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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
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vi
xi
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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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-,,' , ~~ ~'Z! . ..;:
(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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112
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 .................................... .
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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
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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.
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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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