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PROCESS OPTIMIZATION AND PROPERTIES OF PROTEIN CONCENTRATES FROM

BREWERS' SPENT GRAIN

A Thesis by

~ Rosemarie Diptee

Submitted to Faculty of Graduate Studies and Research in partial fulfillment of the requirements

of the delree of Master of Science

Department of Food Science and Agricultural Chemistry, Macdonald Collele of McGill University,

Montreal, Quebec.

March, 1989.

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Suggested Short Title

pqOPERTIES OF PROTEIN CONCENTRA TES FROM BSG

Suggested Short Title

PROPERTIES OF PROTEIN CONCBNTRATBS FROM BSG

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ABSTRACT

Response surface methodology (RSM) was used ta optimize

protein extractability from Brewers' spent .rains (BSO)

prior to the preparation of BSG protein concentrates. RSM

allowed the cletermination of the simultaneous effects of (a)

temperature of extraction, (b) time of extraction, (cl

con~entration of sodium dodecyl sulfate and dibasic sodium

phosphate (Na HPO ) in the extractant solution, (d) BSG: Z 4

Extractant ratio and (e) particle size on protein

solubilisation from dried Brewers' spent grains (DBSG) and

pressed Brewers' spent grains (PBSG). An initial fractional

factorial screening design showed that time, temperature and

particle size of grain were significant variables while

concentration of extractant had no effect on protein yield

from e i ther DBSG or PBSG. The Mean yield of protein

extracted from DBSG was 28.14% and 9.53% for PBSG. A

centr.al composi te rotatable design was applied to fOUI

variables, tempe rature , time, BSG:Extractant ratio and

concentration of dibasic sodium phosphate in extractant

solution; aIl variables had a significant effect on protein

yield from DBSG. The optimum conditions which gave a

protein yield of 60% were a concentration of O. 64X Na. HPO 4

in the extractant solution, a BSG:Extractant ratio of o

2.5:100, an extraction temperature of 90 C and an extraction

time of 98 minutes . .......

The functional, biochemical and nutritional properties

1

of protein concentrates prepared from dried Brewers' spent

grains were studied as a means of determining their

applications in foods. The results indicated the following:

the protein concentrates digestibilities ran.ed from 78.69%

to 80.50%, viscosities ranged from 43.3 cp to 1926 cp, water

absorption values r~nged from 163.3% to 166.7%, fat

absorption values from 166.7% to 193.3%, foam capacity from

120% to 170% and emulsifyin, capacity trom 22 to 23 ml oil

per gram protein sample.

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RBSUME

La mèthode de surface de reponse (M. S. R.) a ét~

utilis~e de fa~on ~ optimiser l'extraction des protéines de

la dr~che de brasserie pour la préparation d'un concentré de

protéines.

La méthode (M.S.R.) a permis la determination des

effets simultanés de (a) ,

la temperature, (b) la durée de

l'extraction, (c) la concentration de dodécyl sulfate de

sodium et de phosphate de sodium dibasique (Na HPO ) dans la a 4

solution extractive, (d) la proportion de solutlon

extractive pour une quantit~ donnée de dr~che. et (e) la

" grosseur des particules, sur la solubilisatIon des proteInes , , , ,

de la dreche de brasserie sechee et pressee. Un plan

experimental factoriel fractionne a démontr~ que la durée

de l'extraction, la ,

tempe rature et la grosseur des

particules de dr~che moulue sont des variables importantes;

tandis que la concentration de solution extractive n'a aucun

effet sur le rendement de protéines extraites de la drèche

séchèe et pressée.

Le rendement moyen de l'extraction de la drèche séchée

ètait de 28.14% et de 9.53% pour la drèche pressée. Un

, ,,, .' \ plan central a composentes rotatives a ete applIque a quatre

, , variables, soit la temperature, la duree de l'extractlon, la

propor t i on de \ dreche pour une quantite' de solutIon

extractive et la concentration de phosphate de sodIum

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dibasique dans cette solution. Toutes ces variables ont

/ ~ / demontre avoir un effet considerable sur le rendement

proteique de l'extraction 'a partir de dr'eche séch~e. Les

condi tians optimales ont donn~ un rendement de 60% et

consistaient en une concentration de 0.64% de Na2

HPO. dans

la solution extractive, une proportion de drèche pour

~ solution extractive de 2.5:100, une temperature d'extraction

de 900 e et une dur~e de 98 minutes.

L'~tude des propriét:s fonctionnelles, biochimiques et

nutritionnelles du ~

concentre de t ~. pro el.nes resultant

permettra de dèterminer son utilit: dans l'industrie

alimentaire.

Les resultats ont ~ /

demontre que la digestibilit~ du

concentré' de protéines varie entre 78.69% et 80.50%, la

viscosite entre 43.3 cp et 1926 cp. D'autre part la

capacité' d'absorbtion d'eau varie entre 163.3% et 166.7%, la

capacité' d'absorbtion de gras entre 167.7% et 193.3%. la

capacité moussante entre 120% et 170% et la capacit~

émulsifiante entre 22 et 23 ml d'huile par gramme de

/ . concentre prote1que.

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ACKNOWLBDGBMBNTS

l would like to thank Dr. Inteaz Alli for his guidance

and assistance throughout the duration of my M.Sc. degree;

the encouragement and motivation were greatly appreciated.

l would like to thank my colleagues of the Department

of Food Science and Agricultural Chemistry, Macdonald

College of McGill University, for their support; special

mention is extended to Jasmine Bourque and Vir~inia

Barraquio for their assistance.

l would like to thank Dr. Jim Smith for his

contribution towards my research work and Mrs. Farida Alli

for her assistance.

l would like to ackLowledge the financial assistance

from Molson Breweries of Canada Ltd. and from the National

Sciences and Engineering Research Council of Canada.

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TABLB OF CONTENTS

Abs tract ................................................... i

Resume ..................................................... .

Acknowledgements ........................................... Table of Contents ••••••••••••••••••••••••••••••••••••••••• t

List of Tables ............................... , ............ . List of Figures ........................................... .

General Introduction

Section 1: Literature Review

A: Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B: Evaluation of Distillers' Spent Grain (DSG)

use in Foods ••........•. . . . . . . . . . . . . . . . . C: Protein Extractability-Effect of Various

Factors

1. 0: Effect of pH of extractant on protein extractabili ty .......••••..••••.....•

for . . . . .

1.1: Effect of ionic strength of extractant on

....

. ...

iii

v

vi

xi

xiv

1

3

3

5

protein extractability ••• .•..••• .•... ..... ••.. 8

1.2: Effect of temperature on protein extractability .•....•.••••••.••• ..........

1.3: Effect of extraction time on protein extractability ••......• ..........

1.4: Effect of particle size of meal on protein extractabili ty ••.••...••.•..•••....••...••

1. 5: Effect of nature of extractant on protein extractabili ty •.••••.••••••••••••.••••••• .....

D: Protein Precipitation Techniques

2.0: Effect of pH on prote in precipitation ......... 2. 1 : Effect of temperature on protein precipi-

tation ................................... • III •••

vi

10

12

13

14

19

22

2.2: Effect of organic solvent on protein precipi ta tion ...................... . ....... 23

E: Nutritional Properties of Distillers' Spent Grain .............................. . ....... 25

F: Functional Properties of Proteins from Spent Cereal Grains .............................. . .... 30

G: Biochemical Properties of Proteins from Brewers' Spent Grain ...................................... 32

H: Response Surface Methodology

3.0: Classical experimental procedure versus response surface methodology (RSM) ...••

3.1: Response surface designs

3.2: Response surfaces ............................. 3.3: Applications of response surface

methodology (RSM) .••..•....•••..

Section II: Haterials and Hetbods

. . . . . . . . . . . . . .

A: Materials ............................................ B: Methods ............................................ 1: Micro-kjeldahl Analysis .............. . . . . . . . . . . .. . . . 2: Sodium Dodecyl Sulfate (SDS) Analysis .............. 3: Functional Properties of Proteins

3.1: Foaming capacity and foaming stability ........ 3.2: Water absorption ..............................

32

33

35

35

39

39

39

40

40

41

3.3: Fat absorption ................................ 41

3.4: Emulsifying capacity and stability •••••..•..•. 42

3.5: Viscosity ...... ., ............................. . 43

·4 : In Vitro Digestibility •• ........................... 44

5: SDS Electrophoresis •••••••••••.••••••.•••••••.••••. 45

5.1: Preparation of gels ••••••.•.••••...•..•..•••.• 45

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5.2: Sample preparation............................ 45

5.3: Electrophoresis •••••••••••• ••••••••••••• •••••• 46

6: Amino acid Analysis •••••••••••••••••••••••••••••••• 47

6.1: Sample preparation •••• ••.•. •••.••• •••••• ••••.. 47

6.2: Reversed phase-HPLC chromatography •••.•• •••••• 48

Section III: Bxperiaental

Experiment 1: Protein extractability from dried brewers' spent grain (DBSO) and pressed brewers' spent grain (PBSO) using a factorial design .••••.•• 51

Experiment 2: SOQium dodecyl sulfate (SDS) extraction of DBSa ............................................. 54

Experiment 3: Extraction of DBSO protein using sodium dodecyl sulfate (SDS) with dibasic sodium phosphate (Naz HPO.) ............................................ 54

Experiment 4: SDS extr~ction of DBSO protein with different levels of dibasic sodium phosphate (Naa HPO.) •••.••••.••••••••••••••••••.••••••.••.••••

Experiment 5: Extraction of DBSG protein using SDS

54

wi th sodium chloride (NaCI) •••.••••••.•••••••••••.• 55

Experiment 6: Preparation (laboratory scale) of DBSG protein concentrates • •••.•• ••••. •••••••••••••• ••••. 55

Experiment 7: Central composite rotatable design for optimization of DBSG prote in extractability ••• •..•• 56

Experiment 8: DBSG protein concentrates (pilot scale preparation) ....................................... 58

Section IV: Results and Discussion

A: Optimization of Prote in Extractability from BSG 60

1: Screening Experiment-Variables affecting BSG Protein Extractability ••••• •••••••••••. ••••••• ••••• 60

2: Dried Brewers'Spent Grain (DBSO) Protein Solubi­lisation

(a) SDS extraction of DBSO ......................... viii

65

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(b) Solubilisation of DBSO protein usina SDS with dibasic sodium phosphate (NazHPO.) •••• •••. 65

(c) Effect of dibasic sodiua phosphate (Na HPO ) on DBSa protein extractability •.•...• ~ ••• ~..... 67

(d) Bffect of concentration of dibasio sodium phosphate (Na HPO ) on extraotability of protein from bBS0

4 • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 69

(e) Effect of presence ,if sodium chloride in SDS solution extractab~lity of protein from DBSO

(f) The effect of sample preparation method on

... 70

protein extractability from DBSO •.••••••••••••• 72

3: Response Surface MethodololY (RSM): Optimization of Conditions for Extraction of Protein trom DBSO .•....••••.••••..•••....••.... fi • • • • • • • • • • • 72

4: DBSO Protein Concentrates

(a) Bffect of extraction temperature on (i) extractability of protein from DBSO and (i~) prote in content and SDS content of prote in concentra tes .................................. .

(b) Reduction of SDS oontents of DBSO protein concentrates ...................................

5: Functional Properties of DBSO Protein Concentrates.

83

84

(a) Foam capacity ••.•••.•••••••.•••••..••...••••••. 91

(b) Foam stabili ty .••••••••••••••••••••.•.••••••.•. 94

(c) Emulsion capacity and stability •••••••••••• •••• 96

(d) Water absorption •••••••••••••••••••••.••••••••• 98

(e) Fat absorption ••••••••••••••••••••••••••••••••. 99

(f) Viscosity .......... ' .............. fi............ 100

6: Characterization of DBSO Protein Concentratea

(a) Sodium dodecyl 8ulfate-polyacrylamide leI electrophoresis ••••••••••.•••••.••••••.•••••••. 105

(b) In vitro dilestibility ••••••••••••••••••••••••• 111

(c) Amino acid composition ••••.•.••••••••••••••••••

Summary .....•.••.....••...••.•..••••...••..••••..•••••.....

References .......................... , ....... , ............. .

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116

119

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LIST OF TABLBS

Table 1. Etfect of pH of solvent on extraction of protein fro. air-classified Arthur flour •••• ••••••••••••• 7

Table 2. Effect of neutral sodium salts on the aolubility of defatted and nondefatted aluten ••••••••••••••• 9

Table 3. Effect of temperature on the extraction of alcohol-soluble stora,e proteins Crom whole milled wheat _rain (cv. Flinor) ••••••••.•••••..•• 12

Table 4. Protein fractions of sorghum and its distillers' grains ....................................... ,... 17

Table 5. Amino acid composition of casein, white wheat, red wheat, corn, and distillera' dried araina with solubles (DDGS) •••• ••••••••.••• •••.••• ••.••• 27

Table 6. Protein efficiency ratio (PER) and net protein ratio (NPR) of distillera' dried arains with solubles (DDSG) •••••••••••••••••••••••.••••••.••• 29

Table 7. In vivo and in vitro digestibility of distillera' dried grains with solubles (DDGS) '" .....•••...•.

Table 8. Emulsification characteristics of various

29

protein sources ••••.•••••••••••••.•••.••.••••.••• 31

Table 9. The composition of the reagents used in amino acid analysis ................... t • • • • • • • • • • • • • • • • 48

Table 10. The ,radient prolram used for amino acid analysis by HPLC ••••••••••••••••• 1 •••• 1 1 • • • • • • • • • 49

Table 11. The run parameters entered into the Waveacan proaram used for amino acid analysis •....• ....••• 50

Table 12. A half fraction of a 2· factorial desiln (coded) to determine factors influencin. extraction of protein from dried brewers' spent arain (DBSG) and pressed brewers' spent arain (DBSG) .1. ....... 52

Table 13. Variable levels and coded values u8ed in a half­fr3ction factorial screenina desian for protein extraction from dried brewers' spent ,rain (DBSG) and pressed brewers' spent arain (PBSG) .•••••.••• 53

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Table 14. Coded level conbinations for a four variable Central Composite Rotatable Design to optimize protein extractability from dried brewers' spent grain (DBSG) ••••••••••..••••••.••.•..•••••• 57

Table 15. Variable levels and coded values used in central Composite Rotatable Design for protein extraction from dried brewers' spent grain (DBSG) •• '.... •••• 58

Table 16. Analysis of variance of preliminary factorial screening design for protein extracted from dried brewers' spent grain (DBSG) ••.••••...•••••••••.•• 63

Table 17: Analysis of variance of preliminary factorial screening design for protein extracted from pressed brewers' spent grain (PBSQ) ... •..•.... ... 64

Table 18. Effect of sodium dodecyl sulfate (SDS) concentration on protein solubilisation from dr ied brewers' spent grain (DBSG) •••.•••.••.•..•• 66

Table 19. Protein extractability from DBSG using SDS solution containing dibasic sodium phosphate and SDS solution only ............................... .

Table 20. Protein extractability of DBSG using SDS

67

extractant ....................................... 68

Table 21. Effect of concentration of dibasic sodium phosphate on extractability of protein from OBSO.. 70

Table 22. Protein extractabilities from OBSO using different SOS solutions containing NaCl and Naz HP04 •••••••••••••••••.••• tt Il........ ......... 71

Table 23. Prote in extractability from sieved DBSG and ,round DSSG ...................................... 72

Table 24. Analysis of variance for second order polynomial model fitted to yield of protein extracted from dried brewers' spent grain (DBSG) •••.••• ••.• 76

Table 25. Coded and uncoded values of variables at stationary point X (point of maximum yield of extracted protein)o............................... 77

Table 26. Protein extractability of DBSG extracts, prote in content and SOS content of DBSG protein concentrates (spray dried) at different tempera tures .................................... .

xii

84

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Table 27. SDS content of DBSG prote in concentrate (laboratory scale preparation) usina optimum extraction conditions ••••••• •••.•••••••••.••••••• 86

Table 28. SDS content of DBSG pl'otein cOllcentrates (pilot scale preparation) usina optimum extraction condi tions ....................................... 88

Table 29. SDS content of DBSG protein concentrates (pilot scale preparation) prepared at temperatures of

o 0 50 C and 100 C ......•.............•.............. 90

Table 30. SDS level of DBSG protein concentrates (pilot scale preparation) prepared at different teaperatures ..................................... 91

Table 31. The effect of pH on the foam capacity of DBSG prote in concentrates and soybean concentrate ••••• 94

Table 32. The effect of pH on the foam stability of DBSG protein concentrates and soybean concentrate •.••• 95

Table 33. Comparison of emulsifying capacity and end-point criteria for DBSG protein concentrates •••••••••••

Table 34. Comparison of emulsion stability of protein

97

concentrates .................. ,.................. 98

Table 35. Fat absorption and water absorption of DBSG protein concentrates and soy concentrate •.•••.•.• 100

Table 36. Viscosities of unbeated protein concentrates ( 10%) at various pH values •••••••.••••••••••••••• 102

Table 37. Viscosities of prote in concentrates (10%) at various pH values after beating •••••••••••••••••• 102

Table 38. Viscosities of unheated protein concentrates ( 15%) at various pH values ••••• ".................. 104

Table 39. Viscosities of prote in concentrates (15%) at various pH values after heatin ••••••••••••••••••• 104

Table 40. Mi.ration distance of four standard proteins and of the proteins of DBSG protein concentrates in SDS-phospha te gel s •••••••••••••••••••••••.•.•••.• 110

Table 41. In vitro di.estibility of casein and D8SG protein concentrates ........................ Il... ........ 112

Table 42. The amino acid co.position of DBSG prote in concentrates (spray dried) ••••••••••••••••••••••• 115

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LIST OF FIGURES

Figure 1. The extractabil1ty of proteins from defatted soybean meal as a function of pH ••••••••••••••.•• 21

Figure 2. Solubility of a globulin-type protein close to its isoelectric point ••• ••••• •••••••••••••••••••• 21

Figure 3. Three-dimensional plot of a "Cradle " response surface .......................................... 36

Figure 4. Three-dimensional plot of a "Saddle point" response surface ••••••••••••••••••••••••••••••••• 37

Fiaure 5. Response surface graph showing the effect of concentration of phosphate and BSG: Extractant ratio on prote in yield .•• ••••• •••• ••• •••••• ••.••• 79

Figure 6. Response surface graph showing the effect of tempe rature and time on protein yield •••.•••••••• 80

Fiaure 7. Contour plot showing the effect of concentration of phosphate and BSG: Extractant ratio on prote in yield ........................................... .

Figure 8. Contour plot showing the effect of temperature and time on prote in yield ••.•••••••..•••.•.•••••.

Figure 9. Plot of molecular weight versus migration

81

82

distance of standard proteins •••••••••••••••••••• 108

Figure 10.Electrophoretic patterns of the proteins of three brewers' protein concentrates •••••••••••••••••••• 109

xiv

GENERAL INTRODUCTION

Brewers' spent grain (BSG) is the principal by-product

from the fermentation of cereal grains for beer production.

Fermentation primarily utilizes starch, whereas other

nutrients, such as protein and fibre are concentrated (Wu,

1986) • BSG commonly contains 26-30% protein (NX 6.25) and

14-17% fibre and might be use fuI in increasing both fibre

and protein in human nutrition. (Prentice et. al. 1978).

The principal use of BSG is as a source of animal feed.

Bsa has been investigated as a possible adjunct for human

food (Prentice et. aL, 1978); Finley and Hanamoto, 1980;

Junnila et. al., 1981). The major limitation of

- incorporating BSG into baked products is the undesirable

organoleptic qualities (Prentice et. al., 1978). The

production of a prote in isolate from BSG May eliminate the

undesirable organoleptic properties (Natarajan, 1980).

The purpose of the present study was to prepare protein

concentrate from BSG on a pilot scale. Optimisation of

protein extractability and precipitation from BSG was

necessary prior to pilot scale preparation. Optimisation of

protein extractability from BSG was determined by

considering the simul taneous effect of several factors

(concentration of extractant, temperature, time t particle

size, meal:solvent ratio) usin. response surface

....... methodology • The nutri tional , functional and biochemical

properties of the protein concentrates (spray-dried) were

1

( investigated with a view of their utilization in foods.

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SECTION 1: LITERATURB RBVIEW

A: Introduction

Several researchers have investigated the potential use

of BSG in fooda (Finley and Hanomoto, 1980; Junnila et. al.,

1981). BSG has a relatively high protein content (25 to

30%). The solubilisation of this nitrogeneous material from

BSG by solvents (water, sodium chloride, aqueoua alcohol,

acidie and alkaline conditions) gave a low nitrogen recovery

(Crowe et. al., 1985). Factors affecting protein

solubilisation (pH, ionic strength, nature of extraetant)

and protein precipitation May be manipulated in order to

improve protein extractability from BSG.

B: Evaluation of Distillers' Spent Grain (DSG) for use in Foods

Distillers' spent grain (DSG), a by-product of the

alcoholie fermentation of cereal grains, are rich in protein

and fibre (Wall et.al., 1984; Miller and Eisenhauer, 1982).

The relatively high nutritional value of DSG has led to

reaearch on i ts potential food appl iea tions (Wu et. al. ,

1987; Morad et.al., 1984; Wall et.al., 1984; Wampler and

Gould, 1984; Taen et.al., 1983, 1982).

Taen et.al. (1982) reported that distillers' dried

grain flour (DDGF) was suitable as a supplement for wheat

flour in the preparation of dark cookies; the DDGF served to

enrieh the protein and fibre contents of the cookies. Bread

3

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supplemented wi th 10% DDGF were superior to whole wheat

bread with respect to loaf volume, crumb grain and color

(Tsen et.al., 1983).

Wampler and Gould (1984) reported that incorporation of

DSG at the 101 and 20% levels in extrusion douahs, produced

extrudates wi th extremely high expansion values. The

authors concluded that DSG can be used as a flour component

for the production of puffed-food products. Wu et. al.

(1987) showed that spaghetti containing up to 10% corn

distillers' grain (CDG) had acceptable flavor, texture and

cooking quality as weIl as enhanced protein, dietary fibre

and essential amino acid contents. Substituting 25% sorghum

DSG for wheat flour in a cookie formulation increased the

protein content by 100% and fibre content approximately

sixfold wi thout affecting cookie quaI i ty (Morad et. al • ,

1984). Prentice et.al. (1978) found that, at the 15%

substi tution level, BSG could be used successfully for

preparation of sugar cookies. The authors observed that

under optimum conditions acceptable physical quali ties of

thu sugar cookies could be attained with 40% BSG. However,

organoleptic evaluations showed that 15% incorporation was

the upper limi t for sugar cookies and special ty cookies as

chocolate chip, oatmeal and raisin. The cookies had an

undesirable brown color with more than 20% BSG.

4

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C: Prote in Bxtractability-Bffect of Various Factors

1.0: Bffect of pH of extractant on protein extractability

The solubility of proteins is influenced markedly by

pH. This is a result of the amphoteric behaviour of

proteins. When the solubility of a given prote in ia plotted

as a function of the pH, a V or U-ahaped curve ia obtained

with minimum aolubility at the isoelectric point (pl). This

behaviour is put to use for dissolving seed proteins.

Solubility and yield of extraction ia generally greater at

alkaline than at acid pH (Fennema, 1985).

Burrows et.al. (1972) reported that with the exception

of wheat gluten and corn zein, most of the proteins from

grains, defatted oil seed meal or leguminous crops can be

extracted quantitatively by use of alkaline solutions.

Smith (1978) reported that the soy proteins (globulins) from

defatted soybean meal have a minimum solubili ty between pH

3.75 and 5.25, and maximum solubilities between pH 1.5-2.5

and above pH 6.3. Wolf et.al. (1975) found that 85% of the

protein in defatted soybean meal is extractable in water at

pH 6.5. The addition of alkali resulted in an increase in

protein extractability by 5-10~. Satterlee et.al. (1976)

reported that the yield of distiller's protein concentrate

(DPC) from fermented wheat and corn was optimal using a pH

12.2. Wu et.al. (1979) developed an alkaline extraction (pH

11.2) procedure for preparing a prote in concentrate from

barley.

5

(,. Several researchers have found that the solubili ty of

the seed proteins is greater at alkaline than at acid pH

(Abdel-Aal, et.al., 1986; Wu and Stringfellow, 1980; Shehata

and Thannoun, 1981; Kazazls and Kalaissakis, 1979). When

proteins are ei ther predominantly posi tively or negatively

charged, at either side of the pl, their solubility

increases (Smith, 1983). Abdel-Aal et.al. (1986)

investigated protein extractabili ty t'rom faba beans, chick

peas and fenugreek flours and found that maximum

solubilities were obtained at pH 8.0 or higher and around pH

2.0. Wu and Stringfellow (1980) studied the effect of pH on

protein extractabi l i ty from air-classi fied wheat flour

(Table 1). The authors concluded that the optimum pH should

be Il.1 (77% protein) rather than pH 11.5 (79% protein),

because the higher pH could lead to increased protein

denaturation. Shehata and Thannoun (1981) reported that

maximum protein extractabili ty from Mung bean flour was

obtained at pH Il.05 (85.3%) and at pH 1.90 (79.4%).

6

....

1

Table 1. Effect of pH of Sol vent on Extraction of Protein fro. Air-Classified Arthur Flour (soivent: solid ratio 6:1)-.

Solvent pH of slurry

0.08N HCI 1.6

0.03N HCI 2.3

0.02N HCI 2.8

IN HOAC 3.1

0.015N HCI 3.3

0.3N HCI 3.5

O. lN HOAC 3.9

O.OlN HCI 3.0

0.05N HOAC 4.2

0.03N HOAC 4.3

O.OlN HOAC 4.6

Water 5.7

0.02N NaOH 10.6

O.03N NaOH 11. 1

0.04N NaOH 11.3

O.05N NaOH 11.5

0.06N NaOH 11.6

0.075N NaOH 11.8

• Source: Wu and Stringfellow (1980).

7

Total protein extracted from flour (%)

28

49

48

53

47

50

51

43

48

31

24

12

69

77

74

79

34

27

(

1.1: Effeet of ionic stren.th of extraetant on protein extractabilit7

The classical effect of salts in increasing the

solubili ty of proteins is called the salting-in effect

(Smith, 1983). Fennema (1985) reported that the ions of

neutral salts, at molarities of O.5-lM May increase the

solubility of proteins. In this range of molari tv the

logari thm of the solubili ty is a function of the ionic

strength (Smith, 1983) where the ionic strength (u) of the

solvent is given by the following equation:

U : 1/2 mZz

U : ionic stren.th m = molarity Z = charge of ion

Several workers have reported that the amount of

protein extracted from seeds by neutral salts de pends on the

nature and concentration of salt used (Preston, 1981;

Finnigan and Lewis. 1985; Kazazis and Kalaissakis, 1979).

Preston (1981) repùrted that low levels of various salts

(O.05-0.2M) significantly reduced wheat gluten

extractabillty when compared ta water; as the salt

concentration .. ,as increased the protein extractabili ty was

highly dependent on the nature of the anion. The author

also showed that the arder of extractability of the defatted

gluten proteins with the anions was as followa: F-. Cl- , Br ,

BrO , :1

C10 . ' 1 -. SCN (Table 2). NaCl solution is

considered to be a good solvent for extracting proteins,

8

- ·. __ ._--------------------

particularly globulins, from dry le.ume seeds (Pant et.al.,

1969) • Changes in the extractabili ty of proteins as a

result of increasing concentrations of simple monovalent

salts have been attributed to effects upon electrostatic and

hydrophobie bonding (Dandliker and De Saussure 1971, Franks

1978, Von Hippel and Schleich 1969). Abdel-Aal et.al. (1986)

reported that the maximum solubility of prote in was reached

at O.5M NaCl for faba bean, chick pea and fenugreek flours.

Table 2. Effect of Neutral Sodi UIIl SaI ts (1. OH) on the Solub\lity CS) of Defatted and Nondefatted Gluten •

Gluten

Salt Detatted Nondefatted ------------------------------------------------------------None (Ha 0)

NaF

NaCI

NaBr03

NaBr

NaClO.

Na!

NaSCN

HAc(O.05 M) Lactic (C.005 M)

: Not deterllined. Source: Preston (1981).

25.0

Trace

5.8

5.4

19.9

31. 3

51. 7

61. 5

70.4 71.4

9

29.5

Trace

5.2

6.4

• ... 28.7

54.6

59.1

77.2 75.8

------------------------------------------------------

Kazazis and Kalaissakis (1979) observed that wh en the

concentration of NaCl was less than or equal to D.01M

minimum quanti ties of ni trogen was extracted (37.2%) from

Vicia sativa, while maximum solubility was achieved at

0.75M. An optimum concentration of O. 2M NaCl resul ted in

maximum nitrogen extractability from rapeseed meal (Finnigan

and Lewis, 1985).

The saI ting-out tendencies at low salt concentrations

may be attributed mainly to electrostatic shielding of ionic

amino acids on the surface of the proteins. This shielding

is dependent primarily on ionic strength. At higher salt

concentrations the ionic strength is sufficient to

neutralize electrostatic interactions (Preston, 1981).

1.2: Bffect of teaperature on protein extractability

The solubili ty of proteins increase with temperature o

between 0 and 40-50 C (Fennema, 1985). Several researchers

have attempted to increase protein extractability from seeds

using an optimum extraction temperature (Alli and Baker,

1981; Finnigan and Lewis, 1985; Shehata and Thannoun, 1981).

Alli and Baker (1981) investigated the effect of

temperature on yield of protein with crystalline

microstructure from Phaseolus beans (white kidney bean, navy

bean and baby lima bean). The authors found that the

protein yield increased to maximum values when the

o 0 temperature of extraction was increased from 27 C to 40 C;

10

... ..,.. '; ~ ,

-. '

above 40°C there d . t i i Id waa a ecrease ln pro e n y e • Djang

et. al. ( 1953) also reported diminished protein yields from

° muni beans at temperatures above 45 C. Heatinl a protein to

above 50°C results in the disruption of secondary and

tertlary structures and is termed denaturation. Such a 10s8

in seoondar'y and tertiary structure often resul ts in a

decrease in solubility (Finnilan and Lewis, 1985). Shehata

and Thannoun (1981) reported a relatively small increase in

° nitrolen extracted from muni bean from 77.63% at 25 e up to

79.85% at 600 e. Satterlee et.al. (1976) observed that

widely varyinl temperatures were required to maximise

protein yield from distillers' fermented wheat and oorn;

extraction temperatures of 23° C and 80° C la\re a hilh yield

of wheat protein oonoentrate (WPC) and oorn protein

concentrate (CPC), respeotively. Finnilan and Lewis (1985)

showed that an inorease in nitrogen extraotion was observed

at 40° C and 50° C as oompared to 20° C and 30° C. Abdel-Aal

et.al. (1985) prepared protein isolated from faba beans;

protein reooveries of 66.2%, 74.0% and 63.9% were obtained

using three different extraction methods in which the

extractions were done at room temperature. Ervin (1986)

reported that SDS extraction of Brewers' spent grain protein

at 27°e gave a lower nitrogen recovery (18.7%) when compared

with that (28%) obtained by extraction at 100°C. Byers

et.al. (1983) studied the effect of tempe rature on the

extraotion of alcohol-soluble storale proteins from whole

Il

(~

(.

(~

mi lled wheat grain wi th and wi thout the addition of 2-

mereaptoethanol (2-ME). A quantitative increase in nitrogen

reeovery was observ~d with an increase in temperature (Table

3) •

Table 3. Effect of Teaperature on the Bxtraction of Alcohol-Soluble Storage Proteins fro. Whole Milled Wheat Grain (cv. Flinor) with an4 without the Addition of 2-Mercaptoethanol (2-MB) •

2-ME Nil

T t ( OC) empera ure 4 20 60

Aleohol systems 70% ethanol 19.3 25.3

50% propan-l-ol 26.7 30.3

50% propan-1-ol + acetie acid ( 1 X) 40.5 46.6

55% propan-2-o1 19.5 27.6

a X N extracted

1% (v/v)

4 20 60

35·.0 33.7 43.0

41.9 44.9 46.9

53.0 53.3 54.4

36.5 36.0 45.8

45.6

52.1

61.5

47.8

aN extracted is expressed as a pereentage of the sum of N recovered from aIl fractions.

b Source: Byers et.al. (1983).

1.3: Rffect of extraction tiae on prote in extractability

Previous work on prote in extractability has shown

marked variation in the amounts of nitrogenous matter

extracted using meal of different particle sizes and

extraction time (Finniaan and Lewis, 1985; Alli et.al.,

1981; Kazazis and Kalaissakis, 1979). Kazazis and

12

Kalaissakis (1979) reported that 71.7% and 83.8% of nitrogen

from vetch seeds (Vicia sativa) was solubilised with

particle size of < 0.5 and < 0.2 mm, respecti vely. The

authors concluded that the seed should be .round as fine as

possible to maximise ni trogen extractabil i ty. Protein

extractabili ty is facili tated by small particle size; the

smaller the particle size the greater the surface area for

contact by extractant (Fellers et.al., 1966). Alli and

Baker (1981) observed that maximum yields of protein 'ü th

crystalline microstructure were obtained from ground beans

(Phaseolus Sp.) with particle sizes in the range 0.50-1.00

mm. Decreased yields were obtained with larger particle

size (1.00-1.41 mm). It was suggested that this decreased

- yield may be the result of incomplete rupture of cellular or

subcellular membranes which surround the protein bodies

(Patel et.al.1974). The grinding of some seeds results in

the denaturation of some proteins which is reflected in

decreased protein extraction from the very fine particles

(Patel et.al., 1974).

1.4: Effect of particle size of aeal on protein extractability

Kazazis and Kalaissakis (1979) reported that with an

extraction time of 90 min. 71.7% and 83.8% of nitrogen from

vetch seeds (Vicia sativa) was solubilised. Alli and Baker

(1981) observed that the yields of protein material from

white kidney beans, navy beans and baby lima beans reached

13

(.

maximum values after extraction periods of 30 min and 20

min. Shehata and Thannoun (1981) found that 76.48% of the

total nitrogen in mung bean was extracted within 5 min; only

a small increase to 79.54% was obtained by extending the

extraction time to 25 min, while longer extraction time (30-

60 min) were found to decrease extractable ni trogen to

77.40% (30 min) and 77.55% (60 min). Finnigan and Lewis

(1985) found that 95% of the extractable nitrogen in

rapeseed meal was removed after 15 min and in most cases,

nitrogen extractability was greater for the ground sample.

A short period of contact between bean meal and extractant

is sufficient for the extraction of maximum amounts of

protein frollt the beans (Finnigan and Lewis, 1985; Shehata

and Thannoun, 1981; Alli and Baker, 1981).

1.5: Rffect of nature of extractant on protein extractability

The diversity in the structural and chemical

composition of plant proteins leads to variability in their

solubilisation in different solvents (Shewry and Miflin,

1985). Osborne (1924) classified the proteins of cereal

seeds based on their solubili ty in water (albumins), saI t

solutions (globulins), ethanol (prolamins) and dilute acids

and bases (glutelins). The glutelins and prolamins

constitute the bulk of the proteins of most kinds of cereal

irains (Altschul, 1958).

The prolamins are rich in uncharged amino acids and poor

14

in acidic and basic amino acids. The low content of charged

amino acids means that prolamins have a low net charge at

any pH (Shewry and Miflin, 1985). Prolamins, such as

gliadin from wheat, hordein from barley, or zein from maize

cau be extracted with 70 to 80% ethanol (ftltschul, 1958).

Neuman et.al. (1984) reported that 70X ethanol extracted

63.5X of the protein in wet corn gluten Meal. Zein

(prolamin) represents 50-60X of the total endosperm protein

(Esen, 1981).

The glutelins are high molecular weight complexes, made

up of subuni ts linked together by disulphide bonds

(Altschul, 1958). Wilson (1981) defined the glutelins as

those proteins that are either soluble in dilute aqueous

alkali or insoluble in neutral aqueous solutions, saline

solutions or alcohol. Wall and Paulis (1978) suglested that

interpolypeptide disulphide bonds make cereal glutelin

poorly soluble, since glutelin extraction requires reducing

and occasionally alkylating agents. Sol vents based on

sodium dodecyl sulphate (SDS), 2-mercaptoethanol (2-ME)

and/or dithiothreitol (DTT) have been investigated as

sui table extractants for glutelin (Landry and Moureaux,

1970; Wall et.al., 1975; Kobrehel and Bushuk, 1978;

Graveland et.al., 1979; EI-Negoumy et.al., 1979; Wilson

et.al., 1981). 2-ME and DTT are used because they reduce

the disulphide linkages of glutelin and facilitate the

extraction of polypeptides (Wall et.al., 1975). Graveland

15

(

et.al. (19791 reported that the disadvantage of using

reducing agents is that chemical conversions take place

which causes high molecular compounds to be degraded into

smaller fragments. By the use of drastic methods such as

extraction wi th strong acid and alk!1li, protein can be

dissolved quantitatively, but its original structure is then

changed (Graveland et.al., 1979, Sathe and Salunkhe, 1981).

Roberts et.al. (19851 used strongly alkaline conditions (pH

12.5) which extracted 83% of the protein from wheat bran.

The utilization of alkaline pH for extraction may cause

changes such as destruction of lysine and cystine, formation

of lysinoalanine and racemization which reduce the protein

quality (Sathe and Salunkhe, 1981).

Severai researchers (Wu et.al., 1981; Wu et.al., 1984);

Wu, 1986) have at tempted to fractionate the proteins from

distillers' grains. The distillers' grain showed a low

protein solubility in the common solvents (water, sodium

chloride and alcohol) as compared to the cereal grains (Wu,

Y.V. 1986; Wu et.al., 1984); the authors suggested that the

low prote in solubility of the distillers' grains was due to

protein denaturation during fermentation and heating.

Wu et. al. (1984) fractionated the proteins of sorghum

and sorghum distillers' grains using different sol vents.

Water, NaCl (1%), t-butanol (60%) and DTT, and borate + SDS

+ DTT extracted albumins, globulins, prolamins, cross-linked

prolamins and giutelins, respectively. With this series of

16

solvents sorghum distillers' ,rains showed a low protein

solubility wh en compared to sorghum (Table 4).

Table 4. Prote in Fractiol\s of Diatillera' Grains •

its

Percent of Total N

Fraction- Sorghum Sorghum

Distillera' Grains

From either method Water extract 1% NaCl extract

60%t-Butanol ext~act 60%t-Butanol + DTT extract

From method 1 Borate + SOS + DTT extract, pH· 11.1 Residue

From method 2 O.lN NaOH + DTT extract, pH Il.8 O.lN NaOH + SDS + OTT extract, pH Il.8 Residue

14 2

20 36

16 la

· .. · .. • ••

1 1 2 3

20 59

30

48 14

:DTT = Dithiothreitol, and SDS = sodium dodecyl sulfate. Source: Wu et.al. (1984)

The inclusion of a reducing agent in the extractant

increased the amount of prolamin nitrogen extracted from

barley, maize or sorghum (Landry and Moureaux, 1970; Miflin

and Shewry 1979; Shewry et.al., 1980). This might be

related to the observation that cross-linked prolamin and

prolamin are the two lar,est protein fractions in sor,hum,

while in sorghum distillers' grain there is li ttle cross-

17

(~

(~

(.

linked prolamin and prolamin (Wu et.aL, 1984). Relatively

strong sol vents (Borate + SDS +DTT, o. IN NaOH +SDS + DTT)

were used to extract Most of the protein from distillers'

grains (Wu, 1986; Wu et.al., 1984).

Detergent solutions (e.g. sodium dodecyl sulfate) have

been found to be effective as an extractant of protein from

distillers' grains and brewers' spent grain (Wu et.al.,

1984; Wu, 1986; Crowe et.al., 198a). Tanford (1968)

reported that SDS causes dissociation and denaturation of

proteins even at very low concentrations. Sodium dodecyl

sulfate solubi 1 i zes prote in aggregates held together by

noncovalent hydrophobic bonds (Graveland et. al., 1979;

Landry and Moureaux, 1970; Landry and Moureaux 1981; Wall

et.al., 1975). Danno (1981) reported that wheat glutenin, a

mixture of high molecular weight proteins containing

interpolypeptide disulfide bonds was almost completely

extracted with 0.5% sodium dodecyl sulfate without prior

reduction of its disulphide bonds.

Van den Berg et.al. (1981) and Baxter and Wainwright

(1979) reported that the major proteins of brewers' spent

grain (glycoproteins, glutelins and hordeins) were

associated in aggregates held together by intermolecular

disulphide bonds and hydrophobie interactions which limi t

their solu~ility in the common solvents. Crowe et.al.

(1985) proposed that the proteins of BSa interacted with

cellulosic material during heating of mashing and drying

18

processes. Detergent solutions could be used to disrupt

protein-cell wall interactions (Van Soest, 1965). A neutral

detergent solution containing SDS as its major component

extracted 84% of the BSa ni trogen (Crowe et. al., 1985).

Kato et.al. (1984) reported that SDS bindina by glycinin

(soy protein) increases when the protein was heat denatured.

Other researchers (Wu et.al., 1981; Wu et.al., 1984j Wu

et.al., 1985j Van den Berg et.al., 1981) have reported that

SDS extractant is selective for heat denatured proteins.

Anionic detergent (e.g. SDS) provides a means of increasing

the negati ve charge on the protein molecule (Sureshchandra

et.al., 1987). The mechanism of protein solubilisation

- using SDS is postulated to involve an interaction of the

hydrophobie groups of the anionic detergent with hydrophobie

sites of the proteinj this increases the negative charges on

the protein surface as a result of the anionic tails, thus

intensifying the repulsive forces between protein Molecules

(Kato et.al., 1984). Kata et.al. (1984) reported that SDS-

binding capacity is linearly proportional to the surface

hydrophobicity of proteins.

D: Prote in Precipitation Techniques

2.0: Effect of pH on protein precipitation

A relationship between pH and protein solubility is

use fuI in determining the pH at which a prote in May be ....... precipitated (Honil et.al., 1987; Abdel-Aal et.al., 1986; Wu

19

and Stringfellow, 1980). Hudson (1983) reported that each

protein has an isoelectric point (pl) which is the pH at

which the net charge on the prote in molecule is zero. At

the isoelectric point, the electrostatic forces of

at traction cause protein aJgregation and precipitation of

the protein (Fig. 2). Isoelectric points vary from one

protein to another (Smith, 1983). Proteins may exhibit

isoelectric points at pH values ranging from 1 to 12 but,

for many proteins this range is reduced to pH 4 to 6. In

their normal environment these proteins tend to have an

overall negatively charged surface (Bell et.al., 1983).

Isoelectric precipitation has been used in the

preparation of several protein concentrates (Nakai et. al. ,

1980; Gheyasuddin et. al., 1970; Wu and Stringfellow, 1980 ;

Satterlee et.al., 1976; Honig et.al., 1987). Hudson (1983)

reported that the soy proteins have a minimum solubility

between pH 3.75 and 5.25 (Fig. 1). The proteins from Iraqi

mung bean flour showed minimum extractabili ties between pH

3.4 and 5.7 (Shehata and Thannoun, 1981 ) • Nakai

et. al. ( 1980) extracted the pro teins of defa tted soy flakes

at pH 10 followed by isoelectric precipitation at pH 4.5.

Honig et.al. (1987) prepared protein isolates from water

extracts of defatted soybean flakes by acid precipitation in

the range of pH 3.5 to pH 5.2. The authors found that the

highest yield of protein isolate was obtained by

20

-

........

�00,-------------------...,

ID

r~ J \

J

ID

.".---------" ~.-

(!) HCI • HID - HaOH

D~~~Z~~~4-~5~~I-~1-+I--~I~I~D~1~1~1~Z pH al latracl

Figure 1. The s07bean meal as

extractabili t7 of proteins from a function of pH. (Hudson, 1983).

@t0~ ~~ +~ ro ~ ~ ~ ~

pH> I.E.P. pH < I.E.P. pH 01 I.E.P. ,

~t

-+ + + - +

I.E.P. pl-!

defatted

Figure 2. Solubilit7 of a alobulin-type protein close to its isoelectric point. (Scopes, 1983) •

21

precipitation at pH 4.5. Sunflower protein isolate bas been

pI'epared by the conventional method of extraction wi th

alkaline solution (pH 10.5) followed by isoelectric

precipitation at pH 5.0 (Gbeyasuddin et.al., 1970). The

extracted proteine of distillers' fermented wheat and corn

were recovered by pH adjustment of the extract ta 4.0

(Satterlee et.al., 1976). Ervin (1983) reported that

isoelectric precipitation was not effective in recovering

the protein extracted from brt:.,ers' spent grains. The

author concluded that a lack of a definite isoelectric point

for the BSG protein may be responsible. Wu and Stringfellow

(1980) studied the effect of precipitation pH on alkaline

extract of air-classified wheat flour at seven pH values

between 4.6 and 7.2. The amount of protein precipitated

ranged from 73-85% with the minimum amount of protein

precipitated near pH 6.3.

A major advantage of isoelectric precipitation lS the

cheapness of mineraI acids and the fact that several such as

phosphorio, hydrochloric and sulphuric are acceptable in

protein food products (Bell et.al., 1983).

2.1: Bffect of teaperature on protein precipitation

Heating i8 often employed to coagulate proteins

(Fennema, 1985). Conversely temperature may be reduced to o

near 0 C to induce insolubility in some proteins (Bell

et.al., 1983). Heat is the moet common physical a.ent

22

.......

o capable of denaturini proteins. Denaturation is very often

followed by a decrease in solubility, as a result of

exposure of hydrophobie iroups and the agireJation of the

unfolded protein molecules (Fennema, 1985). German et. al.

(1982) reported that for oliiomeric proteins (e. a. soy

glycinin) heat May cause association/dissociation of the

oliiomer, and disrupLinn of the quaternary structure itself

May result in aggregation.

Both soy glycinin and oat globulin have high

denaturation temperature (Td) but can be coagulated by heat

below their Td (Ma and Harwalkar, 1987). Mori et.al (1982)

reported that when soy glycinin (0.5-1%) in 0.4-0.5M sodium

chloride solution was heated at 100°C, more than 50% of soy

protein precipitated after 10 min. Ma and Harwalkar (1987)

studied the effect of tempe rature on the rate of

precipitation of a dilute solution of oat globulin in 1.0 M

NaCI. The authors found that less than 10% of the

globulins precipitated after extended heating at 100°C,

while at 110°C the quantity of precipitated protein after 60

min. heating was above 70%. The addition of dithiothreitol

(disulfide reducing agent) to the solution of oat globulin

increased the rate of protein precipitation at 100°C.

2.2: Bffect of organic solvent on protein precipitation

The addition of an orianic solvent such as ethanol or

acetone to an at'lueous extract containing proteins has a

23

(--

variety of effects which lead to protein precipitation;

however, the princ; pal effect is the reduction in the

dielectric constant of the medium (Scopes, 1983). When the

dielectric constant of the aqueous medium is reduced by the

addition of miscible organic solvents, electrostatic

interaction between protein molecules is enhaneed and

precipitation will result (Bell et.al., 1983). Scopes

(1983) reported that the orsanie solvent used must be

completely water-miscible and non-reactive towards proteins.

The sol vents Most widely used are ethanol and acetone. The

use of ethanol as a protein precipitant has been patented in

a proeess for the preparation of a rapeseed protein isolate

(Goodwin, 1977). Glutenin has been obtained by

precipitation with 70% ethanol from whole gluten (Danno,

1981). Precipitation by orgaDic solvents has the advantage

that the factor of altered dieleetric constant when added to

other factors such as pH, temperature, ionie strength and

protein concentration gives a very refined method of protein

fractiona tion (Bell et. al., 1983). Ervin (1986) reported

that in the preparation of protein concentrate from brewers'

spent grain, precipi tation by ethanol alone produced a

protein concentrate with a nitrogen recovery of 28.0~. The

combination of the addition of ethanol followed by

refi,eration at 4° C increased the ni trogen recovery to

49.4%.

24

......

B: Nutritional Propertiea of Diatillera' Spent Grain

Distillers' dried grains with solubles (DDOS) are

generally the major by-products from the fermentation of

whole grains to ethanol (Dong et.aL, 1987); specifically

brewers' spent grain (BSO) is the by-produet of brewing

(Ranhotra et.al., 1982). Several authors have reported that

spent grains (DDG and ODOS) retain the amine aeid profile of

the original uneonverted whole grains (Dong et.al. 1987;

- Sexson et.aL, 1981; Wu et.al., 1984; Wu, Y.V., 1986).

Lysine, the first limi ting amino acid for cereal

grains, is relatively high (3.9-4. 4g/16g N) in barley and

spent grain from barley; this compares with 2.5-3 . .tg

lysine/16g N in spent grains from corn (Wu et.al., 1981).

The nutritional value of barley and its spent grain appears

to be superior to that of the corresponding spent grain from

corn, wheat and sorlhum (Wu et. al., 1981, 1984; Wu and

Sexson 1984). Wu et. al. (1984) reported higher levels of

lysine, threonine and isoleucine levels in wheat distillers'

grains as compared wi th wheat; the authors concluded that

the distillers' grains can be expected to be nutritionally

superior to wheat. The content of lysine was reported to

range from 2.50-3.74g/100g protein in distillers' spent

grain (Ranhotra et.al., 1982) and was 3.51/1001 protein in a

distillers' protein concentrate (Ope) (Scheller and Mohr,

1975). Similar values for lysine content samples of BSO

have been reported (Pomeranz et.a!., 1976; Prentice and

25

(~

(

D'Appolonia, 1977; Kissell and Prentice, 1979).

Don, et.al. (1987) evaluated the protein quality of

di sti llers' dried .rains wi th sol ubles (DDGS) from soft

whi te winter wheat, hard red wheat and corn by amine acid

analysis, prote in efficiency ratio (PER), and net protein

retention bioassays; these authors observed that the

relative amino acid concentrations of the whole arsin

subjected to fermentation were retained in the DDGS; ~hen

compared to reference casein, white wheat DDGS contained

lower concentrations of seven essential amino acids but

equivalent levels of phenylalanine. The contents of

lysinoalanine in wheat DDGS were wi thin the acceptable

levels (maximum 1000 ppm) found in foods (Finot, 1983) and

were considered to be nutritionally safe (Table 5). The

essential amine acid patterns in casein, the whole grain

f lours and DDGS reported by Don, et. al. (1987) were in

agreement wi th those from other reports (Finley, 1981; Wu

et.al., 1981, 1984, 1985; Ranhotra et.al., 1982; Wu and

Strin,fellow, 1982; Sarwar et.al., 1983; Bookwalter et.al.,

1984; Seligson and Mackey 1984; Wall et.al., 1984).

Don, et. al. (1987) reported a PER of less than 1.0

(Table 6) for several DDGS; the PER obtained for corn DDGS

was lower than the values reported by Satterlee et. al.

(1976), Ranhotra et.al. (1982) and Wall et.al. (1984) for

26

Table 5. Aaino Acid Co.position of Casein, White Wheat, Red Wheat, Corn, and Distillers' Dried Grains with Solubles eDDGS) eg a.ino acid/l6g

H)· •

Amino Acid

Alanine Arginine Aspartic acid Cystine 2 Glutamic acid Glycine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Proline Serine Threonine Tryptophan Tyrosine Valine Lysinoalanine (% w/w)

Reference White Casein Wheat

2.85 3.76 3.86 4.96 6.78 5.75 0.29 2.11

21.49 30.69 1. 76 4.14 2.89 2.55 4.87 3.55 9.03 6.92 7.86 2.95 2.37 1. 26 5.01 4.63 8.53 10.34 5.59 4.98 4.10 2.99 1.10 1.01 5.49 3.23 6.~9 4.45 ND <0.04

White liheat DDGS

3.97 5.59 5.70 2.49

29.44 4.45 2.66 3.86 7.61 2.81 d 1. 53 5.22 9.89 4.93 2.96 1.07 3.46 5.04

<0.04

Red Wheat

3.59 4.96 5.04 2.28

32.00 3.98 2.45 3.60 6.93 2.79 1. 26 4.78 9.77 4.98 2.92 0.90 3.35 4.25

<0.04

Red Wheat DDGS

3.82 4.82 5.06 2.20

29.88 4.32 2.42 3.70 7.07 2.46 d 1.96 4.79

10.18 5.03 3.02 0.89 3.34 4.94

<0.04

Corn

7.92 4.08 5.80 2.32

18.74 3.93 2.96 3.58

13.40 2.82 1.62 5.11 9.47 5.16 3.69 0.49 4.08 4.80 ND

Corn DDGS

7.74 4.36 5.60 2.02

18.03 3.99 2.89 3.85

13.50 2.26d 2.00 5.21 8.93 5.34 3.92 0.64 4.57 5.04 ND

Essential Amino Acid Scoring Pattern

2.6 (Met + Cys)

1.7 4.2 7.0 5.1 2.6 (Met + Cys) 7.3 (Phe + Tyr)

3.5 1.1 7.3 (Phe + Tyr) 4.8

------------------------------------------------------------------------------------------------• ~Source: Dong et.al. (1987) dNot determined Methionine + Methionine Sulfone

~~ ~ ~ " 'f r: ,.

...... N

..

similar corn spent grain.

Satterlee et.al. (1976) reported that the PER of a corn

distillers' protein concentrate was 1.45 and apparent

digestibili ty was 78.00% while the PER and apparent

dilestibility of wheat distillers' protein concentrate were

1.26% and 82.80~ respectively. Dong et.al. (1987)

determined the in vivo and in vitro digestibility of wheat

ODOS and corn DDGS (Table 7). These authors demonstrated

that the APD of the two wheat DDGS and corn DDGS were

similar to those reported by Satterlee et. al. (1976) for

wheat and corn distillers' protein concentrates.

Fortification of white wheat DDGS with essential amine acid

increased its digestibility (Dong et.al., 1987). The lack

of essential amine acids, rather than the presence of

antinutri tional components was reported to be the major

cause of the retarded growth observed in rats fed the

unsupplemented white wheat DDGS diet (Dong et. al., 1987).

Ervin (1986) reported that the digestibility of protein

concentrates from aSG were lower than that of casein but

were similar to the digestibility of distillers' protein

concentra tes prepared by Satterlee et.al. (1976). Ranhotra

et.al. (1982) suggested that the relatively low

digestibility of certain protein concentrates has been

attributed to the presence of tannins, phenolics or enzyme

inhibitors.

28

-

Table 6. Protein Btticienc7 Ratio (PBR) and Net Protein Ratio (NPR) of Distillera' Dried Oraina witb Solubles (DDGS)8.

ANRC reference casein 3.0 ± 0.2 2.5 :t 0.2 3.6 :! 0.2

Easential amino acid-fortified white wheat DDOS 1.9 + 0.2 1.7 :!: 0.2 2.5 :t 0.3

White wheat OOGS 0.3 t- 0.1 O.2±0.1 0.9 ~ 0.2 . Red wheat ODOS 0.7 1: 0.1 0.6 ± 0.1 1.3 ± 0.2

Corn ODOS 0.1 t- 0.1 0.1 ± 0.1 0.8 .! 0.2

----------~-------------------------------------------------• (1987) bSource: Don. et.al. Adjusted PER = (PER test prote in ) / (PER Case1n) x 2.5

Table 7. In Vivo and In Vitro Diaeatibilit7 ot Distillers' Dried Grains with Solublea (DDGS) •

X Protein Oi.estibility

Prote in Source In Vivo APDb In Vitro

ANRC reference casein 93.0 + 0.8 8g.6 ± 0.7

Easential amino acid-fortified ND

c white wheat DOaS 88.0 ~ 1.5

White wheat ODOS 84.0 ~ 3.3 79.9 :t 0.8

Red wheat ODOS 84.0 + 2.8 81.0 :,t0.6

Corn ODGS 81. 4 + 3.9 77.9 ... 2.1

White wheat flour ND 88.9 ± 1.0

Red wheat flour ND 86.6 ± 0.6

Corn ND 78.4 ± 0.7 8 bSource: Don. et.al. (1987)

APD = Apparent protein diaestiblilty = (. N in.eated-. N in fecea)/. N inaested) x 100

Not deterJDined

29

F: Functional Properties of Proteins fro. Spent Cereal Grains

The common functionali ty tests (water absorption, fat

absorption, aelation, emulsification and viscosity) used to

characterize vegetable prote in ingredients are aIl attempts

to define or predict the ability of the protein ingredient

to contribute to the texture of the food system (Martinez,

1979). The functional characteristics of distiller's

protein concentrate (DPC) from fermented wheat and corn were

identified for use in bread, extruded puffed snacks and Meat

emulsions (Satterlee et.al., 1976). Kinsella (1976)

reported that soy protein preparations are widely used for

their emulsion capacity (EC) and emulsion stability (ES)

properties in frankfurters, bologna, sausages, soups and

cakes. Satterlee et. al. (1976) compared the emulsifying

characteristics of wheat and corn prote in concentrates with

that of non-fat dry milk (33% protein) and soy isolate (91%

protein). The authors found that the emulsion capacity of

corn protein concentrate exceeds that of non-fat dry milk

and wheat prote in concentrate, but is less than that of soy

isolate, while the_ emulsion stability of CPC was equal to

that of non-fat dry milk, greater than that of wheat protein

concentrate but less than that of soy isolate (Table 8). It

was concluded that the CPC was an excellent emulsifier for

Meat emulsions. Both corn and wheat protein concentrates

exceeded soy protein iaolate in their performance in

extruded products. The protein concentrates gave acceptable

30

u

--

p~ff volumes up to a final protein concentration of 22% for

wheat and 18% for the CPC. Satterlee et.al. (1976) reported

that the addition of either CPC or WPC to a basic bread

formulation resulted in depression of loaf volume as the

amount of added concentrate increased. The WPC had the

least detrimental effect on loaf volume because of i ts

gluten content. Whitaker (1977) reported that the

functional properties of proteins are strongly dependent on

the isolation methods used. Satterlee et.al. (1976)

postulated that the alkaline extraction procedure (pH 12.2)

used to prepare WPC could lower its elasticity properties.

Table 8. E.ulsif~cation Characteristics of Various Protein Sources •

Protein Concentrate

Emulsion Cape~ity

ml Oil emulsified

Emulsion Stability

ml Oil released

ml H 0 and Soliâs released

100 mg Protein 10 g Emulsion 10 g Emulsion

Nonfdt dry lIlilk

Soy isolate

12.00

22.20

Protein concentrates from:

•

Corn

Wheat

18.90

8.90

Source: Satterlee et.al. (1976)

31

0.07

0.03

0.06

0.10

1.60

1. 40

1. 89

2.71

t

(~

G: Bioche.lcal Properties of Proteins fro. Brewers' Spent Grain

Ervin (1986) determined the biochemical properties of

protein concentrates prepared from brewers' spent grains

usina SDS-phosphate electrophoresis. Four components with

average molecular weights of 45,000, 41,000, 36,000 and

27,000 were observed in the prote in concentrates. El-

Negoumy et. a!. ( 1979) reported that the average molecular

wei,hts of the glutelin fraction of barley proteins ranged

from 12,000 to 250,000 daltons. There is little information

on biochemical properties of spent cereal grain in the

literature.

H: Response Surface Methodoloay

3.0: Ciassicai experi.entai procedure versus respODse surface aethodology (RSM)

Response surface methodology (RSM) is widely used in process

optimiza tion studies (Henika, 1982; Giovanni, 1983; Yusof

et.a!., 1988). RSM can be defined as a statistical method

which uses quantitative data from appropriate experimental

designs to determine and simultaneously solve multivariate

equations (Giovanni, 1983). RSM serves 3 primary purposes

(Giovanni, 1983); these are: (1) to determine the

combination of factors which yield the optimum response; (2)

to determine how the response is affected by a given set of

factor levels; and (3) to describe the interrelationship

amon, the test variables. With the classical

32

experimentation procedure only one variable can be tested at

a time and this requires a larger number of experiments. Ma

and Ooraikul (1986) stated that the resul ts of one-factor-

at-a-time experiments do not reflect actual chances in the

environment as the y ignore interactions between factors

which are present simultaneously. RSM can consider several

factors at many different levels in a product and the

corresponding interactions among these factors and levels

(Giovanni, 1983). Thus, RSM enable more accurate

optimization of factors (Ma and Ooraikul, 1986).

3.1: Response surface desi.ns

Giovanni (1983) has described RSM as a four-step

process: Firstly, two or three cri tical factors which are

most important to the product or process under study are

identified. Secondly, the range of factor levels which will

determine the samples to be tested are defined. Thirdly,

the specifie test samples are determined by the experimental

design and then tested. Fourthly, the da ta from these

experiments are analysed by RSM and then interpreted.

Response surface experiments are carried out when a

specifie statistical model for the response is known. Most

response surface experimental designs foeuses on polynomial

models, with emphasis on first and second order desians

(Thompson, 1982). First order models are use fuI for

screenin. experiments. The purpose of screening experiments

33

( is to identi fy the most significant variables. The design

most commonly used to fit first-order models are 2k

factorial designs (Gacula and Singh, 1984). Thompson (1982)

reported that fractional replications of factorial

experiments are recommended for first order designs with

four or more explanatory variables. Gacula and Singh (1984)

stated that first-order models are often inadequate and

provide a paor description of the geometric shape of the

response surface. Thompson (1982) reported that most second

arder response surface experiments utilize central composite

designs which were first proposed by Box and Wilson (1951).

An experimental design having equal predicting pawers in aIl

directions at a constan~ distance from the center of the

design ls called a "rotatable" design (Gacula and Singh,

1984) . The total number of treatment combinations in a

composite design is 2k

+ 2K + 1. By using coded levels for

each variable, the designs are dependent only on the number

of variables and the selected response equation. The center

point for each explanatory variable levei is given a code of

zero. The highest and lowest levels of interes-c. for each

independent variable are coded plus or minus one

respectively for three level designs. For designs with more

than three levels, the highest and lowest levels of interest

are given maximum and minimum codes respectivley (Thompson,

1982) .

34

3.2: Response surfaces

Thompson (1982) reported that the term response surface

has been associated with experiments intended to identify or

evaluate one or more response variables as a function of the

independent variables. When the fitted response function i9

graphed as a function of independent variables, the

resulting graph is called a response surface plot or contour

map (Gacula and Singh, 1984). Giovanni (1983) reported that

response surfaces can be prepared in a wide variety of

shapes. The most commonly generated are the cradle (Figure

3) and saddle point (Figure 4). For the cradle, or bowl,

the optimum response lies along the top edges, while the

saddle point has the optimum response along the sides, or in

each of the four corners.

3.3: Applications of response surface aethodology (RSM)

Ma and Ooraikul (1986) used RSM with central composite

rotatable design to optimize pH, temperature and

enzyme/substrate ratio (E/S) on protein hydrolysis in canola

meal. The three variables were assessed at fi ve l evels

around the optima. The authors observed a closeness in

value of the experimental and calculated yields of total

soluble nitrogen. The experimental result under optimum

condi tions was 0.4882% which agreed wi th t.he calculated

yield of 0.4813%. They concluded that RSM was an efficient

experimental design when several variables affectina the

35

figure 3 _ Three_diaensional l'lot. of a ·cradle

• respon

se

surface.

36

\ 1 , 1

1

1

u

-

, \ \ \

\

--r'

Figure 4_ Three-diaenBional plot. of a "Saddle

point." reBponBe

surface.

37

(~

reaction were evaluated simultaneously. Lin and Zayas

(1987) studied the functional properties of two corn germ

protein preparations using RSM. Yusof et.al. (1988) used

RSM for the determination of optimum pH, processing

temperat Ire and 0 Brix to produce an acceptable guava

concentrate. A central composite rotatable design

configuration for three variables described by Cochran and

Cox (1957) was used. The responses considered were colour,

flavor and overall acceptability.

38

SECTION Il: MATBRIALS AND METRODS

A: Materials

Commercially dried, Brewers' spent ,rain (DBSO; 7.5X

moisture, 4.75% N (dry wei.ht basis» was obtained trom

Molson Breweries of Canada Ltd., Montreal, Quebec. It "las

stored (2°C) in tightly closed plastic bags. A

representative sample was sieved manually, usin, sieves with

apertures of 0.84 mm and 1.41 mm to give two particle sizes.

The sieved samples were stored in plastic baas. These

samples were used in the laboratory scale preparation of

protein concentrates. DBSO was ,round (2 mm sleve) using a

Thomas Wiley Mill (model 4). The ground DBSO "las used in

the pilot scale preparation of protein concentrates.

Pressed Brewers' spent grain (PBSO; 67.2% moisture,

4.04X N (dry weight basis» "las also obtained from Molson

Breweries. PBSO was frozen immediately in plastic baiS to

prevent microbial growth. PBSO was thawed a t room

temperature before use in extraction experiments.

B: Methods

1: Micro-Kjeldahl Analysia

Nitrogen contents of materials to be analysed (DBSO,

PBSG, extracts, protein concentrates, mother liquors) were

determined by the microKj eldahl method (A. O. A. C ., 1980 ;

47.021). AlI analyses were conducted in triplicate.

39

(

(.

2: Sodiua Dodec7l Sulfate (SDS) Anal7sis

The SOS content of the protein concentrates was

determined using the A.O.A.C. procedure (1980; 20.128) for

powdered egg whi te. AlI analyses were conducted in

duplicate.

3: Functional Properties of Proteins

3.1: Foaaing capacit7 and foaaina stability

The foaming properties were determined using the

procedures discussed by Satterlee et.al., (1975) and

Gierhart and Potter, (1978). Dispersions (2%) of the spray

dried protein concentrates were prepared. The pH of the

dispersions were adjusted to 3.0, 5.0, 7.0 and g.O by

dropwise addition of Hel (2N) or NaOH (2N) with continuous

stirring. The total volume of the dispersions were brought

to 20 ml after the pH adjustment.

The protein dispersions were whipped for 2 min. at room

temperature using a Virtis homogenizer (Model 45) at a speed

of 6000 rpm. The whipped slurries were immediately

transferred to graduated cylinders (100 ml) and allowed to

stand for 2 min. Foam volume was recorded at 2 min. and at

30 min. after whipping.

Foam capacity was

(Satterlee et.al., 1975).

calculated using Equation 1

Foam stability was calculated

using Equation 2 (Gierhart and Potter, 1978).

40

Foam Capacity = volume of slurry after whippin. x 100 volume of slurry before whippin.

(Eqn.l)

Foam Stability = Foam volume after standing (30 min) x 100 Initial foam volume

(Eqn. 2 )

3.2: Water absorption

The water absorption characteristics of the OBsa

concentrates and soy concentrate (Acron S) were determined

by the method of Sosuiski (1962). Excess water (20-30 ml)

was added to the sample (1.5 g) in wei,hed centrifuge tubes

( radi us 1. 2 cm). The suspension was mixed vigorously 4

times with a 10 min. rest period between each mixin,. The

suspension was then centrifu,ed (3200 rpm) for 25 min. The

supernatant was decanted and the tube air-dried (10 min)

until no residual liquid could be seen. Water absorption

was expressed in percenta,e as the amount of water absorbed

by 100 Il sample.

3.3: Fat absorption

The fat absorption characteristics of the OBSa

concentrates and soy concentrate (Aaron S) were determined

by the method of Lin et.al. (1974). The sample (0.5 ,) and

corn oil (3.0 ml) were placed in centrifu,e tube (radius 1.2

cm). The contents were stirred for 1 min. with a thin brass

wire ta disperse the sample in the oil. It was left

41

t standing for 30 min. The tube was centrifuged at 3200 rpm.

for 25 min. The oil layer was poured into a graduated glass

tube (15.0 ml) and the volume of ail was read. Fat

absorption was expressed in percentage as the amount of corn

ail bound by 100 g sample.

3.4: E.ulsifyin. capacity and stability

The emulsion capacity and stability were measured using

the procedures of Webb et.al. (1970) and Sathe and Salunkhe

(1981).

The sample (2 g) was blended in a Waring Blendor with

distilled water (50 ml) for 30 sec. at "Hi" speed. Oil

(~ (sunflower) was added in 5 ml portions wi th continuous

blending. Measurement of electrical resistance was obtained

using a Conductivity Bridge (model 31). The instrument was

calibrated according to the A.O.A.C. (1984,31.196). A

sudden increase in resistance upon addi tian of ail was

considered to be the point of discontinuation of oil

addition. The amount of oil added up to this point was

interpreted as the emulsifying capacity of the sample.

The emulsion sa prepared was then allowed to stand in a

graduated cylinder and the volume of water separated at time

intervals of 10 hr. and 96 hr. was noted as a measure of the

emulsion stability. AlI the experiments were conducted at

o ra am tempe rature (25 Cl.

42

-

-

3.5: Viscosit1'

The viscosities of the DBsa concentrates and soy

concentrate (Acron S) were determined by the method of

Flemina et.al. (1974).

Dispersions (10% and 15%) of the proteins were prepared

in small beakers (40 ml) by mixing the protein (2 • and 3 .) .

with distilled water (20 mIl. The protein dispersions were

adjusted to pH 7.0, 9.0, 11.0 and 12.0 by addition of NaOH

(6 N), and were allowed to stand for 10 min. prior to the

viscosity measurements. The 10% pH adjusted protein

dispersions were heated in a boiling water bath for 5 min.

and the 15% dispersions were heated in a water bath

maintained at 55°C for 5 min.

The viscosity of the pH adjusted dispersions were

measured both before and after the heat treatment with a

Brookfield viscometer (LVF model) at room t'emperature using

a dise spindle (No.3). Bach protein dispersion (20 ml) was

transferred to the sample chamber of the small sample

adapter (SC-4) and the torque required to rotate the spindle

at a constant speed (30 rpm) was recorded.

4: In Vitro Digestibilit1'

A multienzyme technique developed by Hsu et.al. (1977)

was used to measure the in vitro protein dilestibility of

the protein concentrates (DBSGIOO, DBSG75, DBSG50) and

sodium caseinate. Sodium caseinate and the followinl

43

(

(~

enzymes were obtained from sigma chemicals (St. Louis,

Missouri): pancreatic trypsin (type II), bovine pancreatic

chymotrypsin (type II) and porcine intestinal peptidase

(Grade III). A suspension conaistin, of trypsin (1.6

mg/ml), chymotrypsin (3.1 mg/ml) and peptidase (1.3 ma/ml)

was prepared using deionised water.

An aqueous suspension (50 ml) of each sample containing

6.25 ma prote in/ml was prepared; the weight of each sample

was calculated on the basis of i ts protein content (% N x

6.25). The suspensions were adjusted to pH 8.0 by addition

of Hel (O.lN) and/or NaOH (O.lN). The multienzyme solution

(5 ml) was added to the suspension of each sample; the

mixture was incubated (20 min; 37DC). The decline in pH

during the 20-minute incubation was determined by measuring

the pH (Fisher accumet selective Ion Analyser Model 750

fi tted wi th a pH combination electrode) at intervals of 5

min. This procedure was repeated three times for each

sample. The in vi tro digesti bili ty of each sample was

calculated accordina to the following equation:

y = 210.46 - 18.10 X

y ia the % in vitro digestibility

X i s the pH recorded after 10 min. of in vitro digestion

(Hsu et.al., 1977)

44

(Eqn.3)

, ," ,

-

5: SDS Blectrophoresis

The procedure descr i bed by Weber et. al. (1972) was

used for sodium dodecyl sulfate (SDS) phosphate

polyacrylamide leI electrophoresis.

5.1: Preparation of le1s

An acrylamide solution (22.2 1 acrylamide; 0.6 i

methylenebisacrylamide in 100 ml aqueous solution) was

prepared. A gel butfer solution (pH 7.2; 7.8 , sodi um

dihydrolen phosphate, 38.6 , disodium hydro,en phosphate and

2 g sodium dodecyl sulfate in 1 L aqueous solution) was also

prepared. Acrylamide solution (10.0 ml), distilled water

(3.4 ml), ,el buffer solution (pH 7.2, 15.0 ml) and N, N,

NI, NI-tetramethylenediamine (TEMED) (0.045 ml) were mix~d

thoroughly. The resul tant solution was refri,erated (5

min). Ammonium persulphate solution (1.5 ml) was mixed with

the cooled solution. The resultant solution was placed in

electrophoresis tubes (internaI diameter = 5 mm, height = 80

mm). A small volume of distilled water was placed on top of

the gel solution to ensure a fIat gel surface. The solution

was allowed to stand (25 min, 2SoC) to allow the gels to

polymerize.

5.2: S .. ple preparation

Protein concentrates (2 m,) and the proteins trypsin,

O(-amylase, lysosyme and e" albumin (1.0 m,) were placed

45

(~

in screw-cap test tubes along with sodium dodecyl sulfate/2-

mercaptoethanol solution (0.5 ml, 2%) and sodium phosphate

buffer solution (0.5 ml, 0.01 M, pH 7.2). The samples and

standard proteins were heated in a boiling water bath for 3

o min. and then at 37 C (2 hours). Approximately 50 mg of

sucrose was added followed by bromophenol blue solution (1

drop, 0.05%). The sample solutions (75 uL) and the

composite standard solution (50 uL) were placed on the top

of the gel.

5.3: Blectrophoresis

An initial current of 1 mamp per tube was used for the

first minute and then increased to 4 mamp per tube for 1

hour. The current was then increased to 8 mamp per tube.

Electrophoresis was allowed to continue until the

bromophenol tracking dye reached the bot tom of the tube.

After electrophoresis, the gels were removed from the

tubes and th en immersed in a fixing solution (40% CH3

0H - 7%

acetic acid) for 24 hours. The solution was changed twice.

The gels were immersed in a staining solution (0.025%

Commassie Brilliant Blue in 40% CH3

0H - 7% acetic acid) for

3 hours. The gels were allowed to stand in a destaining

solution (5% CH,OH - 7.5% acetic acid) until the background

was clear. The gels were stored in the destaininc solution.

46

() 6: !aino Acid Analysis

6.1: Saaple preparation

Protein samples were hydrolysed and derivatized using

the PICO. TAG system. The sample tubes were washed with 6N

HCI, then r.insed thoroulhly with deionised water, and 100%

methanol followed by oven drying.

Samples (2-3 mg) of protein concentrates (DBSGI00,

DBSG75, DBSG50) were weilhed into sample glass tubes (13 x

100 mm). The sample glass tubes were placed in the reaetion

vial, and the samples were dried under vacuum. 200 uL of 6N

HCI/phenol solution was added to the reaetion vial. The

o reaction vial was placed in the oven (120 C) for 24 hours to

permit hydrolysis of the protein samples. On coapletion of

hydrolysis, redryinl aient (10 uL) was added to the

hydrolysates and dried under vacuum. The purpose of the

redrying agent was to remove saI ts from the samples which

might have interferred with the derivatization.

Derivatizing aient (30 uL) was then added to the redried

hydrolysate. The composition of the redrying agent and

derivatizinl agent are given in Table 9. The deri vatizing

reaction was allowed to proceed for 20 minutes at room

temperature. The derivatized hydrolysate was dried under

vacuum.

A quantity (10 uL) of a Pierce H amine aeid hydrolysate

standard (Chromatographie Speeialties, Brockville, Ontario)

was dried, redried, and derivatized usin, the same procedure

47

(~ deseribed for the sample hydrolysates. The derivatized

hydrolysates were stored (_5.0°C) until analysis (HPLC).

Table 9. The Compoai tion of the Rea,enta used in Aaino Acid Analyais.

------------------------------------------------------------Redrying Aient 40 uL of methanol·, 40 uL of deionised

wateIj,' and 20 uL of triethylamine (TEA) ; mixed thoroughly.

Derivatizing Agent

Sample Diluent

Eluent A

Eluent B

210 uL of methanol, 30 uL of deionised water, 30 uL of TEA t and 30 uL of phenylisothioeyanate (PITC)c; mixed thoroughly.

71 mg of Na HPOb dissolved in 100 ml deionized wat~r'b titrated t~ pH 7.4 with 10% H PO (v/v), filtered ; mixed 100 ml of~u/fer with 5 ml of acetonitrile.

19.0 g of sodium acetate band 0.5 ml TEA dissolved in 1 L deionized water, ti trfted to pH 6.4 wi th glacial acetie acid • fil tered; mixed 940 ml of buffer with 60 ml of acetonitrile.

Mixed 400 ml deionized water with 600 ml acetonitrile •

• Caledon Laboratories, Georgetown, Ontario.

bAldrich Chemieal Co., Milwaukee, Wisconsin.

cChromatographic Specialties, Brockville, Ontario.

dMillipore 0.45um filter, Type HA, Millipore Waters Seientific, Mississauga, Ontario.

6.2: Reversed phaae-HPLC chroaatography

The deri vatized standards and samples were thawed and

sample diluent (0.2 ml) was added. The mixtures were

a,i tated usina a vortex mixer. The standards and samples

were filtered (Millipore 0.45 um filter, Type HV, Millipore

48

-

-f '

Waters Scientific, Mississauga, Ontario) prior to injection;

an injection volume of 15 uL was used for the standards and

a volume of 20 uL for the samples.

The chromatographie analysis was performed using a LKB

BROMMA HPLC sys tem ( Bomma , Sweden). The gradient program

used for amine acid analysis by HPLC is shown in Table 10,

and the run parameters used to record the data by means of

the Wavescan program are given in Table Il. The composition

of eluent A and eluent B are given in Table 9. The eluents

were degassed by ul trasonication prior ta use. The column

o oven temperature was set at 44 C. Integration of the peak

areas in the chromatogram obtained at 254 nm was

accomplished usina the Nelson program.

Table 10. The Gradient Progra. used for Aaino Acid Analysis by HPLC.

Time (min) Flow (ml/min) " B

0 1.0 0

10.0 1.0 46

10.5 1.0 100

11.5 1.0 100

12.0 1.5 100

12.5 1.5 0

20.0 1.5 a

20.5 1.0 0

------------------------------------------------------------

49

(

Table Il. The Bun Paraaeters Bntered into the Wavescan Pro.ra. used for Aaino Acid Analysis.

Begin data collection 1.00 min

Stop data collection 12.00 min

Time interval of collection 0.5 sec

Beain UV Scan 250 nm

Stop UV Scan 260 nm

Scan interval 1 nm

Integration interval 0.5 sec

50

~ i.. ~ SBCTION III: BXPBRIMENTAL

.......

" ..

Experiaent 1: Protein extractability fro. dried brewers' spent grain (DBSG) and pressed brewers' Bpent grain (PBSG) uBing a factorial design

A preliminary fractional factorial design was used tu

determine the effects of individual factors on protein

extraction from dried brewers' spent grain (DBSa) and

pressed brewers' spent grain (PBSO). The fractional

factorial design chosen was a half fraction of a 2'

factorial design (2' - 1) as described by Box et. al. (1978).

The factors evaluated were temperature of extraction (50-

100°C), time of extraction (30-90 min), concentration of

sodium dodecyl sulfate (1-3%) and particle si~e of grain (1-

2 mm). The fractional factorial design for both DBSO and

PBSO and coded levels of each factor are shown in Table 12.

Values of coded levels used in the fractional factorlal

design and the method of COdl!lg, as described by Box et.

al. (1978) are shown in Table 13.

Samples (2.5 g) of commercial DBSG and PBSG were mixed

with the SDS extractant (50 ml) and subjected to the

extraction experiments given in Table 12. The SDS extracts

from DBSG and PBSG (filtration through glass wool) were

analysed for protein contents using the microKjeldahl method

(A.O.A.C. 1980; 47.021) .

51

(.

Table 12. A Half Fraction of a 24

Fractorial Design (coded) to Deter.ine Factors Influencing Extraction of Protein rro. Dried Brewers' Spent Orain (DBSO) and Pressed Brewers' Spent Grain (PBSO).

------------------------------------------------------------Variable

b

-------------------Prote in extracted

Run #. X X X3 X. (X)

1 2 ------------------DBSG PBSO

------------------------------------------------------------1 -1 -1 -1 -1 15.81 5.04

2 1 -1 -1 1 36.28 9.86

3 -1 1 -1 1 18.85 7.06

4 1 1 -1 -1 45.99 15.37

5 -1 -1 1 1 14.85 5.37

6 1 -1 1 -1 35.61 11. 43

7 -1 1 1 -1 22.28 8.07

8 1 1 1 1 36.09 14. Il

·Each run replicated twice for a total of 16 runs

b X1 =Temperature DCjXz=Time (mins)jX3

=Conc. of extractant (SDS)

X.=Particle size of grain.

52

.;-

Table 13. Variable Levels and Coded Values used in a Half­Fraction Factorial Screenin. Design for Protein Bxtraction fro. Dried Brewers' Spent Grain (DBSG) and Pressed Brewera' Spent Orain (PBSO).

Coded Levels 1

Variable -1 o +1

Temperature, oC. (Xl) 50 75 100

Time, mins. (Xa ) 30 60 90

Con~entration of SDS , %(X3 ' 1 2 3

Particle size C

of grain, mm(X. ) 1 1.5 2

·Coded variable (-1,1)=Actual value-0.5 (High value+low value)

0.5(High value-Low value'

bSDS=Sodium dodecyl sulfate containing 0.5% Na HPO (pH 7.0) 2 •

C BSG : extractant ratio=5:100(w/v)

53

(

Experiaent 2: Sodiua dodecyl sulfate (SDS) extraction of .DBSO.

Samples (2.5 g) of commercial DBSG were mixed with

different concentrations of extractant (50 ml; 1-5% SDS) in

fIat botto .. flask (250 ml) and refluxed (1.5 h). The

residues were removed by filtration through glass wool. The

filtrates (1.0 ml) were analysed for prote in contents using

the microKjeldahl method (A.O.A.C., 1980; 47.021).

Experi.ent 3: Extraction of DBSG protein using sodiu •. dodecyl sulfate (SDS) with dibasic sodiu. phosphate (Na.HPO.).

Samples (2.5 g) of commercial DBSG were mixed with

different concentrations of the extractant (50 ml); 3% SDS -

0.5% NazHPO., 1% SDS - 0.5% NazHPO.) in fIat bottom flasks

(250 ml) and refluxed (1.5 h). The residues were removed by

filtration through glass wool. The extracts (1.0 ml) were

analysed for protein contents using the microKjeldahl method

(A.O.A.C., 1980; 47.021).

Experiaent 4: SDS extraction of DBSO protein with different levels of dibasic sodiua phosphate (Na.RPO.).

Samples (2.5 g) of commercial DBSG were mixed with SOS

(3% and 1%) combined with different concentrations of sodium

phosphate dibasic (0.5 , 1.0, 1.5 and 2.01) in fIat bottom

flasks (250 ml) and refluxed (1.5 h). The residues were

removed by filtration (throulh glass wool). The extracts

(1.0 ml) were analysed for protein contents using the

54

-

-

microKjeldahl method (A.O.A.C., 1980; 47.021).

Bxperi.ent 5: Bxtraction of DBSG protein usin. SOS wi th aodiu. cbloride (Nacl'.

Sailples (2.5 .) of commercial DBSa were mixed wi th

different concentrations of the extractant (50 ml; 3% SDS -

0.5% Naci t IX SDS - O. 5X Nacl) in fIat bot tom flasks (250

ml) and refluxed (1.5 h). The residues were removed by

filtration through glass wool. The extracts (1.0 ml) were

analysed for protein contents using the microKjeldahl method

(A.O.A.C., 1980; 47.021).

Bxperi.ent 6: Preparation (laboratory scale) of DBSa prote in concentratea.

Samples (5.0 g) of commercial DBsa were mixed wi th

extractant (100 ml; 1% SDS - 0.5% NaaHPO.) in fIat bottom

flasks (250 ml) and refIuxed (1.5 h). The residues were

removed by filtration through .lasa wool, and the filtrates

collected. The proteins in the extracts were precipitated

by the addition of 95% ethanol (0.7 ml C H OH to 1.0 ml of 2 S

extract) followed by refrigeration C4°C) for 17 hours. The

precipi tates were recovered by centri fugation (2000 rpm),

and lyophilized. In some instances precipitates were washed

with ethanol (95X), centrifuged (2000 rpm) and lyophilized.

55

l

(~

Experiaent 7: Central coaposite rotatable design for optiaization of DBsa prote in extractability.

A four factor, 5 level central composi te rota table

design (CCRD) of Box et.al. (1978) was used for optimizina

protein extractability from DBSO. Factors and levels of

each factor in the CCRD (Table 14) were selected on the

basis of significant regression coefficients generated from

the initial screening design as those likely to optimize the

response. In the CCRD, particle size was kept constant at

1.5 mm and the Meal: solvent ratio (w/v) varied from 2.5:100

to 12.5: 100. Temperature varied from 80 to 100°C and time

from 60 to 120 min. The concentration of sodium dodecyl

sulfate was kept constant at 0.5% and the concentration of

NazHPO. in the SDS solution varied from 0 - 1% v/v. The

coded levels of -2, -1, 0, +1, +2 used in a four factor CCRD

(Table 14) were obtained from Box et.al. (1978) and values

of coded levels of variables used in the CCRD are shown in

Table 15. The total number of experimental runs determined

from this design (CCRD) was 25. Triplicate measurements

were taken for each experimental rune On completion of the

extraction experiments the protein contents of the extracts

were determined by the microKjeldahl method (A.O.A.C., 1980;

47.021).

56

Table 14: Coded Level Coabinations for a Four Variable Central Co.poaite Rotable Design to Opti.ize Protein Bxtractabili t7 fro. Dried Brewera' Spent Grain (DBSG).

Runl

1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

•

-1 1

-1 1

-1 1

-1 1

-1 1

-1 1

-1 1

-1 1

-2 2 o o o o o o o

Variableb

-1 -1

1 1

-1 -1

1 1

-1 -1

1 1

-1 -1

1 1 o o

-2 2 o o o o o

-1 -1

1 -1

1 1 1 1

-1 -1 -1 -1

1 1 1 1 o o o o

-2 2 o o o

-1 -1 -1 -1 -1 -1 -1 -1

1 1 1 1 1 1 1 1 o o o o o o

-2 2 o

Protein extracted from DBSG (%)

Predicted

42.39 45.19 44.21 46.28 47.74 50.48 51. 87 51. 28 24.93 28.39 27.84 32.45 32.79 42.54 38.07 36.13 38.86 48.29 37.43 50.34 27.91 44.34 57.41 27.03 51. 46

Observed

45.97 47.36 46.00 47.96 49.84 50.36 53.39 49.73 25.43 27.57 29.82 31.91 33.40 43.28 39.60 39.69 37.76 51. 28 36.29 52.86 25.90 45.78 57.71 26.81 53.05

-Each run replicated twice for a total of 50 runs

b X =TemperatureOC;X =Time,(mins);X =Conc. of phosphate (%) 1 a 3

X.=BSG:extractant ratio.

57

Table 15: Variable Levels and Coded Values used in Central Coaposite Rotable Design for Protein Extraction rroa Dried Brewers' Spent Grain (DBSG).

Levels

Variables -2 -1 o 1 2

------------------------------------------------------------Temperature, Oc (Xl ) 80 85 90 95 100

Time, mins, (Xa ) 60 75 90 105 120

Conc. of phosphate, % in SDS" (X ) 0 0.25 0.5 0.75 1.0

3

BSG:extractant ratio % w/v (X

4 ) 2.5:100 5:100 7.5:100 10:100 12.5:100

'Concentration of Naa HP04

in 0.5% sodium dodecyl sulfate

Experiaent 8: DBSG protein concentrates (pilot scale preparation) •

Commercial DBSG (500 g) was mixed with extractant (10

Lj 0.5% SDS - 0.5% Na HPO ) in an extractor (steam jacketed a 4

kettle, Cooper and Bras Co., Canada) and heated to

extraction tempe ratures of 500

C, 75° C and 100° C for 1.5

hours. The residues were removed by filtration (through

glass wool). The proteins in the extracts were precipitated

by addition of ethanol (0.7 ml 95% CaHsOH to 1.0 ml extract)

followed by refrileration (4°C, 17b). The extract

(containing precipitated proteins) was adjusted to pH 4.5

(12N HCl). The pH adjusted extract was left standing (5b)

58

to allow sedimentation of the proteins. The upper liquid

layer was removed by siphoning. The precipitate was washed

with 50% acetone. Three successive washing steps were

carried out with centrifugation following each washing step.

The precipitates (acetone washed) were washed with water and

recovered by centrifugation (2000 rpm). Water was added to

the precipitate to facilitate spray dryin~. The resultina

product W8S recovered as spray dried protein concentrate.

59

(~

(.

SECTION IV: RESULTS AND DISCUSSION

A: Optiaization of Protein Bxtractability fro. Brewers' Spent Grain (BSG)

The optimization of protein extractability from

brewers' spent grain involved several steps; these are: (1)

conducting a screening experiment to determine cri tical

factors affecting BSG protein solubilisation ( 2 )

modification of the sodium dodecyl sulfate (SDS) extractant

and determination of protein solubilisation rand (3)

determination of optimum extraction conditions using a

central composite rotatable design.

1: Screening Bxperiaent-Variables Atfecting BSG Protein Extractability

The 4 variables (Temperature (Xl)' Extraction time

SDS concentration and Particle size

(X.) affecting BSa protein extractabi li ty were studied

simultaneously using a one-half fraction of a 24

factorial

design. The coded values of +1 and -1 represent the upper

limit and the lower limit respectively for each variable

studied. Table 12 shows the coded values for each variable

and the protein solubilised from DBSG and PBSG using

different treatment combinations. The actual levels of

variables used in each experimental run are shown in Table

13. For aIl the experimental runs, the meal:solvent ratio

was 5.0 g BSG to 100 ml extractant.

60

Linear regression analysis was carried out on the first

order designs (Table 12) for both DBSG and PBSG. Regression

analysis describes a relationship between the response

variable (Y) and the influencing factors (Xl' X.' X" X.).

The regression equations obtained for DBSO and PBSO are

equations 4 and 5 respectively:

y = 28.14 + 10.24X1

+ 2.55X. - 1.04X, - 1.73X.

(Eqn.4)

Y = 9.52 + 3.13XI

+ 1.72X. + 0.23X3

- 0.54X4

(Eqn.5)

where Y represents the prote in extractability. Analysis of

variance (ANOVA) was carried out to determine the

significance of the models for DBSG and PBSa. The ANOVA for

DBSG and PBSO are shown in Tables 16 and 17 respectively.

The F test (mean square regression/mean square residual) for

DBSO (88.20) and PBSO (73.43) showed both mode ls to be

hiihly si,nificant (p< 0.001), with RI values of 0.97 and

0.96 respectively. In order to determine the variables

which have a si,nificant effect on protein yield, the F test

was applied to the fitted coefficients of both equations

(Tables 16 and 17). The siinificant variables (for DBSO and

PBSG) were tempe rature of extraction (Xl)' time of

extraction (Xz) and particle size of grain (X.). The

concentration of the extractant (SDS) had no effect on the

protein extractability • There was an increase in protein

extractabili ty wi th increasina temperature. Ervin (1986)

61

(.

(

reported that BSG protein solubilisation increased with

increasing temperature. The average protein extracted from

DBsa (28.14%) was approximately 3 times greater than the

amount extracted from PBSG (9.53%).

The critical factors (Xl' Xa' X.) determined from this

screening experiment can be used to optimize protein yield

trom DBSG. The hi.her protein solubilisation of DBSG when

compared to PBSG made it more suitable starting material for

subsequent experiments in the optimization of protein

extractability and preparation of protein concentrates.

62

...-,

..... -

-

TlIlble 16. Anal7sis of Variance of Preliainar7 Faotorial Screening Design for Protein Extraoted froa Dried Brewers' Spent Grain (DBSG).

Sourr.:e

Te~perature (Xl)

Time (Xz )

Cone. of SDS (X3

)

Particle size of (X. )

Residual (Error

Total

R2+

1

1

1

grain 1

11

15

0.97

Sum of Squares

57.63

1905.89

Mean Square

1678.54

104.44

17.38

47.88

5.24

F

320.39 b

19.94c

3.3211

9. 14 d

--------------------------------------------~--------- ------• Degrees of freedom b

Level of significance p<O.OOl c

Level of significance p<O.l

ns=non significant

+eoefficient of determination

63

(.

(.

(

Table 17. AnalTais of Variance of Preliainary Factorial Soreenin. Design for Prot.ein Bxt.raoted fro. Pressed Brewers' Spent Grain (PBSQ).

Source Sum of Squares

Mean Square F

------------------------------------------------------------Due to regression 4 209.25 52.31 73.43

b

Temperature (Xl ) 1 156.37 219.51b

Time (X, ) 1 47.26 66.35b

Conc. of SDS (X3

) 1 0.87 1.2311s

Particle size of grain iX. ) 1 4.73 6.64

c

Residual 11 7.84 0.71 (Error

Total 15 217.08

RI + 0.96

------------------------------------------------------------• Degrees of freedom

b Level of significance p<O.OOl

c Level Qf significance p<O.l

ns=non signifieant

+coefficient of determination

64

2: Dried Brewers' Spent Grain (DBSG) Protein Solubilisation

Ca' SDS extraction of DBSG

Table 18 shows the effect of different concentrations

of SDS solution on protein solubilisation from DBSO. Ervin

(1986) and Crowe (1985) reported that SDS was an effective

extractant for brewers' spent grain proteine In order to

determine whether protein extractability from DBSO increased

at higher SDS concentration, the optimum extraction

parameters determined from the preliminary experimental

design (1/2 fraction of 24

factorial design) were lcept

constant and the SDS concentration was varied from 1 ta 5%.

An optimum concentration of 3% SDS ~ave the highest

protein extractability (42.3%). At SDS concentration above

3'. there was a decrease in the DBSO protein solubility

(Table 18). The 3% SDS extract was darker in col our and

contained a higher level of dissolved protein compared to

the 1% SDS extract (30.6%). Wu et.al. (1985) reported that

extractants containing SDS solubilised up to 48% of the

proteins of distillers' grain.

(b) Solubilisation of DBsa protein uainll SDS wi th dibasic sodiu. phosphate (Ha.dPO.,

Addi tion of dibasic sodium phosphate (0.5%) to SDS

solution (3.0%) resulted in an inCIease in the protein

extracted from DBSO; the protein extractability of the

extract was increased by approximately 14.0% (Table 19).

Ervin (1986) reported that a SOS solution (3% SDS + 0.5%

65

(~

(~

Na HPO ) solubilised 62.4% of the DBSG-protein. 2 •

In the

present study, 55.8% of the DBSO protein was solubilised.

The difference in DBSG-N solubilisation (using same

extractant) may be attributed to differences in composition

of the OBSG and the sample preparation. When 0.5% dibasic

sodium phosphate was added to SDS solution (1.0~), the

protein extractabi li ty of the extract was increased by

24 . O~. Comperison of the protein extractabilities of the

3.0% and 1.0% SOS extracts (with 0.5% Na HPO ) showed that Z 4

the protein extractabili ties were similar (Table 19). 'l'he

3.0% SDS extract gave a protein extractability (55.85%) that

was only 1.57% greater than the 1.0% SDS extract (54.28%).

Table 18. Rffect of Sodiua Dodecyl Sulfate (SOS) Concentration on Protein Solubilisation tro. Dried Brewers' Spent Grain (DBSG).

SDS Concentration (X) Prote in Extractability (%)b

------------------------------------------------------------1

2

3

4

5

30.6 (5.13)·

39.8 (1.78)

42.3 (5.03)

39.6 (2.54)

40.7 (2.52)

ï-------------------------~---------------------------------Results are means (standard deviations) of triplicate analyses.

bprotein extractability:(%N x 6.25 x 100)/ % protein in DBSO

66

Table 19. Protein Bxtractability fro. DBsa using SDS Solution Containing Dibasic Sodiua Phosphate and sns Solution only.

Extractant

3.0% SDS + 0.5% Na HPO a •

1.0% SOS + 0.5% NaaHPO.

3.0% SOS

1. 0% SDS

Prote in Extractability (%)

55.85 (1.43)ôI

54.28 (2.47)

42.30 (5.03)

30.60 (5.13)

1 Results are means (standard deviations) of triplicate analyses.

(c) Ettect of dibasic sodiua phosphate (Na HPO ) on DBSO protein extractability 1 •

SOS solution containing dibasic sodium phosphate (0.5%

Na2

HPO.) was a more efficient extractant for OBSa protein

than SDS solution alone (Table 20). SOS solution (3.0%) had

a pH 7.31. Addition of 0.5% dibasic sodium phosphate to

this solution increased the pH to 9.00 (Table 20). Dibasic

sodium phosphate is used in macaroni, cooked breakfast

cereals, and other flour products to raise the pH above 7

and thereby increase the cooking rate (Oeman and Melnychyn,

1971). To determine whether it was the pH effect of

Na2

HPO. that resul ted in the increased solubili ty of OBSG

protein, the SOS solution (3.0%) was adjusted to pH 9.00

using O. IN NaOH and the prote in extractabili ty determined.

There was no increase in the prote in extractability

67

(

(

(Table 20).

responsible for the increase in DBSG protein solubility.

This could be due to phosphate-protein interactions (Deman

and Melnychyn, 1971). Hydro"en bondin. between the unshared

oxygen atoms of the phosphate group and the nitrogen atoms

of amino, guanidino and imidazole groups of proteins have

been sugge&ted (Deman and Melnychyn, 1971). Extraction of

protein from DBSG with sodium phosphate (0.5%) resulted in a

much lower protein extractability (8.48%) than that obtained

with 3.0% SDS (42.30%).

Table 20. Protein Bxtractabili t7 of DBsa Bxtractant.

uaing SDS

Extractant Protein Extractability (%)

3.0% SDS (7.3l)b 42.30 (5.03)·

3.0% SDS + 0.5% Na HPO z • (9.00) 55.85 (1.43)

3.01 SDSc

(9.00) 40.10 (3.54)

0.51 Naz HPO. 8.48 (2.39)

• Resul ts are means (standard deviations) of tri pliea te analyses.

b pH of the extraetant

Cadjusted to pH 9.00 with O.lN NaOH.

68

(d) B~~ect of concentration of dib.sic Bodiu. phosphate (Na.RPO.) on extractabilit7 of protein fro. DBSa

The levels of dibasic sodium phosphate (Na. HPO. ) added

to the SOS solution were 0.51, 1.0%, 1.5% and 2.0%. These

levels of NaIRPO. were added to SOS solution (3.0% and 1.0~)

and the solutions were used to solubilise the protein froll

DBSG. The amount of protein solubilised were deterllined

(Table 21). Several researchers (Deman and Melnychyn, 1971)

have delDonstrated a marked increase in the quanti ty of

water-soluble nitrogen extracted from processed cheese with

increase levels of phosphate. In our experiments, addition

of 1.0% and 1.5% Na RPO 1 •

protein than 0.5% Na RPO 2 •

to sns (3.0%)

( Tables 21).

solubilised leBs

The 2.0% Na HOP 1 •

gave a protein extractability of 56.591 which was only 2.0~

greater than that obtained wi th 0.5% Na. RPO. (54. 28~) •

Ervin (1986) "..ased sns (3.0%) containinll Na. RPO. (O. 5X) as

the major extractant for protein from BSG. Dibasic sodium

phosphate concentrations above 0.5% did not increase the

extractability of protein from DBSa. The 3.0% SDS solution

containinll NaIHPO. (0.5 2.0%) lIave hillher protein

extractabili t ies than the 1.0% SDS solution containing

Na. HPO. ( 0 • 5 - 2.0%). This confirms that DBsa protein

extractability increases with concentration of the

extractant (SOS solution).

69

(~ Table 21. Htfect of Concentration of Dibasic Sodiua Phosphate on Bxtractability of Protein tro. DDBa.

------------------------------------------------------------Extractant Protein Extractability (% )

------------------------------------------------------------1.0% sns + 1. 5% Naz HPO. 54.28 (2.47)1

1.0% ans + 1.0% Naz HPO. 50.97 (0.87)

1.0% sns + 1. 5% NazHPO. 52.24 (0.68)

1.0% sns + 2.0% Naa HPO. 50.85 (0.02)

1.0% sns + 2.0% Naz HPO. 50.85 (0.02)

3.0% sns + 0.5% Naz RPO. 55.85 ( 1. 43)

3.0% sns + 1.0% Na,a RPO. 53.54 (1.18)

3.0% sns + 1. 5% Naz HPO. 54.70 (2.64)

3.0% sns + 2.0% Naz HPO 56.59 (1.91) • ------------------------------------------------------------1

Resul ts are means (standard deviations) of triplicate analyses.

(e) Bffect of presence of sodiua chloride in SDS solution extractability of protein fro. DBBG

sns extracting solution (1.0%, 3.0%) which contained

sodi um chloride (0.5%) Jave prote in extractabili ties of

26.97% and 33.90% respectively (Table 22). This was Iess

than that obtained wi th no addition of sodium chloride to

1.0% SDS (30.60%) and 3.0% sns (42.30X). Abdel-Aal et. al.

(1986) reported that the maximum solubility of prote in was

reached at O. 5M NaCl for faba bean and ohick pea. An

optimum concentration of O. 2M NaCl resul ted in maximum

nitroJen extractability trom rapeseed meal (Finnigan and

Lewis, 1985). In our experiments the concentration of Nacl

70

.... -

-

(0.6%) used may have caused a "salting out" effect. When

the same concentration of dibasic sodium phosphate (O. 5S)

was added (Table 22' to SDS (1.0% and 3.0%', a aharp

increase in protein solubility was observed. Protein

extractabilities of 54.28% and 55.85% were obtained with

1.0% SDS (containing 0.5% Na. HPO. ) and 3.0% SDS (containing

0.5% Na. HPO. ' respectively. It is possible that the

chloride ions (from NaCI) may be competing with the protein

for the solvent molecules, while the phosphate ions (from

Na HPO ) interact with the protein molecule and enhances its a •

solubility (Deman and Melnychyn, 1971).

Table 22.Protein Bxtractabilitiea rro. DBSG using Different SDS Solutions Containing NaCl and Na.HPO •.

Extractant Protein Extractability (%)

1.0% SDS 30.60 (5.13'·

1.0% SDS + 0.5% NaCl 26.97 (1.01)

0% SDS + 0/5% Na.HPO. 54.28 (2.47)

3.0% SDS 42.30 (5.03)

3.0% SDS + 0.5% NaCl 33.90 (2.57)

3.0% SDS + 0.5% Na. HPO. 55.85 ( 1. 43'

• Results are means (standard deviations) of triplicate analyses.

71

(~

(

Cf) The effect of sa.ple preparation .ethod on protein extractability fro. DBSO

The protein extractabilities of SDS extracts of sieved

DBSO (aift with sieve of 1.5 mm) and .round DBSO (Thomaa

Wiley aill - 2m. sieve) are shown in Table 23. The protein

extractability from sieved DBSO (59.23%) was approximately

8.0% .reater than that (51.15%) from ground DBSG. Pool and

shooter (1955) reported that the yield of the glutelin

fraction was lower when finely ground barley was used in the

extraction. The authors suggested that the ~rinding action

may have resulted in the denaturation of some of the

proteine and hence a lower protein solubility.

Table 23. Protein Bxtractability rro. Sieved DBSG and Ground DBSG

Sample

Sieved DBSG

Ground DBSG

Protein Extractability (%)

59.23 (0.76)'

51 . 16 (0.00)

Il Resul ts are means (standard deviations) of triplicate determinations.

3: Response Surface Methodolo'7 (RSM): Opti.ization of Conditions for Bxtraction of Protein fro. DBSO

RSM is a statistical technique that uses quantitative

data to determine and simultaneously solve multivariate

equations which specify the optimum protein yield for a

specified set of factors throu.h mathematical models

(Giovanni, 1983).

72

........

The experimental desi.n used for optimization of BSG

protein extractability was a central composite rotatahle

design (CCRD). This design was used to determine the

optimum levels of temperature, level of Na HPO 1 •

in

extractant, time of extraction and BSG: extractant ratio

which would result in acceptable yield of protein extracted

from DBSG. The SOS concentration was kept constant at 0.5%,

while the particle size was kept constant at 1.5 mm. In the

CCRD each factor (Temperature (Xl)' extraction time (X ), a

concentration of phosphate (X3

), BSG:Extractant ratio (X.)

was considered at 5 levels and the coded and uncoded lev~ls

for these variables (Xl' Xa , X3

, X.' are sho~n in Tables 14

and 15 respectively. The ranae for each variable was

determined from previous experiments (Section IV). The

number of treatment combinations obtained with CCRDs is 1[

given by 2 +2K+l where K equals the number of variables

under study. In this study since 4 variables were

considered, there were 25 treatment combinations. The

amount of protein extracted for each treatment combination

with the corresponding coded values of tempe rature of

extraction (Xl)' time of extraction (Xa ), concentration of

NazHPO. in the extractant (X3

) and BSG:extractant ratio (X.)

is shown in Table 14. This CCRD was used for fittinl second

order p~lynomial model. Multiple regression analysis of the

uncoded data in Table 14 resulted in equation 6:

73

(

y = -463.89 + lO.03X + 1.22X + 26.61X - 208.77X 1 a 3 of

2 - O.06X

1

2 2 a -O.OlXa

- 49.383

- 619.85X. + O.004X1 X2 + O.36X1 X3

+2.53X1

X. + O.03Xa X3

- O.09X2

X. + 28.36X3 X ..

(Eqn.6)

where y represents the predicted prote in yield of the

second order polynomial model.

74

Analysis of variance (ANOVA) was carried out on this

fitted model (equation 6) (Table 24). Khuri and Cornell

(1987) reported that to infer that the fitted model

adequately describes the behavior of the response over the

experimental ranaes of variables would necessitate a testing

of lack of fit of the fitted model. The test for adequacy

of fit of the fitted model (Equation 6) produced an F value

(lack of fit mean square/pure error mean square) of 0.53

(Table 24). This Fo.os value did not exceed the tabulated

val ue 0 f 2.57 ( 9, 26 d. f. ) . Thus, lack of fit was not

significant and the fitted model was adequate ior the

description of the response surface. The coefficient of

determination (R2 = 0.87) indicated that 87% of the total

variation in the protein extractability was explained by the

fi tted model.

The location of the maximum yield value ls the point of

maximum response. Since the fitted model was adequate, it

was used to locate the coordinates of the stationary point.

The statinnary point (Xo ) is the point at which the slope of

the response surface is zero, and may be a maximum, minimum

or saddle point (Khuri and Cornell, 1987). The stationary

point was determined using the equations suggested by Khuri

and Corneli (1987). The values of the variables at the

stationary point are obtained from these equations. These

are the optimum extraction conditions (Table 25) for maximum .......

protein extractabii i ty from DBSG. The predicted yield

75

(.

(58.95% w/w) of extracted protein at the stationary point

(Xo ) closely agreed with the observed yield (58.45% w/w).

The nature of the stationary point (maximum or minimum)

Table 24. Anal7sis of Variance for Second Order Polyno.ial Model Fitted to Yield of Protein (S) Extracted fro. Dried Brewers' Spent Grain (DBSO).

Source dF

Due to regression 14

Residual 35 (Error)

Lack of Fi ta 9

Pure Error 26

Sum of Squares

4214.71

633.06

93.81

539.25

Mean Square

301.05

18.08

10.42

20.74

F

16.85

0.503118

• Lack of Fit Sum of Squares(SS) = Residual SS-Pure Error S8

ns = non significant

was determined by converting the second order polynomial

equation (Equation 6) to its canonical form (Equation 7).

The canonical equation obtained was:

y = 58.95 - 0.008w2

1

(Eqn.7)

where W1

to W. are the variables. The coefficient of the

variables, the eigen values (-0.008, -0.05, -49.03 and -

619.41) were aIl negative indicating that the stationary

point was a maximum.

76

3-dimensional response surface graphs (Figures 5 and 6)

were generated from the fitted second order polynomial model

(Equation 6).

Table 25. Coded and Uncoded Values of Variables at Stationary Point X (point of aaxiaua yield of extracted protein). 0

Variable Coded Uncoded

Temperature, o C(X ) 0.20 95 1

Time, mins (Xa ) 0.53 98

Cone of phosphate, % in SDS (X

3 ) 0.56 0.64

BSG: extractant ratio % w/v (X

t ) -2 2.5:100

Predicted yield of extracted protein at stationary point = 58.95% w/w

Observed yield of extracted protein at stationary point' = 58.45% w/w

These graphs showed that the response surface was a maximum.

In Figure 5, temperature (90°C) and extraction time (95 min)

were kept constant and the protein yield plotted as a

funetion of phosphate concentration and BSG:Extractant (w/v)

ratio. It is seen that an increase in the concentr.' tian of

Na HPO in the extraetant solution and a decrease in the a t

BSG:Extractant ratio (w/v) resulted in an increase in

protein yield. The response surface graph (Fig. 6) shows

the effect of temperature and extraction time on prote in

77

o yield, with the concentration of phosphate (0.65%) and

BSG:Extractant ratio (2.5: 10(') beini he Id constant. An

increase in the temperature and extraction time will result

in an increase in the protein yield CFii. 6). From the

response surface graphs it is difficult to determine the

actual levels of variables that will iive a specifie protein

yield. Contour plots of the response surfaces (Figures 7

and 8) made this easi~r. It entails the plotting of

different surface height values (specifie values of protein

yield) which enables one to focus attention on the levels of

the factors lit which changes occur in surface shape (Khur i

and Cornell, 1987). Contour lines are drawn by connecting

....... points in the experimental region that produces the same

value of protein yield.

Figure~ 7 and 8 are contour plots of the response

surfaces of figures 5 and 6 respectively. When the

o temperature (90 C) and extraction time (95 min) are kept

constant, it is possible to determine the various

combinations of phosphate concentration ( % ) and

BSG:Extractant ratio (w/v) that will ,ive a specifie protein

yield (Figure 7). In the contour plot of Fiiure 8, the

phosphate concentration (0.65%) and BSG:Extractant

(2.5:100) are kept constant, so that various combinat ions of

temperature e.nd extraction time can be determined for a

specifie protein yicld.

78

(~

62.08

_ 42.47 ~ c 'i -o .. a. -o

" 'i >=

22.65

Figure 5.

Temperature (900e)

Time (95 min)

Response surface graph showing the effect of Concentration

of Phosphate and BSG:Extractant ratio on protein yield.

79

0.2~O

-

62.33

49.72

Concentration of Phosphate (0.15%)

BSG:Extractant (2.5: 1 00)

re", 8S.67 " .... ,. ,(J (-'C)

Figure 6. Response surface graph showing the effect of temperature

and time on prote·'n yield.

80

120

-'Ifl. -• -.. .&: Co fil 0 .= a. -0 c: 0 --• --

(# c: • u c: 0 (J

Temperature (9COC)

Tlm. (95min) 1.00~------------------------~--------~----~--~

0.75

0.50

0.25

O.OO+-~~~~~~~~~~~~~~~~~~~~~~~

0.000 0.063 0.125

aSG:!atJ aw:bnt (w/v)

0.188 0.250

Figure 7. Contour ~lot showing the effect of concentration of phosphate and

BSG:Extractant ratio on protein yield.

81

()

-(.) 0 -...... • ..

" = --- .. .. • CI. e (!

-.....

Concentration of Phosphate (0.65%)

BSG:Extractant (2.5: 1 00)

100r.r.~r-~--~----~---------------------

95

• p,: fO

90

85

80~~~~~rrrr~~~~~~~~~~~~-J

60 75 90 105

nme(mln) Figure 8. Contour plot showing the effect of temperature and time

on protein yield. 82

120

(

4: DBSO Protein Concentrates

(a) Bffect of extraction temperature on (i) extractabi1ity of protein fro. DBSO and (ii) protein content and SDS content of protein

concentrates

Protein concentrates were plepared on a pilot scale o 0

(Section III) at extraction temperatures of 50 C, 75 C and

The prote in extractability of the extracts and the

prote in contents and SDS contents of the spray dried

concentrates are shown in Table 26. The extractability cf

protein increased as the temperature of the extraction was

increased. An increase in prote in extractability of 14-15%

o was observed for each 2~ e increase in temperature. The

protein extractabi li ties at temperatures of 500

C, 750 e and

1000 e were 18.44~, 33.61~ and 48.53% respectively (Table

26). Ervin (1986) a1so reported that SDS extraction of OBSO

protein increased with temperature. The prote in content and

SOS content a1so increased as the temperature of extraction

was increased. Protein concentrates with protein content of

56.15%, o

69.65% and 81. 79~ were obtained at 50 C, o

75 C and

1000 e respectively.

o A temperature of 100 e favored the

formation of protein rich DBSG concentrates. The SDS level

of the protein concentrates prepared at extraction o

and 100 C were 1.88%, 7.38% and

9.80~ ?espectively. Kato et.al. (1984) reported that SOS

binding by glycinin increased when the protein was heat

denatured. Extraction of OBSO proteins at the higher

extract.ion temperatures (750

C, 100° C) may have caused

83

-

denaturation of the extracted proteins wi th a resul tina

increase in the SDS binding capacity of the proteins (Table

26) •

Table 26. Prote in Bxtractabili ty of DBSO Bxtraots, Protein Content and SDS Content of DBSO Protein Conoentrates (spray dried) at Different Te.peratures.

Extraction Temferature

( C)

50

75

100

Protein Extractability

(% )

18.44 (0.43)·

33 .61 (O. 20 )

48.53 (0.44)

Protein Concentrate

Protein Content

(X)

56.15 (0.66)·

69.65 (1.91>

81.79 (0.00)

SDS Level

(% )

1.88

7.38

9.80

• Results are means (standard deviations) of triplicate determinations.

(b) Reduction of SDS contents of DBSa protein conoentrates

It was found (Section IV) that SDS combined with

dibasic sodium phosphate (Naz RPO.) was the most efficient

extractant used in the preparation of the DBSO protein

concentrates. Previous studies have indicated that SDS ls

an effective extractant for DBSG protein (Crowe, 1983;

Ervin, 1986). It has been established that SDS forms a

complex with aIl proteins, and that a maximum amount of

84

<- bound SDS is 1.4 g of SDS per gram of protein (Graveland

et.al. 1979). Washing of the OBSG protein concentrates with

organic sol vents such as ethanol and acetone (Section IV)

was a mandatory step for the removal of residual SOS in the

protein concentrates. The SOS level of DBSG protein

concentrates was determined by the standard A.O.A.C.

procedure (1980, method no. 20.127).

Table 27 shows the SOS level of protein concentrates

prepared from laboratory experiments. The prote in

concentrate (OBSGPCl) with a 12.52~ SOS content was not

washed with ethanol, while the prot~in concentrate (OBSG

PC2) subjected to ethanol washing had a lower SOS content

(8.84~) . SOS is soluble in ethanol (Weast, 1975). These

high levels of SOS are not acceptable in foods. The maximum

allowable level of SDS in foods is 0.5% in gelatin intended

for marshmallow composition (Food and Drugs Act).

Extraction of BSG with SOS solution (1.0% with 0.5% Naz HP04

)

gave a protein extractability of 54.28~, while 53.59~

protein was extracted with 0.5% SOS (with 0.5% NazHPO.),

Because of the similarities in prote in extractabilities, a

lower SDS concentration was considered preferable for the

preparation of DBSG concentrates. Use of a lower

concentration of the SOS solution (0.5%) followed by

excessive washing (3 steps) of the protein concentrate (OBSG

PC3) with ethanol (95%) before freeze drying resulted in an

acceptable SDS content of O. 50~ (Table 27). These

85

G

..... '.

....

--, ,

extraction aüd washina procedures which resulted in low SDS

content were subsequently used to prepare protein

concentrates on a pilot scale.

Table 27. SDS Content of DBsa Protein Concentrate (laborator,. scale preparation) usina Optiaua Extraction Conditions.

Protein concentrate

DBSGPCl

lvashing method for protein concentrate

no ethanol washina

Dryina method for protein concentrate

FD

DBSGPC2 washina with ethanol (95") FD

DBSGPC3a

Excessive washina with ethanol (95") FD

optimum extraction conditions:

Temperature

Time - 1. 5 h

Extractant - 1" SDS + 0.5" NaaHPO.

Meal:solvent - 2.5 g:50 ml

FD - freeze dried

• extractant consisted of 0.5" SDS + 0.5" NaaHPO.

SDS content (%)

12.52

8.84

0.50

DBSGPC1, DBSGPC2, DBSGPC3: DBSG protein concentrates prepared usin, different dryin. and washina methods.

86

<-

(

Table 28 shows the SOS content of DBSG concentrates

prepared using a pilot scale equipment. The protein

concentrate (OBSGPC4) which was washed with water and spray

dried had a high content of SOS of 11.95%; this indicated

the ineffectiveness of water for the removal of residual SOS

from the concentrate. Kato et.al. (1984) suggested that the

interaction of proteins with SOS is due to the electrostatic

bond and that the SDS-binding capacity decrease with an

in~rease in ionic strength; this tendency was not observed

in our experiments. Hydrophobie interaction was also

suggested to explain the SOS-binding capaci ty of proteins

(Kato et.al. 1984); it is possible that in our experiments

water was ineffective in severing the bond due to

hydrophobie interaction. The SDS content of the prote in

concentrate (8.56%) washed with 95% ethanol was higher th an

that (5.33% SOS) washed wi th 50% ethanol (Table 28). SDS

was removed from prepared glutenin by washing the

precipi tate several times wi th 75% ethanol, which resul ted

in SDS level of 0.4 - 0.8% (Danno, 1981). Consequently, an

ethanol concentration of 50% was used for the preparation of

the prote in concentrate (DBSGPC7). Excessive washing (3

steps) of the protein concentrate (DBSGPC7) wi th ethanol

(50%) did not result in a marked reduction in the sns

content of the concentrate. The concentrate (DBSGPC7 )

showed SDS contents of 4.62% (freeze dried) and 6.63% (spray

dried) . It was observed that the drying methods used for

87

,\ <. -

....

preparation of the protein concentrate (DBSGPC7) influenced

the SDS content of the concentrate. The spray dried

concentrate had a higher SDS content (6.63%) th an the freeze

dried concentrate (4.62%). Kato et.al. (1984) reported that

SDS binding by glycinin increased when the protein was heat

denatured. The high temperature used for spray drying of

Table 28. SDS Content of DBSG Protein Concentra tes (pilot scale preparation) using Opti.u. Extraction Conditions.

Prote in concentrate

Washing method for protein concentrate

Drying method for protein concentrate

DBSGPC4

DBSGPC5

OBSGPCS

DBSGPC7

washin, with water

50% ethanol

95% ethanol

Excessive washing with ethanol (50%)

SD

SO

FD

FD SD

optimum extraction conditions:

Temperature

Time

Extractant

Meal:solvent

SD FD

- 1.5 h

- 0.5% SDS + 0.5% NaaHPO.

- 2.5 g:50 ml

- spray dried - Freeze dried

sos (%)

level

11. 95

5.33

8.56

4.62 6.63

DBSGPC4, OBSGPC5, DBSGPC6, DBSGPC7: DBSG protein concentrates prepared usina different dryin, and washing methods .

88

(

(

the concentrate may contribute to the higher SOS content of

the spray dried concentrate.

The preparation procedure for OBSG protein concentrates

was modified in an attempt to reduce the SOS content of the

protein concentrates. Protein concentrates were prepared at

o 0 SO C and 100 C; sorne (DBSGPCb, DBSGPC10) were washed with

ethanol (50%) and others (DBSGPC9, DBSGPCll) wi th acetone

(SO%) (Table 29). A centrifugation procedure followed each

washing step to ensure efficient removal of dissolved SDS

(Table 29).

The SDS content was lower for the protein concentrates

prepared at SOoC than for the concentrates at 100°C (Table

29) • It is possible that at 50°C "the SDS binds less

° strongly to the protein when compared to 100 C. Washing of

the protein concentrates with acetone resulted in

consistently lower SDS content when compared with ethanolic

washing (Table 29). The Most efficient solvent for SDS

removal from the protein concentrates was acetone (50%).

This washing procedure wlth acetone was used for the

final preparation of protein concentrates at the pilot scale

level. ° DBSG protein concentrates were prepared P-t 50 C,

7SoC and 1000e and subjected to the same number of washing

steps (3) wi th acetone (SO%) (Table 30). The concentrates

prepared at 50oe, 75°e and 100°C were found to have SDS

levels of 1.88%, 7.38% and 9.80% respectively. The SDS

content increased with increasing temperature of extraction.

89

Table 29. SUS Content of DBSG Protein Concentrates (pilot scale preparation) Prepared at Temperatures of 50°C and 100°C.

Protein concentrate

Temperature of extraction (oC)

Wa~hing method for protein concentra te

DBSGPC8 50 centrifugation and ethanol (50%) washing

DBSGPC9 50 centrifugation and acetone (50%) washing

DBSGPCIO 100 centrifugation and ethanol (50~) washing

DBSGPC11 100 centrifugation and acetone (50%) washing

Extraction conditions:

Temperature o 0

- 100 C, 50 C

Time - 1. 5 h

Extractant - 0.5% SDS + 0.5% NazHPO.

Meal:solvent - 2.5 g:50 ml

FD - freeze dried

Drying method for protein concent::-ate

FD

FD

FD

FD

SDS (~)

level

1. 41

1.05

3.14

2.70

DBSGPC8, DBSGPC9, DBSGPC10, DBSGPC11: DBSG protein concentra tes prepared using different drying and washing methods and extraction temperature.

~) ,. )

0 0-

, >

(~

A laraer number of washin, steps (wi th acetone) May be

required to reduce the high SDS content of the DBSG protein

concentrates.

Table 30. SDS Level of DBSO Prote in ConcentrateB (pilot scale prepara~ion). Prepared. at Different Teaperatures (50 C, 75 C, and 100 C).

Protein concentrate

DBSG50

DBSG75

DBSGIOO

50

75

100

Warjhina method

AW

AW

AW

AW - washing with 50X acetone

SD - spray dried

Dryina method

SD

SD

SD

SDS level (%)

1.88

7.38

9.80

5: Functional PropertieB of DBSO Protein Concentrates

(a) Fo .. capacity

Foam capacity values obtained for protein slurries (21

weiaht/volume) of the DBSG protein concentrates and

commercial soy protein concentrate (Acron S) are shown in

Table 31.

The DBSG protein concentrates prepared at different

tempe ratures exhibited marked

di fferences in their foam capaci ty. This could be due to

differences in the SDS content of the concentrates; DBSG50,

DBSG75 and rBSOIOO had SDS contents of 1.88X, 7.38X and

91

9.80% respectively. The protein concentrate prepared at

° 50 C showed no foam capacity over the pH ran,e studied.

However, the concentrates prepared at hi,her extraction

° ° temperatures (75 C and 100 C) showed foam capacity at pH

val ues above 3. Foamina was negli,ible at pH 3 (DBSG100)

and pH 3 and 5 (DBSG75). Hudson (1987) reported that not

aIl proteins exhibit the same foamin, propsrties;

foamability depends on the molecular wei.ht, surface

hydrophobicity and internaI bonding of the protein Molecule.

The extraction tempera tures used in the study May play an

important role in determining the nature of the proteins

solubilised.

DBSGIOO prote in concentrate showed foam capacity in the "

range 160-170 with maximum foam capacity at pH 5. The foam

capacity were lower (126.6-130.0) for the DBSG concentrate

prepared at 75°C with a maximum at pH 7.0. The resul ts

su"est that the foaming properties of the DBSG protein

concentrates is pH dependent. Since pH affects the overall

char,e on the protein Molecule, it is likely that the DBSG

concentrates May have different isoelectric points.

Townsend and Nakai (1983) reported that numerous factors

including pH, temperature, the presence of salts, sugars and

lipids and the protein source affect the foamin. behavior of

proteins.

The soy prote in concentrate showed foam capacity

ran.in, from 100 to 143 over the ranae of pH values studied

92

( (3 to 9). The foam capacity of the soy concentrate was

lower than that (160-170) of the DBSGI00 concentrate, but

higher than the foam capacity (126-130) of the DBSG75

concentrate.

The DBsa concentrates like the soy protein concentrate

displayed maximum foam capacity at pH 7.0. The foam

capacity (100) of the soy concentrate was significantly

(p < 0.05) lower at pH 5.0 compared to the DBSG prote in o

concentrate prepared at 100 C (170).

93

G, Table 31. The Btfect of pH on the Fo .. Capacity (X) of DBsa

Protein Concentratesa

and Soybean Conoentratea

•

Protein Concentrate pH 3.0

Foam Capacity

pH 5.0 pH 7.0 pH 9.0

------------------------------------------------------------DBSG100 NF 170.0(5.00)b 165.0(8.66) 160.0(6.23)

DBSG75 NF NF 130.0(13.33) 126.6 ( 5.41)

DBSG50 NF NF NF NF

Soy Concentrate (Acron S) 123.3(7.64) 100.0(5.0) 143.3(7.64) 143.3(7.64)

------------------------------------------------------------a 2X protein dispersion

b Resul ts are means (standard devia tions) of tr i pl ica te measurements.

NF: no foaming

(b) FOBa stability

Table 32 shows the foa. stability of the protein

slurries (2X wt./vol) of the DBSG protein concentrates, and

commercial soybean prote in concentrate.

The foam stability rang~d from 80 to 93 for the OBSG

concentrates, and 79 to 100 for the soy protein concentrate.

Maximum foam stability values of 93 and 100 were obtained at

pH 5.0 for DBSG100 concentrate and soy protein concentrate

respectivel,.. Hudson (1987) reported that proteins

stabilise foams by forain. a flexible, cohesive film around

air bubbles; as the pH moves away from the isoelectric point

94

~ ~

l i "

(~

(pl), net charge increases and film strength and foam

stability decrease. It is possible that the isoelectric

point of DBSO concentrates may lie in the pH range 3 to 5.

The DBS075 concentrate showed no foaming properties at pH 3

and 5. Koivurinta et. al. (1980) reported that BSO proteins

did not show an isoelectric point over the pH range of 3 to

12. The BSO protein concentrate (DBS0100) showed high foam

stability (92%) at pH 7.0, whi!e the soy concentrate

demonstrated a sharp drop in foam stability (79%) at this

pH. Both the DBSGI00 concentrate and soy concentrate showed

maximum foam capacity and stability at pH 5.0.

Table 32. The Bffect of pH on Foa. Stability eX) of DBSG Protein Concentrates· and S07bean CODcentrate· •

Foam Stabi!ity

Protein Concentrate pH 3.0 pH 5.0 pH 7.0 pH 9.0

DBSG100 NF 93.0(4.2) b

92.0(2.8) 80.0(3.3)

DBSG75 NF NF 82.0(2.6) 79.0(3.2)

DBSG50 NF NF NF NF

Soy Concentrate (Acron S) 93.0(2.6) 100.0(0.0) 79.0(6.5) 93.0(3.5)

------------------------------------------------------------·2% prote in dispersion b Resu! ts are means (standard deviations) of triplicate measurements.

NF: no foamin,

95

(c) Baulsion capacity and stability

The emulsion capaci ty of DBSO protein concentrates

(prepared at 100oe, 75°e, SOoC) was measured using the

procedure of Webb et. al. (1970), while the emulsion

stabili ty was determined usina the aaethod of Sathe and

Salunkhe (1981a).

The three DBSO protein coneentrates showed somewhat

similar emulsion capacity (22-23 ml oil/g sample), while the

commercial soy concentrate showed a higher emulsion capacity

(30 ml oil/a sample) (Table 33). Satterlee et. al. (1975)

reported the emulsion capaci ty of the Great Northern bean

prote in concentrate to be 131 ml oil emulsified per gram of

sample. Soy protein preparations are widely used for their

emulsion capacity (EC) and emulsion stability (ES)

properties in frankfurters, bologna, cakes and soups

(Kinsella, 1976). In this study, the soy concentrate (Acron

S) showed a low emulsion capaci ty almost comparable to the

DBSG concentrates. It is likely that the method used May

have underestimated the emulsion capacity of the protein

samples. Conditions such as equipllent design, speed of

blendina, rate of oil addition, temperature, pH and kind of

oil used, aIl affect the emulsifying capaci ty of proteins

(Kinsella 1976).

Poor emulsion stability was also characteristic of the

DBSG concentrates sinee 42-48 ml water separated after 10

hours (Table 34). Protein concentrate from the Oreat

96

(-

(~

(~

Northern ù~an showed no separation of water after 120

hours (Sathe and Salunkhe, 1981a). The soy concentrate

emulsion was highly stable since separation of the water

phase occurred after 120 hours (Table 34).

Table 33. Co.parison o~ E.ulsifying Capacity and End-Point Criteria for DBBa Prote in Concentrates·.

Sample

DBSGlOO

DBSG75

DBSG50

Soy concentrate (Acron S)

Oil emulsified (ml/g)

22

23

23

30

14% protein dispersion

Resistance at end point (Ohms)

2.8 x 10' ( 2 .00 ) b

2.5 x 10· (0.35 )

2.6 x 10· (0.49)

5.4 x 103

(0.00)

b Results are means (standard deviation) of duplicate measurements.

97

-

Table 34. Co.parison.of Baulsifyin. Stability of Protein Concentra tes •

------------------------------------------------------------

Sample Initial vol of emulsion (ml)

DBSG75 50

DBSG50 50

Soy concentrate (Acron S) 60

Vol. of H 0 separated (ml) a 0 at room temp (21 C)

after time (hr)

10

42.5 (3.53)

42.5 (3.53)

o (0.00)

120

42.5 (3.53)

42.5 (3.53)

60 (0.00)

·4% protein dispersion b

Results are means (standard deviation) of duplicate measurements.

(d) Water absorption

Water absorption capacity of the DBSO protein

concentrates were determined using the procedure of Sosulski

(1962).

The DBBO concentrates demonstrated somewhat similar

water absorption capacity (Table 35). The water absorption

capaci ties of the DBSG protein concentrates were 163.3%

(DBSGI00) and 166.7% (DBSG75 and DBSG50). A marked

difference in water absorption capacity was observed for the

DBSG concentrates and the soy concentrate (Table 35). The

say concentra te had a water absorption capaci ty (483.3%),

approximately 3 times greater than that of the the DBSG

concentrates. Lin et.al. (1974) reported the water

98

absorption capaci ties of sunflower protein concentrates to

be 137.8%, 166.2% and 203.0% for DE-60, DE-80 and DE-90

respectively, with the high-heated concentrates (DE-80 and

DE-90) having the higher water absorption capacity. This

behavior was not observed for the DBSO concentrates which

were prepared at di fferent temperatures. The DBSO

concentrate prepared at the higheat temperature (100°C) had

a water absorption capacity 3.4% less than that prepared at

the lower temperature (50o e). The higher water absorption

capacity of the soy concentrate suggests that the soy

protein is more hydrophilic in nature than the BSG proteins.

(e) Fat absorption

The fat absorption capacity of the DBSO protein

concentrates was determined by the procedure of Lin et. al.

( 1974) • The DBSG protein concentrates had oil absorption

values ranging from 166.7% to 193.3% (Table 35). The DBSO

concentrates aIl bound more oil than the soybean concentrate

(153.3%). Lin et. al. (1974) reported that soy products had

oil absorption values ranging from 84.4% to 154.5%. It is

possible that the DBSO proteins may be more lipophilic than

the soy proteins and so bound a greater percentage of oil.

Sunflower protein concentra tes were reported to have fat

absorption values in the range 226.5% to 254.9% (Lin et. al.

1974) •

99

-

Table 35. Fat Absorption and Water Absorption of DBSG Protein Concentrates and SOl" Concentrate.

Protein concentrate

DBSG100

DBSG75

DBSG50

Soy concentrate (Acron S)

Fat absorption (X)

173.3 (10.10)·

166.7 (8.63)

193.3 (11.54)

153.3 (11.10)

Water absorption (X)

163.3 (5.73)

166.7 (11.54)

166.7 (5.77)

483.3 (8.55)

a Results are means (standard deviations) of triplicate analyses.

(f) Viscosity

The effect of pH on viscosity of the DBSG protein

concentrates (10% w/v) is shown in Table 36. A 10%

dispersion of the protein concentrates was used to study

viscosity, since for a 5% dispersion the viscosity measured

was almost negligible. Flemin, et. al. (1974) reported that

5% slurries of various prote in concentrates (soy flour,

sunflower concentrates and sunflower isolate) had low

viscosities.

The change in viscos i ty wi th pH varied for the

different protein concentrates. The DBSG100 showed low

viscosities at aIl the pH values. The concentrate (DB8G100)

showed little change in viscosity with pH. Protein

concentrates DBSG75 and DBSG50 which were prepared at lower

100

<-

(~

temperatures than the DBSG100 concentrate displayed higher

viscosities than DBSG100 at pH's above 7. DBSG75 showed

highest viscosity at pH 9.0 while for DBSG50 maximum

viscosity was observed at pH 12.0. The viscosi ties of the

soy concen trate (Acron S) exceeded those of the DBSG

concentrates at higher pH's of Il and 12. The viscosity

difference (at pH Il and pH 12) between the soy concentrate

and DBSG concentra tes was exceedingly large for DBSGIOO and

DBSG75 compared to DBSG50 (Table 36).

Slurries (10% w/v) of the protein concentrates were

heated in a boiling water bath (5 min) in order to study the

effect of tempe rature on viscosity (Table 37). There was

a marked reduction in the viscosities of the DBSG

concentrates and the soy concentrate at higher pH values (9,

11 and 12). However, DBSG75 and DBSG50 show~d an increase

in viscosity at pH 7.0 after beating.

Fleming et. al. (1974) reported that viscosi ty tended

to increase wi th concentration of protein in the product.

In this study DBSGI00 with the hi~hest protein content

(81.8X) compared to DBSG75 (69.6%) and DBSG50 (56.1%) showed

lowest viscosities at comparable pH's. It i8 possible that

the hiah temperature of extraction (lOO·C) used in preparing

the protein concentrate May have affected this functional

property.

101

..,.. ..

-

.-

-

Table 36. Viscosities of Unheated Protein Conoentrates (lOS) at Various pH Values.

------------------------------------------------------------

Protein Concentrate

DBSGI00

DBSG75

DBSG50

Commercial concentrate

pH 7.0

43.3(6.8)

19.3(6.6)

9.6(0.0)

soy 19.3{0.0)

Viscosi ty (cp) 1

pH 9.0 pH 11. 0 pH 12.0

43.3(6.8) b

48. l( 0.0) 43.3(6.8)

582.6(8.3) 312.9(6.8) 77.0(7.2)

168.5(8.3) 808.9(6.2) 919.6(5.3)

38.5(0.0) 1223.0(7.3) 1858.6(9.3)

·Viscosity is expressed in centipoise

b Resul ts are the means of triplicate determinations at the speed setting of 30 rpm •

Table 37. Viscosi ties of Protein Concentrates (10~) at Various pH Values after Heatin ••

Protein Concentrate

DBSGIOO

DBSG75

DBSG50

pH 7.0

19.3(0.0)

216.7(4.4)

19.3(0.0)

Commercial soy concentrate 134.8(7.2)

Viscosi ty (cp) Il

pH 9.0 pH 11.0 pH 12.0

14.4(6.8) b

9.6(0.0) 14.4(6.8)

43.3(6.8) 38.5(3.6) 38.5(0.0)

38.5(0.0) 38.5(0.0) 28.8(0.0)

288.9(0.0) 284.1(6.8) 587.4(5.3)

b Results are the means of triplicate deterllinations at the p~eed setting of 30 rpm •

102

(

As the concentration of the protein samples was

increased from 10% ta 15X, there was a corresponding

increase in the viscosi ties of the protein concentrates at

aIl the pH values studied (Table 38). A general trend

observed for aIl the protein concentrates (15% w/v) was an

increase in viscosi ty wi th an increase in pH. It was also

observed that the protein concentrates prepared at lower

extraction tempe ratures (DBSG7 5 and DBSG50) exhi bi ted

higher viscosities al pH values above 7 compared to DBSGIOO

(Table 38). DBSG50 concentrate was exceedingly viscous at

pH Il and pH 12. It is likely that levels of DBSGIOO above

15% (w/v) are required to achieve high viscosities at

alkaline pH. Fleming et.al. (1974) observed that the

viscosi ty of soy suspensions increased markedly during

alkaline conditions.

Slurries (15% wjv) of the protein concentrates were

heated in a water bath maintained at 55°C (15 min) and their

viscosities determined (Table 39). Heating increased the

viscosities of the DBSGI00 concentrate at the pH values

studied except at pH 9. The concentrate DBSG75 showed a

decrease in viscosity at the pH values examined except at pH

7. It was observed that for DBSG50, heating resul ted in a

103

., .

-

-

Table 38. ViBcosities of Unheated Protein Concentratea (15X) at VariouB pH Values.

Protein Concentrate

DBSGIOO

DBSG75

DBSG50

Viscosity (cp)

pH 7.0 pH 9.0

158.8(4.3)b 207.0(7.6)

96.3(6.6) 852.2(8.9)

19.3(0.0) 760.8(0.0)

pH Il.0 pH 12.0

327.4(3.6) 963.0(6.6)

914.8(8.1) 780.0(4.7)

>1926 >1926

b Results are the means of triplicate determinations at the speed setting of 30 rpm .

Table 39. Viscosi ties of Protein ConcentrateB (15S) a t Various pH Values after heatins.

Protein Concentrate pH 7.0

Viscosity (cp)

pH 9.0 pH 11. 0 pH 12.0

DBSGIOO b

10.4(4.8) 115.5(3.2) 365.9(6.6) 1877.8(6.1)

DBSG75

DBSG50

269.6(4.5) 250.4(0.0) 168.5(6.8) 125.2(3.6)

19.6(0.0) 1020.8(0.0) 1271.2(0.0) 1521.5(0.0)

b ResultB are the means of triplicate determinations at the speed Betting of 30 rpm.

104

l decrease in viscosity at the hiaber pH (pH Il and pH 12) and

an increase in viscosity at pH 9.0.

The effect of tempe rature on viscosi ty varied for the

difterent protein concentratea. Heatin, of prote in slurries

(10%) in a boilina water bath (100· C) reaul ted in a lDarked

'reduction in viacoaity. When protein alurriea (151) we!'e

heated at a lower teaperature (5S·C) the viacoaities of the

protein concentratea were iaproved at specific pH values.

For each protein concentrate there exista particult.:"

condi tions of tellperature, sample concentration and pH in

order to achieve a desired viscoaity. Dependina on the

protein concentration, pH, ionic .tren,th and heat treatment

(time, teaperature) the texture of a protein slurry can

chan,e ta becoae viacous or to fora a solid ael (Hermansson,

1986, 1985, 8athe and Salunkhe, 1981aj flemina et. al.,

1975). The DBSG concentra'tes in this atud1' did not ahow any

aellina properties.

6: Cbaracterization 01 DBSG Prote in CODcentratea

(a) Sodiua dodecyl aulfate~polTacrTl .. ide .el electrophoreais

The ,els used for sodium dodecyl sulfate-polyacrylamide

ael electrophoreais (SDS-PAGE) were 22.2% acrylamide with a

37:1 ratio of acrylaaide to BIS. The protein lysosyme,

t.rypsin, e" albullin and Q(-a_ylase with aolecular weiahts

of 14,300, 23,300, 43,000 and 54,000 daltons respectively,

were used as molecular weiaht lIarkers. A linear re,ression

105

....... , -.

--l 1 ..

equation Cr = -0.990) was generated from the log of the

molecular weiaht of the standard proteine versus their

electrophoretic mobilities (Weber and Osborne, 1969) (Fi~ure

9).

y = 5.735 - 0.030X

y represents the molecular weight

and X the mi.ration distance

SDS-PAGE electrophoresis revealed the presence of 4

8ubuni ts for aIl three protein concentrates prepared at

00 00 0 different tempe ratures (50 C, 75 C, 100 C) (Figure 10). The

molecular weights ranged from 8050 to 59600 daltons (Table

40). Ervin (1986) also reported the presence of 4 protein

components for DBSO prote in concentrates wi th molecular

we ights ranging from 27000 to 45000 daltons. A common

feature for aIl the protein concentrates (DBSGIOO, DBSG75,

DBSG50) was the general appearance of the protein bands.

The first two protein bands (high molecular weight from

42200 to 59600) were very sharp and distinct. while the

four th band was almost indistinguishable (lowest molecular

weight component). The high molecular weight components may

be the major proteins of the DBsa concentrates. Van den

Berg et. al. (1981) and Baxter and Wainwright (1979)

considered the proteins of BSO to be predominantly

glycoproteins, glutelins and high sulfur-containing hordeins

which were associated in allre.ates held together by

intermolecular disulfide bonds and hydrophobie interactions •

106

(~

(

The molecular weight of each protein compone~t was

comparable amon, the OBSG prote in concentrates; this showed

that the tempe rature of extraction did not affect the type

of protein (or polypeptide) that was solubilised.

El-Negoumy et. al. (1979) reported that the average

molecular weights of the ,lutelin fractions of barley

proteins ran.ed from 12000 to 250000 daltons. The three

major protein components of the DBSa concentrates may be

glutelins since their molecular weights ranged form 17200 to

59600 daltons.

107

_ ....

-

-

-.. t:: CI 4.7 ~ -" .. 'a -~ I:! al -u 4.5 a L. .. -:1 .., • -a • ..3 al a .j

Migratian distance ( .. 1

Figure 9. Plot of molecular weight versus migration distance of standard proteins.

108

National Library of Canada

Canadian Theaea Service

NOTICE

THE OUALITY OF THIS MICROFICHE IS HEAVILY DEPENDENT OPON THE OUALITY OF THE THESIS SOBMITTED FOR MICROFILMING.

UNFORTUNATELY THE COLOURED ILLUSTRATIONS OF TRIS THESI~ CAN ONLY YIELD DIFFEREHT TOfJr::S OF GREY.

Bibliothaque nationale du Canada

Service des thlses canadiennes

AVIS

LA OUALITE DE CETTE MICROFICHE DEPEND GRANDEMENT DE LA OUALITE DE LA THESE SOUMISE AU MICROFILMAGE.

MALHEUREUSEMENT, LES DIFFERENTES ILLUSTRATIONS EN COULEURS DE CETTE THESE RE PEUVENT DONNER OUE DES TEINTES DE GRIS.

......

u

-

..,.".

Figure 10. Electrophoretic patterns of the standard proteins and three protein concentrates; l.A.

0\ - &aylase. 1. B. egg albuain. 1. C. trypsin. t.D. lysos~e. 2 DBSGIOO. 3 DBSG75. 4 DBSG50 •

109

(~

<-

Table 40. Mi.ration Distance of Four Standard Proteins and of the Proteins of DBSG Protein Concentrates in SDS-Phosphate Gels.

Molecular Weight

14300

23300

43000

54000

8050

19700

45200

59600

8600

19700

42200

55600

8600

17200

43000

51900

Standard Proteins

DBSG100

DBSG75

DBSG50

Migration Distance (mm)

53

44

38

33

61

48

36

32

60

48

37

33

60

50

38

34

------------------------------------------------------------

110

-

(b) In vitro digestibility

The multienzyme technique reported by Hsu et. al (1977)

was used to determine the in vitro protein digestibility of

DBSG protein concentrates.

Tbe DBSG concentrates prepared at different

showed somewhat similar

digestibility values. The protein concentrates had

digesti bili ties in the range 78.69% to 80.50% (Table -11).

Ervin (1986) reported lower values for DBSO concentrates

prepared at 100°C (73.86%) and 75°C (73.94%). The

preparation procedure for the DBSG protein concentrates in

the present study May attribute to the di(ference in protein

digestibility.

Kakade (1974) reported that the susceptibility of

proteins to proteolytic digestion depends on the

availability of amine acid residues which are compatible

with enzyme specificity. The digestibili ties of the DBSG

protein concentrates were 10lJer than tha t of casei n

(83.21%). Protein diges'libili ty is a rate measurement of

protein hydrolysis by digestive enzyme (Kakade 1974). The

high temperature used in DBSG protein extraction May have

been beneficial to the protein digesti bi 1 i ty, since the

protein concentrate at 1000 e (DBSGI00) had a digestibility

(80.50%) that was significantly different (P < 0.05) than

the protein concentrate at 500 e (78.69%). Heatinl a protein

u to above 50 e results in disruption of secondary and

111

( tertiary structures and is termed denaturation (Finnigan and

Lewis, 1985). A change in the tertiary structure of a

prote in Molecule (by denaturing a.ents e. g. heat) will

expose the enzyme susceptible bonds with a resultant

increased rate of prote in hydrolysis (Kakade, 1974).

Table 41. In Vitro Di.eatibility of Caaein and DBSa Protein Concentratea.

Sample Digestibility (%)

------------------------------------------------------------

•

Casein

DBSGIOO

DBSG75

DBSGSO

83.21 (0.26)·

80.50 (0.25)

79.68 (0.13)

78.69 (0.25)

Resul ts are means (standard deviations) of triplic~te determinations.

(c) Aaino acid co.position

Table 42 shows the amino acid composition of DBSa

protein concentrates (spray dried) which were prepared at

extraction temperatures of 50oe, 7Soe and 100°C. The amino

acid contents of the DBSG concentrates were determined using

reversed phase-HPLC of PITC derivatives (Section II).

The content of each amino acid increased with the

temperature of extraction used for preparation of the

protein concentrates (Table 42). In aIl likelihood, this

112

is related to the fact that DBSG50, DBSG75 and DBSG100 were

found to have protein contents of 56.15~, 69.65~ and 81.79~

respectively (Section IV). On the basis of amine acid

analysis, the protein content of the concentrates were

28.12~ for DBSG50, 39.92~ for DBSG75 and 55.39~ for DBSG100.

The true protein content as determined by amine acid

analysis was approximately 28% less than the protein content

indicated by microKjeldahl analysis. This difference in

protein content May be due to: (1) the microKjeldahl

analysis determines the total nitroaen which includes

ni trogen arising from non-prote in material such as, small

molecular wei,ht peptides and free amine acids, (2) in the

microKjeldahl procedure, the protein content is determined

by multiplication of the factor of 6.25, (3) the amine acid

composition in the present work does not include tryptophan

and (4) the presence of salts durin, sample derivatization

can reduce the amount of aspartic acid and possibly other

acidic and neutral amino acids.

Comparison of the amino acid composit10n of the protein

concentrates (DBSG50, DBSG75, DBSG1 00) wi th the values

recommended by FAO/WHO (1973) showed that the three protein

concentrates were deficient in the amine acids methionine,

cystine and isoleucine. The low content of cystine and

Methionine May have been caused by oxidation of these amine

acids during hydrolysis of samples (Elkin and Wasynczuk,

1987). The essential amino acids (Phenylalanine, tyrosine,

113

l threonine, valine) were present in adequate levels.

The amino acid values obtained for the protein

concentrates (DBSG75, DBSG100) were similar (except for

.lutamic acid, isoleucine and proline) to the results

reported by Ervin (1986) for DBSG prote in concentrates o 0

prepared at the same extraction temperatures (75 C, 100 C).

In our experiments, DBSG75 and DBSG100 showed much lower

levels of .lutamic acid and isoleucine than the protein

concentrates prepared by Ervin (1986). The lower levels of

glutamic acid and isoleucine may be related to the

composition of the brewers' spent grain used in this study.

(

114

...... u

.,. ..

-

-)

Table 42. The A.ino Acid Co.position of DSSa Protein Concentrates (spray dried).

------------------------------------------------------------Amino Acid

a DBSG50 DBSG75 DBSG100

------------------------------------------------------------asp a. 3.07(0.098)b 3.80(0.106) 4.92(0.071)

glu a. 5.14(0.095) 7.31(0.141) 9.48(0.367)

ser 1.54(0.059) 2.14(0.085) 2.94(0.071)

gly 1.36(0.054) 1.73(0.078) 2.35(0.049)

his 0.70(0.029) 0.92(0.000) 1.58(0.262)

arg 1.37(0.036) 1.71(0.056) 2.17(0.191)

thr 1.25(0.052) 1. 70 (0.098 ) 2.48(0.049)

ala 1.36(0.044) 1.87(0.021) 2.56(0.007)

pro 2.89(0.076) 5.76(0.064) 8.42(0.212)

try 0.41(0.092) 0.64(0.042) 1.29(0.134)

val 2.04(0.048) 2.71(0.092) 3.69(0.064)

met 0.56(0.084) 0.75(0.056) 1.27(0.071)

cyst 0.13(0.054) 0.24(0.028) 0.44(0.049)

ile 0.52(0.081) 0.68(0.021) 0.91(0.056)

leu 2.63(0.092) 3.71(0.120) 5.17(0.127)

phe 1.81(0.067) 2.55(0.127) 3.53(0.120)

lys 1.34(0.051) 1.70(0.078) 2.19(0.085)

a Amino acid content is expressed as a amine acid/100 g sample.

b Results are means (standard deviations) of triplicate determinations.

115

(

SUMMARY

The ini t ial screening design showed tha t the cri tical

factors affecting protein extractability from DBSO and

PBSO were time, temperature and particla size of grain.

The concentration of extractant (SDS) between 1% and 5%

had little effect on protein extractability. The

extractability of protein from DBSG (28.14%) was

approximately 3 times higher than the amount (9.53%)

extracted from PBSO.

2. The optimum extraction conditions (determined from the

second order polynomial model) which gave a protein

extractability of 60% were a concentration of 0.64%

Na HPO , a BSG:Extractant a • ratio of 2.5:100, an

extraction temperature of 90° C and extraction time of

98 minutes.

3. Acet.one (50%) was found to be the more efficient

soivent for removal of SDS from the DBSO protein

concentrates than 50% and 95% ethanol. The acetone

washed spray dried procein concentrates, DBSG100,

DBS075 and DBS050 showed SDS contents of 9.80%, 7.38%

and 1.88% respectively.

-le The foaming properties of the three DBSO protein

conc~ntrates (DBSOIOO, DBS075, DBSG50) were pH

dependent. The DBSO concentrates like the soy

116

concentrate, displayed maximum foam capacity at pH 7.0.

DBSGI00 concentrate showed higher foam capaci ty (160-

170) than the soy concentrate (100-143).

5. DBSGIOO, DB3G75 and DBSG50 concentrates showed water

absorption capacities of 166.7",166."" and 163.3%

respectively which were lower than the water absorption

capacity of soy concentrate (483.3%).

6. The DBSO concentrates (OBS0100, DBS075, DBS050) showed

poor emulsion capacity (22-23 ml oil/g sample) and

stability compared to the soy concentrate.

7. Temperature, pH and sample cOTlcentration affected the - viscosity of the OBSO protein concentrates.

Viscosities were maximum at 15% sample concentration

and at alkaline pH (pH 9.0 and pH 11.0) for aIl three

protein concentrates. DBSG75 and DBSOS 0 showed

viscosities of 914.8 cp and> 1926 cp respectively

which were higher than that (327.4 cp) of the OBSG100

concentrate at pH 11.0.

8. DBSGIOü, DBSG75 and DBSG50 concentrates ghowed fat

absorption capacities of 173.3%, 166.7" and 193.3%

respectively, which were higher than that (153.3"> of

soy concentrate.

-.......

117

( 9. DBSGIOO, DBSG75 and DBSG50 concentrates showed protein

digestibilities of 80.50%, 79.68% and 78.69%

respectively which were lower than that (83.211) of

caseine

10. Sodium dodecyl sulfate polyacrylamide

electrophoresis revealed the prepence of fou~ protein

bands for aIl three protein concentrates. The

molecular weights ran.ed from 8050 to 59600 daltons.

11. The prote in concentrates had adequate levels of the

easential amino acids phenylalanine, tyrosine,

threonine and valine but inadequate levels of

(.. methionine, cystine and isoleucine in relation to the

FAO/WHO scores for essential amine acids.

118

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