reviews are presented in 2.5.
2.4 Phytic acid
Phytic acid (myo-inositol hexaphosphate) is a major component of all plant seeds,
constituting 1-3% by weight of many cereals and oilseeds, and typically accounting for 14
60-90% of the total phosphorus (Graf, 1983). It usually occurs as phytin, a mixed
calcium-magnesium-potassium salt in discrete regions of the seeds such as the germ of
corn (OYDell et al., 1972). It serves as the storage of phosphorus (Hall and Hodges, 1966)
and a cell wall precursor (Loewus and Loewus, 1980) during dormancy and germination
of plant seeds.
The structure of phytic acid has been studied extensively (Anderson, 1914;
Johnson and Tate, 1969; Blank et al., 1971; Truter and Tate, 1970; Costello et al., 1976).
So far, it is generally recognized that the structure proposed by Aderson (Figure 3, I) is
most probable. It also has been suggested that phytic acid will be strongly negatively
charged (Figure 3, 11) over the entire range of pH values normally encountered in food
systems (CostelIo et d., 1976). This unique structure of phytic acid indicates tremendous
chelating potential. It reacts with ~ e ~ + and forms insoluble femc-phytate complex at low
pH (Anderson, 1963). At intermediate and high pH, it precipitates all other polyvalent
cations (Maddaiah et al., 1964; Wise and Gilburt, 1981) thus lowering the nutritional
bioavailability of several trace minerals. This interference with intestinal absorption of
cations may lead to mineral deficiencies in humans and animals. Studies showed that
when phytic acid is ingested at 2-8 g per day, which is 0.57-2.2 g of phytate phosphorus.
it interferes with the bioavailability of divalent minerals, notably zinc, iron, calcium and
magnesium (McCance and Widdowson, 1942; Hoff-Jorgensen et al, 1946; Nahapetian
and Bassiri, 1975; Erdman and Forbes, 1977). Nutritional implications of phytic acid
have also been discussed exhaustively in several authoritative reviews (Oberleas, 1973;
Erdman, 1979; O'Dell, 1979; Cheryan, 1980; Cosgrove, 1980; Maga, 1982; Reddy et al.,
1982).
0 I1
HO-P-OH I
HO-P-OH II 0
FIGURE 3. The structures of phytic acid
In addition to interacting with minerals, phytic acid can also react with protein
depending highly on pH. At low pH in the absence of cations phytic acid precipitates
most proteins by binding to protomeated basic residues (Okubo et al., 1976). At a pH
range kom 6 to 10 in the presence of cations it reacts with protein by forming a ternary
protein-metal-phytate complex (Cheryan, 1980). This complex is dissociated at higher pH
and phytic acid becomes insoluble and precipitates out from solution (Saio et al., 1968).
This fact can be used to separate phytate £iom protein solution, thereby obtaining low
phytate protein isolates fiom oilseeds. Details of these aspects are reviewed in 2.6
2.5 Application of solvent extraction technique
2.5.1 Introduction
Solvent extraction is frequently used in the processing of oilseeds. A typical
process includes extracting seed with hexane after suitable treatment to isolate
triglyceride oil and removing polar compounds by subsequently extracting the residue
with a polar solvent such as alcohol. The methods for extracting soybeans generally
consist of contacting the seeds with extracting solvent by percolating the extracting
solvents through the seeds (Haynes and Simms, 1975). Various extractors have been
developed to improve the extraction efficiency @iosady et al., 1989a). The conventional
extraction process for rapeseed is adapted from soybean technology but tailored for small
seed size, high oil and glucosinolate contents. The process utilizes percolating bed
17
extractors in which rapeseed flakes are contacted with hexane. However, this process
gives less satisfactory oil extraction results due to the decreased mechanical strength of
rapeseed flakes, which leads to the compaction of the exmction bed (Diosady et al.,
1987).
Another major problem associated with the conventional extraction method is that
it preserves glucosinolates in the meal. As mentioned in 2.1 and 2.3, oil bearing seeds,
such as rapeseed and mustard, fiom the plants of the Cruciferue type are important
sources of edible oils and potential sources of high quality protein. However the
proteinaceous meal contains unwanted constituents such as glucosinolates, phenolics and
phytates after oil extraction. These substances should be removed or at least reduced in
order for the meal to be acceptable for human consumption. The reduction or removal of
glucosinolates is particularly important, since they can be hydrolyzed by the enzyme
myrosinase to form toxic degradation products that interfere with the production of
thyroxin in the body and adversely affect growth (Hill, 1979; Butler et al., 1982). The
formation of toxic degradation products can be prevented by heat inactivation of
myrosinase (Eapen et al., 1968; Maheshwari et al., 1980; Owusu-Ansah and Marianchuk,
1991 ; Wang et al., 1994), just like the pre-heat treatment employed in the conventional
process to prevent myrosinase fiom hydrolyzing glucosinolates during subsequent
processing. However, the intact glucosinolates remaining in the meal may still be broken
down to toxic products by the enzymes produced by microorganisms in the lower
gastrointestinal tract (Fenwick et al., 1982) if the meal is consumed by animals or human
beings. Moreover, such treatment denatures proteins and affects the colour and the
functional properties of the meal, limiting its usefulness.
18
To overcome these drawbacks, a number of studies have been carried out (Eapen
et al., 1969; Rutkowske and Kozlowska, 1979; Maheshwari et al., 1981; Serraino and
Thompson, 1984). Nevertheless, commercial application of these methods has not been
feasible due to the poor functional properties of the resultant meal, high loss of mass and
protein or high processing cost.
In 1984? a new process was deveioped in our laboratory (Rubin et al., 1984)
aiming to eliminate glucosinolates from oilseed protein products and to attain higher
extraction rates. This process employs a two-phase solvent system in which a polar
solvent such as methanol-ammonia-water extracts glucosinolates and other undesired
components fiom oilseeds, and facilitates the simultaneous oil extraction by a non-polar
solvent such as hexane. The meal thus produced is free of glucosinolates and the oil is
low in phosphorus.
2.5.2 Two-phase solvent extraction process
Work in our group over the past fourteen years has resulted in many significant
advances. Diosady et al. (1985a) studied the effect of alkanol-ammonia-water treatment
on the glucosinolate content of rapeseed meal. It was found that methanol was the most
effective alkanol for glucosinolate removal by the alkanol solution containing ammonia
and water followed by ethanol, isopropanol and t-butanol. Methanol with 10% or more
ammonia decreased the glucosinolate content of rapeseed meal by about 80% @iosady et
al., 1985b) and the addition of water to the rnethanohnmonia solution further reduced
19
the glucosinolate content of canola meal to trace levels @iosady et al, 1985a). The
ammonia present in the solvent has been shown to rupture the cell wall of the seeds,
resulting in an improved rate of extraction (Schlingmann and Vertesy, 1978a; 1978b), and
to reduce the concentration of undesirable lipids such as fatty acid oxidation products,
resulting in improved flavour and reduced residual oil content in the meal (Schlingmann
and von Rymon Lipinski, 1980; 1982).
Subsequently, a two-phase solvent system was developed (Rubin et. al, 1984). In
this process, the methanol-ammonia-water was combined with hexane resulting in an
effective removal of glucosinolates as well as other unwanted components by the
methanol phase and a good recovery of oil in the hexane phase. The resultant meal was
light and bland, free of glucosinolates and contained significantly reduced levels of
polyphenols (Naczk et al., 1986a). The functional properties of the meal produced by the
two-phase solvent extraction process have been reported by Naczk, et al. (1995) and
Diosady et al. (1985).
Later, the process was optimized in our laboratory in terms of solvent
composition, solvent-to-seed ratio and contact time (Rubin et al., 1986). It was concluded
that 1 0% (w/w) ammonia in methanol containing 5% (w/v) water is a good solvent for the
removal of glucosinolates from canola seed. A soivent-to-seed ratio (R) of 6.7, 2 min. of
blending, and 10-15 min. of a quiescent period were required to lower the glucosinolate
content in the meal below the detection level of the analytical method applied.
The process has also been successllly tested on high glucosinolate-containing
rapeseed and mustard seed (Naczk et al., 1986b). The optimum conditions for the
removal of glucosinoiates £?om high-glucosinolate rapeseed and mustard seed included a
20
solvent-to-seed ratio of 6.7 and a quiescent time of 30 min. for both seed, and 10 % and
14 % ammonia in methanol-water respectively. Under these conditions the glucosinolate
content of the meals reduced by 80 %, and a second extraction of the resultant meals with
10 % ammonia in methanol-water resulted in 98 % glucosinolate removal. These studies
were reviewed by Shahidi et ai. (1988). The effect of the solvent treatment on the fate of
glucosinolates was further studied (Shahidi et d., 1990). It was shown that most of the
glucosinolates in the seed were removed by methanol-ammonia-water and passed mainly
unchanged into the gums on recovery of methanol. Only trace amount of glucosinolate
breakdown products were found in the oil or meal, suggesting that most of the
degradation products were also soluble in the polar solvent.
The laboratory process was scaled up for the production of glucosinolate-free
canola meal (Diosady et al., 1987). A semi-pilot-scale Szego mill developed in our
department was used for solvent grinding of canola. The resulting slurry was then
contacted with hexane in liquid cyclones where 97 % of oil was extracted (Diosady et al.,
1989b) and a countercurrent liquid-liquid extractor where the oil was extracted to less
than 0.5% residual oil (Diosady et al., 1989~). The results showed that the methanol-
mmonia-water solution was more effective in the extraction of glucosinolates and
polyphenols on the semi-pilot-scale than in the laboratory. The glucosinolate content of
the resultant meal was below the detection limit (2.2 pmoVg dry meal), and the
polyphenol content of the meal was reduced by 80%. Later the Szego mill was combined
with a hydrocyclone to grind and detoxify canola with a methanol solution containing
10% ammonia and 5% water (Adu-Peasah et al., 1989). The reductions of glucosinolates
and polyphenols were similar, but the meal was more uniform than that produced by the
Szego mill alone. It was also found that the presence of methanol in the meal, prior to the
solvent extraction of oil with hexane, enhanced the oil recovery. Simultaneous grinding
and extraction of canola in the Szego mill with the two-phase solvent was aiso
investigated (Tar et al., 1989). The energy requirement of the process is similar to that of
conventional extraction, since the energy savings in the cooking and flaking steps are
enough to offset the cost of recovering the second solvent (Diosady et al., 1989b; Tar et
at., 1989).
2.6 Preparation of protein isolates
2.6.1 Introduction
Most of the work related to the preparation of Brassica protein isolates was
carried out for rapeseed. Protein isolates can be prepared by extraction of seed, flour or
treated seed meal with various aqueous media. These extraction solutions include
aqueous sodium chloride (Smith et al., 1959; Lo and Hill, 1971), dilute alkali (Sosulski,
1969; Gillberg and Tornell, 1 W6a; Shah et al., l987), dilute acid (Gillberg and Tornell,
1976a), water (Cameron and Myers, 1983), as well as aqueous sodium
hexametaphosphate (Thompson et al., 1982). The dissolved protein can be recovered by
isoelectric precipitation (Shah et al., 1987): or by heat coagulation and acidic polymers
(Gillberg and Tomeell, 1 WBb), or by ultrafiltration (Girault, 1973).
In order to remove undesirable substances, some additional treatments may be
required during the preparation of protein isolates. For example, phytic acid may be
removed by ultraf~ltration with the addition of calcium chloride and proper control of pH
(Serraino and Thompson, 1984) or by ion-exchange (Seo and Morr, 1985); Glucosinolate
hydrolysis products and colour compounds as well as phenolic compounds can be
adsorbed by activated carbon (Woyewoda et al., 1978; Seo and Morr, 1985). These
treatments have been incorporated into various processes to produce high quality protein
products. However, the economical viability of these processes remains unproven due to
high losses of protein.
2.6.2 Dissolution of protein
Dissolution of protein in different solvents has been investigated extensively. It
was found that not all of the protein in seed or meal can be extracted into the liquid phase.
The nitrogen solubility of rapeseed in various solutions was studied by Smith et al.
(1959). 0.6N NaCl was discovered to be most effective for rapeseed protein extraction,
giving a maximum extractability of 68.8%. In another case, Lo and Hill (1971) obtained a
83.5% nitrogen solubility with 10% NaCl solution and a ratio of 30. Other protein
extraction experiments were also conducted with various concentrations of NaCl and
solvent-to-seed ratios, resulting in different protein extractability (Owen et al., 1971). The
variation of the nitrogen solubility was probably due to different seed varieties and
various treatments employed during processing (Gillberg and Tornell, 1976a).
Dilute alkali is another effective solvent for protein extraction. Giraut (1 973) used
0.1N NaOH to extract protein from rapeseed and achieved a nitrogen solubility of 90%. A
similar result was obtained by running the extraction at pH 11.1 (Gillberg and Tornell,
1976a). Blaicher et al. (1983) was able to extract 92% nitrogen from rapeseed meal using
alkaline solution. Tzeng et al. (1990) observed a nitrogen solubility of 80-90% at pH 10-
12.5 for hexane-defatted rapeseed meal and up to 70% for methanol-ammonia-
waterhexme treated defatted meal. Xu and Diosady (1994) also studied the protein
solubility of the two-phase solvent treated Chinese rapeseed meal under strong alkaline
conditions. A nitrogen solubility of 74% was achieved at pH 12. The lower nitrogen
solubility was likely due to the denaturation of protein during the solvent treatment
(Naczk et al., 1985).
Sodium hexametaphosphate (SHMP) was also used for protein extraction. It was
demonstrated that 2% aqueous SHMP was effective in extracting cottonneseed protein
(Shemer et al., 1973). A two-stage extraction of rapeseed flour with 2% SHMP solution
at pH 7 was reported to gave a nitrogen solubility of 97% (Thompson et al., 1976). The
effect of SHMP concentration on nitrogen extractability of canola meal was investigated
by Tzeng et al. (1988a). It was shown that 1% solution resulted in over 70% nitrogen
extracted fiom the meal, and further increase of concentration didn't significantly
enhance the extractability. Subsequently, 1% SHMP solution was used to extract protein
in the preparation of canola protein isolates (Tzeng et al., l988b).
The nitrogen extractability of protein material is not only dependent on the solvent
used but also on other important factors such as the solvent-to-meal ratio and the pH
value. The effects of these factors on rapeseedcanola protein extraction were also
24
investigated in our laboratory. It was found that a solvent-to-meal ratio of 18 was
effective for extracting 80% nitrogen (Tzeng et al., 1988a), and high pH values (12) gave
higher extraction results (Tzeng et al., l988a and 1990; Xu and Diosady, 1994).
2.6.3 Precipitation of protein
The most common method for recovering protein &om the extract is acid
precipitation. The dissolved protein can be partially precipitated at an isoelectric pH or
several isoelectric pH values. A number of isoelectric points were found for different
meals in the acidic range with various protein recoveries. Owen et al. (1971) indicated
that pH 2.5 was suitable to precipitate protein extracted from commercial rapeseed meal.
Girault (1973) used pH 6.5 for isoelectric precipitation of rapeseed protein dissolved in
dilute alkali with a protein recovery of 56%, while El Nockrashy et al. (1977) achieved
65.7% protein recovery by precipitation at pH 3 -6.
The effect of pH on isolation of protein fiom Indian mustard meal was examined
by Shah et al. (1 987). It was shown that the protein recovery reached a maximum of 65%
at pH 5.6 after peptization at pH 9.5. Similar work was done on Chinese rapeseed meal
(Xu and Diosady, 1994). Protein recoveries of over 50% from pH 12 aqueous NaOH
extract were found in a pH range of 5.5 and 8.0 with a maximum of 55% at pH 6.5. The
low protein yield was probably due to the fact that rapeseed and mustard seed have very
complicated protein compositions. These proteins have different molecular weights and
widely spread isoelectric points (Gillberg and Tonneell, 1976a).
25
Consequently multi-stage isoelectric precipitation was investigated to increase
protein yield. Gillberg and Tome1 (1976a) used two successive precipitation steps to
recover protein. A recovery of 45% of dissolved nitrogen was first obtained by lowering
the pH to 6.6. The second step involved the study of the protein recovery from remaining
extract on precipitation at different pH. It was found that at pH 3.5, a maximum of 18%
of the dissolved nitrogen was recovered with a total recovery of 63% from the original
extract. El Nochshy et a1 (1977) also precipitated rapeseed protein at two different
isoelectric points. pH 6.0 and 3.6 were used in sequence, resulting in a 72% nitrogen
recovery from the meal.
The use of other methods to achieve protein yield was also studied. Thompson
(1977) applied heat coagulation to aid isoelectric precipitation, but it turned out not to
enhance protein recovery greatly. Gillberg and Tomell (1 976b) obtained increased protein
yield by using coagulating agents such as polyacids. However, the protein contents of the
isolates were lowered, probably because of increased precipitation of other substances
from the protein extract.
2.6.4 Removal of phytic acid
A number of techniques have been developed for phytic acid removal from
oilseeds. These methods generally include water extraction based on differential
solubility, calcium chloride treatment, dialysis, enzyme treatment, ion exchange,
autoclaving, and membrane processing (Cheryan, 1980).
26
Water extraction is the most common method for phytic acid removal. It is based
on the differential solubility of protein and phytic acid in aqueous alkali. The pH of a
protein extract can be adjusted to 11 or higher where protein remains very soluble while
phytic acid is almost insoluble (Gillberg and Tomeell, 1976a), and phytic acid can be
separated fiom the protein solution by centrifugation or/and filtration. At a moderate pH
range of 6 and 10, a ternary protein-metaI-phytic acid complex forms. To remove phytic
acid in this pH range, addition of competitive chelators is required to break down the
complex. DeRharn and Jost (1979) found that at pH 7.5, an excess of 8.5% or more NaCl
could disassociate the ternary complex, resulting in the separation of insoluble phytic acid
from the protein extract. The addition of EDTA prior to the further processing of rapeseed
also gave the same result (Serraino and Thompson, 1984).
At pH values lower than 5.5, the interaction between protein and phytic acid is
electrostatic in nature. The disruption of protein-phytate complex in this pH range can be
achieved by the addition of CaC12. The calcium ions compete with protein for phytate and
excess of ca2+ will shift the equilibrium, resulting in the formation of calcium phytate
which then can be removed from the protein solution by ultrafiltration (Serraino and
Thompson, 1984). Calcium chloride treatment also has been used in our laboratory to
prepare low phytate rapeseedhinola protein isolates (Diosady et al., 1989a; Chen, 1989;
Tzeng et al., 1990).
Other methods for phytic acid removal have been studied as well. Dialysis can be
used to remove non-associated phytic acid with close control of pH and cation
concentration (Cheryan, 1 9 8 0). Enzymatic hydrolysis by phytase lowered phytate content
in soybean protein (Chang et al., 1977). Ion-exchange appeared effective for phytic acid
removal (Seo and Morr, 1985). Autoclaving at llS°C for 4 hours resulted in the
destruction of phytic acid (O'Dell, 1969), and membrane processing was used to separate
the phytate disassociated from protein by calcium chloride (Tzeng 1987).
2.7 Application of membrane processing technique
2.7.1 Introduction
Membrane filtration, such as ultrafiItration or diafiltration, is a relatively new
separation process based on the ability of membranes to discriminate between dissolved
molecules of different size and shape. The filter used in this process is a thin but tough
membrane made from a polymer such as cellulose acetate, polyamide, polysulfone,
polyvinylidenefluoride, etc. (Cheryan, 1986). The membrane is selectively permeable and
classified by its molecular weight cut-off, defined as the molecular weight of a solute 90-
95% of which is retained by a membrane. A membrane can retain most molecules with
molecular weights higher than its molecular weight cut-off, while allowing most smaller
molecules to permeate it due to pressure, thus achieving separation (Figure 4). Because of
its pressure-driven characteristics, ultrafiltration or difiltration results in a greater flux of
solution than dialysis which is driven by concentration difference.
Since glucosinolates, phytates and phenolic compounds are soluble in water and
are much smaller in motecular size than proteins, it should be possible to use membrane
processing to selectively remove these undesirable components and produce high quality 28
C MEMBRANE PERMEATE
FIGURE 4. Schematic representation of a cross-flow membrane filtration process
protein products. Mustard proteins have molecular weights ranging fiom 10,000 to
490,000 ddtome (Fischer and Schopfer, 1988; Venkatesh and Rao, 1988; Marcone et al.,
1997), a membrane with a molecular weight cut-off of 10,000 should be able to achieve
mustard protein purification and isolation.
Ultrafiltration is one of the membrane processes widely used in food,
pharmaceutical and biological systems due to the fact that its continuous molecular
separation does not involve a phase change or interphase mass transfer. In ultrafiltration,
the pressure gradient across the membrane forces solvent and smaller species through the
pores of the membrane, while the lager molecules are retained. The schematic diagram of
a batch ultrafiltration process is represented in Figure 5. During the process, the volume
of a solution is reduced with the removal of solvents and other small molecules while the
quantity of macromolecules remains unchanged, resulting in the concentration of retained
macromolecular substances and the reduction of unwanted small molecular impurities.
The relationship between the concentration ratio of a solute and the volume ratio of the
solution (Cooper, 1984; Cheryan, 1986) can be expressed as:
CdC, = (v,JV~)~ = C F ~
where Cr is the concentration of the solute in the final solution; C, is the concentration of
the solute in the original solution; V, is the volume of the original solution; Vf is the
volume of the final solution; R is the average rejection coefficient of the solute and CF is
30
Back Pressure Valve Retentate
1 Membrane
Feed Tank Membrane Cartridge Permeate
Prefilter
Peristaltic Pump
V, = the volume of the original solution Vf = the volume of the final solution
FIGURE 5. Schematic diagram of a batch ultrafiltration
the concentration factor defmed as V n f . Rejection at any point in the ultrafiltration
process is defined as:
Rt = I- CPKR
where Cp and CR are the solute concentrations in the permeate and retentate at that time
respectively. R is zero for solutes that will completely pass through the membrane and is
one for solutes that are completely rejected by the membrane.
For example, if the volume of a solution containing a large molecular compound
and a small molecular impurity is reduced to one tenth o f its original volume by
ulMiltration (CF=IO), and presumably the macromolecules are all rejected by the
membrane @=I), the concentration of the large molecular compound will increase ten
fold. Since small molecules normally freely pass through the ultrafiltration membrane,
their concentration on either side of the membrane should be the same during processing
(R=O), resulting in about same concentration as the original feed solution. However, the
quantity of the small molecular impurity will be reduced ten fold with the decrease of the
solution volume. Consequently, the purity of the large molecular compound will be
increased by ten fold as well.
Because the concentration of small molecular substances in solution remains
constant during the entire process, there are minimal changes in the microenvironment
during ultrafiltration, i.e., no changes in pH or ionic strength (Cheryan, 1986). Another
advantage of ultrafiltration processes is that they need fairly low pressures for operation,
which would lower equipment and operating costs by a considerable margin. A fkther
advantage as compared to conventional dewatering processes such as evaporation, fieeze
concentration or freeze drying is the absence of a change in phase or state of the solvent
during the dewatering process, resulting in considerable savings in energy and products
with better fictional properties. Other advantages are that no complicated heat transfer
or heat-generating equipment is needed, and only electrical energy is required to drive the
P-P-
Nevertheless, ultrafiltration can not take the solutes to dryness. In fact,
ultrafiltration at a huge concentration factor is unrealistic due to the low mass transfer
rates obtained with highly concentrated macromolecufes and the high viscosity that
makes pumping of the retentate difficult (Cheryan, 1986). Furthermore, increased
concentrations of a dissolved macromolecule will result in its precipitation fkom the
solution and cause great reduction of permeate flux.
2.7.3 Diafiltration
Diafiltration is a special technique of ultrafiltration. It is also a pressure-driven
membrane process that can separate dissolved components by different molecular sizes
and shapes. The main difference is that diafiltration involves adding water at the
appropriate pH and temperature to the feed tank at the same rate as the permeate flux,
thus keeping feed volume constant during processing (Figure 6). At the same time,
permeable solutes are removed at the same rate as the solvent. The relationship between
solute concentration ratio and dilution ratio (Cooper, 1984; Cheryan, 1986) can be
represented as:
Back Pressure Valve Retentate
Membrane
Feed Tank
Peristaltic Pump
V, = the total volume of water added V, = the volume of original solution
Membrane Cartridge Permeate
Prefilter
FIGURE 6. Schematic diagram of a diafiltration process
CBC, = e VJV. (R-1) = eDV (R-1)
where Cf is the solute concentration in the final solution; C, is the solute concentration in
the original solution; V, is the total volume of water added; V, is the volume of the
original solution; R is the average rejection coefficient of the solute and DV is the
diavolume defined as V, / V,.
For instance, if a solution containing a large molecular compound such as a
protein and a small molecular impurity such as a salt is continuously diluted with water at
a water-to-solution ratio of five @V=5) in a diafiltration process, and if all the salt
molecules permeate through the membrane (R=O), a 99.3% removal of salt £?om the
solution can be achieved, while the concentration and the quantity of protein remains
unchanged during the whole process with the assumption that 100% of the protein is
rejected by the membrane (R= 1).
Diafiltration is particularly useful if the concentration of the retained solute is too
high to permit effective ultrafil~ation for purification. To effectively achieve complete
purification of macromolecular substances, a combined ultrafiltration and diafiltration
process with a certain concentration factor and diavolume should be used to remove the
impurities in the system.
2.7.4 Production of vegetable protein isolates using membrane processing techniques
The use of membrane processes to produce vegetable protein isolates has been
well documented. Okubo et al. (1975) investigated the production of soy protein isolates
35
by ultrafiltration. The recoveries of cottonneseed protein (Lawhon et al., 1977) and
sunflower seed protein (O'Connor, 1971) by membrane processing were also reported.
The production of rapeseed protein isolates has been studied extensively in our
laboratory. Diosady et al. (1984) used a single extraction step followed by two stages of
ultrafiltration to produce a rapeseed meal containing less than 0.2 mg/g glucosinolates
and a rapeseed protein isolate having 80% protein content and 0.4 mg/g glucosinolates.
Further development of this process to obtain purer protein isolate hcludes the use of
other treatment and diafiltration. A process developed by Tzeng et al. (1988a) comprising
aqueous sodium hexametaphosphate extraction, activated carbon treatment,
ultrafiltration diafiltration and ion-exchange purification yielded an isolate containing
about 90% protein, fiee of glucosinolates, low in phytates and fiber, light in colour, and
bland in taste. The protein recovery in the isolate based on the protein content of the meal
was 63%. In order to increase the protein recovery, another process was developed
(Tzeng et al., 1988b), in which isoelectric precipitation was incorporated into the process
after aqueous sodium hexametaphosphate or aqueous sodium hydroxide extraction and
ultrafiltration to produce an isoelectric protein isolate. This step was followed by
did~ltration of the remaining solution at pH 3.5 to produce an acid soluble protein
isolate. The two isolates free of glucosinolates had phytate and protein contents of less
than 2% and close to or higher than 90% respectively. A protein recovery of up to 71 -2%
in the isolates was achieved. However, ultrafiltration of solutions with high protein
concentration at high pH is undesirable because it may cause membrane fouling or reduce
membrane lifetime.
Accordingly, an improved process of treating meal containing vegetable proteins
was developed and patented @iosady et al., L989a). This process includes steps of
extracting meal with a suitable aqueous solvent, precipitating some of the protein, and
membrane processing (ultrafiltering and diafiltering) the unprecipitated protein solution.
The process was used for the production of canola protein isolates (Tzeng et al., 1990).
Isoelectric and soluble protein isolates containing 87-104% protein (Nx6.25) were
obtained with no glucosinolates and low phytate content. The process was modified and
tested successllly on Chinese rapeseed meal (Xu and Diosady, 1994). The obtained
protein isolates were also of very high quality.
The production of mustard seed protein isolates by membrane processing has not
been reported. Therefore, research has been carried out on this subject and the result will
be presented in this thesis.
The objective of this thesis work was to find an optimal process for yellow
mustard seed processing. By combining solvent extraction and membrane processing
techniques, food grade protein isolates from yellow mustard seed were to be produced
economically.
3. MATERIALS AND EXPERIMENTAL METHODS
3.1 Materials
The yellow mustard seed used in this project was a Canadian variety, #lCW,
supplied by G. S. Dunn & Co. Limited, Hamilton, Ontario. The seed was commercially
ground and the ground seed was obtained fiom Hermann Laue Spice Company Ltd,
Scarborough, Ontario. The composition of the ground seed is shown in Table 3.
3.2 Membrane processing unit
An Arnicon CH4 hollow-fiber concentrator manufactured by Amicon Cop.,
Lexington, MA was used in this project and operated in both ultrafiltration and
diafiltration modes. The schematic diagrams of ultrafiltration and diafiltration are
represented in Figures 5 and 6, respectively. In general, a built-in peristaltic pump drew
solution fkom a small container and pumped it through a prefilter to remove the fine
solids in the solution, and then through an Amicon DIAFLO H1 P10-20 hollow-fiber
membrane cartridge with a nominal molecular weight cut-of of 10,000 dalton and a
membrane area of approximately 0.05 m2. A back-pressure valve at the outlet of the
cartridge was used to control the pressure in the cartridge and adjusted to obtain high
permeation rates during operation. The permeate containing dissolved low molecular
TABLE 3. The composition of ground yellow mustard seed
Material
Ground yellow
mustard seed
Hexane defatted
mustard meal
Moisture ("/.I
*Phytic acid ("/.I
* On a moisture-fiee basis
weight components and water was driven through the fiber wall while the retentate
consisting of high molecular weight solutes was circulated back to the sample container.
After each run, the unit was drained and immediately flushed with distilled water. A
detergent solution containing 5 g /L Terg-A-Zyme obtained fiom Alconox Inc., New York
was then recycled through the unit for an hour. After draining the enzyme detergent
solution, the system was again flushed with distilled water until the initial water flux was
fully restored. The cleaned membrane cartridge was stored in 1 % formaldehyde solution.
It was rinsed with distilled water and soaked in the water overnight before use.
3.3 Experimental methods
3.3.1 Solvent treatment of ground yellow mustard seed
In this project, a solvent extraction process was employed to reduce the high level
of glucosinolates in yellow mustard seed. The treated defatted meal of ground yellow
mustard seed was produced by treating ground seed with a solvent consisting of methanol
with 5% water and 15% dissolved ammonia, followed by Soxhlet extraction with hexane.
The solvent used in the system was prepared by mixing pure methanol with 5% water
(v/v) and then bubbling anhydrous ammonia into the methanol-water solution in an ice
bath for 50-60 min until the ammonia concentration reached 15% (w/w). During the
preparation, samples were taken for the determination of ammonia concentration in the
solution by titration against 2.0 N HzS04. If the ammonia concentration was over IS%,
40
dilution with methanol containing 5% water was carried out to bring down the ammonia
concentration to the desired value. The ammonia concentration of the diluted solution
was confirmed by titration before use. In these calculations it was assumed that the
density of the solution during ammonia bubbling remained unchanged.
The solvent thus prepared was used for the production of yellow mustard meal
(Figure 7). A typical batch of the treated defatted meal was prepared as follows: 140g of
ground seed was mixed with 938 mL methaaol-ammonia-water solution (R=6.7) in an
Osterizer Blender (Model B8810, Sunbeam Corporation Ltd.) at its highest speed for 2
min. After a quiescent period of 15 min, the meal was recovered by vacuum filtration
using Whatman No.40 filter paper and washed 3 times with 234 mL pure methanol
(R4.67) each time. Then the meal was dried in a well-ventilated place overnight at room
temperature, while the gum and methanol were recovered using a vacuum rotary
evaporator. The oil-containing meal was then extracted with hexane for 24 h using a
Soxhlet extractor. The defatted meal was dried in the same manner, and the oil was
recovered fiom the hexane by a rotary evaporator under vacuum. The recovered methanol
and hexane were reused. A total of about 1.2 kg of yellow mustard meal was thus
produced and mixed well for fiuther testing and processing.
Ground mustard seed
MeOWl s%m3/5%H20 R=6.7
Mixing 2 min. 2OOOrpm
Quiescent period 15 min.
MeOH
R=l.67
Washing 3 times
I 1
Hexane I Dryh3 I Gum MeOH
I Recovery
Treated defatted meal Oil Hexane
FIGURE 7. Flow diagram of MeOHINI13/H20 treatment for ground yellow mustard seed
3.3.2 Alkaline extraction of protein from yellow mustard meal
The effects of extraction time, residue washing time and pH on &dine extraction
of protein from yeIlow mustard meaI were investigated.
To determine the effect of alkaline extraction time on the recovery of nitrogen,
each 10 g of methanol-ammonia-water treated and hexane extracted meal was dispersed
in 180 g of pH 12 aqueous NaOH solution (solvent-to-seed ratio R of 18)- The mixture
was maintained at pH 12 by adding 25% and 5% NaOH solution using a VWR Scientific
pH meter (model 8000). The contact time was varied fiom 5 to 30 rnin in 5 min
increments. One test was also made at 60 min. At the completion of each extraction the
sluny was centrihged using an ECB-22 centrifuge (International equipment Co., U. S.
A.) at 7500 rpm for 25 min. A 10 mL sample of the supernatant was taken for nitrogen
content analysis, and the extracted nitrogen was calculated.
The effect of washing time on nitrogen extraction was also studied. Three
samples, 10 g each, of treated defatted meal were extracted for 30 min with 180 g o f pH
12 aqueous NaOH as described above. After alkaline extraction and centrifbgation, the
supernatant of each slurry was poured into a 400 mL beaker while the meal residue was
mixed twice by gentle stirring with 60 mL of pH 12 aqueous NaON for a period of 5, 10
and 15 min each time. The resuspended meal was then centrifbged and the supernatant
was combined with the alkaline extract. The combined extract was stirred, and 10 mL of
the mixed extract was withdrawn for nitrogen analysis. The washed meal residue was
oven-dried at 10S°C overnight and was also analyzed for nitrogen. The nitrogen
recoveries in the combined alkaline extracts and in the meal residue were then calculated.
43
For the test of nitrogen extractability at different pH, each 180 g of distilled water
was adjusted by 25% NaOH to a pH value of 10.5 to 12.5 in increments of 0.5. Ten grams
of treated defatted meal was then dispersed into the aqueous alkali with stirring and the
pH was maintained at the desired value by adding 5% NaOH. After 30 rnin extraction, the
mixture was centrifbged for 25 min at 7500 rpm. The supernatant was analyzed for
nitrogen.
3.3.3 Isoelectic precipitation of yellow mustard protein
The precipitation of yellow mustard seed protein &om the combined alkaline
extracts at different pH values was examined. Sixty grams of treated defatted meal was
first mixed with 1080 rnL of pH 12 aqueous NaOH (R=18) and extracted at pH 12 for 30
rnin by the addition of NaOH solution. m e mixture was then centrifuged at 7530 rpm for
25 min. The supernatant was poured into a beaker and the solid was resuspended twice in
360 mL of pH 12 aqueous solution for 15 rnin each time. The washings were combined
with the alkaline extract.
Aliquots of 200 rnL combined extract were withdrawn for protein precipiktion at
different pH values ranging fiom 4 to 7 in increments of 0.5. The pH value of the extract
was adjusted to the desired point by adding 6N HCI solution and maintained at that level
for 15 min. The resulting suspension was centrifuged at 7500 rpm for 20 rnin. After the
supernatant was decanted, the precipitated protein isolate (PPI) was washed with water
(five times of its wet weight) at the pH value of the precipitation. The resultant PPI was
44
oven-dried at 10S°C overnight before analyses for dry matter and protein content. The
fraction of protein precipitated fkom the alkaline extract was calculated.
3.3.4 Preparation of yellow mustard protein isolates
The process developed for the production of canola protein isolates by Tzeng et
al. (1 990) was modified to optimize the operating parameters for the production of yellow
mustard protein isolates. A schematic diagram of the process is shown in Figure 8. The
process consists mainly of alkaline extraction, isoelectric precipitation, ultrafiltration and
diafiltration.
Typically, the treated defatted yellow mustard meal was dispersed in pH 12
aqueous NaOH solution at a solvent-to-meal ratio of 18. The pH of the slurry was
maintained at pH 12 by adding 25% NaOH for 30 min. At the end of the alkaline
extraction, the slurry was centrifuged at 7500 rpm for 25 min. The meal residue was then
resuspended for 5 min twice in pH 12 aqueous NaOH at a solvent-to-meaI ratio of 6 each
time. The twc washings were combined with the extract and the meal residue was freeze-
dried for 72 h by a Labconco Freeze Dryer-5.
The pH of the combined extract was then brought down to 6 by the addition of 6N
HC1 solution with stirring, and was maintained at this value for 15 min for the completion
of isoelectric precipitation. The suspension was then centrifuged at 7500 rpm for 20 min
and the precipitate was washed with 5 times its wet weight of distilled water and
Treated defatted meal
I Alkaline extraction I-, I and washing at pH 12 1
I
I Extraction solution I I
Isoelectric precipitation ~ I protein solution I
I UltrafiItration and diafiltration I
I 1 Freeze-drying 1 I
I Wet residue I
Meal residue (MR)
Protein precipitate T I Water washing 1
Freeze-drying + Soluble protein isolate Precipitated protein isolate
FIGURE 8. Flow diagram for production of yellow mustard protein isolates
centrifuged again to give a precipitated protein isolate (PPI). The PPI was fieeze-dried in
the same manner as the meal residue.
Subsequently, the supernatant was ultrafiltered at a concentration factor of 10
using m Amicon CH4 concentrator with a membrane cartridge having a molecular weight
cut-off of 10,000. The concentrated protein solution was then diafiltered with water at a
diavolume of 5. The soluble protein isolate (SPI) was obtained by fieeze-drying the
retentate resulting from diafiltration.
3.4 Chemical analyses
Moisture content was determined according to AACC Method 44-15A (AACC,
1976). The samples were dried in a forced-air oven for 24 h at 105OC. The moisture
content was determined gravirnetrically after the dried samples were cooled down to
room temperature in a desiccator.
Oil content was determined by extracting ground seed or meal for 24 h using a
Soxhlet extractor. The oil was subsequently desolventized by a rotavapor and dried in an
oven for 2 h. The oil content was then determined gravimetrically.
Crude protein (N x 6.25) was determined by the Kjeldahl method, AACC Method
46-12 (AACC, 19761, using a Buchi 425 digester and a Buchi 320 distillation unit.
Details of this analysis are given in Appendix 8.1.
Phytate content was determined according to the procedure developed by Naczk et
al. (1 986a). Phytate phosphorus was determined colorimetrically according to the AOCS
Offkial Method Ca 12-55 (AOCS, 1980). Phytic acid content was calculated from
phytate phosphorus using a conversion factor of 3.55. Detailed procedure is listed in
Appendix 8.2.
Glucosinolate content was determined colorimetrically as the thiocyanate ion
according to the method developed by McGregor (1978). Details of this method are
presented in Appendix 8.3. Myrosinase used for glucosinolate analysis was prepared
according to the procedure given in Appendix 8.4.
4. RESULTS AND DISCUSSION
4.1 Production of CH301WNHJH20 treated and hexane defatted yellow
mustard meal
The analytical results on the CH30H/NH3/H20 treated hexane defatted yellow
mustard meal are listed in Table 4. The glucosinolate content of the meal was reduced by
92% with the CH30WNH3/H20 treatment, fiom 192 to 15 W g , compared to the result
of an 80% removal obtained by Naczk et al. (1 986) from mustard meal. The protein and
phytate content of the meal increased by more than 16% and 30% respectively after the
treatment. The increase was expected because the solvent removed gum which consisted
of phospholipids, phenolics, carbohydrates, soluble nitrogen compounds, glucosinolates
and their breakdown products from the ground seed, concentrating the protein and phytate
in the treated meal. The treated defatted meal was light yellow in colour and very mild in
taste.
The treatment process was repeated three times, and its mass, protein and phytic acid
balances were obtained. The results were reproducible as shown in the following three
tables. The yield of the treated defatted meal obtained by this process was 5 1.3% after the
removal of oil and gum present in the ground seed (Table 5). The 32.0% oil yield of the
treatment and defatting process was quite close to the 33.3% obtained fiom the defatting
of the ground seed (Table 3), suggesting that CH30H/NH3/H20 Weatment did not remove
much oil. Compared to the results obtained by Rubin et al. (1986) for canola treatment,
49
TABLE 4. The effect of the CH30H/NHm20 treatment on the composition of yellow mustard meal
-- -
(All results are on moisture and oil free bases)
Material
Ground seed
Treated defatted meal
Phytic acid content ("/.I
Glucosinolate content
(PW~
192.7 + 1.2
15.1 f 0.7
Protein content (%I
43.5 t 0.6
50.7 t 0.2
TABLE 5. Mass balance of treatment and defatting
Mass recovery in treated defatted meal
(W
Mass recovery in
oil (%I
Mass recovery in
gum ("/.I
Loss of mass (%I
(All results were on moisture free bases and as % of the mass of ground seed)
the percentage of gum (14.6) being removed from yellow mustard ground seed by the
treatment was much greater than that fiom canola (8.8%). This could be due to the fact
that yellow mustard seed contained more carbohydrates and glucosinolates than canola,
which resulted in much more dissolved gum in CH30H/NH3/&0. The gum should be
further tested for its composition and the possibility of utilization. The 2.2%
unaccountable loss was the same as the result obtained for canola treatment. This loss
was probably due to the experimental losses during transfers of samples.
The recovery of ground seed protein in the treated defatted meal was 89.2% (Table
6). This result was similar to the protein recovery from canola varieties which ranged
fiom 87.1 to 93 -7% (Rubin et d-, 1986). Around 10.8% of nitrogen was lost during the
treatment, but much of this was polar non-protein nitrogen, in the form of free amino
acids, peptides, and other nitrogen-containing compounds such as glucosinolates and their
breakdown products in yellow mustard seed. The overall loss of protein material was
probably less than 10%.
The resuft of a 99.1% phytic acid recovery in the treated defatted meal (Table 7)
indicated that CH30H/NH3/H20 treatment did not remove phytate fiom the ground seed.
This is consistent with the result obtained by Naczk et al. (1986a) for canola treatment.
The low solubility of phytic acid was due to the high proportion of methanol in the
solvent.
TABLE 6. Nitrogen balance of treatment and defatting
Run Nitrogen recovery in treated defatted meal (%I
Loss of nitrogen (%)
A
B
C
Average + S. D.
(All results were as % of nitrogen in ground seed)
89.8
88.4
89.5
89.2 + 0.7
TABLE 7. Phytic acid balance of treatment and defatting
Phytic acid recovery in treated defatted meal (%>
Loss of phytic acid (%)
(All results were as % of phytic acic in ground seed)
4.2 Protein extractability of CH30H/NHJE120 treated and hexane
defatted yellow mustard meal
The protein extractability of CH30H/NH3/H20 treated and hexane defatted yellow
mustard meal in aqueous NaOH was examined in terms of alkaline extraction time, meal
residue washing time and pH. The results of the effect of alkaline extraction time on
protein recovery in the extract are presented in Table 8 and Figure 9. It was clearly shown
that the amount of dissolved protein in the extract increased first rapidly then more slowly
with the extension of the extraction time fi-om 5 to 30 min in increments of 5 min. Further
increase of extraction time to 60 min did not improve the protein extractability of the
treated defatted meal. Therefore, 30 min was chosen as extraction time for alkaline
extraction in all hrther studies.
After the alkaline extraction and centrifugation, part of the protein solution remained
in the meal residue. To maximize the protein recovery, the meal residue was washed
twice with pH 12 aqueous NaOH. The effect of meal residue washing time on the
recovery of protein in both combined extract and meal residue was studied (Table 9). The
increase of washing time did not significantly decrease the protein content of the resultant
meal residue. The protein recovery in meal residue also decreased slightly with the
increase of washing time while the protein recovery in the combined alkaline extracts
respectively increased but not to any great extent. It seemed that 5 min washing time was
enough for extraction of the protein trapped in the meal residue. The total protein
recovery exceeded 100% due to the analytical error.
55
TABLE 8. The effect of alkaline extraction time on the recovery of protein in extract
*As % of protein in treated defatted meal (Ail extractions were done at pH 12 with a soIvent-to-meal ratio of 1 8)
Extraction time (min)
*Protein recovery in extract (%) +
5
64.8
10
76.9
15
79.9
20
8 1.8
25
82.4
30
83.2
60 I
83.2
60 --------- - 5 10 15 20 25 30 35 40 45 50 55 60
Extraction time (min)
FIGURE 9. Protein recovery in extract at different extraction times
TABLE 9. The effect of meal residue washing time on the recovery of protein in both combined extract and meal residue
Washing time (rnin)
*Protein content of MR (%)
Protein recovery in MR (%)
Protein recovery in combined extract (%)
Total protein recovery ( O h )
*On moisture-free basis (All extractions were done at pH 12 for 30 min and all recoveries were calculated as % of protein in treated defatted meal).
The effect of pH on the nitrogen extractability of the treated defatted meal was
investigated subsequently, as pH was known to be a very important factor that affected
protein extraction in previous studies on rapeseed/canola The results are shown in Table
10 and Figure 10. As expected, the nitrogen extractability increased with increased
extraction pH. The nitrogen extractability increased remarkably from 42.6% at pH 10.5 to
73.0% at pH 1 1.5. Nitrogen extractability was increased to 8 1.1 % when the pH was
raised to 12.0. Further increase of pH to 12.5 resulted in only a slight increase of the
extractability. Compared with the results obtained by Tzeng et al. (1990), the nitrogen
solubility of the treated defatted meal was much higher than that of the two-phase solvent
extracted canola meal at dl pK values. At pH 12.0, 8 1.1% of the meal nitrogen was
extracted into the alkaline solution, which was significantly higher than the 60% obtained
with the two-phase solvent extracted canola meal at the same pH (Tzeng et al., 1990), and
was also higher than the 75% obtained with the solvent extracted Chinese rapeseed meal
(Xu and Diosady, 1994). Nevertheless, this value was still lower than the 90% nitrogen
extractability of hexane defatted canola meal at pH 12.0, which was probably due to the
formation of much less soluble protein aggregates resulting fiom the denaturation and/or
the dehydration of protein by the methanol-ammonia solution during meal preparation
(Tzeng et al., 1990).
Another factor, solvent-to-meal ratio, was not studied in this case for its effect on the
protein extractability of the treated defatted yellow mustard meal. At pH 12, a solvent-to-
meal ratio of 1 8 gave a protein solution with high viscosity due to the dissolved mucilage.
A decrease in the ratio would result in difficulties during the alkaline extraction, and an
TABLE 10. Nitrogen extractability of CH30H/NEflz0 treated and hexane defatted yellow mustard meal at different pH values
*Protein recovery in extract (%)
*As % of protein in treated defatted meal
-- -- - ---- 0.5 11 11.5 12 12.5
Extraction pH
FIGURE 10. Effect of pH on nitrogen extractability of CH30H/N&/H20 treated and hexane defatted yeliow mustard meal
increase in the ratio would not be economical. Therefore, a ratio of 18 was used for the
dispersion of the meal and the extraction of the protein.
According to Pfaender (1983), alkaline treatment of proteinaceow materids
produces potentially toxic lysinoalanine. De Groote et al. (1 976) exhibited microscopic
lesions of kidney in rats fed diets containing lysinoalanine, raising questions about its
toxicity to humans. The formation of the potentially harmful levels of lysinodanine could
be prevented by carefid control of reaction conditions during protein extraction, and the
canola protein products prepared by the process developed in our laboratory (extracted at
pH 12) were found not to represent a significant health hazard due to the lysinoalanine
content (Deng et al., 1990). However, long contact at pH higher than 12 is not
recommended for protein extraction. Therefore, pH 12 appeared appropriate for achieving
maximum protein extraction while obtaining good quality protein products, and was
chosen as the point for alkaline extraction of the treated defatted meal.
4.3 Precipitability of yellow mustard protein
Similarly to rapeseed, mustard seed contains proteins with different isoelectric points
and molecular weights (Shah et al., 1987; Fischer and Schopfer, 1988; Venkatesh and
Rao, 1988; Marcone et al., 1997). The detailed protein composition is specific to each
variety, as is the precipitability profile of protein from solution. The recovery of yellow
mustard protein was investigated in this project and the results are presented in Table 11.
TABLE 11. Effect of pH on precipitation of yellow mustard protein
5.5
6.0
6.5
7.0
*On dry basis
Dry matter of precipitate (g)
*Protein content of PPI (%I
# Protein recovery ("/.I
# As % of dissolved protein in extract
The protein content of precipitated protein isolate (PPI) and the recovery of PPI fiom
protein solution at different acidic pH values were plotted in Figure 1 1.
As indicated in Table 11, the amount of dry matter increased steadily from pH 4 to 5,
as did the protein recovery and the protein content of PPI. In the pH range from 5 to 6, the
amount of recovered dry matter remained almost constant while the protein recovery
increased slightly due to the increase of the PPI protein content. At pH 6, a maximum
protein recovery of 72.8% based on the solution was obtained with a PPI of 97.4% in
purity. Above pH 6, the protein recovery started to decrease along with the dry matter
amount. However, the protein content of PPI continued to increase with an increase in
pH.
The observed precipitation behavior of yellow mustard seed protein could be
explained by the protein distribution itself, and also by the possible role played by the
interactions between proteins and other seed components such as carbohydrates and
nucleic acids in the extract (Gillberg and Tomell, 1976a). Since yellow mustard seed
contains water-soluble mucilage and most of its composition is
carbohydrates/polysaccharides (Cui et al., 1993a), it is possible that the two polymers
interact with each other in the extract, and that the interaction may lead to precipitation
depending upon pH. When the pH of the protein solution was lowered to 7.0, pure protein
isolate was obtained (Table ll) , suggesting that there were no other components co-
precipitated with the protein. When the pH decreased to 6.0 for isoelectric precipitation,
the recovery of protein reached the maximum value (Figure 11). However, the protein
content of the resultant PPI decreased slightly due to the increased precipitation of other
components such as polysaccharides. Further decrease of pH fiom 6 to 5 resulted in a
64
+ Protein contentof 6b1 -W- Protein precipitated
FIGURE 11. Effects of isoelectric precipitation at different pH on yellow mustard protein recovery from alkaline extract and on protein content of PPI
small reduction in the protein recovered &om the extract while the amount of dry matter
did not change much. It was possible that the precipitation of polysaccharides and/or
other carbohydrates increased slightly and the protein precipitation decreased. From pH 5
to 4, both the dry matter amount and the protein content decreased more rapidly,
suggesting a significant reduction of protein precipitation. Further lowering of the pH
from 4 was not attempted because of the trend of rapid decrease of protein recovery in
PPI and the trend of lower protein content of PPI. The effect of pH on the solubility of
yellow mustard mucilage in protein solution and the effect of the interactions between
polysaccharides and proteins on the precipitation of protein are not fully understood so
far. Further investigation should be carried out and the carbohydrate content of the PPI
should be determined directly.
The maximum protein recovery of 72.8% (equivalent to 57.8% of the total treated
defatted meal protein) was achieved in the precipitation of yellow mustard protein, which
was much higher than that obtained in canola and Chinese rapeseed protein isolation.
Tzeng (1987) reported a PPI recovery of 47.1% (equivalent to 25.8% of the total meal
protein) from CH30WNH31HrO-hexane extracted canola meal. Xu ( 1 993) obtained a
ma.ximum protein recovery of 54.9% in the precipitation of the dissolved Chinese
rapeseed protein extracted from the two-phase solvent treated meal. The yellow mustard
PPI recovery is the highest seen so far, and the protein content of PPI is also high. In
addition, yellow mustard protein can be isoelectrically precipitated at a nearly neutral pH
and thus the precipitates can be washed with tap water instead of acidic water. These facts
indicate that the process could be used to produce yellow mustard protein isolates
economically.
4.4 Production of yellow mustard protein isolates
The process optimized for the production of yellow mustard protein isolates was
based on that developed by Tzeng (1987) for canola processing. It consists of four major
steps: extraction and washing at an optimal pH, pH adjustment for isoelectric
precipitation, ultrafiltration and diafiltration of the remaining solution at an appropriate
concentration factor and diavolume, and fieeze-drying. The reproducibility of the process
was examined with respect to mass, protein and phytic acid balances. The minimum cost
of the produced yellow mustard protein isolates was also estimated based on a mass
balance of the process.
4.4.1 Recovery of protein products
The process was repeated three times and the results are shown in the following
tables. The mass recoveries of three products are listed in Table 12. The total yield of two
isolates was 38.0% of the treated defatted meal (30.0% in PPI and 8.0% in SPI), which
was higher than those previously reported on the production of canola and Chinese
rapeseed protein isolates using similar processes. Chen (1989) obtained a total protein
isolate yield of 29.6% with CH30H/NH3/H20-hexane extracted canola meal. Tzeng et al.
(1990) accomplished a 32.5% total isolate yield with hexane defatted canola meal, and
Xu (1993) achieved 34.1% with CH30H/NH3/H20-hexane extracted Chinese rapeseed
meal. In the meal residue (MR), 55.6% of the initial mass was recovered, which was 67
TABLE 12. Mass balance among products in the production of yellow mustard protein isolates
Run Mass recovery
in PPI (%)
Mass recovery in SPI
Mass recovery in -MR
(%I
(All results were as % of the mass of treated defatted meal)
Loss of mass (%I
PPI: precipitated protein isolate SPI: soluble protein isolate MR: meal residue
lower than the 58.5% and 69.1% obtained from the treated Chinese rapeseed and canola
meal, respectively. The total loss of solids during processing was 6.4%, which was
similar or less than the values reported earlier. This mass loss was probably due to the
actual loss of small molecular weight components into the permeate and experimental
losses during the fiequent transfer of samples.
The protein content of the products is given in Table 13. Precipitated protein isolate
(PPI) was found to have 98% protein content while soluble protein isolate (SPI) contained
about 86% protein. Xu (1993) also reported a higher PPI protein content (100%) than SPI
protein content (9 1 %) for Chinese rapeseed protein. Contrary results were obtained for
canola protein, where PPI and SPI contained 97% and 100% protein, respectively (Chen,
1989). This could be explained by the variation in composition in protein and other
components such as carbohydrates among different seeds and seed varieties. The pH at
which PPI was precipitated and SPI w a recovered might also play a role. In the case of
yellow mustard protein isolation, a pH of 6.0 was used for the isoelectric precipitation
and the membrane processing to recover PPI and SPI. This is close to the pH of 6.5 used
for recovering the Chinese rapeseed protein isolates. On the other hand, canola processing
was carried out at pH 3.5. It should be noted that the protein contents of PPI and SPI from
yellow mustard meal were lower than those fiom canola and rapeseed meal treated by
similar processes. Probably yellow mustard seed contained much more mucilage or
carbohydrates than the other seeds (Weber et d., 1974), resulting in more carbohydrates
in the protein solution. At pH 6.0, only a small portion of the soluble yellow mustard
carbohydrates was recovered in PPI and most of them remained in the solution. These
soluble carbohydrates must be of high molecular weight due to the interactions between
69
TABLE 13. Protein content of product (N% x 6.25)
*Pooled estimated standard deviation (All results were on moisture-fiee bases)
PPI: precipitated protein isolate SPI: soluble protein isolate MR: meal residue
themselves and/or their attachment to the soluble protein by interaction with the protein.
Another product, the meal residue, contained 19.8% protein (Table 13), and could be
considered for food or animal feed depending on whether other components such as
phytates and glucosinolates were present.
The nitrogen balance among the products is presented in Table 14. In all runs, about
93% nitrogen in the treated defatted meal was recovered as three usable products,
including two protein isolates and a meal residue. A recovery of 71 -6% of the nitrogen in
the meal was obtained in two isolates (Table IS), which was the highest percentage
among those obtained fiom different C H 3 0 m / H 2 0 treated meals. About 21 -7% of
the nitrogen remained in meal residue. The total nitrogen recovery in the three products
based on the meal was significantly higher than that from Chinese rapeseed meal. Xu
(1993) reported a 87% total nitrogen recovery with 60% in the two isolates and 27% in
the meal residue. Although Tzeng (1 987) obtained a total of 93% nitrogen recovery, the
nitrogen recovery of the two isolates was only around 25%. The higher protein recovery
in yellow mustard protein isolates seems more attractive economically as it is desirable to
have higher nitrogen recovery in the isolates rather than in the meal residue.
The protein recovery ratio of PPI to SPI from yellow mustard was 4.28 (Table 15),
which was significantly higher than those obtained fiom Chinese rapeseed (1.65) by Xu
(1993) and fiom canola (1.13) by Chen (1989).
About 6.7% of the nitrogen in the meal was lost in either the permeate or during
transfers (Table 14). Most of the material in the permeate was probably non-protein
nitrogen which is a complex fiaction containing free amino acids, peptide, and other
nitrogen-containing compounds (Bhatty and Finlayson, 1973). These compounds have
72
TABLE 14. Nitrogen balance among products in the production of yellow mustard protein isolates
Nitrogen recovery in PPI
Nitrogen recovery in SPI
(%>
Nitrogen recovery in MR
(%I
Loss of nitrogen
(%)
(All results were as % of nitrogen in treated defatted meal)
PPI: precipitated protein isolate SPI: soluble protein isolate MR: meal residue
TABLE 15. Total protein recovery in isolates
Run
A
B
(*As % of protein in treated defatted meal)
C
Average f S . D.
PPI: precipitated protein isolate SPI: soluble protein isolate
*Protein recovery in both isolates (96)
72.0
71.8
PPI-to-SPI recovery ratio
4.33
4.17
71.1
71.6 -t 0.5
4.35
4.28 -+ 0.10
relatively low molecular weights, hence can pass through the membrane into the
permeate. Nitrogen loss couid also be attributed to the error in the nitrogen analyses and
the mass loss during processing.
4.4.2 Removal of phytic acid
As reviewed earlier in Section 2.4, interactions between protein and phytic acid are
highly dependent on pH (Cheryan, 1980). At a pH below the isoelectric point (5.9,
protein has a net positive charge while phytic acid has a strong negative charge at all pH
values (Costello et al., 1976), resulting in electrostatic interaction between these two
components and the formation of a protein-phytic acid complex (Okubo et al., 1976).
Therefore, the removal of phytic acid from a protein system at low pH depends on the
ability to dissociate this complex. Okubo et al. (1976) observed that the presence of
calcium chloride at low pH interfered with the formation of the protein-phytate complex
and explained that the calcium ion was able to compete with the protein for phytic acid.
Chen (1989) studied the effect of treatment at different calcium ion levels prior to
isoelectric precipitation of canola protien at pH 3.5 and concluded that with the addition
of 15% CaC12 (by weight of the starting meal) into the protein solution, 60% of the
dissolved phytic acid was removed by membrane processing. The phytic acid contents of
the resultant PPI and SPI were 1.10% and 0.65% respectively.
In the case of yellow mustard protein isolation, isoelectric precipitation was carried
out at pH 6.0. It was found that even when no CaClz was added to the alkaline extract 74
before isoelectric precipitation, the PPI contained no phytic acid at all and the SPI only
had 0.43% (Table 16). According to Cheryan (1980), at intermediate pH values (6 to lo),
the protein as a whole is negatively charged and in the presence of multivalent cations the
negatively charged phytic acid reacts with protein by forming a ternary protein-cation-
phytate complex. This complex started to dissociate at pH higher than 10 with the
addition of NaOH and the phytic acid became insoluble while the protein remained in
solution (Saio et al., 1968; deRham and Jost, 1979). The reaction was postdated to be:
Protein-cation-phytate + ~ a + = Protein-Na + Cation-phytic acid
A possible explanation was that the addition of excess sodium ions shifted this
equilibrium to the right, resulting in the disassociation o f the protein-cation-phytate
complex. In the production of yellow mustard protein isolates, extraction of protein from
the treated defatted meal was conducted at pH 12.0, at which the concentration of ~ a '
increased with the addition of NaOH. The presence of the excess ~ a + prevented the
formation of the ternary complex when the pH of the protein solution was brought down
to 6.0 for protein precipitation. Subsequently, free phytates in the protein solution were
removed by membrane processing. Similar results were attained for Chinese rapeseed
protein isolates which were prepared at pH 6.5 without CaC12 addition (Xu, 1993).
The recovery of phytic acid is shown in Table 17. Almost 90% of the total phytic
acid present in the treated defatted meal remained in the meal residue. Less than 1 I% was
extracted into the protein solution. This result confmed those obtained from canola and
Chinese rapeseed protein production; namely, that pH 12.0 was an appropriate point for 75
TABLE 16. Phytic acid content of proc
Run
B
C
Average + S. D.*
Phytic acid content of PPI (%) - -
Not detectable
Not detectable
Not detectable p- --
Not detectable
"Pooled estimated standard deviation (All results were on moisture-free basis)
Phytic acid content of SPI (%)
Phytic acid content of MR (%)
PPI: precipitated protein isolate SPI: soluble protein isolate MR: meal residue
TABLE 17. Phytic acid balance among products in the production of yellow mustard protein isolates
(All results were as % of phytic acid in treated defatted meal)
Run
A
B
C
PPI: precipitated protein isolate SPI: soluble protein isolate MR: meal residue
Total loss of phytic acid
(%I
Phytic acid recovery in PPI
(%)
None
None
None
i Average f S. D. I None I 0.8 t 0.1 89.3 + 0.6
Phytic acid recovery in
SPI (%)
0.8
0.9
0.8
Phytic acid recovery in
MR (%)
88.9
88.9
90.0
protein extraction, at which pH protein extractability was high and phytic acid
extractability was low. The meal residue contained 6.5% phytic acid (Table 16), which is
too high for food utilization. However, the meal residue could be used as a protein
supplement to animal feed. It could also be W e r extracted with aqueous HCI to obtain a
food-grade material and a by-product phytic acid. A total of 9.9% of phytic acid was lost
during the processing (Table 17), either via permeate or due to the errors in the phytic
acid analyses and/or losses during the frequent transfers of products.
4.4.3 Removal of glucosinolates
As discussed earlier, 92% of the glucosinolates in the ground seed was removed by
the MeOWM13/H20 treatment. The remaining glucosinolates should be removed by the
membrane processing, since glucosinolates are small molecules which can fieely pass
through the membrane. To confirm this, the glucosinolate contents of the three products
(PPI, SPI and MR) were analyzed, and the results are shown in Table 18. It can been seen
that the glucosinolate contents of the three products were all < 2.2 p o V g . According to
the analytical method developed by Wetter and Young (1976) for the analysis of
glucosinolates in rapeseed, the lower detection limit is 0.25 mg/g or 2.2 pmoVg. A lower
glucosinolate content could not be detected. Thus, the yellow mus&ad protein isolates and
the meal residue can be considered as glucosinolate-free products. The PPI was light in
colour and had a bland taste. The SPI was off-white and had a slightly astringent taste.
These isolates can be considered as protein ingredients for human consumption. The meal
78
TABLE 18. Glucosinolate content of products
Material *GlucosinoIate content (POW
PPI I < 2.2
SPI
*On moisture-free basis
PPI: precipitated protein isolate SPI: soluble protein isolate MR: meal residue
residue (MR) was very light in colour and could be used for animal feed or as a low grade
food ingredient. The analytical results indicated that all of the glucosinolates in the
treated defatted meal were extracted into the protein solution since no glucosinolates were
found in the MR. The removal of glucosinolates by membrane processing was then
confirmed as little glucosinoIates were present in either the PPI or the SPI.
4.4-4 Cost evaluation
The minimum cost of the produced yellow mustard protein isolates was estimated
based on the mass balance of the whole process. The result is illustrated in Figure 12. The
cost of mustard seed is $360/tonne. Mustard oil could be sold for $500/tome, equal to
other commodity oils and the meal residue was priced as feed-grade at $200/tonne.
Assuming that the selling prices of both isolates are the same, the minimum cost of each
isolate based solely on the mass balance of the whole process is $734/ to~e. If each
isolate can be sold at a price of $ 3 k g ($3000home), the processing margin is $442 per
tonne of seed or $861 per tonne of treated defatted meal. The minimum sale price for
isolates was cheap. However, the total cost to produce yellow mustard protein isolates
should also include the cost of other materials such as water and other solvents and
chemicals, the cost of labour and energy, and other expenses such as the rental cost of
equipment. The processing costs per tonne for canola are typically $20-30, and thus in
this complicated process for yellow mustard seed processing costs of $150-200 per tome
Yellow mustard seed Cost: $360
I 0.320 tonne
Treated defatted meal Sale: $200 Sale: $160
($390/ton ne)' ($500/ton neIA
I 1-
0.285 tonne Meal residue
Sale: $57 ($200/ton ne)"
L
A assumed * calculated
I
Precipitated protein isolate Sale: $1 I 3
($734/tonne)*
0.041 tonne Soluble protein isolate
Sale: $30 ($734/ton ne)*
FIGURE 12. Cost evaluation of yellow mustard protein isolates based on the mass balance of the whole process
should be more than sufficient. This indicates a potential profit in the range of $200-300
per tonne of seed processed.
In summary, the modified process for yellow mustard protein isolation was
reproducible and yielded two protein isolates and a meal residue with high protein
recovery. The precipitated protein isolate was high in protein, fkee of glucosinolates and
phytic acid, light in colour and bland in taste. The soluble protein isolate contained less
protein and had a slightly astringent taste, but was free of glucosinolates, very low in
phytic acid, and very light in colour. These isolates could be considered as protein
ingredients for human consumption. The meat residue was free of glucosinolates and light
in appearance, and was suitable for animal feed, or as a low grade food ingredient. The
process appears to be economical, and has the potential for commercial utilization.
5. CONCLUSIONS
The modified processes are effective in removing glucosinolates and phytates, and
produce two protein isolates with a protein recovery of 71 % and a meal residue with 2 1%
protein recovery based on treated defatted meal. Starting with ground seed, the protein
recoveries in the isolates and the meal residue are 64% and 19% respectively.
The precipitated yellow mustard protein isolate (PPI) has a 98% protein content
and the soluble protein isolate (SPI) contains 86% protein. Both isolates are free of
glucosinolates (< 2.2 pM/g), very low in phytic acid (none and 0.4%) and very light in
colour. The PPI has a bland taste while the SPI tastes slightly astringent. Both isolates can
be considered as protein ingredients for human consumption.
The meal residue obtained after the recovery of the isolates contains
approximately 20% protein and is free of glucosinolates. Thus it can be used as an animal
feed or a food ingredient.
The processes are potentially profitable, with processing margins of $200-300 per
tonne of seed treated.
6. RECOMMENDATIONS
1. The functional properties of both yellow mustard protein isolates should be studied.
2. The carbohydrate content of the products should be determined.
3. The gum should be f d e r investigated for its composition and the possibility of
utilization, or disposal.
4. The amino acid composition of both yellow mustard protein isolates should be
analyzed to confirm the high nutritional value of the isolates indicated by the
1i terature .
5. The process should be scaled up to assess the feasibility for commercial production.
6 . The process water seearns should be recycled to reduce the water usage and the
wastewater treatment required.
7. Mucilage extracted or dehulled yellow mustard seed shoulc
material to enhance the protein content of the isolates.
be tested as the starting
8. The isolation process should be modified to further improve the taste of both isolates,
but especially that of the SPI.
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8. APPENDICES
8.1 Protein analysis-The Kjeldahl Method ( & K C Method 46-12, 1976)
Weigh out each sample into boat formed nitrogen-free paper. Select the weight of the sample so that the total quantity of nitrogen in the sample is not less than 10 rng.
Put one sample into each digestion tube.
Put a nitrogen-free paper with no samples into a digestion tube as a blank.
Add 3 pellets of FOSS-TABS and approximately 25 ml of concentrated aqueous &So4 solution into each tube.
Preheat Buchi 425 Digester at #4 setting.
Clamp suction tube onto the digestion tubes and turn on suction.
Place digestion tubes in heater and heat at #4 for 20 min. Raise heat to #6 and maintain for 10 min. Increase temperature setting to #10 and heat for 35 min or until the mixture in the tube becomes clear.
Turn off the digester. Remove the digestion tubes and allow them to cool for 20 rnin.
Carefully add 100 ml of distilled water to each digestion tube. Mix the solution well by gently shaking the tube and allow to cool for 15 min.
10. Turn on the water flow to Buchi 320 Distillation Unit and warm up the unit.
11. Add 50 ml 4% boric acid solution and 3 drops of N-Point indicator to a 500 ml Erlenmeyer flask.
12. Insert the flask into the Buchi 320 Distillation Unit and make sure that the end of the glass tube is as far below the surface of boric acid solution as possible.
13. Install the digestion tube which contains the digested sample and add 150 ml of aqueous 25% NaOH solution to it.
14. Carry out distillation until 350 ml of distillate is collected.
15. Lower the collection flask so that the glass tube is not immersed in the distillate and carefully rinse the glass tube with distilled water while the distillation is still taking place.
97
16. Turn off steam and empty the digestion tube by aspiration.
17. Immediately titrate the collected distillate with O.1N H2S04 solution.
18. Calculate nitrogen and crude protein content as follows:
% nitrogen = [(V-Vb) x N x 14 / WJ x 100%
where V = volume of HzS04 titrated for sample in ml Vb = volume of HzSOj titrated for blank in rnl N = normality of HzS04 solution (=0.1) W = weight of sample in mg
% crude protein = % nitrogen x 6.25
8.2 Phytic acid analysis (Naczk et al., 1986a)
1. A 0.25g sample is extracted with 50 ml of 1.2% HCI solution containing 10% NazS04, (both constituents wlv), for 2 hr using a wrist action shaker (Burrell Corp.) at the maximum stoke length.
2. After centrifugation at 2000 rpm for 5 rnin, the mixture is filtered using Whatman No. 41 filter paper.
3. Exactly 5 rnl of the filtrate is pipetted into a 50 ml glass centrifuge tube and then mixed with 5 ml of distilled water and 6 mf of O.67N HC1 containing 0.4% (wlv) FeCl3.6H20.
4. The mixture is heated in a boiling water bath for 75 min to complete the precipitation.
5. The ferric phytate is collected by centrifugation at 2000 rpm for 15 min. The precipitate is then washed with 5 ml of 0.07N HC1 containing 4% (wlv) Na2S04 and centrifbged again.
6. The washed ferric phytate precipitate is digested with 6 ml of a 1 :1 (vlv) mixture of concentrated H2S04 and HNO;. The digestion is completed when white fumes hang over the liquid, (some longer than others but around 1.5 hr).
7. The digested solution is removed fiom the heat and allowed to cool for 15 min.
8. Approximately 12 ml of distilled water is slowly added to the digested solution and the solution is then heated in a boiling water bath for 40 min.
9. The solution is allowed to cool to room temperature and diluted to exactly I00 ml. The phytate phosphorous is then determined calorimetrically according to AOCS Oficial Method Ca 12-55.
8.3 Glucosinolate analysis (McGregor, 1978)
Weigh out approximately 50 mg of sample into the weighing boat and transfer to a 4 rnl screw-cap vial. Add one glass bead into the vial.
Add 1 ml of citrate-phosphate buffer (pH4.5) containing 6 mg of freshly dissolved yellow mustard myrosinase isolate. Cap the vial immediately with a Teflon-lined screw cap and mix the contents on a Vortex mixer to thoroughly wet the sample.
Lmmediately add 2 ml of methylene chloride. Cap and start shaking as soon as possible on a wrist action shaker at maximum stroke length for 2hr-
Centrifuge at 5000 rpm for 10 m i . .
With a 250 pl Hamilton syringe immediately transfer 125 p1 of the methylene chloride layer to a test tube containing 1.5 ml of distilled water.
Heat the test tube under a hot water tap repeatedly, interspersed with mixing on the Vortex mixer until the methylene chloride has evaporated.
Add 0.5 rn1 of O.lN NaOH solution, mix on the Vortex mixer and let stand for 15 rnm.
Add 0.5 ml of 0. lN HN03 solution and mix on the Vortex mixer.
Add 2.5 rnl of freshly prepared femc nitrate reagent, mix on the Vortex mixer and let stand in the dark for exactly 15 min.
Quickly and carefblly pipet 3 rnl of ferric thiocyanate solution from the test tube and transfer to a cuvette. Immediately read the absorbance (optical density) at 470 nm.
Add 45 pl of 5% mercuric chloride solution to the cuvette using a 100 p1 Hamilton syringe and mix with a small plastic stick. Read the background absorbance at 470 m.
Calculate glucosinolate content as follows:
Glucosinolate content ( p W g sample) = [(ODs - ODb) 1 (K x W,)] x 2 1 0.125
Where ODs = the optical density of the sample solution at 470 nm ODb = the optical density of the background solution at 470 nm K = the slope of the standard curve of 0.00 IN standard thiocyanate solution W, = the weight of the sample in g
8.4 Preparation of myrosinase (Jones, 1979)
Cool approximately 200 g of yellow mustard seeds in a refrigerator overnight.
Precool distilled water containing 30% (v/v) acetone overnight at 1-2'C.
Macerate the mustard seeds in 50 g proportions in a 500 ml polyethylene centrifuge bottle in an ice bath with 200 ml of the cold 30 % acetone solution using Brinkman's Polytron PT 20 macerator until a smooth paste is formed.
Place the macerate in the refigerator at 1-2*C for 1 hr.
Centrifuge the macerate in a refrigerated centrifige at 5 O C for 50 min at 2500 rpm.
Transfer the supernatant to a 2 L beaker.
Precool pure acetone to -20 OC with dry ice.
Add 140 rnl of the pure acetone to each 100 rnl of supernatant to give a final acetone concentration of 70% (v/v).
Mix the solution thoroughly by stirring.
10. Leave the solution to stand for 30 min in an ice bath.
1 I. Pour off the acetone solution and collect the precipitate using a stimng rod.
12. Mix the precipitate with 500 ml of cold acetone and stir the mixture for 15 min in an ice bath. Break the precipitate using the glass rod if necessary.
13. Separate the precipitate by vacuum filtration on a Buchner funnel with a Whatman No. 41 filter paper.
14. Dry the precipitate over drying agent in a vacuum desiccator overnight.
15. Store the myrosinase in a dry and cool place of -5 to - 10 O C .
