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THX PRODUCTION OF PROTEIN ISOLATES FROM HEXANE-DEFATTED GROUND YELLOW MNSTARD MEAL

Flora Y.H. Lui

A thesis submitted in conformity with the requirements for the degree of MASTER OF APPLIED SCIENCE

Graduate Department of Chemical Engineering and Applied Chemistry University of Toronto

O Copyright by Flora Y.H. Lui 1998

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Flora Y.H. Lui Master of Applied Science 1998 Department of Chemical Engineering and Applied Chemistry, University of Toronto

Production of Protein Isolates from ffexane-Defatted Ground Yellow Mustard Meal

ABSTRACT

A process for the production of high-quality yellow mustard protein isolates was

developed. The process consists of the extraction of oil-free meal at pH 12, followed by

ultrafiltration and diafiltration to remove glucosinolates, and then isoelectric precipitation

at pH 4.75 to recover some 50% of the dissolved protein. The acid-soluble protein is

concentrated by ultrafiltration and purified by diafiltration prior to recovery by fieeze-

drying. These steps complement one another to yield three products with good protein

recovery. A precipitated protein isolate containing about 89% protein (Nx6.25). a soluble

protein isolate containing 98% protein and a meal residue with 19% protein were

obtained. The two isolates recovered about 66% of the nitrogen in the meal, and were

low in glucosinolates, essentially free of phytates, light in colour, and mostly bland in

taste. The process is simple and has good potential for commercial application.

. -. P roducrion of Prorein Isola~es from Haane-Defatted Ground Yellow -2.fustard Meal l

ACKNOWLEDGMENTS

I would like to express my deepest gratitude to Professor Diosady for his excellent

guidance and advice throughout the course of this work, without which. the whole project

would be impossible.

I am also gratefid to Hermann Laue Spice Co. Inc. (Canada) for providing the

seed and technical support.

My sincere thanks also go to all my colleagues in the Food Engineering Group.

especially to Lei Xu. whose help is very much appreciated.

I would also like to thank Haoqun Luo for her companionship during this project.

I would never forget the triumphant and the distressful moments that we have shared in

the last two years.

Finally. many thanks to Kelvin for his love and support.

Producrion of Prorein Isolaresfrom Defarted Ground Yellow Mustard Meal iv

TABLE OF CONTENTS

ABSTRACT

ACKNOWLEDGMENT

TABLE OF CONTENTS

LIST OF TAE3LES

LIST OF FIGURES

1. INTRODUCTION

2. LITERATURE REVIEW

2.1 Overview of mustard seed

2.2 Utilization of mustard seed

2.3 Preparation of protein isdates

2.3.1 Introduction

2.3.2 Dissolution of Protein

2.3 -3 Precipitation of Protein

2.3 -4 Recovery of Protein Isolates by Membrane Processing

2.4 Glucosinolates

2.4.1 Chemistry of Glucosinolates

2.4.2 Method of Detoxification 2.4.2.1 Inactivation of Myrosinase 2.4.2.2 Removal of Glucosinolates and their Hydrolytic Products 2.4.2.3 Other Methods of Detoxification

2.5 Membrane technology

vii

2.5.1 Introduction

Production of Protein Isolates f rom Defatted Ground Yellow lMustard Meal

3. MATERIALS AND METHODS

3 - 1 Ground Yellow Mustard Seed and Starting Meal

3 2 Membrane Processing Unit

3 -3 Experimental Methods

3.3 - 1 Extractability of Yellow Mustard Protein 3.3.1.1 Effect of pH on Nitrogen Extractability of Yellow Mustard

Meal 3.3.1.2 Effect of Extraction Time on Nitrogen ExtractabiIity of

Yellow Mustard Meal 3.3.1 -3 Effect of Solvent-to-Meal Ratio on Nitrogen Extractability

of YeIlow Mustard Meal

3 -3 -2 Removal of Glucosinolate by Membrane Processing

3.3 -3 Isoelectric Precipitation of Yellow Mustard Protein

3.3.4 Recovery of Yellow Mustard Protein Isolates

3 -3 -5 Chemical Analyses

4. RESULTS AND DISCUSSION

4.1 Protein Extractability of Defatted Yellow Mustard Meal

4.1.1 Effect of pH on Protein Extractability

4.1.2 Effect of Extraction Time on Protein Extractability

4.1.3 Effect of Solvent-to-Meal Ratio on Protein Extractability

4.2 Precipitation of Yellow Mustard Protein

4.3 Recovery of Yellow Mustard Protein Isolates by Membrane Processing

4.3.1 Mass and Protein Recovery of Products

Production of Protein Isolitres from Defotred Ground Yellow Mustard Meal vi

4.3.2 Removal of Phytic Acid

4.3 -3 Removal of Glucosinolates

4.4 Economic Assessment

5. CONCLUSIONS

6. RECOMMENDATIONS

7. REFERENCES

8. APPENDICES

8.1 Moisture Analysis

8.3 Protein Analysis

8.3 Phytic acid Analysis

8.4 Glucosinolate Analysis

8.5 Economic Assessment

Production of Prorein Isolates from Defatted Ground Yellow Mustard Meal vii

LIST OF TABLES

TABLE 1. Seeded Area and Production for Western Canadian Mustard

TABLE 2. Comparison of FAO/WHO/UMJ (1 985) - Suggested Pattern of Amino Acid Requirements with the Composition of Yellow Mustard Meal

TABLE 3. GIucosinolates in Mustard Seed

TABLE 4. Composition of Yellow Mustard Seed and Its Meal

TABLE 5 . Effect of pH on Extractability of Yellow Mustard Protein

TABLE 6. Effect of Extraction Time on Extractability of Yellow Mustard Protein

TABLE 7. Effect of Solvent-to-Meal Ratio on Extractability of Yellow Mustard Protein

TABLE 8. Effect of pH on Precipitation of Yellow Mustard Protein

TABLE 9. Protein Content of the Products

TABLE 10. Nitrogen Balance among Products

TABLE 11. Mass Balance among Products

TABLE 12. Phytic acid Balance among Products

TABLE 13. Phytic acid Content of Products and Starting Meal

TABLE 14. Glucosinolate Contents of Protein Isolates Produced from -4ctual and ControI Processes

TABLE 15. Glucosinolate Balance among Products

TABLE 16. Material Distribution of Protein Isolation Process using Yellow Mustard MeaI

Producrion of Protein Isofares from Defatted Ground Yellow Mustard Meal viii

LIST OF FIGURES

FIGURE 1.

FIGURE 2.

FIGURE 3.

FIGURE 4.

FIGURE 5.

FIGURE 6.

FIGURE 7.

FIGURE 8.

FIGURE 9.

FIGURE 10.

FIGURE

FIGURE

FIGURE 13.

FIGURE 14.

FIGURE 15.

FIGURE 16.

FIGURE 17.

FIGURE 18.

Specialty Crop Areas in Canada

Myo-Inositol 1,2,3,4,5,6 Hexakis Dihydrogen Phosphate (C6HI8P6o2~)- Possible Structure of Phytic acid at Neutral pH based on the Anderson Model

General Structure of Glucosinolates

The Normal Products of Myrosinase Hydrolysis

The Products of Myrosinase Hydrolysis of 2-Hydroxy-3 -Butenyl Glucosinolates

Cross-flow Filtration

Batch Ultrafiltration

Diafiltration with Continuous Addition of Fresh Solvent

Process Flowsheet

Effect of pH on Extractability of Yellow Mustard Protein

Effect of Extraction Time on Extractability of Yellow Mustard Protein

Effect of Solvent-to-Meal Ratio on Extractability of Yellow Mustard Protein

Effect of pH on Dry Matter of Yellow Mustard Protein Precipitate

Effect of pH on the Protein Content of Yellow Mustard Protein Precipitate

Effect of pH on Precipitation of Yellow Mustard Protein

Structure of Parahydroxybenzyl Glucosinolate

Reaction of Phenol with Dilute Sodium Hydroxide

Price Breakdown of Products

Production of Protein Isolares from Hexane-Dejarted Ground Yellow .Clusrard Meal I

1. INTRODUCTION

The rapeseed/canola crop is the world's third most important edible oil source.

after soybean and palm (Downey. 1990). The rapeseed oil of world commerce comes to a

minor extent From the mustards, especially brown and yellow mustards (Sovero. 1993).

Canada is one of the major world suppliers of mustard with an annual seed production of

243 thousand tomes in ! 997 (Canadian Grain Commission, 1998).

Mustard is a spice that has been widely used as a condiment to enhance the

flavour and colour of many different foods. Today. mustard is the largest volume spice in

international trade (Hemmingway. 1993). The dry mustard seeds do not possess any

characteristic odour but produce a pungent taste when crushed and moistened. It is

known that the unique sharp taste of mustard is mainly caused by isothiocyanates. one of

the hydrolysis products of glucosinolates present in the seed. In addition to its spiciness.

the emulsifying and stabilizing properties of mustard seed allow it to find application in

the pickling and meat processing industries.

Mustard seed contains 2846% protein with a fairly well-balanced amino acid

composition (VanEtten. 1967): which forms a potential source of proteins in food

products. However. the presence of toxic and antinutritional substances such as

glucosinolates. phytates and phenolics in the seeds limits their use as food or feed.

Glucosinolates can be toxic to livestock when consumed in Iarge amounts over extended

periods. They hydrolyze to form toxic compounds that can interfere with thyroid function

and cause goiters (VanEtten et al., 1969). Phpic acid is another undesirable compound of

Producrion of Prorein Isolates *om He-rane-Defatted Ground Yellow .tfustard Meal

the seed as it has a potential for binding positively charged molecules such as metal

cations and proteins (Thompson and Serraino. 1986). The formation of insoluble

complexes reduces the mineral bioavailability (Erdman. 1979) and alters the protein

functionality (Kratzer, 1965). Pheno!ic compounds contribute to a bitter flavour and give

a dark colour in the protein products since they readily undergo enzymatic and non-

enzymatic oxidation to produce quinones which can react with the protein (Blouin et al..

1982). Therefore. in order for the meal or other mustard protein products to be used for

human consumption as a protein supplement, the level of these undesired substances must

be reduced.

Much research has been focused on the preparation of mustarcUrapeseed protein

concentrates and isolates suitable for human consumption (Gillberg and Tornell. 1976:

Maheshwari et al.. 198 1 ; Niazi et al.. 1989; Thompson et al.. 1976). However. none of

these techniques have been commercially employed in the production of protein products.

Some studies only examined the physico-chemical properties and the characteristics of

the protein (Marcone et al.. 1997; Venkatesh and Appu Rao. 1988). while the results of

others were undesirable due to low product recovery. poor quality. or high cost.

A novel process designed for the characteristics of rapeseed and canola was

developed in our laboratories to produce protein isolates using a membrane-based

process. This process comprised of four main steps: alkaline extraction of oil-free meal.

isoelectric precipitation to recover proteins and ultrafiltration followed by did~ltration to

concentrate and purify the remaining acid-soluble proteins (Tzeng et al.. 1990). The

process proved to be effective with "canola" varieties that contain low lev& of

Production of Protein Isolates from Hexane-Defatted Ground )'ellow .Clustard .Weal 3

glucosinolates and erucic acid. The products were free of glucosinolates. low in phytates.

light in colour. and bland in taste.

Accordingly, the objective of this project was to adapt the membrane-based

protein isolation process to defatted ground yellow mustard meal. thus producing two

high-quality protein isolates: precipitated protein isolate (PPI) and soluble protein isolate

(SPI). As the products will be designed to be used as a protein supplement for human

consumption. purity and recovery are not the only factors to be considered. A low level

of glucosinolates and phytates is essential for the products. As a result. the process

should have a good potential for commercial application and the mustard protein products

should be able to compete with well-established soy protein products.

Production of Prorein Isolates from Herane-Defitted Ground Yeiiorv . t i ~ ~ t ~ d .Meal 4

2. LITERATURE REVIEW

2.1 Overview of Mustard Seed

Mustard is a cool season crop that is grown in China India Canada France.

Pakistan. United States. United Kingdom. and other European countries

(Shankaranarayana et al.. 1 978). In Canada it is produced in the more northerly areas of

the Great Plains. Its production increased to 243 thousand metric tonnes in 1997 fiom the

mean production of 194 thousand tonnes in 1987-96 (Table 1). Mustard is classified as a

specialty crop in Canada. The seeded area was doubled between 1991 to 1996 and is

currently ranked fourth among other specialty crops (Figure 1).

Mustard is a member of the Brassica of cruciferae family. Carbonized seed of

Brassica juncea and B. rapa have been unearthed at Ban Po village in China and dated to

approximately 4000 B.C (Fenwick et al.. 1983). The cultivation of these crops was

primarily for medicinal purposes in ancient times. It is amongst the oldest recorded

spices. with Sanskrit records dating back to about 3000 B.C. (Mehra. 1968) and an

extensive literature fiom Greek and Roman times onwards (Rosengarten. 1969).

There are three kinds of mustard seeds: black (Brassica nigra L.). brodoriental

(Brassica juncea Coss.) and yellow (Brassica hirta M. or Sinapis alba L.). The seed of

black mustard is small. broadly oblong, dark brown or black in colour and mucilaginous.

Brown mustard seed is spherical with a smooth surface, reddish brown in colour. Yellow

mustard is large seeded, with the seed being light yellow in colour, spherical in shape and

mucilaginous. The chemical composition of mustard seed varies between varieties. crop

Production of Protein kolares from Haane-Defatted Ground Yellow :l.iusrard Meal 5

Region Seeded Area ' Seeded Area ' ~roduction' ~roduction' Mean production'

1997 1996 1997 1996 198 7-96

'000 hectares '000 hectares '000 tonnes '000 tomes '000 tomes

Manitoba 6.9 4.4 6.3 4.9 6.8

Saskatchewan 226.6 194.2 186.4 193.3 154.9

Alberta 58.7 41.4 50.6 39.3 50.6

W. Canada 292.2 240.0 243.3 237.5 193.8

I Field Crop Reporting Series No. 8, December 5, 1997; Statistics Canada

2 Field Crop Reporting Series No. 8, revised estimates for 1986-96

TABLE 1. Seeded Area and Production for Western Canadian Mustard (Canadian

Grain Commission - Quality of Western Canadian Mustard 1997).

Prodzrcrion of Protein Isolates from Herane-Defatred Ground Yellow Mustard Meal 6

Specialty crop areas, Canada

Millions of acres ~ 1 9 9 1 1 9 9 6

FIGURE 1. Specialty Crop Areas in Canada (Farming Facts 1997, Statistics

Canada, Catalogue no. 21-522-XPE)

Production of Protein isolates from Hexane-Defatred Ground Yellow* .tfustard Meal - /

years and regions of cultivation (Shankaranarayana et al., 1978). Typical yellow mustard

seeds are composed of 2842% protein, 2946% oil, 2025% carbohydrates. 4% minerals

and 2-3% phytates as well as glucosinolates, phenolic compounds and dithiolthiones.

The interest towards seed storage proteins has recently increased because of the

high cost and limited availability of animal proteins in many developing countries.

Mustard seed contains about 30% protein with a fairly well-balanced amino acid

composition which compares favorably with the FAO/WHONMJ (1985) suggested

pattern of amino acid requirements for adults, school children. and preschool children

(Table 2). Compared to the standards for infants, mustard meal only has a modest

deficiency in the leucine and lysine content. Therefore, mustard protein's high nutritional

value should be able to compete with well-established soy protein products as a food

ingredient in the human diet. However, the use of mustard protein is currently hindered

by the presence of some undesirable compounds. such as glucosinolates and phytic acid.

Glucosinolates are sulphur-containing secondary plant metabolites. They are

readily hydrolyzed by an enzyme, rnyrosinase, to produce isothiocyanates.

oxazolidinethiones and nitriles. Although isothiocyanates are known to contribute to the

unique taste of mustard. they have been found to have toxic effects on animals. When a

large enough amount is consumed, they can interfere with thyroid function and cause

goiter (VanEtten et al., 1969). Thus, glucosinolates and their hydrolysis products must be

removed or substantially reduced before mustard protein can be considered as a food

ingredient. Heat, chemical treatment and selective extraction procedures can achieve the

detoxification of the seed. The details of these detoxification methods will be

Production of Prorein Isolates from Hexane-Defatzed Ground Yellow .2lusturd Meal 8

Suggested pattern of requirement

Amino acid Infanf Preschool School Adult Composition (g/ 1 6g nitrogen) childb childc mustard meald

Histidine Isoleucine

Leucine

Lysine

Methionine + cysteine

Pheny lalanine + tyrosine

Threonine

Tryptophan

Valine

a Average amino acid composition of human milk.

2-5 years. C

10- I2 years. d

Source: VanEtten et al. (1967). e

1.7 refers to methionine only; data not determined for cysteine.

Not determined.

TABLE 2. Comparison of FAOIWHONNU (1985) - Suggested Pattern of Amino

Acid Requirements with the Composition of Yellow Mustard Meal

(Sinapis alba).

Producrion of Protein Isolares from Hexane-Defaired Ground Yellow .Cfusrard Meal 9

summarized in Section 2.42.

Phytic acid is another undesirable component in mustard seed due to its

interaction with minerals and/or protein in high-phytate diets. It has been recoazed that

phytic acid and its salts are the primary means for the storage of phosphorus in many

plant seeds (Webster, 1928). Phytates constitute 1 to 2% by weight of many cereals and

oilseeds and typically account for 60-90% of the total phosphorus (Cheryan. 1980). The

structure of phytic acid proposed by Anderson (1914) as shown in Figure 2 is generally

accepted as correct. The structure suggests that phytic acid is strongly negatively charged

with tremendous chelating potential. Due to the presence of six reactive phosphate

groups. phytic acid tends to complex or bind positively charged molecules such as

minerals or proteins. The formation of insoluble complexes with minerals such as ~ a ?

M~". ~ n ? ~e". etc.. reduces their bioavailability (Erdman. 1979: Erdman and Forbes.

1977). Besides interacting with metal cations. phytic acid can also complex with proteins

at low pH. thus affecting pepsin digestibility (Kratzer, 1965). Although phytate is

generally considered as an antinutrient in foods. it has found wide applications in other

industries such as dentifrices, mouth rinses. dental cements. and coating materials for

metals and aIloys (Graf. 1983).

Mustard seed contains mustard oil, which has high commercial value. Mustard

oil is nutritionally similar to other vegetable oils and makes up 2846% of the seed.

Tocopherols (vitamin E) present in mustard help to protect the oil from rancidity. thus

contributing to a long shelf life (Minn-dak Growers. Ltd.). Mustard oil was reported to

exhibit antifungal activity and inhibit the growth of several yeasts. Studies showed that

Prodztcrion of Prorein Isolatesfrom Hexane-Defaited Ground Yellow .Ctusrard Meal I0

FIGURE 2. Myo-Inositol 1,2,3,4,5,6 Hexakis Dihydrogen Phosphate (C6Hl8P6024).

Possible Structure of Phytic Acid at Neutral pH Based on the Anderson

Model. (Cheryan, 1980)

Producrion of Prorein kolares from Hexune-Defarted Ground Yellow Jfusmrd .Veal / I

the antibacterial activity in mustard seed was related exclusively to the ally1

isothiocyanate (Beuchat. 1976; Hoffman and Evans. 19 1 1 ; Isshiki et al., 19%: Mari et al..

1993; Tressler and Joslyn, 1954; Webb and Tanner. 1946).

Mustard oil is used as an edible oil in India especially from the spring types of

Brassica juncea However, the high level of erucic acid in mustard oil has limited its use

as a potential source of edible oil in many countries. Feeding studies with laboratory rats

showed that erucic acid may cause heart. adrenal and ovarian tissue lesions (Thomasson.

1955; Carroll and Noble, 1957; Beare et al., 1963). Erucic acid is a long chain

monounsaturated fatty acid with a high fire and smoke point. This property permits

erucic acid to withstand high temperatures and remain liquid at room temperature

(Nieschiag et al.. 1964). Oils containing high levels of erucic acid have found extensive

use as lubricants or in lubricant formulations (Van Dyne et al.. 1990). The oxidative

products of erucic acid are used in the production of plastics. resins and nylons

(Nieschlag et al.. 1967).

The outer seed coat of mustard seeds, especially yellow mustard seeds. has been

found to be rich in mucilaginous material (Bailey and Norris, 1932). Yellow mustard

mucilage is a heterogeneous mixture of polysaccharides which consist of six neutral

sugars and two uronic acids. The monosaccharides include glucose. galactose. mannose,

rhamnose. xylose and arabinose. The uronic acids are composed of both glucuronic and

galacturonic acids (Cui et al., 1993a). The consistency of prepared mustard products.

such as salad dressings and food pastes. was attributed to the presence of mucilage

(Weber et al., 1974). Mucilage is relatively easy to extract since it is deposited in the

Producrion of Protein Isolates from He-rane-Defarred Ground Yellow :lfusrard .Weal 12

epidermal layer of the seed coat (Siddiqui et al., 1986; Vaughan, 1970). However. it is

not economically feasible to use whole yellow mustard seeds a s a source of mucilage

since there are no practical applications for the seeds after the extraction of mucilage. On

the contrary. extraction of mucilage from mustard bran appeared to be a better way of

producing the gum (Vose. 1974; Weber, 2 974).

2.2 Utilization of Mustard Seed

Mustard seed has been used as a spice and medicine since ancient times. The use

of mustard as a condiment and a remedy for scorpion stings was recorded by Pythagoras

about 530 B.C. and Hippocrates about 400 B.C. (Fenwick et al.. 1983). Canada is by far

the largest single prodilcer of high quality mustard seed for condiment uses. growing

approximately 90% of the world's requirements (Mancan Ingredients. 1998).

As mentioned in section 2.1, the unique taste of mustard is caused by

isothiocyanates. the hydrolysis products of glucosinolates. The differences in mustard

flavours are a result of different types of glucosinolates associated with the specific

species of mustard. Black and brown/oriental mustards release a volatile compound. ally1

isothiocyanate. which possesses a sharp and pungent flavour. Yellow mustard releases a

non-volatile compound. p-hydroxybenzyl isothiocyanate, which has a sharp taste without

a pungent aroma.

Traditional commercial mustard products include ground mustard. prepared

mustard and mustard flour. Ground mustard is a powder obtained by grinding whole

Production of Protein isolates from Herane-Defatted Ground Yellow Musrard .Weal 13

mustard seeds. Yellow arid oriental mustards are the most common species used for

ground mustard. Ground yellow mustard is used widely in the meat processing industry

at levels fiom below 1% to 3%. Besides its high protein value, it also contains

mucilaginous material which allows it to act as an emulsifier, flavour enhancer. water

binder and texture aid in meat products. Ground yellow mustard is also used in many

seasoning blends and in lower priced prepared mustards. Ground oriental mustard is used

mainly in Chinese style mustards. It also finds major application in spice blends when a

variety of 'heat' sensations are desirable, such as Cajun and East Indian cuisine (M~M-

dak Growers Ltd.. 1998).

Besides ground mustard. whole mustard seed also has commercial value. Whole

yellow mustard seed is used primady in the pickling industry and in the manufacturing of

many sausages. Brown mustard is often used in its whole seed form in specialty granular

prepared mustards. Prepared mustard is a smooth paste ccnsisting of mustard seed.

vinegar. spices or other condiments. The famous 'Dijon' table mustard is made of brown

mustard seed.

Mustard flour is a fine powder prepared fiom the interior portion of the seed. It is

prepared by milling yellow and/or oriental mustard seed, and then blending the different

flours to exact specifications. Mustard flour is an essential ingredient in mayonnaise.

sdad dressing, sauces and related products, since it stabilizes oil-in-water emulsions.

Mustard absorbs liquid in foods including fat and vegetable oils to an extent of 1.5 times

its weight in salad oil and two times its weight in water. Thus, it prevents undesirable

separation and aids in product consistency (Minn-dak Growers Ltd.. 1998).

Prodrccfion of Protein Isolates from Hexane-Defarred Ground Yellow .blusrard Meal 14

Mustard bran is a by-product of the milling process during the production of

mustard flour. Previously ignored, the bran has currently found increasing demand due to

its emulsifying and stabilizing properties. Although all mustard seeds contain mucilage,

only yellow mustard mucilage is significant because of its high yield and hctionality

(Cui et al., 1993qb). Yellow mustard bran contains about 25-30% water-soluble

mucilage and is used for viscosity control in many pourable type mustards. It is also

responsible for the stabilizing, emulsifying, water-binding and fat absorbing properties of

other bran-containing products.

The nutritional and functional contributions from mustard have been proven

through the mentioned products; however, the main application of mustard is to enhance

the flavour of many foods. Mustard oils are the characteristic flavour components of

whole seed, ground mustard, prepared mustard and mustard flour.

Although the spiciness of mustard is the characteristic of many mustard products.

it limits the use of mustard in many other food products. For example. the amount of

ground mustard added to processed meats is limited to a level of less than 1%. Therefore.

in order to take advantage of the natural functional properties of mustard without the

development of its characteristic flavour. deheated mustard is created by permanently

destroying the 'heat' of the mustard. The seed is subjected to controlled time.

temperature, and moisture conditions, where the enzyme myrosinase is inactivated and

the development of the inherent isothiocyanates is interrupted. This results in a bland

flavoured product, which retains the desirable functional characteristics of conventional

mustard. Deheated mustard can be a natural source of cold water-soluble gums. protein

Production of Protein Isolates from Hexane-Defutted Ground Yellow lCfusrard .Weal I S

and oil and finds use as a thickener. emulsion stabilizer. antioxidant and replacement for

some food gums. It can be added to any food products.

2.3 Preparation of protein isolates

2.3.1 Introduction

Protein isolates are usually referred to as products containing 90% or more

protein. During the protein purification procedure, the most important stage is the

isolation of protein. Extraction of oilseed protein isolates has been accomplished fiom

meal, flour or concentrate, which were produced by a variety of treatments. Protein can

be extracted using water (Cameron and Myers, 1983): aqueous sodium chloride (Girault.

1973; Lo and Hill, 1971; MacKenzie and Blakely, 1972; Owen et al., 1971), aqueous

sodium hexametaphosphate (Thompson et al.. 1976; Tzeng et al., 1988% b), dilute alkali

(Gillberg and Tornell, 1975a; Sosulski et al., 1976; Tzeng et al., 19881, and 1990) and

dilute acid (Gillberg and Tomell, 1 W6a: Kesharvarz. 1977). After the initial isolation

from the starting material, the next step is to recover the protein fiom the extraction

solutions. Recovery of protein can be achieved by isoelectric precipitation (Gillberg and

Tornell, 1976a; Tzeng et al.. 1988b and 1990), precipitation with precipitating reagents

(Gururaj Rao et al., 1978), dialysis (Bhatty et al., 1968). and ultrafiltration (Diosady et al..

1984; Kroll et al., 1985; Tzeng et al., 1988). Additional treatments may be incorporated

into the process to remove undesirable components, such as phytates, glucosinolates and

colour compounds. Phytates can be removed through ion-exchange treatment (Tzeng et

Production offrotein Isolatesjiom Hexane-Defatted Ground Yellow .Liustard Meal 16

al.. 1988a) while glucosinolates and colour compounds can be eliminated using activated

carbon adsorption (Woyewoda et al., 1978). Therefore, the above protein purification

methods can be adapted to produce mustard protein isolates with high purity and low

levels of undesirable compounds.

2.3.2 Dissolution of protein

Interest in the isolation of protein fiom mustard seed has only recently arisen and

therefore research in this area to date, has been limited. Contrarily. protein isolation fiom

rapeseed has a much longer history. A number of workers have described techniques for

solubilization and extraction of protein from rapeseed meal. Sodium chloride and dilute

alkali are two examples of the common solvents used for the extraction of protein. An

extractability of 68.890 was obtained with 0.6N NaCl (Smith et al.. 1959). while only

60% of the nitrogen was extracted with 1 N NaCl (Owen et al.. 197 1 ). Bhatty et al. (1 968)

extracted 67% of the total nitrogen with 10% (w/v) NaCl in two successive extractions;

however, 75 to 80% nitrogen extractability was achieved with 10% (wh) NaCL by Lo and

Hi11 (1971). These variabilities in rapeseed protein solubility were probably associated

with the seed varieties and the different extraction conditions such as temperature and

solvent-to-meal ratio.

Dilute alkali has been shown effective in extracting protein from rapeseed. A

0.2% NaOH solution is capable of extracting 85% nitrogen with two successive

extractions at a combined solvent-to-meal ratio of 20 (Sosulski and Bakal, 1969). Girault

(1973) attained about 89% nitrogen solubility fiom defatted rapeseed flour by extracting

Production of Prorein lsolares from Hexnne-Dejatted Ground Yellow .Musrard Meal 17

with 0.1N NaOH. Several researchers have investigated the effect of pH on protein

extractability of rapeseed meal using dilute NaOH solution (Tzeng et al.. 1988b. 1990:

Yin et al., 1990). Reports indicated that nitrogen extractability increases with increasing

pH. At pH values greater than or equal to 11, nitrogen extractability above 85% was

obtained. Gillberg and Tornell (1976) achieved a nitrogen solubility of 94% by carrying

out the extraction at pH 1 1.1.

The use of sodium hexametaphosphate (SHMP) to extract rapeseed protein has

also been reported by several workers. Polyphosphates are well known as effective

complexing agents in protein isolation (Briggs. 1940; Finley et al.. 1973: Hidalgo et al..

1973). Shemer et al. (1 973) extended the application of SHMP From a complexing agent

to an extraction agent. They demonstrated that 2% aqueous SHMP solution is effective

in extracting cottonseed protein. Later. a process was successfully developed by using

2% aqueous SHMP solution to extract 97% protein fiom rapeseed flour (Thompson et al..

1976). The effect of SHMP concentration on nitrogen extractability was studied in our

laboratories (Tzeng et al.. 1988a). It was reported that the nitrogen extractability was not

significantly increased by SHMP concentrations above 1%.

The variabilities in protein extractability of rapeseed meal depend upon the seed

species, the extraction conditions. and the processing treatments used to produce the

meal. Rapeseed meal without heat treatment has a higher protein solubility than the meal

prepared fiom seed heated in a rotating drum at 9 0 ' ~ to inactivate myrosinase (Gillberg

and Tomell. 1976). Similar evidence showed that nitrogen extractability of

methanol/arnmonia/hexane-extracted rapeseed meal was much lower than that of hexane-

Production of Protein Isolates from Hexane-Defined Ground Yellow Jhtard Meal 18

defaned meal (Tzeng et al., 1990). This decrease is probably due to the denaturation

and/or the dehydration of protein during the treatment.

Various studies on the extraction of protein ficm mustard seed have been

undertaken. MacKenzie and Blakely (1 972) found that the extractabilities of protein from

the three types of mustard seeds (black, brown, and yellow) are much lower than that

reported for rapeseed using the same procedure. The pH solubility profiles of proteins

fkom B. juncea in water and 1M NaCl solution were studied (Gururaj Rao et al.. 1978).

Results showed that the solubility in 1M NaCl solution was higher at all pH values above

4 as compared to water. However, the maximum solubility was 92% at pH 11 in both

cases.

2.3.3 Precipitation of Protein

Protein dissolved in the extraction solution after separation from the insoluble

solids is usually recovered by precipitation at its isoeIectric pH. The isoelectric point of a

protein is defined as the pH at which a protein does not migrate in the presence of an

electrical field (Regenstein and Regenstein, 1984). However, there are usually substances

present other than water and protein in a real solution. Proteins may specificaIly bind

certain ions and as a result cannot be isolated without some salt attached. This salt

maintains electrical neutrality either in the form of counterions or bound ions. Thus. the

protein bound with salt would have a different isoelectric point than the free protein.

Therefore, the point of minimum solubility is used to represent the isoelectric point of

protein in actual cases.

Production of Prorein isolates from Hexane-Defarted Ground Yellow .Musrard Meal 19

Many studies have shown the feasibility of recovering the protein isolates through

isoelectric precipitation. Pokorny et al. (1963) showed that the dissolved rapeseed protein

was precipitated at around pH 6.0, with a protein recovery of 49% while Girault (1973)

obtained a protein recovery of 56% by precipitating protein at pH 6.6 from alkaline

extract. The variations of the isoelectric points and the protein recovery through

precipitation of rapeseed protein were probably due to the differences among rapeseed

varieties and extraction methods for dissolution of protein from starting material. Girault

(1 973) showed that acid precipitation of 0. IN NaOH extract could recover 56% of the

solubilised nitrogen, while only 3 1% was recovered from the 10% (w/v) NaCl extract. In

addition. the variations could attribute to the fact that rapeseed contains a mixture of

proteins with widely differing isoelectric points (pH 4-1 1) and molecular weights

(1 3,000-320,000Da) (Lonnerdal et al.. 1977).

Some researchers have studied multi-step isoelectric precipitation to improve

yield. Kodagoda et al. (1973) used pH 2.6, 3.8, and 4.2 as the isoelectric points to

precipitate the rapeseed protein extracted at pH 5.7, 2.0, and 10.0 respectively. resulting

in a total protein recovery of 59%. However, the isolates were generally low in protein

content. This three-stage extraction process was later moditied by Keshavarz et al.

(1977) to obtain colourless protein products. Protein was successively extracted at pH

8.0. 2.0, and 10.0 which was then precipitated at pH 2.6, 3.8 followed by 7.0. and 4.2

respectively, with a 62% protein recovery. A protein content of 92% was achieved in the

isolate extracted at pH 10.0 and precipitated at pH 4.2.

As mentioned in section 2.3.1, precipitation of protein isolates can be achieved by

Production of Protein Isolates from Herane-Defarted Ground Yellow Mustard Meal 20

adding precipitating reagents. Ammonium sulphate and sodium sulphate are often used.

The precipitating reagent is usually added to reach some specific percent of saturation of

the salt. As different proteins precipitate at different percentages of saturation. a

particular fraction of protein can be isolated. Gururaj Rao et al. (1978) first extracted

protein from B. juncea with 1M NaCl, and then, ammonium sulphate was added to the

extract to complete saturation- followed by subsequent adjustment of pH to 3.8. This

resulted in about 88% recovery of proteins. However, the precipitated protein did not

readily redissolve. The precipitation was then done at pH 7.8; 94% protein recovery was

obtained and the precipitate was dissolved readily in 1 M NaCI. The disadvantage of this

method is that ammonium sulphate needs to be removed from the protein. This can be

done by dialysis.

Apart from removing impurities, dialysis can also be used to recover protein from

the extraction solution (Bhatty et al., 1968). Protein was extracted fiom rapeseed meal

with 10% NaCl solution. The supernatant was then dialyzed for 36 hours against distilled

water. A precipitate was formed during the dialysis and was found to recover 45% of the

extracted protein. This method not only recovered the precipitated protein, but also

dialyzed out small molecules such as salt, phytates and glucosinolates. However. the long

operation time of dialysis reduces the feasibility of usage.

2.3.4 Recovery of Protein Isolates by Membrane Processing

As shown in section 2.3.2, recovery of protein through precipitation gives rather

Iow protein yield. This means that a significant amount of extracted protein remains in

Production of Protein Isolates from Herane-Defatted Ground Yellow Mustard Meal 21

the solution. In order to recover this soluble protein, membrane processing can be used.

Ultrafiltration is a membrane processing mode which has been used notably for the

recovery of protein. The theory of ultrafiltration technology will be reviewed in section

Ultrafiltration processes have been used commercially for a number of years in the

dairy industry for the recovery of whey proteins while simultaneously reducing pollution

loads (GIover et al., 1978). intensive research on the use of ultmfdtration for processing

soy (Lawhon et al., 1977; Omosaiye and Cheryan. 1979) and cottonseed proteins

(Lawhon et al., 1980) have been conducted.

Membrane processing of the aqueous solution of rapeseed protein has also been

proposed for preparing rapeseed protein isolates. Maubois et al. (1976) produced an

isolate containing 76% protein. Von Bockelrnann et al. (1977) applied a single-stage

ultrafiltration process, which removes 93% of the glucosinolates. However. the protein

content of the final dry product was only 30%.

A series of membrane-based processes have been developed in our laboratories

for the production of rapeseed protein isolates. Diosady et al. (1984) developed a novel

process based on water leaching and ultrafiltration, which removed approximateIy 9 1 % of

the glucosinolates. This process produced a meal containing 0.2 mg/g glucosinolates and

an isolate containing 0.42 mg/g glucosinolates and 80.4% protein. Later. further studies

on membrane processing showed that ultrafiltration alone had little effect on the yield and

quality of the final products (Tzeng, 1987). An additional purification procedure needed

to be incorporated in order to refine the products. A processing scheme was then

Production of Protein Isolates from Herane-Defatted Ground Yeflow Mustard :Ueal 7 7 - -

developed which included extraction using aqueous sodium hexametaphosphate solution

followed by activated carbon treatment, ultrafiltration. diafiltration. and purification by

ion-exchange (Tzeng et al., 1988a). The process resulted in a protein isolate with about

90% protein content and 63% protein recovery. Two years later, a process designed for

the characteristics of rapeseed and canola proreins was developed. This process consisted

of alkaline extraction. isoelectric precipitation. and ultrafiltration followed by diafiltration

(Tzeng et al.. 1990). These steps complemented one another to yield products of good

quality with high protein recovery. The total isolates yield was 32.5% of the solids.

accounting for 75.4% of the nitrogen in the starting meal.

2.4 Glucosinolates

2.4.1 Chemistry of Glucosinolates

Glucosinolates are sulphur-containing secondary plant metabolites. The basic

structure for glucosinolates is shown in Figure 3 (Ettlinger and Lundeen. 1956). More

than 100 glucosinolates are known and the differences between them depend upon the

chemical nature of their sidechain (R). The mustard species are not identical with respect

to their glucosinolate content. The structures of glucosinolates occurring in mustard seed

are listed in Table 3.

Glucosinolates are generally considered undesirable in concentrated feeds. both

for palatability and nutritional reasons. It was reported that goiter enlargement and

improper functioning of the thyroid gland were consistently produced if Brassica seeds

Production of Prorein Isolares from Hexane-Defatred Ground Yellow Mustard Meal 23

Mustard seed Glucosinolate species

sidechain (R)

Black mustard

(Brassica n i p ) Sinigrin

Gluconapin

Gluconasturtiin

Brown mustard Sinigrin

(Brassica j uncea) Gluconapin

Glucobrassicanapin

Progoitrin/epiprogoitrin

Yellow mustard Sinalbin

(Sinapis alba) Glucoputranjivin

TABLE 3. Glucosinolates in Mustard Seed (Fenwick et al.).

Production of Protein Isolates from Hexane-Defarted Ground Yellow Mustard Meal 24

were fed to experimental animals (VanEtten, 1969). The intact glucosinolates are

apparently free from toxicity, but on hydrolysis by myrosinase, an endogenous enzyme,

they release potentially toxic products such as isothiocyanates, nitriles, thiocyanates and

oxazolidinethione according to the mechanism shown in Figures 4 and 5 (Tookey et al..

1980). The pathway followed depends on pH, temperature, the structure of the side chain

and the presence of cofactors. some of which are still unknown (Dietz and Harris. 1990).

As listed in Table 3. there are two types of glucosinolates in yellow mustard seed.

It has been shown that p-hydroxybenzyl glucosinolate content is at least 2000 times

higher than that of isopropyl glucosinolate (Fenwick et al.. 1983; Kjaer et d.. 1953: Teny

and Corran, 1939). Thus, p-hydroxybenzyl glucosinolate is considered to be the

dominant glucosinolate in yellow mustard seed. Josefsson (1968) studied the yields of

isothiocyanate obtained after hydrolysis of p-hydroxybenzyl glucosinolate at different pH

values. In addition. it has been shown that when p-hydroxybenzyl isothiocyanate is

treated with alkali. it is rapidly split into p-hydroxybenzyl alcohol and thiocyanate ion

(Gmelin and Virtanen, 1960). From this. some researchers have developed methods for

quantitative determination of p-hydroxybenzyl isothiocyanate by colourirnetric

measurement of the thiocyanate ion, which was released by quantitative reaction with

diiute NaOH(aq) (Josefsson. 1968: McGregor, 1978).

2.4.2 Method of Detoxification

2.4.2.1 Inactivation of Myrosinase

Intact glucosinolates are not toxic if they remain unhydrolyzed in the digestive

Producrion of Protein isolares from Herane-Defitted Ground Yellow .\.lustard Meal -75

\\ N- 0 SOT

FIGURE 3. General Structure of Glucosinolates (Ettlinger and Lundeen. 1956).

S - 13 -D -Glucose

R-C /

\\ NOSO;

R-C

\\ NOSO,

R - N = C = S R - C E N R - S - C N

Isothiocyanate Nitrile Thiocyanate

FIGURE 4. The Normal Products of Myrosinase Hydrolysis (Tookey et al.. 1980).

Production of Protein Isolates from Hernne-Defatred Ground Yellow .tiustard Meal 26

Epithionitrile

* ESP = epithiospecifer protein

Oxazolidine - 2 - thione

FIGURE 5. The Products of Myrosinase Hydrolysis of 2-Hydroxy-3-Butenyl

Glucosinolates (Tookey et al., 1980).

Production of Protein Isolatesfiom Hexane-Defatted Ground Yellow ~Mustard Meal 27

system. Therefore, if the myrosinase was inactivated completely within the seed prior to

processing, the meal and other mustard products would be safe for human consumption.

Tlermal inactivation of myrosinase has been reported for mustard seed, rapeseed. turnip

rapeseed and canola seed.

In 1964, it was reported that myrosinase activity was destroyed by cooking

crushed rapeseed. without addition of water. at temperatures greater than 8 0 ' ~ (Reynolds

and Youngs. 1964). Appleqvist and Josefsson (1967) found that the myrosinase was

completely inactivated by heating seeds with 8% moisture content at 90'~ for 15 minutes.

Myrosinase inactivation can also be achieved by microwave-heating of whole seeds at

their original moisture content of 5.2 to 6% for three minutes (Eapen et d.. 1968). The

efffectiveness of microwave treatment has also been suggested by Armstrong (1975) and

Maheshwari et al. (1980).

Besides dry heat treatment, inactivation of myrosinase using wet heat treatment

was also reported. Eapen et al. (1968) found that immersion of seeds in boiling water

was effective in inactivating the enzyme. In addition, they also reported that steam

blanching treatments of seeds for five to thirty minutes were effective in completely

inactivating the enzyme. However. most of these e w e inactivation studies have

neglected to investigate the full effects of these procedures on the nutritional and

functional properties of the meal proteins and the quality of the resulting meal and oil

produced.

Recently, various researchers have developed a simple process for complete

thermal inactivation of myrosinase without degrading the seed (Wang et al.. 1994). The

Production o f Protein Lsolates from Hexane-Defatted Ground Yellow Musrard Meal 8

process consists of a steam-jacketed screw conveyor for preheating, followed by a stearn-

injection system. A steam line pressure of 140 kPa which corresponded to a temperature

of 1 0 5 ~ ~ . and a mean residence time of 12.8 minutes appeared to achieve complete

inactivation of myrosinase in yellow mustard seed.

2.4.2.2 Removal of Glucosinolates and their Hydrolytic Products

Inactivation of myrosinase leaves the glucosinolates intact in the meal. I f this

meal is further processed for food purposes. the compounds may be hydrolyzed to toxic

isothiocyanates and oxazolidinethiones by the myrosinase reintroduced by other dietary

ingredients or common microflora of the gastrointestinal tract of humans or other animals

(Greer and Deeney, 1959; Oginsky et al.. 1965). Therefore. removal of glucosinolates

and their hydrolytic products is a better approach to detoxify the seed and its meal before

they can be fdly utilized in the human diet.

A simple diffusion extraction method was developed based on the principle that

only low molecular weight glucosinolates would passively diffuse through the membrane

of the seed. It has been reported that five or six one-hour extractions of the intact seeds

with 0.01 N NaOH at 6 0 ' ~ ~ after inactivating the myrosinase by boiling in water for three

minutes. were sufficient to reduce the glucosinolates to trace levels (Sosulski et al..

1972). Later, Bhatty and Sosulski (1972) successfully used ethanolic NaOH to extract

rapeseed glucosinolates as well as inhibiting the myrosinase activity by four two-hour or

six one-hour diffusion extractions. However, the disadvantages of these methods were

the reduced solubility of the rapeseed proteins, the increased fiber levels of the meals and

Production of Protein isolates porn Hexane-Defarted Ground Yeilow Mustard .Meal 2 9

the long extraction times required.

Various researchers have studied the extraction of glucosinolates from crushed

seed for better contact. One of the methods used the boiling water treatment to inactivate

myrosinase and then aqueous extraction of glucosinolates from wet-grind seeds followed

by air classification of the dried defatted rapeseed meal. This method was found to

produce rapeseed flour suitable for human consumption (Eapen et al., 1969). The process

removed up to 98% glucosinolates but protein losses were reported as high as 40%. This

process was fkrther improved to reduce losses of soluble solids (Tape et al.. 1970) and

later optimized by employing finer screens and counter-current equipment for extraction

(Eapen et aI., 1973). Two types of treatments of presscake rapeseed meal, steaming and

water extraction by single and double stages, were developed (Ballester et al., 1970). In

1973, the same researchers reported that a two hour continuous water extraction

procedure with stirring was sufficient to remove isothiocyanates completely and reduce

oxazolidinethiones by 97% (Ballester et al., 1973).

Combinations of polar solvents have been successful in the detoxification of

rapeseed and its meal. Several workers have reported that glucosinolates could be

effectively extracted with aqueous mixtures of methanol, ethanol or acetone, without

extracting much of the meal solids (Mukherjee et al., 1976; Perlroth et d., 1976).

Diosady et al. (1985) reported that methanol was the most effective alkanol for the

removal of glucosinolates. Moreover, the presence of ammonia and water in alkanols

was found to significantly increase the effectiveness of the detoxification process. A two-

phase solvent extraction system, consisting of hexane and methanol containing dissolved

Producrion of Prorein Isolares from Hexane-Defatred Ground Yellow Mustard Meal 30

ammonia was developed in our laboratories. The glucosinolates content in rapeseed meal

was reported to decrease with increasing ammonia concentration (Diosady et d.. 1985).

The residue meal was low in glucosinolate (< 0.2 rng/g), light in colour and bland in

flavour (Rubin et al., 1986). Later, the two-phase solvent extraction system was applied

to remove glucosinolates from high-glucosinolate rapeseed and mustard seed. Over 98%

of the glucosinolates was removed after a second extraction of the meal (Naczk et al..

1986). while Ponzani (1996) obtained 99.9% removal under optimum conditions.

2.4.2.3 Other Methods of Detoxification

Various studies involving destruction of glucosinolates and their hydrolytic

products in rapeseed and its meal by heat, chemicals and microorganisms have been

reported. Temperatures of 1 2 0 ~ ~ and above were reported to destroy more than 50% of

the isothiocyanates and oxazolidinethione (Rutkowski. 1970). Several researchers have

demonstrated that salts of iron. copper and nickel were the most active in decomposing

glucosinolates (Bell et al.. 1970; Kirk et al., 197 1). Microbial fermentation techniques

have been successful in the destruction of glucosinolates through the action of the fungus

Geotrichurn candidurn on rapeseed presscake (Staron, 1970). However, the above

methods of destroying glucosinolates resulted in a loss of nutritive value of the protein

and the formation of toxic by-product in some instances, while others are not economical

in large scale industrial applications.

Rapeseed breeding programs lauched in the early 1960's have produced some low

glucosinolate rapeseed varieties through genetic selection. These methods would also

Production of Protein Isolaresfrom Hexane-Defarted Ground Yellow ~Musrard Meal 3 1

improve meal palatability and eliminate any hazards from reintroductions of myrosinase

somewhere along the food chain. The discovery of the summer rape variety which had a

glucosinolate content well below average in 1967 made it possible for the genetic

development of low-glucosinolate varieties (Appelqvist and Ohlson, 1972). Presently.

the development of low glucosinolate mustard is achieved through an interspecific cross

between an Indian type B. juncea and a "Bronowski-gene(s)" containing low

glucosinolate B. carnpestris L. followed by backcrossing to the B. juncea parent (Love et

al.. 1990). This achievement should allow the breeding of canola quality oilseed B.

juncea mustard.

2.5 Membrane Technology

2.5.1 Introduction

Membrane filtration refers to the separation of dissolved solutes in liquid streams.

or the separation of gas mixtures by passing the fluid through a selectively permeable

membrane. Membranes of varying "molecular weight cut-offs" (MWCO) or pore sizes

are available. Solutes of molecular weight higher than MWCO are retained while the

lower molecular weight solutes are removed through the membrane. Examples of some

technically relevant membrane separation processes are dialysis, electrodialysis.

microfiltration, gas permeation, reverse osmosis, and ultrafiltration.

Conventional filtration alIows the process fluid to pass through the filter by

Production of Protein Isolates from Hexane-Defatted Ground Yellow Mutard Meal 32

gravity, which leads to an increasing solid build-up adjacent to the filter surface. AS a

result, the rate of filtration decreases and consequently increases the filtration time. With

cross-flow filtration. the fluid is diffused through the membrane and any molecules that

are retained inside the membrane will be pushed along in the direction of flow. A

diagram of this process is illustrated in Figure 6. This type of filtration ensures that the

accumulation of the retained molecules at the surface of the membrane is minimized.

Therefore, the concentration polarization on the membrane is effectively reduced.

Membrane separation processes are usually performed at ambient temperature.

Thus, temperature-sensitive solutions can be treated without causing thermal degradation

or chemical alteration of their constituents. This is one of the main reasons why this

technique is commonly used in the food, pharmaceutical. and biological industries

(Strathmann, 1986).

2.5.2 Ultrafiltration

Ultrafiltration is a cross-flow membrane separation process in which the

solvent is removed from solutions containing high-molecule-weight solutes such as

proteins. The principle is similar to that of most other membrane separation processes, in

that pressure is applied to the solution on the feed side of a supported membrane. The

solvent passes through the membrane while high-molecular-weight solutes larger than the

MWCO are retained and pushed along in direction of flow. Since glucosinolates,

phytates, and other micromolecular components have significantly lower molecular

weights than proteins, it should be possible to separate them from proteins in aqueous

Production of Protein Isolates from Herane-Defarred Ground kellow :tlustard .Cfea[ 33

permeate

direction of flow

membrane

FIGURE 6. Cross-flow Filtration.

Prohrction of Protein Isolatesfiorn Herune-Defatted Ground Yellow ;Mustard Meal 34

solution by using ultrafiItration.

Frequently, molecules larger than the molecular weight cut-off are not completely

rejected, whilst molecules much smaller than molecular weight cut-off are partially

rejected (Paterson, 1993). In order to quantitatively estimate the relative degree of

purification of a process fluid in a given ultrafiltration process, or calculate the extent of

ultrafiltration processing required to attain a certain degree of separation the

ultrafiltration must be mathematically modelled.

The rejection of solutes is usually used to

membrane and is defined as

where R = rejection coefficient

C, = solute concentration in permeate

C, = solute concentration in retentate

The characteristic equation of the ultrafiltration

where CV, = initial solute concentration in feed tank

C V ~ = final solute concentration in feed tank

V, = initial volume in feed tank

measure the performance of the

Vf = final volume in feed tank

R = rejection coefficient

Production of Prorein lsolares from He-~ane-Defalted Ground Yellow Mustard Meal 33

CF = concentration factor

The schematic diagram of a laboratory-scale batch ultrafiltration unit is shown in

Figure 7.

If the process solution contains a high-molecular-weight solute A and a low-

molecular-weight solute B, having rejection coefficients RA = 1 and RB = 0 respectively.

the ratio of final concentration of A to initial concentration of A is equal to the ratio of

initial volume to final volume in the feed tank. Therefore, the concentration of high-

molecular-weight solute in the retentate is increased by a concentration factor (CF) and

the concentration of low-molecular-weight solute is not influenced by the ultrafiltration

system. In this way, ultrafiltration concentrates the retained species but the

micromolecular impurities are still maintained at their initial concentration. To reduce

these impurities to trace levels. diafiltration can be employed.

2.5.3 Diafiltration

Diafiltration is used to purify the process solution by diluting the concentration of

the low-molecular-weight solute. This is done by adding fresh solvent, continuously or

discontinuously, to replace the permeate that has been removed. Fresh solvent is added to

the feed tank at a rate that maintains a constant retentate volume. The process diagram of

a laboratory-scale diafiltration with continuous addition of fresh solvent is shown in

Figure 8. This mode of diafiltration is particularly useful if b h e r purification of the

process solution is required since ultrafiltration can never remove micromolecules to very

low levels.

Producrion of Protein Isolatesfrorn Hexane-Defatted Ground Yellow Mustard .%leal 36

Back - pressure Valve

Feed Tank Permeate

Membrane Cartridge

FIGURE 7. Batch Ultrafiltration.

Production of Protein Isolates from Hexane-Defatted Ground Yellow ,Mustard Meal 37

Back - pressure Fresh Valve

S y t (Zetentate

FIGURE 8. DiafiItration with Continuous Addition of Fresh Solvent.

Production of Protein Isolates from Herane-Defarred Ground Yellow Muturd .Cleal 38

The characteristic equation of the diafi~ltration system is expressed as

where CK = solute concentration in the retentate

Co = solute concentration in the original feed

Vw = volume of fresh solvent added

Vo = initial volume in feed tank

DV = diavolume, the ratio of the two volumes

If the process solution contains a low-molecular-weight solute B with a rejection

coefficient of R = 0, a five-volume exchange of fresh solvent, at constant process sample

volume, concentration of solute B is reduced to e-' ~ 0 . 0 0 7 times the original

concentration. This means that diafrltration effects a 99.3% removal of solute B from the

system. Therefore, the remaining solution in the feed tank will be concentrated with the

high-molecular-weight solute and essentially free of micromolecular impurities.

Although a greater diavoiurne will induce a higher purity, diafiltration with a very

large diavolume is not economically feasible because of the long processing time and the

enormous amount of fresh solvent required. Therefore, from an economic point of view.

a combination of ultrafiltration and diafiltration is generally used.

Production of Protein /solatesjFom Hexarze-Defarted Ground )hilow .tfustard Meal 3 9

3. MATERIALS AND METHODS

3.1 Ground yellow mustard seed and starting meal

Whole ground yellow mustard seed (Sinapis alba) was obtained from Hemann

Laue (Hela) Spice Co. Inc., Scarborough. ON., Canada.

The starting meal was prepared by extracting the oil from ground seed with

hexane for 24 hour, using a Soxhlet extractor. For the preparation of each batch. 200g of

seed was evenly extracted using four Soxhlet extractors. The defatted meal was then

dried in the h e hood overnight at room temperature. The process was repeated eight

times and approximately lkg defatted ground yellow mustard meal was produced. The

eight batches of meal were mixed thoroughly and their compositions are shown in Table

4.

Production of Prorein /solatesfrom Herane-Defatted Ground Yellow :Mustard .Weal 40

protein* content

[%I

+ Data reported as average k one standard deviation of three measurements

* results reported on oil-free and moisture-fiee basis

" oil is not detectable in the meal

glucosinolate content*

[ P o l k 1

TABLE 4. Composition of Ground Yellow Mustard Seed and Its Meal.

phytate* content

[%I

Production of Protein Isolares from Herane-De fatted Ground Yellow .Iluslard .Meal 4 1

3.2 Membrane Processing Unit

A bench-scale Arnicon CH4 hollow-fiber concentrator was used. which was

manufactured by Arnicon Corporation, Lexington, MA., U.S.A. It can be operated in

either the ultrafiltration or the diafiltration mode. The unit included a built-in peristaltic

pump. which forced the solution through a prefilter to remove fine solids. and then

through a hollow-fiber membrane cartridge. The cartridge employed in this project was

the Amicon DIAFLOTM H 1 P 10-20 with the following characteristics:

Nominal molecular weight cut-off = 10,000 Ddtons

Membrane area = 0.05 rn'

Height of cartridge = 200 rnm

Diameter of cartridge = 25.4 mrn

The pressure in the cartridge was controlled by a back-pressure valve at its outlet.

The permeate. consisting of water and dissolved low-molecular-weight species whose

molecular weights were below the molecular weight cut-off of the membrane. was driven

through the fiber walls. while the retentate, containing the high-molecular-weight species.

was recirculated to the feed tank. Upon completion of an experiment, the unit was

immediately drained and flushed with distilled water. A 5 g/L commercial enzyme-

containing detergent solution (Terg-A-Zyme. Alconox Inc., New York. U.S.A.) was then

recycled through the unit for about one hour. After draining, the unit was again flushed

with distilled water until the initial water flux was fully restored. A volume of 8-10 liters

of distilled water was usually used. The cleaned membrane cartridge was stored in a

Producrion of Protein isolates from Hexane-Defatred Ground Fe'ellorv .Mustard Meal 42

0.5% (wlv) formaldehyde solution. One day prior to use. the cartridge was rinsed and

soaked in distilled water.

3.3 Experimental Methods

3.3.1 Extractability of Yellow Mustard Protein

The extractability of yellow mustard protein was studied with respect to three

extraction conditions, they are: pH, solvent-to-meal ratio, and extraction time. The

following conditions for each of the variables were studied:

pH: 3 to 13, with increments of 1

Solvent-to-Meal Ratio: 12 to 22 (v/w), with increments of 2

Extraction Time: 10,20, 30. 4 - 6 0 ? 120 minutes

3.3.1.1 Effect of pH on Nitrogen Extractability of Yellow Mustard Meal

For each test, log of meal (on -as is' basis) was dispersed in distilled water at a

pre-selected pH with a solvent-to-meal ratio of 18 at room temperature in a 250 mL

beaker. The extraction was carried out using a magnetic stirring plate for 30 minutes.

The pH of the solution was maintained at the target value by adding 25% or 5% (wlw)

NaOH as base or 6N or 1N HCI as acid. A VWR Scientific model 8000 pH-meter

manufactured by Orion Research Inc. was used. As soon as the extraction was

completed, the supernatant was separated fiom the wet residue by centrifbgation at 7000

rpm for 20 minutes using an IEC 8-22 centrihge (International Equipment Co., Needham

Production of Prorein /solaresfrum Herane-Defarred Ground Yellow Mzarurd .Weal 43

Hts.. MA.. U.S.A.). The wet residue was then washed once with distilled water. which

was at the same pH as the extraction solution, at a solvent-to-meal ratio of 6 (vlw) for 10

minutes. The washing liquid was again separated from the residue by centrifugation and

then combined with the supernatant obtained from the first extraction. The volume of the

mixture was recorded and aliquots were taken to determine the dissolved nitrogen

content.

As pH 12 was determined to be the optimum condition for protein extraction (see

section 4.1. I), the tests on extraction time and solvent-to-meal ratio would be carried out

at this pH value.

3.3.1.2 Effect of Extraction Time on Nitrogen Extractability of Yellow Mustard

Meal

For each determination, 10 g of meal was mixed with 180 mL pH 12 distilled

water at room temperature and the extraction was carried out for a specific duration

which varied as follows: 10. 20, 30. 45, 60 and 120 minutes. The pH of the extraction

solution was maintained at 12 by the addition of 5% (w/w) NaOH solution. At the end of

the specified time interval. the sluny was separated from the extraction solution by

centrifugation and was then washed once with 60 mL pH 12 distilled water for 10

minutes. The residue was again separated from the washing liquid by centrifugation. The

extraction solution and washing liquid were combined and the volume of the mixture was

recorded. Aliquots were taken from the mixture to determine the dissolved nitrogen

content.

Prodztcrion of Protein lsolates from Hexane-Defatted Ground Yellow :Wustard Meal 44

As 30 minutes was chosen to be the optimum time for the extraction, the tests on

solvent-to-meal ratio would be carried out using this contact time.

3.3.1.3 Effect of Solvent-to-Meal Ratio on Nitrogen Extractability of Yellow

Mustard Meal

Tests were carried out using solvent-to-meal ratios of 12. 14, 16, 18. 20 and 22

(v/w). For each test, 10 g of meal was mixed with the corresponding amount of pH 12

distilled water in a 250 mL beaker and the extraction was carried out by stirring on a

magnetic stimng plate at room temperature for 30 minutes. The pH of the extraction

solution was maintained at 12 by the addition of 5% (w/w) NaOH solution. The slurry

was separated from the extraction solution by centrifugation and was then washed once

with 60 mL of pH 12 distilled water for 10 minutes. The residue was again separated

from the washing liquid by centrifugation. The extraction solution and washing liquid

were combined and the volume of the mixture was recorded. Aliquots were taken from

the mixture to determine the dissolved nitrogen content.

3.3.2 Removal of Glucosinolate by Membrane Processing

The meal was extracted at the optimum extraction pH, exaaction time. and

solvent-to-meal ratio as determined from the aforementioned experiments. Therefore.

25g of meal was extracted with 450mL aqueous alkali at pH 12 for 30 minutes. The

sluny was centrifuged and the supernatant was then fiitered using Whatrnan No. 41 filter

paper. The residue was washed once with pH 12 aqueous alkali at a solvent-to-meal ratio

Production of Prorein Isolates from Hexane-Defutred Ground Yellow .Mustard .Veal 45

of 6 (v/w) for 10 minutes. Centrihgation and filtration recovered the washing liquid.

which was then combined with the supernatant to form the alkaline extract.

The combined alkaline extract was then ultrafiltered at a concentration factor of 2.

Immediately after ultrafiltration, the retentate was purified by diafiltration at a diavolume

of 5, in which aqueous alkali at pH 12 was used as the fresh solvent to replace the

removed permeate. This two-stage membrane processing should remove the undesirable

components such as glucosinolates, phytates and phenolics. A control run without

membrane processing was made in order to determine the effectiveness of the removal of

glucosinolates by ultrafiltration and diafiltration.

3.3.3 Isoelectric Precipitation of Yellow Mustard Protein

The precipitation of extracted yellow mustard protein at various different pH

values was examined. The protein was first extracted with aqueous alkali as described

above, without carrying on to the membrane processing. Samples of exactly 200 rnL of

alkaline extract were precipitated at pH values ranging from 3.5 to 7.0 in increments of

0.5. The pH was adjusted with 6N or IN HC1 to the target value and maintained for 15

minutes. After centrihgation at 7000 rpm for 20 minutes. the precipitates were washed

with distilled water which was acidified to the precipitation pH, for 10 minutes and

centrihged again, then oven-dried at 105' overnight. The dry matter and nitrogen content

were determined.

Production of Protein Isolates from He-rane-Defatred Ground Yellow ,Mustard .Meal 46

3.3.4 Recovery of Yellow Mustard Protein Isolates

The determination of the optimum extraction conditions combined with the

confirmed necessity of the membrane processing prior to isoelectric precipitation. led to

the development of the process for preparation of protein isolates from mustard meal.

The flowsheet for the process is shown in Figure 9. Typically, 25g of defatted ground

yellow mustard meal was extracted with 450 rnL aqueous NaOH solution at pH 12 for 30

minutes. After centrifugation and filtration, the residue was washed once with pH 12

aqueous NaOH solution at a solvent-to-meal ratio of 6 (v/w) for 10 minutes. The

washing liquid was combined with the original extract, while the washed meai residue

was freeze-dried with a Labconco Model 5 Freeze Dryer.

The combined alkaline extract was ultrafiltered at a concentration factor of 2 and

then diafiltered at a diavolume of 5 , in which pH 12 distilled water was used as the fresh

solvent to replace the removed permeate. The pH of the retentate was then adjusted to

4.75. where the isoelectric precipitation of yellow mustard protein occurred. The pH was

maintained for 15 minutes by the addition of 6N or 1N HC1. After centrifugation at 7000

rpm for 20 minutes, the supernatant was vacuum-filtered using Whatman No. 42. filter

paper. The precipitate was then washed once with five times its weight (on wet basis) of

distilled water acidified to pH 4.75 for 10 minutes. The washing liquid was combined

with the original supernatant after centrifugation and filtration, while the precipitate was

fieeze-dried to recover the precipitated protein isolate (PPI) .

The combined acidic extract was subsequently ultrafiltered at a concentration

factor of 5 and diafiltered at a diavolume of 5, in which distilled water was used as the

Production of Protein Isolates from Haane-Defarted Ground Iellow .blustard Meal 4 7

( Starting Meai 1

& washing

I Alkalineextract I

Centrifirgation & filtration Wet residue

* Retentate

I I

* Freeze-drying

Permeate Meal Residue c3 Uhrafiltration & diafiltration

I

( Acidic extract 1

Centrifugation & filtration protein precipitate

I Retentate I

I 1

Permeate

Freeze-drying

Ukrafiltration & diafiltration -

Precipitated Protein I

FIGURE 9. Process Flowsheet.

* Freeze-drying Soluble Protein Isolate

Production of Protein Isolates from Heme-Defatred Ground Yellow .Clustard Meal 48

fiesh solvent to replace the removed permeate. The retentate was keeze-dried to recover

the soluble protein isolate (SPI) .

3.3.5 Chemical Analyses

The moisture content was determined according to AACC Method 44-15A

(AACC, 1976). The moisture-containing samples were dried at 1 0 . 5 ~ ~ in a forced-air

oven (Blue M Electric Co., Blue Island, IL., U.S.A.) overnight, and the moisture content

was then determined gravimetrically. Details are given in Appendix 8.1.

Crude protein (N x 6.25) was determined by the Kjeldahl method according to

AACC Method 46-12 (AACC. 1976). A Biichi model 425 digester and Biichi 325

distillation unit (Biichi Laboratorium-Technik AG, FlawiI, Switzerland) were used.

Details of this analysis are given in Appendix 8.2.

Phytate was determined according to a procedure developed by Naczk et al.

(I 986), which was modified from the methods published by Wheeler and FerreI (1 97 1 ).

Ellis et al. (1977), and Tangkongchitr et aI. (1981). The phytate phosphorus was

determined colourimetricalIy according to the AOCS Offkial Method Ca 12-55 (AOCS.

1980). The phytic acid content of the sample was calculated from the measured phytate

phosphorus value multiplied by a conversion factor of 3.55. Details are given in

Appendix 8.3.

Production of Protein Isolates/rom Hevane-Defatted Ground Yellow .Vh.stard .Meal -19

Glucosinolate content was determined according to the unpublished method

developed by McGregor (1978): that was modified from the method published by

Iosefsson (1968). The method is used to determine parahydroxybenzyl glucosinolate

content. the major glucosinolate of yellow mustard. The free thiocyanate icn was

measured colourimetrically, and the result was expressed as pnole of free thiocyanate

ion. parahydroxybenzyl isothiocyanate or parahydroxybenzyl glucosinolate. per gram oil-

free. moisture-free meal. Details are given in Appendix 8.4.

Producrion of Prorein Isolares from He~ane-Defarted Ground Yellow .Liusrard Meal 50

4. RESULTS AND DISCUSSION

4.1 Protein Extractability of Defatted Yellow Mustard Meal

4.1.1 Effect of pH on Protein Extractability

The pH solubility profile of yellow mustard proteins in aqueous solution was

studied. The results are summarized in Table 5 and Figure 10.

As expected. the nitrogen extractability increased as pH went up over the alkaline

region. Extractability increased steadily from about 25% until it leveled off at 85% as pH

rose fiom 7 to 12. A s pH increased fiom 22 to 13, no further increase in extractability

was observed. It can be concluded that nitrogen extractability of yellow mustard meal

reached a maximum of 85% at pH 12. However. it is lower than the nitrogen

extractability of hexane-defatted brown mustard meal, which was 92% at pH 11.0

(Gururaj Rao et al.. 1978). By comparing the pH solubility profiles of both mustard

meals, the nitrogen extractability of yellow mustard was found to be generally lower than

that of brown mustard. At pH 7, 55% of the nitrogen was extracted fiom brown mustard.

while only 25% was extracted &om yellow mustard. This variation is due to the different

mustard species, as well as the difference in meal preparation. Although hexane-defatted

meal was used in both cases, the brown mustard meal was dehulled, As some fraction of

the nitrogen is bound to the hull of the seed, dehulling probably allowed a better

dissolution of the nitrogen, thereby increasing its extractability.

The pH solubility profile of yellow mustard protein showed two minima at pH 4

Prodzxrion of Prorein lsolates from Hexane- De farred Ground Yellow Musrard Meal 51

Extraction pH Nitrogen Extractability [Oh]

* Average k one standard deviation of three measurements

TABLE 5. Effect of pH on Extractability of Yellow Mustard Protein.

Production of Protein Isolates from Herune-Defatted Ground Yellow Mustard Meal 52

Protein Extractability as a Function of pH

Error bar represents standard deviation of three measurements

FIGURE 10. Effect of pH on Extractability of Yellow Mustard Protein.

Production of Protein lsolaresfiorn Hexane-Defatted Ground kellow .bfustard Meal 53

and 6. At these pH values, about 17% and 22% of the total nitrogen were extracted fiom

the meal respectively. These results indicated that the yellow mustard protein had

minimum extractability over the acidic range, which was in close agreement with the

result obtained by Gururaj Rao et al. (1978). They also reported two minima in the

solubility profile of brown mustard protein. However, the minima occurred at pH 3.8-4.0

and 7.8-8.0. The differences could be attributed to the different mustard species. which

would probably contain proteins with different molecular weights. compositions and

isoelectric points.

As mentioned in the Introduction, the protein isolation procedure was adapted

from the novel process developed for rapeseed and canola; therefore, it may be interesting

to compare the nitrogen extractabilities of these meals. Tzeng et al. (1990) have studied

the nitrogen extractability of hexane-extracted canola meal. The results showed that 80-

90% nitrogen extractability at pH 10-12.5 was obtained. Canola meal has an overall high

nitrogen solubility in the alkaline range. However, in the case of mustard, the nitrogen

extractability increased from 57 to 85% as pH increased fiom 10 to 12. A maximum

increase of 17% was achieved as pH increased by one increment fiom pH 10 to 1 1. This

suggested that the influence of pH on the mustard protein extractability is greater than

that on the canola protein.

Tzeng et al. (1990) have also studied the effect of high-pH extraction on nitrogen

extractability in methanol-ammonia-waterhexme extracted canola meal. As mentioned

in Section 2.4.2.2, methanol-ammonia-waterhexane was found effective in removing

glucosinolates from rapeseed, so that a low glucosinolate content, light coloured and

Production of Prorein Isolares from Hexane-Defarted Ground Yellow Mustard .Meal 54

bland flavoured meal was produced. By comparing the nitrogen solubility profiles,

mustard behaved similarly to this methanol-ammonia-waterlhexane extracted canola.

The pH value greatly influenced both their nitrogen extractabilities. However. a

maximum of 70% of the nitrogen was extracted fiom the methanol-ammonia-

waterhexme extracted cano la meal while 90% was extracted from hexane-extracted

canola meal. This suggested that the protein was probably denatured and/or dehydrated

during the methanol-ammonia-water treatment of the meal.

As a conclusion, pH 12 is found to be appropriate for the alkaline extraction of

mustard protein.

4.1.2 Effect of Extraction Time on Protein Extractability

The effect of the extraction time on the nitrogen extractability of yellow mustard

meal is shown in Table 6 and Figure 11. Since the optimum pH condition for protein

extraction was determined to be pH 12 in the previous section. the studies on nitrogen

extractability influenced by extraction time were carried out at pH 12.

The extraction time represents the duration of the extraction. After extraction, the

residue was washed for 10 minutes. Therefore, the actual extraction time is the

experimental extraction time plus the washing time. The nitrogen extractability of the

meal before the washing step was also included in Table 6 as a comparison. As shown

from the results, the nitrogen extractability was not only dependent on the length of the

extraction time, but also the existence of the washing step. It can be seen that nitrogen

extractability increased rapidly as extraction time rose from 5 to 30 minutes without

Production ofprotein Isolates from Hexane-Defatted Ground Yellow ,Mustard .Meal 55

Extraction time Protein Extractability Overall Protein

bin1 [%I " Extractability [%I

a Protein extractability of the meal after the indicated extraction time period.

b TotaI protein extractability of the meal during the indicated extraction time period plus 10

min washing.

* Average t one standard deviation of three measurements

TABLE 6. Effect of Extraction Time on Extractability of Yellow Mustard Protein.

Production of Protein Isolates from Hexane-Defafted Ground Yellow .Mustard Meal 56

Protein Extractability as a Function of Extraction Time

washing

4 extraction only -

Extraction Time [min]

Error bar represents standard deviation of three measurements

FIGURE 11. Effect of Extraction Time on Extractability of Yellow Mustard

Protein.

Production of Protein Isolates from Herane-Defatted Ground Yellow .Mustard Meal 57

washing. After an extraction period of 30 minutes, about 77% of nitrogen was extracted

fkom the meal. However, a longer extraction period showed no significant increase in

extractability. As a compromise between the nitrogen extractability and the processing

time, an extraction period of 30 minutes was chosen to be the optimum time for the

entirekomplete extraction to be carried out. In addition, it has been proclaimed that

degradation of some amino acids and formation of potentially harmful levels of

lysinolalanine during the alkaline treatment of proteinaceous materials may occur at high

pH (Pfaender, 1983; Deng et al., 1990). Therefore, for alkaline extraction at high pH.

extended extraction times should be avoided.

It is also interesting to compare the nitrogen extractabilities of mustard protein

with and without the washing step. Results showed that about 10% more nitrogen could

be extracted from the meal during the washing. However. the effect of washing

diminished as the extraction time went beyond 10 minutes. The increase in nitrogen

extraction during the washing step may be explained by two possible mechanisms. In the

first mechanism. the excess nitrogen may be extracted from the solid residue during

washing while in the second mechanism. washing displaces the trapped protein solution

from the matrix with fresh solvent. These two mechanisms can easily be verified by

examining the results. In an extraction period shorter than 20 minutes, the increase of

nitrogen extractability was mainly due to the first mechanism while the second

mechanism was mostly related to the period longer than 45 minutes. Results showed that

the increase in extracted nitrogen after washing was greater at periods shorter than 20

minutes. This means that excess nitrogen was actually extracted besides washing off the

Production o f Protein Isolates from Hexone-Debtred Ground Yellow ,1.lusrard Meal 38

trapped solution from the residue. During the period of 20 to 45 minutes. both

mechanisms brought about similar effects on the increase of extractability. Therefore.

this indicated that washing was an essential step subsequent to the alkaline extraction of

mustard protein.

4.1.3 Effect of Solvent-to-Meal Ratio on Protein Extractability

Protein was extracted at pH 12 for 30 minutes at various solvent-to-meal ratios.

Its effect on protein extractability is shown in Table 7 and Figure 12.

The solvent-to-meal ratio (R) showed little effect on nitrogen extractability.

Increased solvent-to-meal ratios from 12 to 18 resulted in only slightly increased nitrogen

extractability. When the ratio was increased to 18. nitrogen extractability reached a

maximum of 85%. As the ratio further increased to 22, nitrogen extractability dropped

slightly or it can be considered as about the same. During the extraction with a solvent-

to-meal ratio of 12 to 16, the slurry was too thick to stir, which resulted in inefficient

mixing. Therefore. the relatively low nitrogen extractability was probably due to the non-

homogeneous condition of the extraction solution, in which protein could not be evenly

extracted from the meal. Moreover. the high viscosity of the protein extract made it

difficult to recover after centrifugation, where a certain fraction of extracted protein

would still be adsorbed onto the solids. Consequently, R was set at 18 for subsequent

runs as this ratio yielded the highest nitrogen extractability.

Accordingly, in preparing yellow mustard protein isolates, it is appropriate to

extract meal proteins at pH 12 with a solvent-to-meal ratio of 18 for 30 minutes. Under

Production offrotein Isolares/rorn Hexane-Defatted Ground Yelfow Mustard Meal j9

SoIvent-to-Meal Ratio (v/w) Protein Extractability [%I *

-- -

* Single measurement for each test

TABLE 7. Effect of Solvent-to-Meal Ratio on Extractability of Yellow Mustard

Protein.

Producrion of Protein Isolares from Hexane-Defarred Ground Yellow .Musrard Meal 60

Protein Extractability as a Function of Solvent-to-Meal Ratio

Solvent-to-Meal Ratio (dw)

+ Single measurement for each test

FIGURE 12. Effect of Solvent-to-Meal Ratio on Extractability of Yellow Mustard

Protein.

Production of Protein Isolates porn Hexane-Defitted Ground Yellow :Mustard Meal 61

these conditions at which protein extractability of hexane-defatted ground mustard meal

was found to be 85%.

4.2 Precipitation of Yellow Mustard Protein

Proteins can be classified either as simple or conjugated. They can also be

classified on the basis of their solubility. As a result. more than one category of proteins

usually exist in oilseeds. The detailed protein composition is specific to each species.

which each have widely different isoelectric points and molecular weights. As mentioned

in section 2-33, protein bound with salt would have a different isoelectric point than the

free protein. Thus, the effect of pH on the precipitation of extracted yellow mustard

protein was investigated in order to find the optimal conditions for the recovery of protein

dissolved in the alkaline solution. The results are shown in Table 8. The dry matter. the

protein content and the recovery of precipitated protein are plotted against pH in Figures

1 3, 14 and I 5 respectively.

At pH lower than 4.0 and higher than 6.0, only a small fraction of the dissolved

yellow mustard protein was precipitated. As pH increased from 3.5 to 4.5, both the dry

matter and the protein recovery increased rapidly. The results showed a precipitation

maximum covering a narrow pH range from 4.5 to 5.0, within which over 50% of the

extracted protein was recovered in the precipitate. In this pH range, the protein recovery

remained almost constant, with a slightly higher yield of 54% at pH 4.75. This maximum

protein yield was close to most results reported for rapeseed. Gillberg and Tomell (1 970)

Producrion of Prorein lsolares from Herane-Defatted Ground Yellow .Mustard Meal 62

PH Mass Recovery Protein Content Protein Recovery (%) " (%I (%)

a As % of dissolved solid

As % of dissolved protein

* Average + one standard deviation of three measurements

TABLE 8. Effect of pH on Precipitation of Yellow Mustard Protein.

Production of Protein Isolafes from Hexane-Defarred Ground Yellow .Mustard .Cleal 63

Mass Recovery of Precipitate as a Function of pH

Single measurement for each data point

FIGURE 13. Effect of pH on Mass Recovery of Yellow Mustard Protein Precipitate.

Production of Protein /solares from Haane-Defatted Ground Yellow .Cfusrard Meal 64

Protein Content of Precipitate as a Function of pH

Error bar represents standard deviation of three measurements

FIGURE 14. Effect of pH on the Protein Content of Yellow Mustard Protein

Precipitate.

Production of Prorein Isolares from He-rane-Defatted Ground I'ellow .Mustard Meal 65

Protein Recovery of Precipitate as a Function of pH

+ Error bar represents standard deviation of three measurements

FIGURE 15. Effect of pH on Protein Recovery of Yellow Mustard Protein

Precipitate.

Producrion of Prorein lsalares from Herane-Defatted Ground Y e l h Mustard Meal 66

and Girault (1973) reported similar recoveries of 55% and 56% of the dissolved protein

respectively. The result was also similar to the protein recovery of 54.9% obtained in our

laboratory with Chinese rapeseed protein, which has its isoelectric point in the neutral pH

range (Xu, 1993).

In terms of the protein content of the precipitates, yellow mustard behaved very

different fi-om Chinese rapeseed. Chinese rapeseed attained the highest protein content at

its isoelectric point (Xu, 1993). However, as shown in Fig. 14, the protein content of

yellow mustard protein precipitate continued to increase as pH went up. This trend can

be explained by the presence of one of the seed components - carbohydrates. The outer

seed coat of yellow mustard seed contains mucilaginous materials, of which 56% are the

water-soluble polysaccharides (Cui et al., 1 993 b). Therefore, during the protein

extraction of the meal, the water soluble fiaction of mucilages was also extracted into the

aqueous solution. Due to the possible electrostatic interaction between proteins and

anionic polysaccharides, protein may precipitate with the polysaccharides during the

isoelectric precipitation, which consequentIy lower the protein content of the precipitate.

On the other hand, in terms of the content of the non-protein components, it varied from 9

to 20% as pH value decreased (Table 8). This could be explained by the ampfroteric

behaviour of the mucilages such as -COOH group of uronic acids, which may also have

isoelectric points in the acidic pH region as proteins do. Therefore, the trend with the

increasing protein content as pH went up from 3.5 to 7 was possibly due to the mentioned

behaviours of polysaccharides.

Polysaccharides can have molecular weight as high as or higher than proteins and

Production of Protein Isolates from Hexane-Defatted Ground Yellow :Cfustard Meal 67

thus they would be retained rather than removed during ultrafiltration and diafiltration. If

most of the pofysaccharides have not been precipitated, they would become concentrated-

The viscosity of the solution would then be increased during ultrafiltration or

diafiltration, and eventually led to an increase in processing time. Accordingly, pH 4.75

was chosen for isoelectric precipitation of yellow mustard protein in subsequent

experiments. This pH value was a reasonable compromise between the need to decrease

the polysaccharide content to prevent serious gel-layer formation and concentration

polarization during the subsequent membrane processing steps and the desire to maintain

a reasonably high purity of the precipitated protein.

4.3 Recovery of Yellow Mustard Protein Isolates by Membrane Processing

Isoelectric precipitation of protein recovered 54% of the total extracted protein:

thus, 46% of the protein still remained dissolved in the aqueous solution. As mentioned

in section 2.3.3, this soluble protein can be recovered through membrane processing. A

process consisting of alkaline extraction, isoelectric precipitation, ultrafiltration followed

by diafiltration was developed in our laboratories for the characteristics of rapeseed and

canola. The process was successfilly tested with the hexane-defatted canoIa meal,

commercial meal, methanol-ammonia-waterhexme extracted canola meal (Tzeng et al.,

1990), and methanol-ammonia-waterhexane extracted Chinese rapeseed meal (Xu,

1993). Therefors, it was used as a model for the recovery of soluble protein from mustard

meal.

Production of Protein Isolates from Hexane-Defatted Ground Yeilow .Cfustard Meal 68

4.3.1 Mass and Protein Recovery of Products

The process mentioned above was modified and the process flowsheet is shown in

section 3.3.4. The modified process was run three times with the results shown in Tables

9-1 1. In all cases, most of the nitrogen in the starting meal was recovered as three usable

products, including two high-quality protein isolates and the meal residue. Both isolates

are light ivory colour. with a bland taste in precipitated protein isolate and slightly

astringent in the soluble protein isolate.

Precipitated protein isolate (PPI) was found to have a protein content of 89% (N%

x 6.25, Table 9) while the soluble protein isolate (SPI) contained about 98% protein.

This was contrary to the results reported for Chinese rapeseed protein where precipitated

protein isolate had a higher protein content than the soluble protein isolate. Xu (1993)

obtained a PPI containing nearly 100% protein and a SPI with a protein content of about

90%. This difference is not only due to the variation in composition of protein among

rapeseed and mustard seed, but also related to the presence of mucilage. As mentioned in

the previous section, the interaction between mucilage and protein, as well as the

amphoteric property of the mucilage lowered the protein content of the precipitated

protein isolate, while soluble protein isolate was essentially free of polysaccharides. The

third product, meal residue (MR), contained 19% protein that could be suitable for animal

feed or for human consumption after the removal of phytic acid.

The protein recovery of the isolates was about 66.5%. which was higher than that

achieved with methanol-ammonia-waterhexme extracted canola meai and Chinese

rapeseed meal; 46.8% (Tzeng et al., 1990) and 60.0% (Xu, 1993) respectively. However.

Production of Protein Isolares from Hexane-Defarred Ground Yellow :\.lustard Meal 69

Run number MR PPI SPI

Average k S.D.* 19.1 * 1.0 89.1 k 0.5 98.0 * 0.4

MR:

PPI:

SPI:

meal residue

precipitated protein isotate

soluble protein isolate

results reported as average one standard deviation of three measurements

* pooled estimate of standard deviation of nine measurements

TABLE 9. Protein Content of the Products.

Production of Protein Isolates from Herane-Defarted Ground Yellow Musrard Meal 70

Run number PPI SPI PMT OTHER LOSS

Average * S.D.* 19.6 h 0.9 37.0 * 0.4 29.5 * 0.2 13.7 0.5 0 -3

MR: meal residue

PPI: precipitated protein isolate

SPI: soIubIe protein isolate

PMT: permeate combined from Is' and 2"d membrane processing

' results reported as average * one standard deviation of three measurements

* pooled estimate of standard deviation of nine measurements

TABLE 10. Nitrogen Balance among Products (as % of total nitrogen in the meal).

Production of Protein Isolates from Hexane-Defatted Ground Yellow Mustard Meal 71

Run number MR PPI SPI LOSS

MR: meal residue

PPI: precipitated protein isolate

SPI: soluble protein isolate

* average + one standard deviation of three samples

TABLE 11. Mass Balance among Products (as % of total mass in the meal).

Producrion of'Protein Isolates from Hexane-Defatted Ground Yellow .Chrard .Meal 72

the result was lower than that achieved with hexane defatted canola meal, where 75.4%

was recovered in the two protein isolates (Tzeng et al.. 1990). The protein recovery in the

two isolates from yellow mustard was quite evenly distributed. Precipitated protein

isolate recovered 37.0% of the total protein in the meal while 29.5% was recovered in the

soluble protein isolate. In comparison to Chinese rapeseed protein isolates, PPI had a

similar recovery of 37.4%; however. only 22.6% was recovered in SPI. As shown in

Table 10, about 14% of the total nitrogen was lost in the permeate; most likely non-

protein nitrogen. This fiaction presumably contained free amino acids, peptides. and

many other nitrogen-containing compounds such as melanoid compounds (Bhatty and

Finlayson, 1973), all of which have relatively lower molecular weights than the molecular

weight cut-off of the membrane cartridge. The other 0.3% loss can be attributed to the

errors in the nitrogen analyses as well as to mass loss due to the frequent transfer of

samples during processing.

The mass recovery of the protein isolates was 31 -0% of meal solids (Table 11).

The result, however, was lower than the 34.1% mass yield obtained from methanol-

ammonia-waterhexme extracted Chinese rapeseed meal (Xu, 1993) and the 32.5%

reported by Tzeng et al. (1990) with hexane defatted canola meal as the starting material.

The total mass Ioss accounted for 25% of the meal solids. which mainly consisted of the

low molecular weight substances in the permeate, such as non-protein nitrogen and low

molecular weight water-soluble fiaction of the hulls. The meal residue accounted for

44% of the meal solids, which consisted mainly of the water-insoluble fraction of hulls

and 19% protein.

Production of Protein lsolates from He-rane-Defaited Ground Yellow .Cfustard Meal 73

4.3.2 Removal of Phytic Acid

As mentioned in section 2.1, phytic acid is a strong chelating agent as it is

negatively charged at all pH values. There is a possibility that phytic acid will be bound

to protein during isoelectric precipitation since protein has a net positive charge at pH

values below its isoelectric point. Therefore. the phytic acid content of the products and

the starting meal was analyzed and summarized in Tables 12 and 13.

Results showed that the majority of the phytate remained in the meal residue. only

10.7% of the total phytate was dissolved and removed through the membrane processing

(Table 12). This suggested that the extractability of phytic acid was only about 11% at

pH 12. in the work by Xu (1993), the phytic acid extractability of methano-arnrnonia-

waterhexme extracted Chinese rapeseed meal was 2 1.3% at pH 12. Earlier. Chen (1 989)

reported an extractability of 18.1% for methanol-ammonia-waterhexane extracted Westar

canola meal at pH 12.

As shown in Table 13. phytic acid could not be detected in the isolates; however.

the meal residue contained 4.37% phytic acid. which was approximately double the

amount in the starting meal. This is because the phytic acid content in the meal residue

was concentrated as other components, such as protein and soluble hulls, were extracted

from the meal. This result was superior to that of the isolates produced from canola and

Chinese rapeseed. Xu (1993) achieved less than 1% phytic acid in both isolates from

methanol-ammonia-waterhexme extracted Chinese rapeseed meal. while Chen (1989)

obtained a phytic acid content of 0.9% in PPI and 0.5% in SPI with an addition of 40%

CaC12 by weight of the methanol-ammonia-waterhexme extracted canola meal.

Producrion of Protein Isofares from Hexane-Defatted Ground Yellow Mustard Meal 74

Phytic acid recovery (as % of starting meal)

- - -

Hexane-defatted meal

PPI

SPI

LOSS 10.7

MR: meal residue

PPI: precipitated protein isolate

SPI: soluble protein isolate

4 Average + one standard deviation of three measurements

* Phytic acid is not detectable in PPI and SPI

TABLE 12. Phytic acid Balance among Products (as % of starting meal).

Production of Protein Isolates from Hexane-Defatted Ground Yellow .Mustard Meal 75

Phytic acid content [%I

PPI

SPI

MR: meal residue

PPI: precipitated protein isolate

SPI: soluble protein isolate

(I Average * one standard deviation o f three measurements

* Phytic acid is not detectable in PPI and SPI

TABLE 13. Phytic acid Content of Products and Starting Meal (wt %).

Production of Prorein Isolares from Hexane-Defarred Ground Yellow .Clustard Meal 76

4.3.3 Removal of Glucosinolates

In order to study the effectiveness of the membrane processing prior to isoelectric

precipitation, a control run without this membrane processing was carried out and the

products were subjected to glucosinolate analysis. The control experiment was conducted

as follows: alkaline extraction at pH 12. isoelectric precipitation at pH 4.75. then.

~ l t r ~ l t r a t i o n and diafiltration. A comparison of glucosinolate contents of the products

through the actual and control processes is shown in Table 14.

Glucosinolates are hydrophilic so that they are readily dissolved in aqueous

solution during the protein extraction step. It was shown that 98.5% of glucosinolates

have been extracted from the meal (Table 15), leaving a meal residue with low

glucosinolate content. Most of the dissolved glucosinolates were passed through the

membrane and were discarded with the permeate during the membrane processing.

As shown in Table 14, the glucosinolate content of the protein isolates produced

from the actual process was 1.13 and 2.95 pmoYg in PPI and SPI respectively. which was

much lower than that from the control process. This suggested that most of the

glucosinolates were removed through the membrane processing prior to isoelectric

precipitation. In addition, this also suggested that there was interaction between the

protein and the glucosinolates in the acidic pH region. From the other point of view. it

can be said that the interaction between them was minimal in the alkdine region. The pH

dependence of the interaction between protein and glucosinolate can be explained by the

structure of glucosinolate. As mentioned in section 2.4.1. the predominant glucosinolate

in yellow mustard seed is parahydroxybenzyl glucosinolate, with its structure shown in

Production of Proteirt Isolates from Hexane-Defutted Ground Yellow Mustard Meal 7 7

-- - -

Glucosinolate Content [pnoVg] * I

with membrane processing

without membrane processing

prior to isoelectric precipitation I

PPI

* glucosinolate content reported as pmoles of free thiocyanate ion, equivalent

to pmoles of parahydroxybenzyl isothiocyanate or parahydroxybenzyl

glucosinolate per g oil-free, moisture-free meal. Results reported as average

+ one standard deviation of three measurements.

TABLE 14. Glucosinolate Contents of Protein Isolates Produced from Actual and

Control Processes.

Production of Protein Isolates from Herane-Defatted Ground Yellow .Cfusrard Meal 78

Glucosinolate recovery (as % of starting meal)

I I

with membrane 1 without membrane processing j 1 processing

prior to isoelectric precipitation I

Hexane-defatted meal 100 1 100

I I

MR* 1.52 0.02 i 1.44 + 0.01

SPI* 0.19 a 0.01 , 3-61 + 0.05

I LOSS 98 -2 I , 91.6

I

MR: meal residue

PPI: precipitated protein isolate

SPI: soluble protein isolate

* results reported as average t one standard deviation of three measurements

TABLE 15. Glucosinolate Balance among Products (as % of starting meal).

Production of Protein lsolates from Haane-Defatted Ground Yeflow .Cfustard :Meal 79

Figure 16. Due to the presence of the -OH functional group bonded to a benzene ring,

this specific type of glucosinolate can also be classified as a phenolic compound. During

the extraction of protein, the phenol group of the glucosinolate was converted to its

sodium salt, which was then extracted into the aqueous phase (Figure 17). As a result.

both protein and glucosinolate possessed negative charges in aqueous NaOH, where the

potential for interaction between them would be insignificant due to electrostatic

repulsion. As a result, glucosinolates were removed through the permeate during the

membrane processing.

Accordingly, membrane processing prior to isoelectric precipitation is essential to

remove glucosinolates from the alkaline extract. It prevents the interaction between

protein and glucosinolates, thus, producing two high quality protein isolates with low

glucosinolate content.

4.4 Economic Assessment

The process for the isolation of protein from yellow mustard meal was

successfblly developed, producing two protein isolates that are high in protein content.

low in glucosinolates and essentially free of phytates. However, in order to ascertain

whether the process has a potential for commericial application and whether the products

have the ability to compete with the well-established soy protein products, it is important

to determine the production cost of the two mustard products and compare them with that

of commercial protein products. First, the minimum production cost of the isolates was

Production of Protein Isolates from Hexane-Defatted Ground Yellow ;Mustard Meal 80

FIGURE 16. Structure of Parahydroxybenzyl Glucosiaolate.

Phenol Hydroxide ion Phenoxide ion Water

FIGURE 17. Reaction of Phenol with Dilute Sodium Hydroxide.

Producrion of Protein kolares~orn Hexane-Defatted Ground Yellow .thsrard Meal 81

calculated based on a process where the sale of the products was just enough to break-

even with the cost of the seed and the cost of processing was neglected. The cost of

1000 kg of seed is known to be $ 360, whereas the oil and the meal residue can be sold

for $ 0.50 and $ 0.20 per kg respectively under current market conditions. The cost

breakdown of the products is shown in Figure 18 and the details of the calculation are

shown in Appendix 8.5.

As shown in Figure 18, the minimum production cost of mustard protein isolates

is calculated to be $ 0.66 per kg. The current average cost of isolated soy protein and

casein is $ 5.24 and $ 8.27 per kg respectively (U.S. Soyfoods Directory, !998). In order

to find out whether the cost of mustard proteins are comparable to those of the

commercial protein products, the processing margin should be estimated. By comparing

with the cost of soy protein, the processing margin of mustard protein could be as high as

$ 4.58 per kg ($5.24 - $0.66) of protein isolate produced. By minimizing the processing

cost as much as possible, the difference between the processing margin and the minimum

processing cost will be the maximum profit that can be made. As the processing margin

is almost seven times higher than the material cost ($0.66/kg) of the isolates. the process

should be able to make profit. The actual processing cost would include the cost of

capital, as well as all manufacturing cost including power, labour and materials used. and

the material distribution of the isolation process f?om 1 tome of yellow mustard seed is

shown in Table 16.

T?~oughout the process, a significant amount of water was used (approximately

86 kg per kg of seed), thus, the recycling of the process water streams is important. Since

Production of Protein Isolates from Hexane-Defarred Ground Yellow .Mustard 'Meal 82

1 tonne of seed ($ 3601 tonne) Cost: $360

332 kg oil ($ 0.5 I kg)

Value: $ 166

292 kg MR ($ 0.2 1 kg)

Value: $ 58.4

119 kg PPI ($0.66 / kg)

Value: $ 78.6

86.2 kg SPI ($0.66 /kg)

Value: $ 56.9

FIGURE 18. Cost Breakdown of Products.

Production of Protein Isolates from Hexane-Defatted Ground Yellow .+frcstard Meal 83

Material Input

1000 kg seed

I 4 100 L hexane -

170 L 25% NaOH

86000 L water

72 L 6N HCI

pp

Material Output

332 kg oil

3700 L recovered hexane - -

292 kg meal residue - - -- -- - - - - - --

1 19 kg precipitated protein isolate -- --

86.2 kg soluble protein isolate

8 1000 L permeate

TABLE 16. Material Distribution of Protein Isolation Process using Yellow

Mustard Meal.

Production of Protein Isolates from Hexane-Defatted Ground Yellow Mustard Meal 84

the washing solutions of meal residue and the precipitated protein were reintroduced into

the main stream, the only process water stream that would need to be recycled originates

from the two permeate streams produced during membrane processing. Most of the

permeate could be directly recycled; however, the ultrafiltration permeate from the

membrane processing prior to isoelectric precipitation contains glucosinolates and

coloured component. These compounds should be removed before recycling. The

recycling of the rest of the permeate, without further treatment, is practical as all the

water streams were low in soIids and glucosinolates contents.

Production of Protein /solares from Herune-Defarted Ground Yellow :Mustard .Meal 83

In summary, a modified membrane-based process comprising of four main steps

was developed: alkaline extraction and washing at pH 12; ultrafiltration and diafiltration

of the alkaline protein extract; isoelectric precipitation and washing at pH 4.75; and

finally ultrafiltration and diafiltration of the soluble protein solution. The process was

successfully adapted to yellow mustard meal, which yielded two protein isolates and a

meal residue with good protein recovery. The precipitated protein isoiate and the soluble

protein isoIate were high in protein. low in glucosinolate, free of phytic acid and have low

selling price which was comparable to those of soybean protein isolate. The protein

isolates from mustard seed can be considered acceptable as protein ingredients for human

consumption while the meal residue is suitable enough for animal feed or for human

consumption after the removal of phytic acid.

Production of Prorein ~solates/rom Herme-Defatted Ground Yellow Mustard Meal 86

5. CONCLUSIONS

1. The modified process was successfblly developed which yielded two protein isolates

and a meal residue, with a protein recovery of 66.5% in the isolates. The protein

content of precipitated and soluble protein isolates is 89.1% and 98.0% respectively.

The meal residue with 19.1 % protein content can be used in animal feeds.

2. Both protein isolates were light in colour and bland in taste. They were lighter in

colour and blander in taste compare to the isolates produced from the process without

membrane processing prior to isoelectric precipitation. Therefore, membrane

treatment is effective in reducing colour and flavour of the products.

3. Both protein isolates were free of phytates and low in glucosinolates.

4. The minimum production cost of the two isolates was found to be $0.66 per kg. The

process should be able to make profit.

5. The process was run three times with low standard deviation for the results. This

suggests that the process is reproducible.

Production of Protein isolates from Hexane-Defatted Ground Yellow .tlusrard Meal 57

6. RECOMMENDATIONS

I. The concentration factor for the ultrafiltration prior to isoelectric precipitation should

be optimized to reduce water usage during the upcoming diafiltration step.

2. The necessity of the diafiltration subsequent to isoelectric precipitation should be

confirmed in order to reduce water usage and processing time.

3. The recycling of the processed water streams should be attempted in order to reduce

the water usage and environment impact of the process.

4. The dehulled yellow mustard seed instead of whole seed should be used as it would

reduce the amount of polysaccharides present in the aqueous extract. which would

consequently lead to an increase in protein content of the precipitated protein isolate.

5. The scale-up bench or semi-pilot scale study for the modified membrane-based

yellow mustard protein production process should be conducted for the future

comrnericdization of the process.

6. Removal of phytic acid fkorn the meal residue should be attempted so that it can be

used for human consumption as it still contained 19% protein.

Production of Protein Isolates from Het-ane-Defatred Ground Yellow Mustard Meal 88

7. The functional properties of both protein isolates must be determined prior to their

practical use in food formulations.

8. The amino acid composition of both yellow mustard protein isolates must be

determined to confirm their nutritional value.

9. A complete chemical analysis should be conducted to confirm the composition of the

1 1% non-protein fraction in the precipitated protein isolate.

10. A complete economic assessment of the process should be made.

Protiucrion of Prorein isolares from Hexane-Defated Ground Yellow ,Wcitard Meal 89

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Production of Protein Isolates from Hexane-Defatted Ground Yellow .bfusrard .Weal 95

Toronto. Toronto, ON-, Canada.

Regeostein, J.M. and Regenstein, C.E. (1984). Food Protein chemistry. Academic Press, Inc.. Orlando, p. 30.

Reynolds, J.R. and Youngs, C.G. (1964). EfEect of Seed Preparation on Efficiency and Oil Quality in Filtration Extraction of Rapeseed. J. Am. Oil Chem. Soc. 41 : 63- 65.

Rosengarten, F. (1969). Mustard seed. In The Book of Spices. Philadelphia, pp. 295- 305.

Rubin, L.J., Diosady, L.L., Naczk, M., and Halfani, M. (1986). The Alkanol- Ammonia-WaterHexane Treatment of Canola, Can. Inst. Food Sci. Technol. J. 19: 57-6 1.

Rutkowski, A. (1970). Effect of Processing on the Chemical Composition of Rapeseed Meal. Proc. Int. Conf: on the Sci., Tech. and Marketing of Rapeseed and Rapeseed Products. Quebec. Canada, l97-1. Rapeseed Association of Canada and the Department of Industry, Trade and Commerce, Ottawa, Canada p.496.

Shankaranarayana, M.L., Raghavan, B. and Natarajan, C.P. (1978). Mustard. In Encyclopedia of Food Science, Peterson, M. and Johnson, A. ed., The Avi Publishing Co. Inc., Connecticut. pp. 530-533.

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Siddiqui, I.R., Yiu, S.H., Jones, J.D. and Kalab, M. (1986). Mucilage in Yellow Mustard (Brassica hirta) Seeds. Food Microstructure. 5: 157- 162.

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Sosulski, F.W. and Bakal, A. (1969). Isolated Proteins from Rapeseed, Flax and Sunflower Meals. Can. Inst. Food Sci Technol. J. 2: 28-32.

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Sosulski, F.W., Soliman, F.S. and Bhatty, R.S. (1972). Diffision Extraction of Glucosinoiates fcom Rapeseed. Can. Inst. Food Sci Technol. J. 5 : 10 1 - 104.

Production of Protein isolates from Hcxane-Defatred Ground Yeilorv Mustard Meal 96

Sovero, M. (1993). Rapeseed, a New Oilseed Crop for the United States. In New Crops. J. Janick and J.E. Simon eds., Wiley, New York, pp. 302-307.

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Production of Protein Isolates from He-rune-Defarred Ground Yellow .blwtard Meal 97

Tzeng, Y.M., Diosady, L.L., and Rubin, L.J. (1988b). Preparation of Rapeseed Protein Isolates Using Ultrafiltration, Precipitation and Diafi~ltration. Can. Insr. Food Sci. Technol. J. 2 l(4): 4 19-424.

Tzeng, Y.M., Diosady, L.L., and Rubin, L.J. (1990). Production of Canola Protein Materials by Alkaline Extraction. Precipitation, and Membrane Processing. J. Food Sci. 55(4): 1 147-1 156.

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VanEtten, C.H., Kwolek, W.F., Peters, J.E. and Barclay, A.S. (1967). Plant Seeds as Protein Sources for Food or Feed. Evaluation Based on Amino Acid Composition of 379 Species. J. Agric. Food Chem. 15: 1077-1089.

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Production of Protein Isolates from Herane-Defarted Ground Yellow .tfusrard Meal 98

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Yin, J., Xie, J.K., and Li, D.F. (1990). Extractabilities of rapeseed meal components. Jiangszl Food and Fermentation. 4: 1 .

Production of Protein Isolates from Hexane-Defatted Ground YeNow Musrard .\fed 99

8. APPENDICES

8.1 Moisture Analysis

Weigh out each sample into an aluminum plate.

Cover the plate with a sheet of aluminum foil and punch several small holes on the

foil.

Put the sample in a forced-air oven to dry overnight at 1 0 5 ~ ~ .

Put the sample in a desiccator to cool down to room temperature.

Measure the weight difference.

Calculate the moisture content by the following equation:

rnoistzrre loss moistztre content PA] = x 100%

original weight

Sample calculations:

hexane-defatted mustard meal:

weight of wet sample = 2.29 13g

weight of dried container = 1.5644g

weight of dried container and dried sample = 3.7055g

:. weight of dried sample = 3.7055 - 1.5644 g = 2.141 lg

rnoistvre !ass = 2.29 13 - 2.14 1 1 g = 0.1502 g

0.1 502g moisture content = ~ 1 0 0 % = 6.56%

2.29 1 3g

Production of Protein Isolates from Haane-Defatred Ground Yellow :Cfustard Meal 100

Raw data: Note: Products from the 3 actual processes were thoroughly mixed and analyzed.

meal residue tiom actuai_process: ___~~.~~~.~~~~~~~~~~~~~~~~~. . .~~~.~.~~~~. . .~ .~~. . . . . . * . . . . . . . . . . . . . .~~~.~~~~~.~~~.~.~. .~~. . . -~~~~.~~.-~.~- -* -~~-~*~~--~.*~~.~*~~***-~~* . . 1.9049 $1 1.5823 i + 0.3508 0.3226 : 8.039

#2 1.5816 / . 0.3582 1.9121 0.3305 7.733

avg. t s.d. 7.88 + 0.15

I avg. 2 s.d. 6.28 4 0.41

SPI from control process: ..*-...--.---..*..-......-......**.**~~~** .____.-___._...~.-.-.-------..-.-.--.----....~--.-.------*-----.~~..-.*~~~------~-----...~.~~.~~.~..~~~~-~~-...~~.~~.~~.~~.~...~.~~.~~~.....-------------~.*.-*---**-*-------..* # 1 1.5724 i . 0.1887 1.7482 0.1758 i 6.836

$2 1.5648 . / 0.1896 i 1.7412 / 0.1764 6.962

#3 1.5230 / - 0.1880 1.6997 / 0.1767 j 6.01 1

avg. f s.d. i 6.60 t 0.52

Producrion of Protein Isolares from Herane-Defarted Ground Yellow Mustard Meal I0 I

8.2 Protein Analysis (Kjeldahl Method)

Weigh out each solid sample into boat-formed of nitrogen f?ee paper or measure the

volume of liquid sample using pipette and put one sample into each digestion tube.

Add 3 pellets of KJELTABS (mercuric oxide type) and approximately 25 rnL

concentrated &SO3 to the tube.

Preheat Biichi 425 Digester at #4 setting.

Clamp suction tube onto the digestion tubes. put a piece of glass wool into one end

of the suction tube, connect the other end to the aspirator and turn on the suction.

Place digestion tube in the digester and heat at #4 for 20 min. Raise heat to #6 for 10

min. Turn the setting to #10 and heat for 35 min. Digestion is completed when the

mixture is clear or light yellow.

Turn off the digester, remove the digestion tube and allow it to coo1 to room

temperature.

Add approximately lOOmL distilled water to each tube and shake it until the sample

is rotally dissolved. Add 25mL 8% Na2S2O3(aq) to each tube and shake.

Turn on the water flow to Biichi 3 15 distillation unit and warm it up.

Add 50mL 4% boric acid and 3 drops of endpoint indicator to a 500mL erlenmeyer

flask.

Place the flask on the right side of Buchi 3 15 unit and the digestion tube on the left

side. Make sure the plastic tube is put deep into the boric acid solution.

Add approximatelv 150mL 25% NaOH(as1 to the digestion tube.

Production of Protein Isolares from Hexme-Defatted Ground Yellow .tfusrard .Weal 102

12. Distill until the total volume of solution in the flask is approximately 325mL.

13. Lower the flask and rinse the plastic tube with distilled water into the flask-

14. Tun off the steam valve and empty the digestion tube by suction.

15. Immediately titrate the collected condensate in the flask with 0.1N H2S04(aq) until

endpoint (light pink colour) is reached.

16. Calculate the nitrogen content of the sample by the following equation:

where V, = volume of titrant for the sample (mL)

Vb = volume of titrant for the blank (mL)

W, = weight of sample (rng) or volume of sample (mL)

N = normality of H2S0j = O.1N

17. Calculate the protein content of the sample by

% protein = O/jN * 6.25

where oilseed proteins were assumed to have 16% N. 6.25 is the reciprocal of 16%.

Sample calculations:

Hexane-defatted mustard meal:

% protein = 6.45% * 6.25 = 40.3%

Production of Protein Isolatesfrorn Herane-Defarted Ground Yellow .Clustard .$leal 103

Raw data:

Protein analysis of products: Wstan,nE meal in each run = 25g; W,,,,, in 2Sg starting meal = 10.08g

sample ) ~ , , , $ ~ l ' i titran~,,bb.k[mLl protein eontent[%l* ) protein recovery(%]

avg. k s.d. ; 89.5 i 0.6 36.8 4 0.3 .--..---.----.--.--..-....----*-- i-r.--.--.*t-.*--~~-~~*-~~~-~-~:~-~----.-r.---..-..-----------.-.--.------ii-...iiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiii~--~--~~~-...--~-~-~~*.~~~-*~*-.~---...

SPI (ITI.C.= 3.6 1%). WSpl = 3.0 1 g* 97.9 1 I : 0.1 158 i 12.49 29.23

avg. k s.d. 98.2 k 0.5 29.3 k 0.1 --------.------------*---..----. A--........----.-.------L.--...--.-.----.*-*-.*---*-..--.---.------------L-.------..-.----...--.----------..--------.----....-..-L.--..-..-.-.---....---.-------.-.---------.----------.

Actual run 72:

, 6

avg. f s.d. ! 19.0 + 0.3 19.3 k 0.3

3 : 0.1097

avg. t s.d. .-~--~~~~~~~~.~~~~~~~~~.....*.~*-*.....**..-...*-..-*.*.-~-*.~.....-~--..***.*--.---..----...-..-----...-.-..------------.--.-**....*....~.-..*-.-**------------------------.----------..-*--...-.--.*.---.*-.-----*----*---.- SPI (m.c.= 4.62%), Wspl = 3.04g*

1 1.79 # 1 : i 0.1107 i 97.7 1 29.46

avg. + s.d. ; 97.8 + 0.1 29.5 2 0.04 --------------- --------rr......ii...~.~~~............*.-.-.--L.t..--...rr--r-.------r----.r.+r-r----*---...r--....--.--..-.....---.-.----.-..-r.---.--*-..----...--.---.-.-..-----r.-.-.....---.----------------.-----------.

Actual run #3:

Production of Protein Isolates from Hcrane-Defutred Ground Yellow Mustard Meal I04

MR (m.c.= 7.95%), WxIR = 10-4 1 g* 1 : 0.1819 i 3 -47 18.13 18.72

18-15 $3: i 0.1817 3.47 18.74

18.14 #3: ! 0.1823 3 -48 18.73

avg. i s-d. ; 18.1 f 0.01 18.7 I: 0.0 1 ................................. A........... ............................................................................................................................................................ PP[ (m.c.= 5.34%), Wppr = 4.24g*

89.15 I : 0.1 153 11-12 37.50 4 9 . :

I

89.33 w . 0.1162 1 1 .23 37.57

1 1 .06 88.97 #3:! 0.1149 : 37.42 I

avg.ks.d. 89.2 t 0.2 37.5 k 0.1

SPI (m.c.= 7.20%), Wspr = 3 .OSg* 1 : 0.1 120 i 1 1.68

avg. f s.d. i

Control run:

MR (m.c.= 12.2%), WSIR = 1 O.37g* 19.13 1 : 0.1724 ; 3-3 I 19.92

$2: 0.1727 : 3 -09 17.83 18.57 1

1 8.46 3 : 0.1727 3 2 0 1 8.99

18.5 t 0.6 avg. 4 s.d. ! 19.2 + 0.7 ................................ d............................................. ............................................................................................................................................... PPI (m.c.= 7.90%). Wppl = 4.60g*

10.90 87.09 1 : 0.1189 ; 39.74 "7. i 88.69 c,.: 0.1189 I !.I0 40.47

avg. 2 sad. 87.7 k 0.8 40.0 f 0.4 ..-..........*.*................ A. ............................................................................. A............................ ---+--...---..-.-.----..- -.....-...........--~-~ ............................. SPI (m.c.= 6.60%), Wspl = 2,96g*

95.80 1 : 0.1193 13.30 28.13

97.78 2:: 0.1143 ! 1 1.93 28.7 1

1 1.66 94.98 #3:: 0.1152 27.89

avg. + s.d. 96.2 + 1.4 28.2 f 0.4

i

' as is basis * moisture-free basis

Protein analysis on extractabilities of yellow mustard meal: Note: Wstb,,, ,,I in each run = I Og; WPmtei,, in log starting meal = 3.983g, 1 OmL of each sample

was taken.

Production of Protein Isolates from Herune-Defu~ted Ground Yellow .t(ustard Meal 105

1. Effect of pH on nitrogen extractability, R= 18, time=30rnin

sample

2. Effet

sample

....**--..*--*... 5 min

10

20

20

20

30

3 0

30

45

5 0

120

of extraction time on nitrogen extractability i protein

Vex,,,, titrant (mLj i extractability

i (sarnp. - blank) i [ m u ; ["/.I

Protein extractability [ O h I

i overall protein titrant [mLI ; . extractability (sarnp. - blank) i ["/.I

Producrion of Protein Isolates from Hexane-Defatted Ground Yellow .C/ustard Meal 106

Protein analysis on precipitation of yellow mustard protein: Note: 200mL alkaline extract was used for the precipitation of protein in each test. W,,,,,, =

4.074g in each 200mL extract.

sample dry weight titrant [mLl

13.40

14-03

14.02

13.94

14.2 1

14.20

14.29

14.47

14.27

14-46

14.53

14.48

14-39

16.58

16-63

16-57

16-36

16.48

16-34

protein content [%I

79.54

83 -06

83.12

83.01

84.53

84 -42

84.6 1

85.55

85.26

85.58

85.65

85.6 1

85.38

88.73

88.64

88.8 1

90.32

90.8 1

90.95

protein yield[%]

Production of Protein Isolates from Hexane-Defatted Ground Yellow .tIustard .Weal 107

8.3 Phytic acid Analysis

1. Weigh out -0.40g sample into a centrifuge tube and extract with 5OmL 1.2% HCI

containing 10% Na2S04(aq) (both constituents w/v) for 2 hours using a Wrist Action

Shaker (Burrell Corporation, Pittsburgh. PA; Model 75).

2. Centrifuge the sample for 10 rnin at 2500 rpm.

3. Filter the solution using Whatman No. 41 filter paper.

4. Pipet 5.0mL of the filtrate into a digestion tube and mix with 5.0mL distilled water

and 6.0mL 0.07N HCI containing 0.4% (wh) FeC13 - 6H20.

5 . Heat the digestion tube in a boiling water bath for 75 min to complete the

precipitation.

6. Collect the precipitate by centrifugation (International Clinical Centrikge) at 2500

rpm for 15 rnin. Dispose the supernatant.

7. Add 5mL 0.07N HC1 containing 4% (w/v) NazS04(aq) to the precipitate and

centrifkge for 15 min again. Dispose the supernatant.

8. Add 7mL of a 1 : 1 (vlv) mixture of conc. H2SO;r and m03.

9. Turn on the digester to #4 setting and warm it up.

10. Put the digestion tube into the digester and heat at #4 setting for 5 min. Raise heat to

#6 setting for 5 rnin. Raise heat to # lo setting and heat for about 1.5 hour. White

fumes will hang over the liquid when digestion is completed.

1 1. Remove the tube and allow it to cool for 10 min.

1 2. Add approximately 10 mL distilled water to the tube SLOWLY.

Production of Protein Isolates from He-rane-Defatted Ground Yellow .tfusrurd .Ileal 108

1 3 Heat the tube in a boiling water bath for 30 min.

14. Cool the solution to room temperature and dilute it to 1 OOmL with distilled water in a

1 OOmL volumetric flask.

15. Determine the content of phytate phosphorus colourimetrically according to AOCS

Oficial Method Ca 12-55: Pipet l OmL of the solution to a 50mL volumetric flask.

add 8mL hydrazine sulfate and 2mL sodium molybdate to the flask in this order.

Shake well and Iossen the stopper. Heat it in a vigorously boiling water bath for 10

min. Cool down the solution to 2 5 * 5 O ~ and dilute it to the mark.

16. Prepare the calibration curve by analyzing 0. 1. 2. 4. 6, 8. 10 ml aliquots of the 0.0 1

g /L standard phosphorus solution. Add distilled water to 10 rnL to the aliquots in 50

mL volumetric flasks. Then. repeat step 15.

Sample caiculations:

hexane-defatted mustard meal:

weight = 0.4386g

absorbance at 6 5 0 m = 0.1616

equation of the calibration curve:

y = 0.29 1 1 x + 0.0061

where x = mass of phosphorus in the sample, y = absorbance

thus. x = (0.16 16 - 0.006 1) / 0.291 1 = 0.5342

phosphorus content = (0.5342rng/438.6mg)*5* 100% = 0.609 %

where '5' is the dilution factor

Production of Prorein fsolares from Haane- De fatred Ground Yellow .Mustard :Weal 109

phytic acid content = 3 -55 * 0.609% = 2.16%

molecular weight of phytic acid where 3 5 5 =

weight of phosphorus in 1 mole of phytic acid

Determination of calibration curve:

Calibration curve for phosphorus

0.00 0.20 0.40 0.60 0.80 1-00 1.20 1.10 1.60 1.80 2.00

conc. of phosphorus [rniJL]

+ only one measurement was made for each sample

+ caIibration curve was fieshly prepared each time: slope ranges from 0.29 1 1 to 0.2943 and y- intercept ranges from 0.0037 to 0.0093.

Production of Protein Isolates from Hexane-Defatted Ground Yellow .tiusturd .%leal 110

Statistical analysis on calibration data:

Standard error of estimation: S,,, = 0.00 I 637

let equation of calibration curve be y = box + b,

Standard error of y-intercept (bl ):

hypothesis testing for bl: Ho: p, = 0 HA: Pt * O tn-p.m,l - - t j , ~ Ool = 5.893

b, 0.0061 =-- - = 6.04 S,, 0.00101

,and P>99.8%. Ho should be rejected. It can be concluded that bt * 0 Since It *I > t,_,,uj-

and p is statistically significant.

Raw data: Phytate analysis for products:

sample f W,m,I. lglU absorbance phytate content [ O h I* SiL

hexane-defaned mustard meal: *

$ 1 0.3528 0.2429 4.443

4.400 $2 0.3 562 0.2429

0.3680 $3 0.2439 4.277 4

4.37 k 0.08 avg. + s.d. 0.004 .-.-..-.-...-.-.-..-.---.-.---.......~~~~__.~.~~.~---.-.---.*~-.*-...--.-...-..~~~~-~.~..~.~._____._.._._.~~~.~~~~~~-~-~~-~.-..-~~.~~~.~-..~~.~~..~-~~....~~---~~.**.*..**_.~*-~.-~.--~*~~~~~.~----~~--~.-..~.~.~..._.~..~.-_-...

PPI from actual run:

# 1 0.3975 - 0

0.40 13 - 0 $2

0.4003 - 0 $3

Production of Protein Isolates from Herune-Defutted Ground Yellow .2.fusturd Meal 112

8.4 Glucosinolate Analysis (McGregor Method)

Weigh out 50mg of oil-free sample into a 4 mL screw-cap vial.

Add 1 glass bead and imL of pH 4.5 citrate-phosphate buffer containing 6mg of

fieshly dissolved yellow mustard myrosinase into each vial.

Mix the contents in the vial on the Vortex mixer.

Immediately add 2mL of methylene chloride into each vial.

Mix the contents in the vial on the Vortex mixer and shake for 2 hrs as soon as

possible using Wrist Action Shaker.

Centrifuge the sample for 30 min.

Take l2SpL of the methylene chloride layer from the sample using a 250yL syringe

and add it to a test tube containing 1.5ml of distilled water.

Heat the test tube under a hot water tap and mix the sample on the Vortex mixer

interspersingly until the methylene chloride has evaporated.

Add 0.5ml of O.1N sodium hydroxide to the test tube. Mix on the Vortex mixer and

stand for 2 5 min.

Add 0.5ml of O.1N nitric acid to the tube and mix on the Vortex mixer.

Add 23ml of freshly prepared ferric nitrate to the tube. Mix on the Vortex mixer

and stand in the dark for 15 min.

Pipet 3ml of the mixture to a cuvette and measure the absorbance at 470 nm.

Add 45p1 of 5% mercuric chloride to the cuvette using a 100pL syringe and mix with

the tip of the syringe. Measure the absorbance again at 470 nm. This is the

Producrion of Protein Isolates from Hexane-Defatted Ground Yellow Musrard Meal 113

background absorbance.

14. Calculate the glucosinolate content of the sample as follows:

glucosinolate content [ p o l / g] = @,-Ah) Vo I

X-X m V, W,(I-M.C.)

where As = sample absorbance

Ab = background absorbance

m = slope of calibration curve

V, = volume of methylene chloride added at step 4 [mL]

V, = volume of methylene chloride taken at step 7 [rnL]

W, = weight of sample [g]

M.C. = moisture content [%I / 100

Prepare the calibration curve by analyzing 0.1. 0.2. 0.3. 0.4. 0.5. 0.6. 0.7 and 0.8 mL

aliquots of the 0.001 N standard thiocyanate solution. Add distilled water to 1.5 rnL

of the aliquots in test tubes. Then, repeat steps 9 - 13.

Sample calculations:

hexane-defatted mustard meal:

weight of sample = 0.0586g, moisture content = 6.569

background absorbance = 0.1030, sample absorbance = 1.3049

equation of calibration curve : y = 0.863 1 x

where y = absorbance at 470~1, x = moles of thiocyanate ion (pmol)

thus, moles of thiocyanate ion = (1 .jO49 - 0.1030) / 0.863 1 = 1 .XU5 pmol

Production of Prorein Isolates/rorn Hexane-Defarted Ground Yellow .Cfustard .Weal ! I 4

glucosmolate content = I

O.O586g(l- 0.0656) 025mL

Determination of calibration curve:

conc. of thiocyanate ion absorbance at 470nrn

Calibration curve for thiocyanate ion

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8

conc. of thiocyanate ion [pmoVSrnL]

4 single measurement for each sample

4 calibration curve was freshly prepared each time; slope ranges from 0.8593 to 0.8775 and y- intercept ranges from 0.0009 to 0.0022.

Producrion of Protein Isolares/rorn .Yexane-Defarted Ground Yellow .Mustard .Weal 115

Statistical analysis on calibration data:

Standard error of estimation: S,, = 0.002985

let equation of calibration curve be y = box + b1

Standard error of y-intercept (bl):

hypothesis testing for bl : Ho: pl = 0 H A : PI ?t 0 tn-p,aQ = t6.0 I = 1.440

Since It */ < tn-pp12 and P<80%, Ho should be accepted. It can be concluded that bl = 0

and is statistically insignificant. The new calibration curve will be:

Calibration curve for thiocyanate ion with y-intercept = 0

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8

conc. of thiocyanate ion (pmoV5mL)

Raw data: Glucosinolate analysis on products:

Production of Protein isolares from Hexane-Defarted Ground Yellow .tfusrard Meal 116

hexane-defatted mustard meal:

# 1 0.0673 i 0.0414 6 -49

0.067 1 6.58 $2 0.0418 g3 0.0677 6.55 / 0.0420

6.54 t 0.04 avg. k s.d. ; 0.003 ...*-..-.......................... &..-........r+.................~~~.-.~*~~...~...................................r..~...............................................-...+.-...................................r.-.....*.---**--.

PPI 60m control process:

# 1 0.0538 0.1833 34.29

0.0558 $2 0.1902 34.30 1

0.0555 0.1834 $3 33.26 I

34.0 2 0.6 avg. 5 s.d. 0.002 ..---.--*---..--.*..*._~.-.~~...- ~~.-.*~...~..~~.-.*...~1-.~.~~.~..*.*~*~~.*~5~~~~~*.~..~..~..~~~~....~~~~~*i~~*.~~.~*...*-~**~-.-.lf-.*~*-..--.-.--------.-----.---------~..*.*~~*~..-*~.*..~*-----*~-------------------------.

SPI from connol process:

56.58 # 1 0.0532 0.3033

0.0553 $2 0.3233 58.02

#3 0.056 1 0.3273 57.90

57.5 + 0.8 avg. k s.d. 0.002

Production of Protein Isolatesfrorn Hexane-Defatted Ground Yellow :Cfustard Meal I17

- as is basis * moisture-free basis Sik is defined as standard error of a particular x value read from calibration curve

and m = number of replicates for the sample n = number of data points in regression y k = absorbance of a particular sample - y = mean of the calibration data corresponding to y x, = a particular calibration data corresponding to x - x = mean of the calibration data corresponding to x

7 / where S i =-

1 I -+-i

(YI -B2 b , m n R

b ; x ( x i - K ) ~

Production ofmProfein Isolates from Hevane-Defafted Ground Yellow Mustard .Weal I18

8.5 Economic Assessment

Determination of minimum production cost of products:

Given: Cost of mustard seed = $ 360/tome Selling price of mustard oil = $ 0.50kg Selling price of meal residue used for animal feed = $ O.?O/kg

Find: Cost of protein isolates according to the mass balance of the process

Assume PPI and SPI have the same production cost $x per kg

Mass balance:

1. Soxhlet extraction

1000 kg seed a 663 kg meat + 332 kg oil + 5 kg loss

2. Protein isolation

663 kg meal 292 kg meal residue + 1 19 kg PPI t 86.2 kg SPI + 166 kg loss

1000 kg seed e 332 kg oil + 292 kg MR + 1 19 kg PPI + 86.2 kg SPI + 17 1 kg loss

Cost balance:

I . Cost = 1000 kg * $360 / tonne = $360

2. Sales = 332 kg*$0.50/kg + 292 kg*$ 0.20kg + ( 1 19 + 86.2)kg*$ x = $224.4 i- $205.2 x

3. Cost = Sates $360 = $224.4 + $205.2 x

x = $0.66 / kg

Therefore, PPI and SPI have a minimum production cost of $ 0.66kg.