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FUNCTIONAL AND STRUCTURAL PROPERTIES OF

MOLECULAR SOY PROTEIN FRACTIONS

TAY SOK LI

(B.Sc. (HONS.), NUS)

A THESIS SUBMITTED

FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

DEPARTMENT OF CHEMISTRY

NATIONAL UNIVERSITY OF SINGAPORE

2005

ACKNOWLEDGEMENTS

My deepest thanks go to my supervisor, A/P Stefan Kasapis for his guidance and

invaluable advice. His interest and encouragement had motivated me to excel and

made this research project very rewarding.

I would like express my gratitude and heartfelt thank you to my co-supervisor, A/P

Conrad O. Perera for his advice and teaching.

My special thanks go to my co-supervisor, A/P Philip J. Barlow for his constant

encouragement and always having a ready smile to offer.

I wish to thank A/P Xu Guo Qin, for his assistance in the usage of atomic force

microscopy and special thanks to Mr Darius Oh Hop Auk and Ms Dai Xue Ni for

their technical support in the operation of AFM. I would also like to thank Dr Yuan

Ze Liang for his technical support in the scanning electron microscopy. I must also

thank all the staff in the department of Chemistry and Food Science Technology

Programme for all the help.

Finally I am very grateful to all the people for making this project possible and

enjoyable.

TABLE OF CONTENTS

SUMMARY

LIST OF TABLES

LIST OF FIGURES

LIST OF ABBERVIATIONS

LIST OF PUBLICATIONS

LIST OF ABSTRACTS AND PRESENTATIONS

CHAPTER 1 Introduction

CHAPTER 2 The 7S and 11S proteins mixtures coagulated by

glucono-δ-lactone (GDL)

CHAPTER 3 The 7S and 11S proteins mixtures coagulated by

chloride or sulphate salt

CHAPTER 4 The effect of κ-carrageenan on the foaming,

gelling and isoflavone content of 11S

CHAPTER 5 The aggregation profile of 2S, 7S and 11S

coagulated by glucono-δ-lactone (GDL)

CHAPTER 6 The functional and structural properties of 2S soy

protein in relation to other molecular protein

fractions

CHAPTER 7 Conclusions and future studies

APPENDICES

I

III

IV

VII

VIII

IX

1

43

67

84

100

117

149

154

I

SUMMARY

Commercially available defatted soy flour was used in order to extract the three

major fractions of the protein (11S, 7S, and 2S). The functional and structural

properties of soy protein fractions were studied.

The gelling and aggregation behavior of the mixed protein systems of the two major

protein fractions with acid and salt coagulants were investigated. It was found that

mixtures of 11S:7S when reacted with glucono-δ-lactone (GDL) will produce

quantifiable gelation behavior based on the premise that higher levels of 7S in the

composite would require longer times of thermal treatment to achieve comparable

physicochemical properties. The mixtures of 11S:7S when reacted with salt

coagulants will result in different types of curd formation. Based on these differences,

the coagulating powers of various salts were determined and found to be in the order

of CaCl2 > MgCl2 > CaSO4 > MgSO4.

One of the ways to improve the nutritional and functional properties of soy protein

was the addition of hydrocolloid to the soy flour before the extraction of protein. It

was found that the addition of κ-carrageenan during the extraction of protein was

able to improve both the nutritional and functional properties. κ-Carrageenan when

added to soy flour during the extraction of 11S caused the 11S to have higher level

of isoflavone, better foaming properties and formed harder gels.

II

The functional and structural properties of 2S soy protein in relation to other

molecular protein fractions were investigated. 2S corresponds to the least percentage

composition in soy protein as compared with 7S and 11S. It was found that 2S

exhibits higher foaming and emulsification properties than 7S, and the latter faired

better than the 11S. We believe that this is due to 2S able to rapidly adsorb into the

air/water or oil/water interface and have higher surface hydrophobicity as compared

with the other soy fraction. The structural properties were monitored using texture

profile analysis (TPA), rheometer, scanning electron microscopy (SEM) and atomic

force microscopy (AFM). The size of the aggregates formed were in the order of 11S

>2S > 7S. This is due to the buffering capacity of 11S which is weaker than 7S thus

maintaining a lower value of pH in the solution (4.5), as opposed to 5.3 for 7S, and

reduced aggregation. It was found that the physical interactions were responsible for

aggregation process of 2S to be faster than 7S. Faster aggregation does not always

leads to harder gel. The large deformation, small deformation modulus and water

holding capacity (WHC) of the protein fractions gels were in the order of 11S > 7S >

2S. The ability to hold water in the 2S gel is the poorest due to the weaker gel

network formed as compared to the other two protein gels. Given time, 7S will

produce a firmer network with a better water holding capacity than that of 2S.

Physical interactions, as opposed to disulphide bridging, were found to be largely

responsible for the changing functionality of the 7S.

III

LIST OF TABLES

Table 1.1. Soy protein fractions

Table 1.2. Functional properties of soy protein (Kinsella, 1979)

Table 2.1. Hardness of soy gel heated for 60, 80 and 100 minutes

Table 2.2. Comparision of L*, hardness and pH of various ratio of 7S:11S

Table 3.1. Descriptions of the protein mixtures with coagulants

Table 3.2. Coagulation results of protein mixtures (4%, w/v) after addition of various

coagulants (0.008M)

Table 3.3. Physico chemical properties of protein mixture (4%, w/v) after coagulated

by calcium sulphate and magnesium sulphate

Table 4.1. Isoflavone Contents in 11S and 11S with κ-carrageenan

Table 4.2. Surface hydrophobicity of 11S and 11S + κ-carrageenan mixture

Table 4.3. The effect of carrageenan on gelling with various salt-coagulants

Table 4.4. Analysis results of “gel” mixtures with addition of carrageenan

Table 5.1. Average size of protein particles formed when 4 % protein solution heated

at 100oC for 10 minutes was deposited onto mica for 1, 2 and 4 minutes.

IV

LIST OF FIGURES

Figure 1.1. Chemical structure of aglycones

Figure 1.2. Chemical structures glucosides

Figure 1.3. Protein solubility profiles of soy protein isolate and soy protein

hydrolysate

Figure 1.4. The conversion of native protein into a protein network according to

heat-induced or cold gelation process

Figure 1.5. Schematic of the two type of network, (a) fine-stranded network; (b)

coarse network

Figure 1.6. The gelation mechanism of soy protein with glucono delta lactone (GDL)

or Ca2+

Figure 1.7 Three types of disaccharides repeating sequence for carrageenans

Figure 2.1. SDS-PAGE of the soy protein fractions, lane (A) is 2S protein fraction;

lane (B) is 7S protein fraction; lane (C) is 11S protein fraction

Figure 2.2a. Comparison of hardness of soy gels heated for 20 and 40 min

Figure 2.2b. Comparison of hardness of soy gels heated for 60, 80 and 100 min

Figure 2.3. Comparison of gumminess of soy gels heated for 20, 40, 60, 80 and 100

min

Figure 2.4. Cohesiveness of soy gels heated for 20, 40, 60, 80 and 100 min

Figure 2.5. L* of soy gels heated for 20, 40, 60, 80 and 100 min

Figure 2.6 pH of soy gels heated for 20, 40, 60, 80 and 100 min

Figure 2.7. WHC of soy gels heated for 20, 40, 60, 80 and 100 min

V

Figure 2.8. Plot of various protein fractions against different lengths of heating time

Figure 3.1. Relationship of coagulating power and various state of curds

Figure 3.2. Turbidity of various slats with 11S protein

Figure 3.3. Turbidity of various slats with 7S protein

Figure 4.1. The chromatograms of the separation of the isoflavones of soy protein

Figure 4.2. Foaming properties of 11S with κ-carrageenan and 11S

Figure 5.1. Images of 11S protein (a) before the addition of GDL, (b-d) after addition

of 0.4% GDL: (b) 11S & GDL deposited onto mica for 1min. (c) 11S &

GDL deposited onto mica for 2 min (d) 11S & GDL deposited onto mica

for 4min




for 4min. Scan size: 3µm by 3µm




for 4min. Scan size: 3µm by 3µm

Figure 5.4. Turbidity measurement of 11S, 7S and 2S protein solutions

Figure 6.1. The interfacial behaviour of the three soy protein fractions, i.e., 11S, 7S

and 2S, as demonstrated for (a) the foaming and (b) the emulsifying

properties

VI

Figure 6.2. pH variation as a function of time following GDL addition at ambient

temperature for the three soy protein fractions.

Figure 6.3. Atomic force microscopy images of the three types of soy protein

aggregates: (a) 11S, (b) 7S, and (c) 2S following addition of 0.4% GDL

and deposition onto mica for 4 min

Figure 6.4. Absorbance readings at 600 nm due to the development of turbidity in the

three types of soy protein aggregates following GDL addition: (a) overall

profile, (b) in the presence of urea, and (c) in the presence of NEM

Figure 6.5. Electron microscopy images of the three types of soy protein gels: (a)

11S, (b) 7S, and (c) 2S


three types of soy protein gels following GDL addition: (a) overall profile,

and (b) in the presence of urea

Figure 6.7. Time course of G' for the three soy fractions at 25°C, frequency of 1 rad/s,

and strain of 0.1%

VII

LIST OF ABBTRVIATIONS

A: Absorbance

AFM: Atomic force microscopy (AFM)

ANS: 1-anilino-8- naphthalene sulfonate (ANS)

BSA: Bovine serine albumin

FI: Fluorescence intensities

G’: Storage modulus

G”: Loss modulus

GDL: Glucono-δ-lactone

H0: Surface hydrophobicity

HPLC: High pressure liquid chromatography

Liq: Liquid

Mr: Molecular weight

NEM: N-ethylmaleimide

pI: Isoelectric point

Ppt: Precipitate

S: Svedberg units

SDS- PAGE: Sodium dodecylsulphate polyacrylamide gel electrophoresis

SEM: Scanning electron microscopy

UV: Ultra violet

WHC: Water-holding capacity

VIII

LIST OF PUBLICATIONS

1. Tay, S.L., Perera, C.O. Effects of glucono-δ-lactone on 7S, 11S proteins and

their protein mixture. Journal of Food Science. 2004, 69(4): FEP139-143.

2. Tay, S.L., Xu, G.Q., Perera, C.O. Aggregation profile of 11S, 7S and 2S

coagulated with GDL. Food Chemistry. 2005, 91, 457-462.

3. Tay, S.L., Tan, Han Yao, Perera, C.O. The coagulating effects of cations and

anions on soy protein. International Journal of Food Properties, in press.

4. Tay, S.L., Perera, C.O., Barlow, P.J. & Kasapis, S. 2006. Foaming,

emulsification and gelation properties of the molecular fractions of a soy

isolate. In Gums and Stabilisers for the Food Industry 13, eds. P.A. Williams &

G.O. Philips, The Royal Society of Chemistry, Cambridge, in press.

5. Sok Li Tay, Stefan Kasapis, Conrad O. Perera & Philip J. Barlow. Functional

and structural properties of 2S soy protein in relation to other molecular protein

fractions. (Submitted)

IX

LIST OF ABSTRACTS AND PRESENTATIONS

1. Singapore International Chemical Conference – 3 (SICC-3). “Frontiers in

Physical and Analytical Chemistry”. 15-17 December 2003. National

University of Singapore, and the Singapore National Institute of Chemistry

(SNIC), Singapore.

Poster presentation: Soy proteins – A study of aggregation process.

2. Regional Conference for Young Chemists 2004 (RCYC 2004). 13-14 April

2004. Universiti Sains Malaysia. Penang, Malaysia.

Oral presentation: The coagulating effects of cations and anions of coagulants

on 7S and 11S protein fractions.

3. Health Science Authority – National University of Singapore (HAS – NUS)

Scientific Seminar. “Health Through Scientific Research”. 19 May 2004.

Health Science Authority and National University of Singapore, Singapore

Oral presentation: Functional Properties of Soy Protein Fractions.

4. Institute of Food Technologists (IFT) Annual Meeting + Food Expo®.12-16

July 2004. Institute of Food Technologists. Las Vegas, Nevada, USA.

Oral presentation: Soy protein - Aggregation and gelation;

X

5. Institute of Food Technologists (IFT) Annual Meeting + Food Expo®.12-16

July 2004. Institute of Food Technologists. Las Vegas, Nevada, USA.

Poster presentation: Effect of extraction and UHT treatment conditions on

isoflavones and protein-quality during soymilk manufacture.

6. Gums and Stabilizers for the food industry. 20-24 June 2005. The Food

Hydrocolloid Trust. The North East Wales Institute. Wrexham, United

Kingdom.

Oral presentation: Foaming, emulsifying and gelation properties of the

molecular fractions of soy fractions.

1

Chapter 1

Introduction

2

1.1 SOY

The soybean belongs to the family Leguminosae and the genus name is Glycine L.

(Clarke and Wiseman, 2000a). It is the source of inexpensive and high quality

protein. Soybeans have long been a staple of the human diet in Asia, especially as

soymilk or tofu (Poysa and Woodrow, 2002). The consumption of soy based

products is increasing in North America due to an increase in Asian immigrants and

an increase in the recognition of the health benefits that this food product (Murphy et

al., 1997). In recent years, interest in animal free foods has increased due to concern

such as mad cow disease, and denying animal intakes of any kind due to ethic and

religious reasons. The food industry is moving towards developing food products

that can be substitute with soy (Nunes et al., 2003).

Among the legumes, soybean has high protein content. Soybean is made up of

approximately 38% protein; 18% oil; 30% carbohydrate and 14% ash and moisture.

In addition, soy beans also contain minerals such as iron, copper, manganese,

calcium, magnesium, zinc, cobalt and potassium; vitamins such as thiamin (B1) and

riboflavin (B2); phosphorus; and minor components such as protease inhibitors,

phenolic compounds and lectin (García et al., 1997).

It is increasingly recognized that certain foods and their components may have health

benefits in additional to their nutritional value. Soy foods provide protein of equal

quality to other proteins and without saturated fats and cholesterol. Furthermore, soy

3

protein is acknowledged by the Food and Drug Administration (FDA), to lower

serum cholesterol level (Stein, 2000). Anderson et al., (1995) found that soy protein

is able to lower lipid content in blood. FDA allows food manufacturers to place a

health claim (healthy heart) on the package labels of food products containing more

than 6.25g of soy protein per serving. In order to reduce the risk of heart disease,

FDA recommends that consumers incorporate four serving of at least 6.25g of soy

protein into the diet for a total of at least 25g of soy protein per day (Stein, 2000).

Traditional soy foods include soymilk, miso (fermented soybean paste), natto

(fermented whole soybeans), soy sauce, tofu, soy milk and dried bean curd sheet

(Fukushima, 1991). Modern technology has created more interesting ways to include

soy and soy protein in the daily diet. Now there are more food products that can be

substituted with soy and are known as soy based products. Examples of these

products include soy infant formulae, meat alternatives and non diary soy desserts

such as soy ice-cream, cheese, yogurt. Soy ingredients are becoming more popular

due to their desirable functional properties; including gelling, emulsifying, fat-

absorbing and water binding properties (Nunes et al., 2003).

4

1.2 SOY ISOFLAVONE

The major phytoestrogens in food are isoflavones in soy food (Murphy et al., 1999).

There are three main types of isoflavones in soybeans, which exist in four chemical

forms (Figure 1.1 and 1.2). They are the aglycons, daidzein, glycitein and genistein;

the glucosides, daidzin, glycitin and genistin; the acetylglucosides 6”-O-

acetyldaidzin, 6”-O-acetylglycitin and 6”-O-acetylgenistin; the malonyl glucosides

6”-O-malonyldaidzin, 6”-O-malonylglycitin and 6”-O-malonyl genistin (Wang and

Murphy 1994).

R1

R2

Compounds

H

H

Daidzein

OH H Genistein

H OCH3 glycitein

Figure 1.1. Chemical structure of aglycones (Wang and Murphy 1994).

5

Figure 1.2. Chemical structures glucosides (Wang and Murphy 1994).

R3

R4

R5

Compounds

H

H

H

Daidzin

OH H H Genistin

H OCH3 H Glycitin

H H COCH3 6”-O-Acetyldaidzin

OH H COCH3 6”-O-Acetylgenistin

H OCH3 COCH3 6”-O-Acetylglycitin

H H COCH2COOH 6”-O-malonyldaidzin

OH H COCH2COOH 6”-O-Malonylgenistin

H OCH3 COCH2COOH 6”-O-Malonylglycitin

6

There is a growing literature on the health protective effects of soy foods.

Epidemiological studies have suggested that the consumption of soybeans and soy

foods is associated with lowered risks for several types of cancers, including breast,

prostate and colon (Messina et al., 1997; Messina 1995; Coward et al., 1993),

cardiovascular diseases (Schultz, 1998; Anderson and Johnstone, 1995) and bone

health (Bahram et al., 1996; Nurmi et al., 2002).

The concentrations of the twelve chemical forms of isoflavones vary in soy foods as

they are affected by the processing methods. The unprocessed soybean will contain

mainly 6”-O-malonyl forms. The malonyl forms will convert to β-glycosides during

extraction process at room temperature as well as during heat treatment during the

production of soymilk and tofu. However, heat treatment during the toasting of

hexane extracted soy flours will produced 6”-O-Acetyl forms (Murphy et al., 2002).

Thus in soy milk and tofu there are mostly glycosides and in toasted defatted soy

flour there are mainly acetyl forms.

7

1.3 SOY PROTEIN

The bulk of soy proteins are globulins, characterized by their solubility in salt

solutions. The solubility of soy proteins in water is strongly affected by the pH.

About 80% of the protein in raw seeds or unheated meal can be extracted at neutral

or alkaline conditions (Kinsella, 1979). As the acidity is increased, solubility drops

rapidly and the proteins precipitate at pH 4.5 – 4.8. This is the isoelectric region of

soybean proteins, taken as a whole, and these proteins are often called acid-

precipitable proteins (García et al., 1997). Figure 1.3 shows a typical solubility curve

for soy protein isolate and soy protein hydrolysate. As seen in the solubility profiles,

high solubility can be observed at pH ≥ 6 and pH ≤ 3 for soy protein isolate

(Achouri et al., 1998).

Figure 1.3. Protein solubility profiles of soy protein isolate ( ) and soy protein

hydrolysate ( ) (Achouri et al., 1998).

8

Generally the current protein fractionation techniques make use of the differences

between the isoelectric points between the various protein fractions. Both bench-

scale and pilot-plant-scale methods for fractionating soy protein fractions from

defatted flour is based on this technique (Nagano et al., 1992; Wu et al., 1999; Wu et

al., 2000). After fractionating the soy protein fractions, electrophoretic analysis is

commonly used to identify the soy protein fractions (Riblett et al., 2001; Roesch et

al., 2005) based on its molecular weight.

Ultracentrifugation, gel filtration and electrophoresis can also be used for more

precise fractionation (Kinsella, 1979). The soy protein fractions have also been

characterised by their sedimentation constants. (S stands for Svedberg units). The

numerical coefficient is the characteristic sedimentation constant in water at 20 °C

(Kinsella, 1979). The composition (Fukushima, 1991), molecular weight (Fukushima,

1991), the pI (Dreau. et al., 1994), methionine and cysteine content (Clarke and

Wiseman, 2000a) of the soy protein fractions are summarized in Table 1.1.

9

Table 1.1. Soy protein fractions

Protein

fraction

Content

(%)

Molecular weight

(thousands)

pI

Methionine

(mg/g)

Cysteine

(mg/g)

2S

15.0

18-33

4.5

50

48

7S 34.0 180-210 4.8 14 22

11S 41.9 300-350 6.4 15 30

15S 9.1 600 - - -

The 11S and 7S fraction contents have been found to vary with soybean variety and

environment (Cai and Chang, 1999). Cai and Chang (1999) reported that 11S and 7S

contents from 13 soybean varieties were 7.3-9.9 and 14.1-22.9% on the dry matter

basis, respectively.

10

1.3.1 2S fraction

The 2S soy protein fraction consists of low molecular mass polypeptides (in the

range of 8000–20,000 Da) and consists of Bowman-Birk and Kunitz trypsin

inhibitors, cytochrome C, and α-conglycinin (Catsimpoolas and Ekenstam, 1969;

Wolf, 1970).

Trypsin inhibitor is an allergen (Lin et al., 2004). The Bowman-Birk trypsin inhibitor

has 7 disulphide bonds and the Kunitz trypsin inhibitor has 2 disulphide bonds per

molecule (Clarke and Wiseman, 2000b).

The 2S fraction in soybean are unusually rich in charged residues like aspartic acid

(Koshiyama et al., 1981; Lin et al., 2004), they are very stable to temperature and

chemical denaturants. 2S soy protein was found to retain its secondary structure at

temperature as high as 97oC (Lin et al., 2004). It is stable between pH 3 to pH 10

((Koshiyama et al., 1981).

11

1.3.2 7S fraction

The 7S fraction is highly heterogeneous. 7S contains β-conglycinin (major form), γ-

conglycinin, α-amylase, lipoxygenase and hemagglutinin (Nielsen, 1985). It has a

molecular mass in the order of 150–190 kDa. Three different β-conglycinin are

known (α’, α and β with molecular mass of 65, 62 and 47 kDa, respectively). All the

three subunits are rich in aspartate, glutamate, leucine and arginine. All of the

subunits are glycoproteins and contain 40-50g carbohydrate per kg (Clarke and

Wiseman, 2000a). Generally, β-conglycinin forms a trimer (7S with seven possible

combinations) at ionic strength of 0.5, and a hexameric form (9S) at low ion

concentration of 0.1 ionic strength (Koshiyama, 1983). The 9S is a dimer of two

trimers facing each other.

The glass transition temperature of 7S is found to be around 70oC (Wagner et al.,

1996). The extent of disulphide crosslinking of 7S is limited because 7S fraction

contain up to 4 sulphur atom (Chronakis et al., 1995). High pressure will denature

protein and it was found that the high pressure denaturation of 7S was 300 MPa, and

this pressure is lower than the pressure to denature 11S. It was suggested that the

lower pressure value was due to the lack of disulphide bond in 7S as compared to

11S (Zhang et al., 2005).

12

1.3.3 11S fraction

The 11S fraction consists of glycinin, the principal storage protein of soybeans. 11S

has a molecular mass of 320–360 kDa. At ambient temperatures and pH 7.6, 11S is a

hexameric structure composed of six acidic (Mr 20 000 - 22 000) and six basic (Mr

35 000 – 40 000) subunits (García et al., 1997). The monomeric subunits have a

generalized structure A-S S-B, where A represent the acidic polypeptide; B present

the basic polypeptide and S-S is the single disulphide bond that links the two

polypeptides (Clarke and Wiseman, 2000a). Each acidic and basic polypeptide is

linked by a single disulphide bridge, except for the acidic polypeptide A4 (Staswick

et al., 1984).

The glass transition temperature of 11S is found to be around 86oC (Wagner et al.,

1996). The 11S soy fraction in a 0.5 ionic strength buffer appears to be stable to

temperature of up to 70oC. Above 70oC, 11S will become increasingly turbid and

will precipitate at 90oC (Yamauchi et al., 1991). The thermal denaturation of 11S is

very sensitive to ionic strength. Increasing sodium chloride concentration from 0 to

1M increased the denatured temperature of 11S by 20oC (Brooks and Morr, 1985).

The 11S fraction was denatured after treatment at 400 MPa and this mechanism

might involve the rupture of the disulphide bonds (Zhang et al., 2005). The 11S is

able to have significant crosslinking during gelation could be due to the presence of

42 sulphydryl groups per molecule (Chronakis et al., 1995).

13

1.4 FUNCTIONALITY OF SOY PROTEIN

Functional property of protein is defined as any property of the protein, except its

nutritional ones that affect their performance and behaviour in food systems

(Kinsella and Whitehead, 1989). Generally, the functional properties of food proteins

may be classified into three main groups: (a) viscosity and water holding; (b)

emulsification and foaming; and (c) aggregation and gelation properties (Galazka et

al., 2000). The functional properties performed by soy protein in prepared food

systems are shown in Table 1.2 (Kinsella, 1979).

The functional properties of proteins are impaired near their isoelectric points, as is

the case of most acidic foods (Kinsella and Whitehead, 1989). Protein functionality

is affected by changes in native state during processing because of protein unfolding

and exposure of the interior hydrophobic regions. Ionic strength and pH of the food

systems in which the ingredients are used also further influence the protein

functionality (Rickert et al., 2004.)

14

Table 1.2. Functional properties of soy protein (Kinsella, 1979).

Functional property

Mode of action

Food system

Solubility

Protein solvation, pH

dependent

Beverages

Gelation Protein matrix formation

and setting

Meat, curds, cheese

Foaming Forms stable films to trap

gas

Whipping toppings,

chiffon dessert

Emulsification Formation and

stabilization of fat

emulsion

Sausage, soup and cakes

15

1.4.1 FORMING AND EMULSIFYING PROPERTIES

For the formation of foams and emulsions, proteins should be water soluble, and it

must rapidly diffuse to the interface, adsorb, reduce the interfacial tension, and then

reorient to form a cohesive film at the air-water or oil-water interface (Diftis and

Kiosseoglou, 2003). Foaming and emulsifying are important properties of proteins

essential in many food formulations.

It was found that generally the foaming and emulsifying of 7S is better than 11S

fractions (Bian et al., 2003). 11S was found to be a rather poor foaming and

emulsifying agent due to the difficulty in adsorbing at the air-water interface (Rickert

et al., 2004). This was attributed to low surface hydrophobicity, low chain flexibility

and high molecular weight of the protein (Wagner and Guéguen, 1995). Utsumi et al.

modified 11S at a molecular level thus designing recombinant systems where the

extent of hydrophobicity of the C-terminal region would determine largely the

emulsifying properties (Utsumi et al., 2002). In the case of 7S, the extension region

found in subunits α and α’ possessed the required functionality for good solubility

and emulsification.

The processing method will affect the functional properties of the soy protein

fractions. Bian et el. (2003) found that even though the emulsifying properties of the

7S is better than 11S in both modified Nagano process and simplified pilot-plant

process, the emulsifying properties and the foam stability of the soy fractions

16

produced by the simplified pilot-plant process were better than the modified Nagano

process.

Individual subunits of 11S may posse different functional properties. The

emulsifying properties of the acidic subunits of 11S (AS11S) is better than native

11S or heat-denatured 11S (Liu et al., 1999). Liu et al. (1999) found that the

emulsion formed by the acidic subunits remained very stable for more than a month

as compared to native 11S which last for only 2 days.

The foaming properties of glycinin can be enhanced by modification of its structure.

Wagner and Guéguen (1999) reported that dissociation, deamination and reduction

of 11S improved its ability to adsorb at the interface and make it’s a better foaming

and emulsifying agent; and Wagner and Guéguen (1995) found that with mild acid

treatment, the foaming ability of 11S was increased. Kim and Kinsella (1987)

showed that reduction of glycinin with DTT (dithiothreitol) increased the foam

stability of 11S.

It was found that pH plays an important role in the interfacial properties of soy

protein fractions. For example at pH 5, 11S and 7S were found to have better

emulsifying properties than the soy protein isolate (Rickert et al., 2004).

17

1.4.2 GELLING PROPERTIES

In food products such as sausages, cheese and tofu, protein gelation is important in

order to obtain desirable textural properties (Alting, 2003). Gelation is defined as

aggregation of denatured molecules with a certain degree of order and resulting in

the formation of a continuous network (Wong, 1989). The general definition of the

gel point is the point where storage modulus, G’, becomes greater than the

background noise (Renkema et al., 2001).

Gelation of a solution of proteins can be induced in various ways. Heat-induced

gelation is responsible for the structure present in many heat-set foods (Totosaus et

al., 2002). Besides heat-induce gelation, hydrostatic-pressure-induced gelation is the

second type of physically induced gelation. Both gelation methods are single-step

methods. Under the conditions applied, the processes of the denaturation of the

protein molecules and subsequent aggregation to a space-filling protein network

proceed simultaneously. Other gelation methods are salt-induced gelation and acid-

induced gelation. Salt- or acid-induced types of gelation consist of two steps. The

direct addition of acid or salt usually does not result in the formation of a protein

network as the gelation step has to be preceded by an activation step in which the

protein molecule denatures and forms soluble protein aggregates. This process is

known as cold gelation of globular proteins (Atling, 2003). The gelation steps of

both hot and cold gelation are illustrated in Figure 1.4.

18

Figure 1.4. The conversion of native protein into a protein network according to

heat-induced or cold gelation process (Alting, 2003).

Generally there are two types of gel networks, the fine-stranded and the coarse

networks. Figure 1.5 shows the two type of network. In the fine-stranded gels, the

proteins are unfolded and attached to each other like a ‘string of beads’. The coarse

gels are formed by random aggregation of the protein (Hermannsson, 1994). It was

reported that heat induced gelation of 11S formed gels like the ‘string of beads’

while cold gelation of 11S, 7S and its mixture formed gels by random aggregation

(Kohyama et al., 1995).

19

Figure 1.5. Schematic of the two type of network, (a) fine-stranded network; (b)

coarse network (Hermannsson, 1994).

Protein-protein interactions responsible for the formation of protein gel consists of

network of protein molecules make of covalent (disulphide bonds) and/or non-

covalent bonds (hydrogen bonds, electrostatic interactions and hydrophobic

interactions). Factors like the protein concentration, pH, temperature, ionic strength,

type of ions and pressure will after the type of bonds formed (Totosaus et al, 2002).

Doi and Kitabatake (1997) reported that at low ionic strength or at pH value far from

the isoelectric point (pI) of the protein, the electrostatic repulsive forces were

predominant. Electrostatic repulsive forces will not favor the formation of random

aggregation and will favor more linear network to form, thus resulting in transparent

gel (Figure 1.5a). And when the gelation occurs at high ionic strength or at pH near

pI of the protein, hydrophobic and van der Waals interaction will predominant and

20

allowed the denarured protein to aggregate randomly and result in the formation of

turbid gel (Figure 1.5b). The arrangement of the protein network will have impact on

the gel properties like rheological behavior, sensory quality and water-holding

capacity (Alting 2003).

21

1.4.2.1 Heat induced gelation

Heating- set gels of soy protein fractions – 7S and 11S have different hardness and is

especially affected by the heating temperature. Nakamura et al., (1985) reported that

11S formed harder gels than 7S when heated at 100oC, pH 7.6 and ionic strength of

0.5. Shimada and Matsushita (1980) reported that 7S formed harder gels than 11S

when heated at 80oC for 30min in water at pH 7.5. Thus the ability of β-conglycinin

to form gels at lower temperature is an important property of protein food ingredients

used to make food such as sausage and ham as high temperature will affect the

texture of these products (Nagano et al., 1996).

It was found that the network of the heat induced soy protein gel were formed

through a combination of forces – hydrogen bonding, hydrophobic interactions and

disulfide bonding (Nakamura et al., 1986). The water holding capacity of the heat

induced gels decreased with increasing salt concentration (NaCl and CaCl2) due to

the increasing open matrix of the gel network (Puppo and Añón, 1998).

22

1.4.2.2 Cold induced gelation

Glucono delta lactone (GDL) induced gelation has been extensively studied.

Kohyama et al. (1995) postulated that heat will cause the soy protein to become more

negatively charged. Then the release of protons induced by GDL will neutralizes the

net charge of the protein. As a result, the hydrophobic interaction of the neutralized

protein molecules becomes more predominant and induces the random aggregation

of proteins, leading to gel formation. This gelation mechanism is shown in Figure 1.6.

Figure 1.6 The gelation mechanism of soy protein with glucono delta lactone (GDL)

or Ca2+. The blue portions denote the hydrophobic regions and e denote electron

(Liu, 1997).

23

Kohyama and Nishinari (1993) reported that the breaking stress of the 7S gel was

smaller than for 11S. Similarly, the rate of gelation for the 7S was slower than 11S.

In addition, the 7S gel possessed a higher pH value but lower cohesiveness,

gumminess and lightness than the 11S preparation at 4% solids (Kohyama et al.,

1995; Tay and Perera, 2004). Scanning electron microscopy (SEM) showed that 11S

produced a coarse network of 2-3 µm in pore size whereas 7S exhibited a finer

structure of about 0.5 µm; and all the gels formed with GDL belonged to the random

aggregation type (Kohyama et al., 1995).

In addition to the study of the cold gelation of soy protein fractions, the cold

gelation of hydrolysed soy protein was also studied. Kuipers et al. (2005) had found

that hydrolysed soy protein with subtilisin Carlserg will gelled with GDL at a higher

pH. The nonhydrolyzed soy protein gels at pH ~6 while the hydrolysate gelled at pH

7.6.

Tofu (soybean curd) is a gel-like food made by addition of coagulants to heated

soybean milk to produce a soy protein gel which traps water, lipids, and other

constituents in the matrix (Poysa and Woodrow, 2002). Salts like magnesium

chloride or calcium sulfate are traditionally used as coagulants, but recently,

glucono-δ-lactone (GDL) has been widely used in tofu-processing because of the

advantage of easily formed homogeneous gels (Kohyama and Nishinari, 1993).

24

Several studies found that cations and anions will affect the gelation properties. Salts

and proteins form coagulates when metal ions such as Ca2+ or Mg2+ form bridges

with the negatively charged protein. This cross-link is due to the electrostatic

interactions between the cations and the proteins (Karim, 1998), this gelation

mechanism is shown in Figure 1.6.

Saio et al. (1969) reported that 11S gels made in the presence of calcium sulfate

were much harder than crude 7S gels. It was not mentioned how the anions played a

role in the gelation mechanism, it had been noted by Wang and Hesseltine, (1982)

that anions had a strong effect on the water-holding capacity of the gels.

Studies found that there is a positive correlation between 11S content and the 11S/7S

protein ratio with the tofu firmness (Cai and Chang, 1999). They also found that tofu

formed from soybeans having different subunits of 11S have different breaking stress

value. 11S fraction has five different subunits: group 1are the A1aB1b, A2B1a, and

A1bB2; group IIa is A5A4B3 and group IIb is A3B4 (Tezuka et al., 2000). Tezuka et

al. (2000) found that the breaking stress value of soy protein curds made from

soybeans of subunits IIa was the highest followed by soy protein curds made from

soybeans of subunits IIb and soy protein curds made from soybeans of subunits I was

the weakest. 11S subunits were found to play an important role in the firmness of the

tofu. Group 1 subunits (Tezuka et al., 2000) and A5A4B3 (Fukushima, 1991) were

found to be related to the firmness of tofu.

25

1.4.2.3 AGGREGATION

Gelation and aggregations are closely relate, Mills et al. (2001) reported that at 1%

concentration, 7S was able to form in soluble macroaggregates which may be the

precursors of protein gel formation. It had been found that depending on the

conditions, both glycinin (11S) and β-conglycinin (7S) are able to form large

aggregates when heated (Mills et al., 2001).

Aggregation is generally referred as the formation of complexes of higher molecular

weights due to protein-protein interactions (Gossett, 1984). Lakemond et al. (2003)

reported that aggregation size is related to the thickness of the strands of the gel.

Hence protein aggregation plays an important part in gelation. In the past the effects

of cations like calcium and magnesium on aggregation were studied but only a few

studies have been done on the effects of anions on aggregation. Lately, Molina and

Wagner (1999) found that anions like chloride and citrate also play an important role

in the protein aggregation process.

26

1.5 POLYSACCHARIDES

Polysaccharides play a key role in modifying the textural properties of food systems

(Karium et al., 1998). The polysaccharides used in food can be referred to as gelling

and thickening agents, gums and stabilizers or hydrocolloid. Protein–polysaccharide

mixed systems have been extensively studied and widely used in the food industry in

the last decades, because the biopolymers interactions are of great importance to

develop products with specific textural characteristics (Kasapis and Al-Marhoobi,

2005; Tolstoguzov, 1998; Braudo, 1998).

Hydrocolloids such as κ-carrageenan, xanthan gum and propylene glycol alginate are

able to hold and maintain water content in food and have found to result in greater

genistein retention during soy protein concentrate production (Pandjaitan et al.,

2000).

Carrageenans are present in numerous species of red seaweed and are a group of

sulphated linear polysaccharides of D-galactose, and 3,6 anhydro-D-galactose (Trius

and Sebranek, 1996). Carrageenans can exist as negatively charged polymers over a

wide range of pH, and are capable of forming complexes with proteins in the

presence and absence of calcium ions (Bernal et al., 1987). Carrageenans have strong

electrolyte characteristic because of their sulphate groups and are classified into three

types: keppa (κ-), iota (ι-) and lamda (λ-) carrageenans according to the number (one,

two or three) sulphate groups per repeated units of disaccharides, respectively

27

(Molina Ortiz et al., 2004). The three types of disaccharides repeating sequence for

carrageenans are shown in Figure 1.7. The free acid form of carrageenan is unstable,

and caraageenans are commonly sold as a mixture of sodium, potassium and calcium

salts (Nussinovitch, 1992).

Carrageenans are extensively in food industry as gelling, thickening and stabilizing

agents (Trius and Sebranek 1996). Examples of dairy applications of carrageenans

are puddings, whipped products and milks (Nussinovitch, 1992). Carrageenans and

soy bean proteins can also be used together in food industry as gelling and viscous

agents (Molina Ortiz et al., 2004). Baeza et al. (2002) found that there is an

improvement in the texture and viscoelasticity of the mixed gels formed by κ-

carrageenan and soy protein due to the synergistic effects between them.

Figure 1.7 Three types of disaccharides repeating sequence for carrageenans

28

1.6 OUTLINE OF THE THESIS

This thesis developed understanding of the various interaction processes,

physicochemical and structural/functional properties of the soy protein fractions (2S,

7S and 11S). The protein-protein and the protein-coagulant interactions of the two

major protein fractions (11S and 7S) were studied first to investigate the effects of

these two fractions on the cold-induced gelation mechanism. In addition, the protein-

polysaccharide and isoflavone-polysaccharide interactions were also investigated.

Thus the effect of the addition of κ-carrageenan during the extraction of protein to

improve the nutritional and functional properties of the major protein fraction was

mapped out. Finally, the functional and structural properties of the 11S and 7S were

contrasted with those of the 2S fraction of soy protein that has not been considered in

earnest by the research community. Considerable advance was achieved by unveiling

the distinct foaming, emulsification, water holding, aggregation and gelation

properties of 2S as compared with those of the more established molecular

counterparts. Results argue for the increasing significance of including 2S in

preparation for tailor-made applications of soy proteins.

Soybean proteins contain two major globulins, 7S and 11S, which show different

thermal transition temperature and gel-forming properties (Kohyama and Nishinari,

1993). Due the differences in the gelation properties of soy protein fractions, many

researchers have attempted to correlate these proteins with tofu quality (Mujoo et al.,

2003). In chapter 3, the correlation of these two protein fractions was carried out by

29

studying the physicochemical properties of the 7S and 11S proteins fractions

coagulated by glucono-δ-lactone (GDL).

GDL was used in the earlier part of the study because it is able to form a

homogenous gel with the protein upon heating. Different type of coagulants will

form gels of different physicochemical properties, thus the effects of other

coagulants should also be studied. Chapter 4 of the study involved the study of the

effect of chloride-type and sulphate-type coagulants on 7S and 11S proteins fractions.

The consumption of soy proteins and its associated isoflavones may provide health

benefits such as prevention of cardiovascular diseases and cancer. Although

isoflavones have low hydrophilic properties, some isoflavones are lost during

aqueous extraction of soy protein fractions. Hydrocolloids such as κ-carrageenan

when added to soy flour have found to improve the retention of isoflavones during

the extraction of soy protein concentrates. The addition of carrageenan during the

extraction of the protein might affect the functional properties of the protein. In

chapter 5 we are interested to investigate if the addition of κ-carrageenan to soy flour

during the extraction of soy protein fraction improves the nutritional and functional

properties of the soy protein fraction. The isoflavones content, the foaming and

gelling properties of 11S with κ-carrageenan added during the extraction of 11S were

studied.

30

The formation of aggregates could be essential or unwanted for the food industry.

Aggregation is essential as it is a prerequisite for the formation of gels (Alting, 2003).

However, the formation of aggregates in certain instances is not desirable. Formation

of aggregates after certain food processing operations could cause undesirable mouth

feel, textural changes and changes in physical properties of the foods (Persson and

Gekas, 1994). Chapter 6 of the study involved the study of the protein aggregation of

the three soy protein fractions - 11S, 7S and 2S.

Different soy protein fractions will have different functional properties. Many studies

have been carried out on the major soy protein fractions, namely, the 7S and 11S, but

very little information is available on the 2S fraction. Although, 2S is presents as a

minor protein fraction, it might have significant effect on the foaming and

emulsifying properties of the protein system. The chapter 7 of the study involved the

study of the foaming and emulsifying properties of 2S soy protein in relation to other

molecular protein fractions soy protein fractions – 11S and 7S. Gelation and

aggregations are closely related. Gelation takes place when protein aggregates form a

network (Lakemond et al., 2003). In this chapter the gelation and structural

properties of the soy protein fraction were also studied. In addition, the relationship

between aggregation and gelation and the study of the chemical and physical

interactions during gelation and aggregation of the soy protein fractions were

investigated.

31

1.7 OBJECTIVES

The overall objective of this thesis is to develop understanding of the physico-chemical

properties of single and mixed soy protein fractions, and in particular the 2S fraction that

has not been considered in earnest by the research community.

The specific objectives are:

• To study the foaming and emulsifying properties of individual fractions

• To study the aggregation profile of individual fractions

• To study the gelation profile relation of individual fractions

• To study the relationship of gelation with aggregation

• To study the interplay of gelation properties of 7S and 11S mixture by

varying the polymeric composition and time of thermal treatment

32

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their protein mixture. Journal of Food Science. 2004, 6, FEP 139-143.

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67) Tezuka, M.; Taira, H.; Igarashi, Y.; Yagasaki, K.; Ono, T. Properties of tofus

and soy milks prepared from soybeans having different subunits of glycinin,

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behavior, hydrophobicity, and some functional properties of acid-treated soy

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reducing treatment on structural and surface active properties of soy glycinin.

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75) Wang, H. L.; Hesseltine, C. W. Coagulation conditions in tofu processing.

Process Biochemistry. 1982, 1, 8-12.

76) Wang, H.J.; Murphy, P.A. Isoflavone content in commercial soybean foods.

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77) Wolf, W. J. Soybean proteins: Their functional, chemical and physical

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78) Wong, D.W.S. mechanism and theory in food chemistry. Van Nostrand

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14.

43

Chapter 2

The 7S and 11S proteins mixtures coagulated by


44

2.1 ABSTRACT

Blends of 7S and 11S proteins with added glucono-δ-lactone (GDL) were

investigated to study the effects of protein composition on gelation. The pH, water-

holding capacity (WHC), textural and optical properties of the gels formed were

studied at a constant temperature as a function of time. Generally, high 11S to 7S

ratios produced gels of higher hardness, cohesiveness, gumminess and +L* values

than those of the rest. 11S formed faster acid-induced gels compared to those

containing low proportions of 11S. Certain proportion of 7S:11S proteins heated for

different lengths of time formed gels of similar pH having similar physical properties.

From the data it was predicted that fractions of 11S:7S differing by 1:10 form gels

with similar physico-chemical properties when the heating time differed by 20

minutes.

45

2.2 INTRODUCTION

Soybeans have long been a staple of the human diet in Asia, especially as soymilk or

tofu, which is prepared from soymilk (Poysa and Woodrow, 2002). Soybeans are an

inexpensive, high quality protein source. Soymilk and tofu consumption is increasing

in North America due to an increase in Asian immigrants, greater acceptance of soy

foods by the general population, and increased recognition of the health benefits of

soy foods, especially by those who wish to reduce their consumption of animal

products (Murphy et al., 1997).

Tofu (soybean curd) is a gel-like food made by addition of coagulants to heated

soybean milk to produce a soy protein gel which traps water, lipids, and other

constituents in the matrix (Poysa and Woodrow, 2002). Salts like magnesium

chloride or calcium sulfate are traditionally used as coagulants, but recently,

glucono-δ-lactone (GDL) has been widely used in tofu-processing because of the

advantage of easily formed homogeneous gels (Kohyama and Nishinari, 1993).

Different coagulants used in soy gels result in products with textural characteristics

varying from soft to firm and with moisture content ranging from 70 to 90% (de Man

et al., 1986). These give rise to different textural and flavor properties. The texture of

tofu should be smooth, firm, and coherent, but not hard and rubbery. The amount of

soy protein used to make the soymilk is critical for tofu yield and quality (Poysa and

Woodrow, 2002).

46

Soybean proteins contain two major globulins, 7S and 11S, which show different

thermal transition temperatures and gel-forming properties (Kohyama and Nishinari,

1993). The isoelectric point of 7S globulin (4.5-5.0) is lower than that of 11S (6.3-

7.0) (Brooks and Morr, 1985). Due to the differences in the gelation properties of

soybean protein fractions, many researchers have attempted to correlate these

proteins with tofu quality (Mujoo et al., 2003). Several studies found that glycinin

(11S) and β-conglycinin (7S) had some relationships with tofu texture (Cai and

Chang, 1999). Saio et al. (1969) reported that 11S gels made in the presence of

calcium sulfate were much harder than crude 7S gels and that 11S mainly determined

the hardness of tofu gels. Hashizume et al. (1975) also observed that 11S gels

coagulated by GDL showed greater breaking force than crude 7S gels.

The pH of the protein gels can be changed by changing the length of heating of the

gels, and/or by changing the ratio of 7S and11S. During the period of heating, GDL

hydrates to become acidic and hydrolysis is accelerated by heat. The pH of the gels

can be altered by controlling the length of heating.

The buffering effect against protons is stronger in 7S than in 11S in the pH range

from 4 to 5. When the same amount of protons was added to both, 7S and 11S

solutions, the pH of 7S systems remained higher than that of 11S (Kohyama et al.,

1995). Hence different proportion of 7S and 11S will form gels with different pH.

47

Thus controlling the time of heating and the ratio of 7S to 11S proteins, coagulation

by GDL can be brought about by heating the mixtures for different lengths of time to

form gels of similar pH. Since pH affect the properties of gels, those with same pH

values could also have similar physical properties like texture, optical and water

holding capacity. Hence different ratios of 7S:11S coagulated by GDL heated for

different lengths of time can produce gels with the similar physico-chemical

properties.

In this study, 7S and 11S globulins were mixed in various ratios; and the physico-

chemical properties of the 7S and 11S protein blends after the addition of glucono-δ-

lactone (GDL) were investigated at a constant temperature as a function of time. The

relationship between the ratio of 7S:11S and the time of heating was used to predict

what protein fractions and for how long they need to be heated to produce GDL

induced gels having similar physiochemical properties.

48

2.3 MATERIALS AND METHOD

2.3.1 Isolation of 7S and 11S globulin

The 7S and 11S protein fractions were isolated by the method of Nagano et al.

(1992). Defatted soybean flour was mixed with 15-fold volume of distilled water,

and then pH was adjusted to 7.5 with 1 M of NaOH. The water-extractable soybean

protein was obtained by centrifugation (9000g x 30min) at 20oC. Sodium bisulfite

(SBS) (0.98 g of SBS/L) was added to the supernatant and the pH was adjusted to

6.4 with 0.1 M of HCL, and the mixture was kept in an ice bath overnight. The

following procedure was performed at 4oC. The insoluble 11S fraction was obtained

by centrifugation at 6500g for 20min. The supernatant was adjusted to contain 0.25

M NaCl and to be pH 5.0 with HCl. After 1 hour, the insoluble fraction was removed

by centrifugation at 9000g for 30min. The supernatant was diluted 2-fold with ice

water, adjusted to pH 4.8 with HCl, and then centrifuged again at 6500g for 20min.

The 7S globulin was obtained as a sediment. Both 11S and 7S fractions were

washed, adjusted to pH 7.5 with NaOH, and then freeze-dried. The protein contents

of the 11S and 7S globulines were determined as 95% and 92% respectively,

according to the method of Kjeldahl using a conversion factor of 6.25 (Kohyama and

Nishinari, 1993).

49

2.3.2 Preparation of gels

The formation of the protein gels was modified based on the method of Kohyama

and Nishinari (1993). The 7S, 11S and mixtures of the two protein solutions (4.35%,

w/v) at different ratios were heated for 10min at 100oC. Then a freshly prepared

GDL solution (5%, w/v) was added to these protein solutions such that the resultant

mixture contained a protein content of 4.0% (w/v) and GDL of 0.4% (w/v). These

mixtures were allowed to stand at 60oC for 20 to 100 minutes after addition of GDL

after which, they were cooled in tap water (25oC) for 60min. The gels were then aged

at room temperature (25oC) for 60min before the analysis were carried out.

2.3.3 Texture profile analyses

Analysis of the force/deformation curve generated by compressing a gel was used to

evaluate the textural properties. Gel cylinders of 15mm diameter and 10mm height

were compressed twice to 30% deformation, with a 35mm diameter probe using a

Stable Micro Systems model TA.XT2i texture analyzer (Godalming, Surrey, UK).

Each sample analysis was replicated six times. The mean values for the hardness,

adhesive, cohesiveness and gumminess were calculated and noted.

50

2.3.4 Color analysis

The color was analyzed using Minolta spectrophometer model CM-3500d (Osaka,

Japan). The measurements were replicated six times on each gel sample. The mean

values for L*, a*, b*, C* and h were calculated and noted.

2.3.5 pH of the gel

Gel cylinders of 15mm diameter and 10mm height, were placed on a plain nylon

membrane (4.5µm) maintained in the middle position of a 50ml centrifuge tube and

centrifuged at 120×g for 5min. The water collected in the centrifuge tube was used

to measure the pH of the gel. . Each value is the mean of six determinations.

2.3.6 Water-holding capacity (WHC) of gels

The WHC of the gels was determined according to the method of Puppo and Añón

(1998) modified as given below. Gel cylinders of 15mm diameter and 10mm height

were placed on a 50 ml Maxi-Spin® centrifuge tube with a plain nylon membrane

(4.5µm) in the middle position (Vivascience AG, Hanover, Germany). Water loss

was determined by weighing the water collected in the lower part of the centrifuge

tube after centrifugation at 120×g for 5min at 15oC. WHC was expressed as percent

51

of the water remaining in the gel after centrifugation. Each value is the mean of six

determinations.

2.3.7 Statistical analysis

Six replicates per treatment were used for the analysis of texture, colour, WHC and

pH. All data points represent the mean ± standard deviation. Comparison of the

means of two samples were carried out by paired T-test with a significance level of

P<0.05. Comparisons of variance of more than two samples were carried out by

single factor ANOVA.

52

2.4 RESULTS AND DISCUSSION

2.4.1 Textural properties of the GDL-induced gels

Hardness is defined as the force necessary to attain a given deformation (Szczesniak,

1962). In both Figures 2.1a and 2.1b (soy proteins and GDL heated for 20 to 100

minutes) showed that gels with higher 11S protein fractions were much harder than

gels with higher 7S protein fractions. 11S protein fractions formed harder gels could

be because 11S formed faster gels than 7S. Kohyama et al. (1995) reported that

faster gelation was observed in mixtures containing 11S in high proportions

compared to those of low proportions of 11S.

In Figure 2.1a (soy proteins and GDL heated for 20 minutes), the hardness of the soy

gels increased as the proportion of 11S increased. However, in Figure 2.1b (soy

proteins and GDL heated for 40, 60, 80 and 100 minutes), the hardness increased,

decreased and then increased, with the minimum hardness of gel forming at the

7S:11S ratio of 5:5. This trend was more obvious in samples heated for longer period

of time that for shorter period of time.

53

Hardness

0

2

4

6

8Fo

rce

( N

)

20min40min

10:0 9:1 8:2 7:3 6:4 5:5 4:6 3:7 2:8 1:9 0:10Ratio of 7S:11S

Figure 2.1a. Comparison of hardness of soy gels heated for 20 and 40 min.

Hardness

0

2

4

6

8

Forc

e / N 60min

80min100min

10:0 9:1 8:2 7:3 6:4 5:5 4:6 3:7 2:8 1:9 0:10

Ratio of 7S:11S

Figure 2.1b. Comparison of hardness of soy gels heated for 60, 80 and 100 min.

54

For 7S:11S ratio of 5:5, the hardness of the gels heated for 80 and 100 min were

similar to those of 10:0 (Table 2.1). Unlike other protein ratios, the gels formed at

5:5 ratio did not increase in hardness with time of heating. Kohyama et al. (1995)

reported similar trends for 7S:11S ratio of 5:5. At 7S:11S ratio of 5:5, 11S does not

seem to play a role in affecting the hardness of the gel.

Table 2.1. Hardness of soy gel heated for 60, 80 and 100 minutes

Hardness / g

Ratio of 7S:11S

60 min heating

80min heating

100 min heating

10:0

30.87 ± 5.90

37.79 ± 2.32 a

41.88 ± 1.05 a

5:5

45.82 ± 1.16

43.15 ± 3.79 a

40.69 ± 3.45 a

a denote that the two mean values are not significant different at P<0.05. Paired-t test

was carried out

Gumminess is defined as the energy required to disintegrate a semisolid food product

to a state ready for swallowing (Szczesniak, 1962). The gumminess increased as the

proportion of 11S increased. This is illustrated in Figure 2.2. For ratio of 7S:11S

55

from 10:0 to 5:5, gumminess increased with the time of heating; and for ratios of

7S:11S from 5:5 to 0:10, gumminess is not affected by the time of heating. Soy

proteins and GDL heated for 20 minutes generally had lower gumminess at all ratios.

It seems that time of heating has little effect on the gumminess of gels containing

higher proportion of 11S fraction.

Gumminess

01234567

Forc

e (

N ) 20min

40min60min80min100min

10:0 9:1 8:2 7:3 6:4 5:5 4:6 3:7 2:8 1:9 0:10Ratio of 7S:11S

Figure 2.2. Comparison of gumminess of soy gels heated for 20, 40, 60, 80 and 100

min

Cohesiveness is defined as the strength of the internal bonds making up the body of

the product (Szczesniak, 1962). For ratio of 7S:11S from 10:0 to 5:5, cohesiveness

increased with the duration of heating as the proportion of 11S increased, (Figure 2.3)

and reached a maximum value at a ratio of 5:5. Beyond this point, the time of

56

heating or the ratio of 7S:11S had no affect on the cohesiveness. It seems that time of

heating had little effect on the cohesiveness of gel containing higher proportion of

11S fraction.

Cohesiveness

0

0.2

0.4

0.6

0.8

1

Rat

io o

f pos

itive

forc

e ar

eas

unde

r the

firs

t and

sec

ond

com

pres

sion

s

20min40min60min80min100min

10:0 9:1 8:2 7:3 6:4 5:5 4:6 3:7 2:8 1:9 0:10Ratio of 7S:11S

Figure 2.3. Cohesiveness of soy gels heated for 20, 40, 60, 80 and 100 min

Based on the above results, it may be concluded that the textural properties such as

gumminess and cohesiveness of acid-induced soy protein gels obtained from ratio of

7S:11S from 5:5 to 0:10 were not affected by the time of heating. This is probably

due to the fact that mixtures containing higher proportion of 11S protein formed

acid-induced gel faster.

57

2.4.2 Optical properties of the GDL-induced gels

The +L* value (lightness) increased with the proportion of 11S and with the time of

heating (Figure 2.4). This is probably due to the fact that mixtures containing higher

proportion of 11S protein form acid-induced gel faster than those containing lower

proportions of 11S.

L*

65

70

75

80

85

posi

tive

valu

e 20min40min60min80min100min

10:0 9:1 8:2 7:3 6:4 5:5 4:6 3:7 2:8 1:9 0:10

Ratio of 7S:11S

Figure 2.4. L* of soy gels heated for 20, 40, 60, 80 and 100 min

The L* values of gels formed after heating for 40, 60, 80 and 100 min were closer to

each other and greater, than those of gels formed by heating for 20 minutes. The

optical results suggested that mixtures containing higher proportion of 11S protein

form faster acid-induced gel.

58

2.4.3 pH of the GDL-induced gels

The textural and optical properties of gels showed that mixtures containing higher

proportion of 11S protein formed faster acid-induced gels. This difference in gel

properties could be due to the differences in their pH (Figure 2.5).

Acidity of gel

4.24.44.64.8

55.25.4

pH

20min40min60min80min100min

10:0 9:1 8:2 7:3 6:4 5:5 4:6 3:7 2:8 1:9 0:10

Ratio of 7S:11S

Figure 2.5 pH of soy gels heated for 20, 40, 60, 80 and 100 min.

Figure 2.5 showed that mixtures of 7S and 11S proteins containing higher proportion

of 11S had lower pH values. When the length of heating increased, the pH values for

all fractions decreased. The texture analysis showed that higher proportion of 11S

formed harder gels (Figures 2.1a and 2.1b). Hence lower pH values generally

correspond to harder gels.

59

The isoelectric point of 7S globulin (4.5-5.0) is lower than that of 11S (6.3-7.0)

(Brooks and Morr, 1985). Therefore, the 7S protein contains more negatively

charged groups (-COO-) at pH higher than the isoelectric point. Therefore, the

buffering effect against protons is stronger in 7S than 11S in the pH range from 4 to

5. When the same amount of protons was added to both the 7S and 11S solutions, the

pH of 7S systems remained higher than that of 11S (Kohyama and Nishinari, 1993).

The smaller pH depression may be attributed to softer 7S gels. Hence 7S or mixtures

containing higher proportion of 7S will be softer and will have higher pH values as

indeed observed experimentally (Figure 2.5).

2.4.4 Water holding capacity (WHC) of the acid-induced gels

For soy proteins and GDL heated for 20 minutes (Figure 2.6), the WHC of the soy

gels containing higher proportion of 11S was higher than those containing higher

proportion of 7S. The soy gels for ratios of 7S:11S from 10:0 to 7:3, were much

softer than those for ratios of 7S:11S from 3:7 to 0:10 (Figure 2.1a and 2.1b). For 20

minutes of heating, the softer gels (ratio of 7S:11S from 10:0 to 7:3) had poor ability

to hold water than the harder gels (ratio of 7S:11S from 0:10 to 3:7). Kohyama et al.

(1995) reported that GDL-induced soy gels containing high proportion of 7S formed

gels slower as compared to those containing lower proportion of 7S. Thus softer gels

(ratio of 7S:11S from 10:0 to 7:3) could form lesser amount of network to trap water

molecule as compared to harder gels (ratio of 7S:11S from 10:0 to 7:3).

60

WHC

20

30

40

50

60

70W

HC

( %

)20min40min60min80min100min

10:0 9:1 8:2 7:3 6:4 5:5 4:6 3:7 2:8 1:9 0:10Ratio of 7S:11S

Figure 2.6. WHC of soy gels heated for 20, 40, 60, 80 and 100 min.

As the heating duration increased from 40 to 100 min, the WHC of the soy gels

containing higher proportion of 11S was lower than those containing higher

proportion of 7S. The harder soy gels (ratio of 7S:11S from 0:10 to 3:7) trap lesser

amount of water molecules than softer soy gels (ratio of 7S:11S from 10:0 to 7:3).

Harder gels should trap more water according to the results for WHC of gels heated

for 20minutes. However, in this case, these harder soy gels trap less amount of water

molecule when heated for a longer period of time (40minutes to 100 minutes). A

possible explanation could be when the heating duration increased, the gap in the

network of gels containing high proportion of 11S could “shrink” and thus entrap

less amount of water. Since GDL-induced soy gels containing high proportion of 7S

formed gels slower as compared to those containing lower proportion of 7S, as

61

reported by Kohyama et al. (1995). The gap in the network of gels containing higher

proportion of 7S could “shrink” less and thus entrap more water in the network. This

would explain the higher WHC observed for acid-induced soy gel containing higher

proportion of 7S (Figure 2.6) when heated for a longer period of time (from 40 to

100 minutes).

2.4.5 Relationship of pH, hardness, L* value and WHC

Table 2.2 showed that except for proteins fractions heated for 20 minutes, certain

proportion of 7S and 11S proteins heated at different length of time will form gels of

similar pH at confidence level of 95%. These gels with similar mean value of pH,

will have similar mean value of L*, hardness and WHC at confidence level of 95%.

These similarities in the physical properties of the gels suggested that gel formation

is strongly influence by pH.

62

Table 2.2. Comparision of L*, hardness and pH of various ratio of 7S:11S

Ratio of 7S:11S

pH

Hardness /g

L*

WHC/ %

8:2 heated for 40min

4.89 b

35.39c

74.93 d

52.54e


4.90 b

38.38 c

74.56 d

55.09 e


4.92 b

37.79 c

74.37 d

53.71 e

b,c,d,e denote that the mean values are not significant different at P<0.05. Single factor

ANOVA was carried out.

Figure 2.7 illustrates that certain proportion of 7S and 11S proteins heated at

different length of time will form gels of similar properties at confidence level of

95%. Respective lines (1 to 10) join points (denote various protein fraction) that

show similar gel properties. For example: Fraction of 10:0 heated for 100 minutes

will have gel of similar properties as fraction of 9:1 heated for 80 minutes, fraction

of 8:2 heated for 60 minutes and fraction of 7:3 heated for 40 minutes. That is when

the heating time of the gel differ by 20 minutes (from 40 to 100 minutes), the

fraction of 11S/7S differ by 1/10, could formed protein gels (GDL coagulated) that

have similar physico-chemical properties.

63

Protein fraction vs heating time

0

20

40

60

80

100

Protein fraction, 7S:11S

incu

batio

n tim

e ( m

in )

12345678910

10:0 9:1 8:2 7:3 6:4 5:5 4:6 3:7 2:8 1:9 0:10

f,g,h,i

f,g f

f f

f

g

f

g

g,i g g

g

h

h

i

i i

Figure 2.7. Plot of various protein fractions against different lengths of heating time.

f denote that the mean values are significant different at P<0.05 for hardness. g

denote that the mean values are significant different at P<0.05 for WHC. h denote

that the mean values are significant different at P<0.05 for L*. i denote that the mean

values are significant different at P<0.05 for pH. Single factor ANOVA was carried

out.

Some exception to the trend occurred mostly in the region of protein fractions around

5:5 and protein fractions heated for 100 minutes. As discuss early, unlike other

protein ratio, ratio of gel at 5:5 did not increased in hardness with time of heating. As

for gels heated for 20 and 100 minutes, the gels might form much too slowly or

rapidly and thus its properties are different from other fractions.

64

2.5 Conclusion

Generally, higher proportion of 11S protein in the gels, higher the hardness,

cohesiveness, gumminess and +L*. The difference in gel properties could be due to

the difference in pH of the finished gels. Specific proportions of 7S and 11S proteins

heated for different lengths of time form gels of similar pH which in turn will have

similar hardness, L* and WHC at confidence level of 95%. From the data it can be

deduced that when the heating time of the gels differ by 20 min (from 40 to 100 min),

the fraction of 11S:7S differing by 1/10 would form GDL coagulated gels of similar

physico-chemical properties.

65

REFERENCE

1) Brooks, J. R.; Morr, C. V. Current aspect of soy protein fractionation and

nomenclature. Journal of American Oil Chemistry Society. 1985, 62, 1347-

1357.

2) Cai, T.; Chang, K. C. Processing effect on soybean storage proteins and their

relationship with tofu quality. Journal of Agricultural and Food Chemistry.

1999, 47,720-727.

3) de Man, J. M.; de Man, L.; Gupa, S. Textural and microstructure of soybean

curd (tofu) as affected by different coagulants. Food Microstruture. 1986, 5,

83-89.

4) Hashizume, K.; Nakamura, N.; Watanabe, T. Influence of ionic strength on

conformation changes of soybean proteins caused by heating and relationship

of its conformation changes to gel formation. Agricultural Biology Chemistry.

1975, 39, 1339-1347.

5) Kohyama, K.; Murata, M.; Tani, F.; Sano, Y.; Doi, E. Effects of protein


globulins. Bioscience Biotechnology Biochemistry. 1995, 59, 240-245.




66

7) Mujoo, R.; Dianne, T. T.; Ng, P. K. W. Characterization of storage proteins

in different soybean varieties and their relationship to tofu yield and texture.

Food Chemistry. 2003, 82, 265-273.

8) Murphy. P. A.; Chen, H. P.; Hauck, C. C.; Wilson, L. A. Soybean protein

composition and tofu quality. Food Technology. 1997, 51(3), 86–88.




10) Poysa, V.; Woodrow, L. Stability of soybean seed composition and its effect

on soymilk and tofu yield and quality. Food Research International. 2002,

35(4), 337-345.

11) Puppo, M. C.; Añón , M. C. Structural properties of heat-induced soy protein

gels as affected by ionic strength and pH. J Agric Food Chemistry. 1998, 46,

3583-3589.

12) Szczesniak, A.S. Classification of textural characteristic. Journal of Food

Science. 1962, 28. 385-289.

67

Chapter 3

The 7S and 11S proteins mixtures coagulated by chloride or

sulphate salt

68

3.1 ABSTRACT

7S and 11S soy proteins were mixed with various salts to investigate the coagulating

effects of cations and anions of the coagulants. When coagulants at concentrations

of 0.008M were added to mixture of 7S and 11S proteins (ratio of 7S:11S from 0:10

to 10:0), different states of curds were formed. Depending on the coagulants, the

mixture would remain as liquid, form uniform curds or precipitate. Coagulants which

result in the formation of precipitates in most protein ratios are defined as those with

the strongest coagulating power. Based on the different states of curds, the

coagulating powers of various salts were in the order of CaCl2 > MgCl2 > CaSO4 >

MgSO4. Similar order was found by turbidity measurements. Coagulants with strong

coagulating power were able to form uniform curds of firmer texture, higher L* and

lower pH values compared those with weak coagulating power.

69

3.2 INTRODUCTION

Tofu or soybean curd is a gel-like food made by adding coagulants to soybean milk.

The four basic types of coagulants that are used to make tofu are: (1) “nigari-type” or

chloride-type coagulants such as magnesium chloride (MgCl2) and calcium chloride

(CaCl2); (2) sulfate-type coagulants such as calcium sulfate (CaSO4) and magnesium

sulfate (MgSO4); (3) Glucono delta lactone (GDL); and (4) acidic coagulants

including citrus juices, vinegar, and lactic acid (Shurtleff and Aoyagi, 2000).

Each of these coagulants will produce tofu of different flavour and texture (de Man

et al., 1986; Karimet al., 1998). In a previous study, various calcium salts, as well as

some non-calcium compounds were tested at various concentrations on a fixed

soybean variety for their suitability as coagulants. It was found that while calcium

chloride, calcium acetate, calcium gluconate, calcium lactate, glucono-δ-lactone, and

acetic acid coagulated the soy milk, calcium phosphate, calcium hydroxide, and

calcium carbonate did not (Lu et al., 1980).

Protein is one of the major components in tofu and the two major proteins in soybean

are 7S and 11S. These two proteins have different molecular weights, isoelectric

points, thermal transition temperatures and gel-forming properties (Yuan et al., 2002).

Salts and proteins form coagulates when metal ions such as Ca2+ or Mg2+ form

bridges with the negatively charged protein. This cross-link is due to the electrostatic

interactions between the cations and the proteins (Karim et al., 1998).

70

In our work, we investigated the gelation effects by various coagulants on soy

protein mixtures made up of varying ratios of 7S:11S globulins. Different ratio of 7S

and 11S proteins will form different extent of aggregations with coagulants.

Different extent of aggregations will result in different types of curd formation.

Based on these differences we will be able to determine the coagulating power of the

coagulants. In the past the effects of cations like Ca2+ and Mg2+ on aggregation were

studied but only a few studies have been done on the effects of anions on aggregation.

Lately, researchers have found that anions like chloride and citrate also play an

important role in the protein aggregation process (Molina and Wagner, 1999). Thus,

further tests were carried out to investigate the coagulating effects of cations and

anions of different salts.

71

3.3 Materials and Methods

3.3.1 Protein Assay

The protein contents of soy foods were determined by the BioRad assay (Boyer,

2000). A calibration curve was prepared by using a series of standard solution of

bovine serum albumin. Absorbance was measured at 595 nm. The protein

concentrations of 7S and 11S were determined by comparison with the calibration

curve.

3.3.2 Preparation of gels

The formation of the protein gels was modified based on the method of Kohyama

and Nishinari (1993). The 7S, 11S and mixtures of the two protein solutions (4.35%,

w/v) at different ratios were heated for 10min at 100oC and then were cooled in tap

water (25oC) for 30min to 25oC. Four coagulants were chosen and they were calcium

chloride calcium sulphate, magnesium chloride and magnesium sulphate. The

coagulants solution (0.1M) was added to these protein solutions such that the

resultant mixture contained a protein content of 4.0% (w/v) and a coagulant

concentration of 0.008M. These mixtures taken in a round glass bottle with a

diameter of 5cm were allowed to incubate at 60oC for 20 min. After which, they

72

were cooled in tap water (25oC) for 60min. The gels were then aged at room

temperature (25oC) for 60min before the analyses were carried out.

3.3.3 Turbidity measurement

Turbidity due to aggregation was determined according to the method of Molina and

Wagner (1999) modified as given below. Turbidity was measured using Shimadzu

UV-visible spectrophotometer (Japan). The absorbance of 1ml of 7S and 11S protein

solutions at 0.5mg/ml in a semi micro cuvette with successive addition of 10 µl of

1M of salt solution was measured. Readings were performed 1min after each

addition with agitation

73


3.4.1 Coagulating power

To facilitate the discussion of the results conveniently, the following five

descriptions of the protein mixtures with coagulants - “ppt”, “gel”, “soft”, “semi”

and “liq” were used and defined in Table 3.1.

Table 3.1. Descriptions of the protein mixtures with coagulants.

ppt

Curds are in the form of precipitates rather than gel.

gel

Curds form complete, uniform gels which are able to retain their

shapes upon detachment from the moulds.

soft

Curds form complete, uniform gels in the moulds, but are unable to

retain their shapes upon detachment

semi

Products are the in-between of gels and liquids; they are generally

much more viscous than liquids, but unable to take on definite shapes

like gels do.

liq

Products remain in the liquid form with varying degrees of

whiteness/clearness.

74

Figure 3.1 illustrates the relationship of various states of curds and their coagulating

powers. A coagulant that readily form precipitates or aggregates with a protein

solution was defined in this paper as one that had a good coagulating power while a

coagulant that remains in solution when added to a protein solution was defines as

one that had a poor coagulating power. Using this guideline, the coagulating power

of coagulants for various states of curds were in the order of “ppt” > “gel” > “soft” >

“semi” > “liq”.

Figure 3.1. Relationship of coagulating power and various state of curds.

ppt gel soft semi liquid (strong) (weak) Coagulating power

75

3.4.2 Different states of curds

Different coagulants have different coagulating powers. Table 3.2 shows that the

coagulating power of the salts varied across 7S and 11S protein ratios. Salts often

cause protein mixtures with higher proportions of 11S to aggregate more and form

precipitates or gels, than protein ratios with higher proportion of 7S. Salts often

cause the protein mixtures with high proportions of 7S to aggregate less and form

mostly liquid states. Thus by varying the ratio of 7S and 11S protein, the

coagulating power of different salts can be found by comparing the state of curds at

various ratios of 7S and11S. By comparing the state of curdling at various ratios of

7S and11S, as shown in Table 3.2, the coagulating power of the salts were found to

be in the order CaCl2 > MgCl2 > CaSO4 > MgSO4. The coagulating power seems to

be affected by both the cations as wells as the anions.

Table 3.2. Coagulation results of protein mixtures (4%, w/v) after addition of various

coagulants (0.008M).

Ratio of 7S : 11S

Coagulant 0:10 1:9 2:8 3:7 4:6 5:5 6:4 7:3 8:2 9:1 10:0

CaCl2 ppt gel liq

CaSO4 gel semi liq liq

MgCl2 ppt gel semi liq

MgSO4 gel semi liq liq

76

Table 3.3 shows that CaSO4 can result in the formation of curds that are firm, with

low pH and high L* value. Based on the earlier results, the coagulating power of

CaSO4 was greater than that of MgSO4. This shows that sulfate salts having stronger

coagulating power could give rise to protein gels with firmer texture, lower pH and

higher L* value than those having weaker coagulating power.

Table 3.3. Physico chemical properties of protein mixture (4%, w/v) after coagulated

by calcium sulphate and magnesium sulphate.

Coagulant

Ratio of

7S:11S

Hardness / N

pH

L*

CaSO4

0:10

0.574 ± 0.029

6.84 ± 0.08

73.42 ± 0.24

MgSO4

0:10

0.412 ± 0.017

7.08 ± 0.11

71.19 ± 0.15

77


Further investigation was carried out to verify that for the same concentration of

coagulant used, the coagulating power was affected by both the cations and anions.

Researchers have found that turbidity (A600 nm) may be used to estimate the degree of

protein aggregation (Molina and Wagner, 1999). When CaCl2, MgCl2 and MgSO4

were added to 7S and 11S protein solutions, the turbidity of the mixture increased to

a maximum and then decreased as the concentration of salts increased. This general

trend was consistent with what was found earlier by other researchers (Molina and

Wagner, 1999; Sorgentini et al., 1995). When a small amount of salt was added to a

proteins solution, increased in turbidity could be due to the electrostatic interactions

between the cations and the proteins. These electrostatic interactions will cause the

cations to form bridges with the proteins, which result in the formation of aggregates.

This process was defined as the “aggregation effect” (Molina and Wagner, 1999).

The decreased in turbidity as the concentration of salts increased could be due to the

decrease in electrostatic interaction between the cations and proteins caused by the

anions. And this process was defined as the “dissociation effect” (Molina and

Wagner, 1999). The decrease in the electrostatic interaction will result in lower

amount of cross linking and thus fewer aggregates. The turbidity measurement was

carried out to investigate the effects of cations as well as anions when added to 7S

and 11S proteins.

78

Figure 3.2 shows that the turbidity of calcium chloride from concentration of 40mM

to 150mM was higher than that of magnesium chloride, except at 70mM (p<0.05).

The only difference between these two salts were the cations, hence, the difference in

the turbidity was due to the cations. The reason why the turbidity of calcium chloride

with 11S protein solution was higher than that of magnesium chloride might be due

to stronger electrostatic interaction between Ca2+ and protein than with Mg2+.

According to the “aggregation effect” (Molina and Wagner, 1999), the electrostatic

interaction between cations and proteins can be used to explain turbidity of protein

solution when a salt was added.

In Figure 3.2, the turbidity of magnesium chloride from concentration of 60mM to

110mM was lower than magnesium sulfate (p<0.05). This showed that the Cl- ion

caused the decrease of electrostatic interaction more than SO42- ion. Similarly,

according to the “dissociation effect” (Molina and Wagner, 1999), the decrease in

the electrostatic interaction caused by anions can be used to explain turbidity of

protein solutions when salt was added. Similar trend was observed for 7S protein

with the various salts as shown in Figure 3.3. Anion was found to affect the turbidity

of both protein and thus likely to affect the gelation properties of these two proteins.

Wang and Hesseltine (1982) had reported that anions have a strong effect on the

water-holding capacity of the gels.

79

Turbidity

0

0.2

0.4

0.6

0.8

1

1.2

1.4

0 20 40 60 80 100 120 140 160salt concentration (mM)

A 60

0nm

calcium chloridemagnesium chloridemagnesium sulphate

* *

^* ^

^* ^

* ^ * ^ * * * *

Figure 3.2. Turbidity of various salts with 11S protein.

* denote that the two mean values for calcium chloride and magnesium chloride are

not significantly different at P<0.05. ^ denote that the two mean values for

magnesium chloride and magnesium sulphate are not significantly different at

P<0.05. Paired-t test was carried out. n = 6.

80

Turbidity

0

0.2

0.4

0.6

0.8

1

1.2

1.4

0 20 40 60 80 100 120 140 160salt concentration (mM)

A 60

0nm

calcium chloridemagnesium chloridemagnesium sulphate

* * ^* ^

* ^ * ^* ^ *

Figure 3.3. Turbidity of various slats with 7S protein.

* denote that the two mean values for calcium chloride and magnesium chloride are

not significantly different at P<0.05. ^ denote that the two mean values for

magnesium chloride and magnesium sulphate are not significantly different at

P<0.05. Paired-t test was carried out. n = 6.

81

3.4.4 CONCLUSION

Based on the results obtained, both the cation and anion species influence the

coagulation of salts and proteins. MgSO4 when added to 11S protein solutions

formed curds that were softer, with higher pH and lower L* value than those formed

with CaSO4. MgCl2 when added to both 11S and 7S protein solutions gave lower

turbidity than when CaCl2 was added to the same solutions. These results suggest

that the coagulating power of Mg2+ salts were weaker than that of Ca2+ salts. CaSO4

and MgSO4 when added to protein solutions were found to form precipitates in fewer

7S:11S protein ratios than CaC12 and MgCl2. MgSO4 when added to both 7S and

11S protein solutions gave higher turbidity than MgCl2 added to the same 7S and

11S protein solutions. These results suggest that the coagulating power of SO42- salts

are weaker than Cl- salts.

82

REFERENCE

1) Bourne, M. C. Food Texture and viscosity: Concept and measurement. 2nd

edition, Academic Press: New York, 2002; 400.

2) Boyer, R. Modern Experimental Biochemistry, 3rd edition, Addison Wesley

Longman: San Francisco, CA, 2000; 43-45.

3) de Man, J. M.; de Man, L.; Gupa, S. Textural and microstructure of soybean

curd (tofu) as affected by different coagulants. Food Microstructure. 1986, 5,

83-89.

4) Karim, A.A.; Sulebele, G.A.; Azhar, M.E.; Ping, C.Y. Effect of carrageenan on

yield and properties of tofu. Food Chemistry. 1998, 66, 159-165.



Agricultural Food Chemistry. 1993, 41, 8-14.

6) Lu, J. Y.; Carter, E.; Chung, R. A. Use of calcium salts for soybean curd

preparation. Journal of Food Science, 1980, 45, 32-34.

7) Molina, M.I.; Wagner, J.R. The effects of diavalent cations in the presence of

phosphate, cirate and chloride on the aggregation of soy protein isolate. Food

Research International. 1999, 32, 135-143.

8) Nagano, T.; Hirotsuka, M.; Mori, H.; Kohyama, K; Nishinar, K. Dynamic



83

9) Shurtleff, W.; Aoyagi, A. Tofu and Soymilk Production. The book of tofu,

volume II, 3rd edition, Soyfood center, Lafayette, C.A.; 2000; 150.

10) Sorgentini, D. A., Wagner, J. R. & Añón, M. C. Effects of thermal treatment of

soy protein isolate on the characteristics and structure-function relationship of

soluble and insoluble fractions. Journal of Agricultural Food Chemistry. 1995,

43, 2471-2479.

11) Wang, H. L.; Hesseltine, C. W. Coagulation Conditions in Tofu Processing.

Process Biochemistry. 1982, 1, 8-12.

12) Yuan, Y. J.; Velev, O. D.; Chen, K.; Campbell, B. E.; Kaller, E. W.; Lenoff, A.

M. Effect of pH and Ca2+-induced associations of soybean proteins. Journal of


84

Chapter 4

The effect of κ-carrageenan on the foaming, gelling and isoflavone

content of 11S

85

4.1 ABSTRACT

The effects of κ-carrageenan addition to soy flour during the 11S extraction were

investigated. The foaming properties, gelling and isoflavone content of 11S before

and after addition of κ-carrageenan were compared. κ-carrageenan when added to

soy flour during the extraction of 11S caused the 11S to have higher level of

isoflavone, better foaming properties and formed harder gels.

86

4.2 INTRODUCTION


benefits such as prevention of cardiovascular diseases and cancer. Although

isoflavones have low hydrophilic properties, some isoflavones are lost during

aqueous extraction of soy protein fractions. Polysaccharides added during the soy

fraction extraction could result in an increased in the isoflavone content of the soy

fractions.

Hydrocolloids such as κ-carraggenan, xanthan gum and propylene glycol alginate are

able to hold and maintain water content in food and have found to result in greater

genistein retention during soy protein concentrate production (Pandjaitan et al.,

2000).

Polysaccharides play a key role in modifying the textural properties of protein food

systems (Karim et al., 1998). The polysaccaride used was carrageenan. Karim et al.

(1998) reported that addition of carrageenan brought about a significant decrease in

hardness of calcium sulphate and calcium acetate tofu but no significant effect on

GDL tofu.

The objective of this study was to improve the retention of isoflavones of glycinin

during the extraction. The foaming properties and the gelling properties of the

improved isoflavone protein fractions were also studied.

87

4.3 Materials and Methods

4.3.1 Foaming properties

The foaming properties were determined by using the method of Molina and Wagner

(2002), modified as follows. Air at a flow rate of 90cm3/min was introduced into 6ml

of 0.1% protein solution in a graduated column of 10mm diameter with a coarse

glass frit at the bottom. Bubbling was continued for 140s. The volume of liquid

incorporated to the form was determined by measuring the volume in the liquid

remained in the column.

4.3.2 HPLC

The isoflavone content of the samples were determined by using the method of

Pandjaitan et al. (2000), modified as follows. One gram of finely ground freeze-

dried samples of the soy protein fraction was stirred in 15ml of 80% aqueous

methanol solvent for 2 h at ambient temperature. The extract was filtered through

Whatman no 42 filtered paper. The filtrate was dried and redissolved in 2.5ml of

80% aqueous methanol solvent. The isoflavone content was determined by Waters

515 HPLC (Milford, USA) using YMC J’sphere ODS-H80 (Milford, USA) column.

A linear gradient was composed of solvent (A) 0.1% glacial acetic acid in ultra pure

88

water, and solvent (B) 0.1% glacial acetic acid in acetoniltrile. After the sample

injection, solvent (B) was increased from 15% to 35% and the solvent flow rate was

1.0 ml/min. The chromatogram was detected using Waters 2996 photodiode array

dectector (Milford, USA) at 254nm.

4.3.3 Surface hydrophobicity

Surface hydrophobicity was determined by a hydrophobic fluorescence probe, using

1-anilino-8- naphthalene sulfonate (ANS) (Hayakawa & Nakai, 1985). Protein

samples with concentrations from 0.01% to 0.06% were prepared by serial dilution

from the 0.1% stock solution using 0.035M potassium phosphate buffer at pH 7.6.

Forty microliters of ANS (8mM in 0.035M phosphate buffer) were added to 3ml

aliquots of each protein solution and samples were incubated in dark for 1 hour.

Fluorescence intensities (FI) of ANS-protein conjugates were measured using a

Perkim-Elmer fluorescence detector, at excitation wavelength of 365nm and

emission of 484nm. Fluorescence reading was corrected using a blank solution of

40µl ANS solution in 3ml of phosphate buffer as the zero point. The initial slope of

fluorescence intensity versus percent protein concentration was calculated by linear

regression, and this was used as an index of protein surface hydrophobicity (H0).

Samples were evaluated in triplicates for each prepared solution

89

4.3.4 Gelling properties

The formation of the protein gels were formed based on the method of Kohyama and

Nishinari (1993), modified as follows. The 11S (with and without carrageenan)

protein solutions (4.35%, w/v) at different ratios were heated for 10min at 100oC.

Then a freshly prepared GDL solution or salt solution was added to these protein

solutions in such a way that the resultant mixture had the following composition:

protein 4.0% (w/v) and coagulant concentration at 0.008M. These mixtures were

allowed to stand for 20 minutes at 60oC after addition of salts or GDL and cooled in

tap water (25oC) for 60min. The gels were then aged at room temperature (25oC) for

60min before the analysis were carried out. Analysis of the force/deformation curve

generated by compressing a gel was used to evaluate the textural properties. Gel

cylinders of 15mm diameter and 10mm height were compressed twice to 30%

deformation, with a 35mm diameter probe in a TA.XT2i texture analyzer.

90

4.4 Results and Discussion

4.4.1 Isoflavone content

Figure 5.1 is the chromatograms of the separation of the isoflavones in 11S. The

respective peaks were label and the quantity was tabulated in Table 5.1. Table 5.1

shows the isoflavone content of 11S and 11S with κ-carrageenan. The total

isoflavone content is the sum of the total daidzein, total genistein and total glycitein

content. The total diadzein content is the sum of the glucosides, malonyl glucoside,

acetyl glucoside and aglycon content, and is the same for total genistein and total

glycitein content.

Figure 4.1. The chromatograms of the separation of the isoflavones of soy protein.

91

Table 4.1. Isoflavone Contents in 11S and 11S with κ-carrageenan

Isoflavone 11S (µg/g) 11S with κ-

carrageenan (µg/g)

Daidzin 2.22 3.73

Glycitin 1.38 1.99

Genistin 7.57 10.10

Malonyl Daidzin 22.02 22.16

Malonyl Glycitin 4.86 4.61

Malonyl Genistin 46.08 46.34

Daidzein 6.51 5.98

Glycitein 1.39 1.21

Acetyl Genistein 0.78 0.80

Genistein 13.67 13.76

Total Daidzein 30.76 31.86

Total genistein 68.11 71.00

Total glycitein 7.63 7.80

Total isoflavone 106.50 110.66

92

The isoflavone content in the 11S soy protein contained mainly the malonyl forms.

The malonyl forms made up 71.6% of the total daidzein content, 58.2% of the total

glycitein content and 67.7% of the total genistein content. It was found that

unprocessed soybean also contained mainly the malonyl forms (Murphy et al., 2002).

The 11S soy proteins undergo only pH extraction process at 4oC, thus the malonyl

forms should be the major forms in 11S. Murphyl et al. (2002) also reported that heat

treatment during the toasting of hexane extracted soy flours, to remove fats, will

produced acetyl forms. The 11S that was obtained for our experiment was found to

contain less than 1% of the acetyl forms out of the total isoflavone. The small

percentage of acetyl forms could be due to the minimal heat treatment process used

during the toasting of hexane extracted soy flours (7-B soy flour, Archer Daniels

Midland, U.S.A).

It can be seen that total isoflavone content of 11S with κ-carrageenan added during

the extraction have higher isoflavone content as compared to without the addition of

κ-carrageenan. The total daidzein content increased by 3.6%, the total glycitein

content increased by 2.3% and the total genistein content increased by 4.3% when κ-

carrageenan was added during the extraction of 11S.

93

4.4.2 Foaming properties

Figure 4.2 shows the profile of the formation and destabilization of foam. Air is

pumped to the protein solution until 140s (dotted line). From Figure 4.2, Vmax, the

maximum liquid incorporated into the foam of 11S with κ-carrageenan added during

the extraction process were higher than glycinin without κ-carrageenan added. The

presence of κ-carrageenan in 11S protein fraction result in an increase of 21.7% in

the maximum amount of liquid incorporated for 11S. Foam stability of 11S was

studied by the half life of the foam at the end of the bubbling period (t1/2). In the

presence of κ-carrageenan, a marked increase in t1/2 (40%) was observed.

Effects of the carrageenan on the foaming properties of 11S

0

1

2

3

4

0 50 100 150 200 250 300Time (s)

Volu

me

(ml)

11S

11S andcarrageenan

Figure 4.2. Foaming properties of 11S with κ-carrageenan and 11S

94

The surface hydrophobicities of control and κ-carrageenan added 11S fractions were

compared as shown in Table 4.2. The addition of κ-carrageenan had lowered the

surface hydrophobicity of 11S globulin. This results show that the 1-anilino-8-

naphthalene sulfonate (ANS) could not join the hydrophilic hydrocolloids (Galazka

et al., 1999), therefore the decrease in hydrophobicity is probably due to the binding

of negatively charged polysaccharides to the protein surface and thus blocking the

hydrophobic sites from binding to ANS. In addition to the binding and blocking

effect, Ortiz et al. (2004) suggested that the site of protein not joined to the κ-

carrageenan might be more hydrophilic. This was in agreement with the appearance

of additional peaks at lower retention times eluted from the Reverse-Phase HPLC,

which indicated the presence of more hydrophilic species in protein-carrageenan mix

(Ortiz et al., 2004).

Table 4.2. Surface hydrophobicity of 11S and 11S + κ-carrageenan mixture.

Protein fraction

Surface hydrophobicity (H0)

11S

388.0 ± 28.0

11S + κ-carrageenan

328 ± 26.6

95

The surface hydrophobicity of a protein generally has a direct correlation with its

foaming properties. Higher surface hydrophobicity leads to a higher chance for the

protein macromolecules to rearrange themselves at the air-water interface, showing

enhanced foam formation and foam stability. However, the increase in 11S

foamability and stability with presence of polysaccharide could not be attributed to

this factor because the surface hydrophobicity of 11S actually decreased due to the

aforementioned protein-polysaccharide interactions. Therefore the measurements of

protein surface hydrophobicity in bulk solution could not represent its behavior at the

air-water interface. Instead, the change in foaming properties was most likely due to

the presence of κ-carrageenan.

4.4.3 Gelling properties

The addition of carrageenan generally did not affect the state of curding for the

various salts exception for the CaSO4. In Table 4.3, for CaSO4 (highlight in blue), the

state of curding changed from gel to ppt in the presence of carrageenan. Polyvalent

metal ions such as Ca2+ can form bridges between negatively charged carboxyl

groups of the protein and the ester sulphate groups of the polysaccharide (Karim et

al., 1998). In such protein-polysaccharide-calcium systems, both the protein and the

polysaccharide can interact independently with calcium ions (Hughes et al., 1980).

Hence the addition of carrageenan improved gelling of proteins mixtures with metal

coagulants.

96

Table 4.3. The effect of carrageenan on gelling with various salt-coagulants.

Types of salts

State of protein mixture

With CaCl2

ppt

With CaCl2 and carageenan

ppt

With CaSO4

gel

With CaSO4 and carrageenan

ppt

With Ca(C2H3O2)2

ppt

With Ca(C2H3O2)2 and carrageenan

ppt

With MgCl2

ppt

With MgCl2 and carrageenan

ppt

With MgSO4

gel

With MgSO4 and carrageenan

gel

The definition of the state of curding was as follows: The “gel” state was when

mixtures of protein solutions after the addition of coagulants, formed complete and

uniform gels. The “ppt” state was when aggregation occurs rapidly and often led to

non homogenous precipitation.

97

For MgSO4 as shown in Table 4.3 (highlighted in blue), the sate of curding remained

the same in the presence of carrageenan. However, because the state of curding was

‘gel’, the samples of the ‘gel’ state were suitable for TPA, colorimetric tests, as well

as water holding capacity tests.

Table 4.4: Analysis results of “gel” mixtures with addition of carrageenan

From Table 4.4, we can see that the presence of carrageenan will not change the pH

of the gel but it will change the hardness, WHC and L* value. The WHC of the gel

with carageenan wass very high and this could caused the gel to become softer. It

was expected that higher water retention would reduce hardness (Karim et al., 1998).

Karim et al. (1998) noted that being a sulfated polysaccharide, carrageenan could

exist as a negatively-charged polymer over a wide range of pH. Above the

isoelectronic point, polyvalent metal ions could form bridges between both the

negatively-charged denatured protein molecules as well as the carrageenan

polysaccharides. As such, the interaction of the protein molecules and the

carrageenan polysaccharides with the cations formed an extensive network structure,

Salt

Coagulant

Mixture Composition

Hardness / N

WHC / %

pH

L*

11S

without carrageenan

0.402

31.08

7.02

70.19

MgSO4

11S with carrageenan

0.332

90.32

7.00

72.09

98

with which the curd could retain more water and hence making it softer. They also

noted the possibility of the protein molecules binding with the carrageenan

polysaccharides without the bridging by the cations in conditions when the pH of the

curd was higher than the isoelectronic point, thus forming a coupled gel network of

carrageenan and soy protein around the aggregated gel curd and together, these

helped to retain even more water.

4.5 CONCLUSION

κ-Carrageenan when added to soy flour have found to improve the retention of

isoflavones during the extraction of 11S. Hydrocolloids not only are able to

enhancement of the isoflavone constituent in 11S but also able to modify the

functional properties of 11S. κ-Carrageenan when added to soy flour to extract 11S

caused 11S to have better foaming properties. The presence of κ-carrageenan

improved the gelling of proteins mixtures with calcium sulphate and the water-

holding capacity of the gels formed with magnesium sulphate.

ACKNOWLEDGEMENT

The work on the foaming was assisted by Qian Tang and the work on gelation was

assisted by Han Yao Tan.

99

REFERENCES

1) Hughes, L. F.; Ledward, D. A.; Mitchell, J. R.; Summerlin, C. The effects of

some meat proteins on the rheoogica properties of pectate and aginate gels.

Journal of Textural Studies. 1980, 11, 247-256.

2) Karim, A. A.; Sulebele, G.A.; Azhar, M. E.; Ping, C.Y. Effect of

Carrageenan on Yield and Properties of Tofu. Food Chemistry. 1998, 66,

159-165.

3) Fitzpatrick, L. A. Soy isoflavone: hope or hype? Maturitas. 2003, 1, S21-S29.

4) Molina, O. S. E.; Wagner, J. R. Hydrolysates of native and modified soy

protein isolates: structural characteristics, solubility and foaming properties.

Food Research International. 2002, 35, 511-518.

5) Murphy, P. A.; Song, T. T.; Buseman, G.; Barua, K.; Beecher, G. R.; Trainer,

D.; Holden, J. Isoflavones in retail and institutional soy foods. Journal of




agricultural and food chemistry. 1992, 40, 941-944.

7) Pandjaitan, N.; Hettiarachchy, N.; Ju, Z. Y.; Crandall, P.; Sneller, C.;

Dombek, D. Enrichment of genistein in soy protein concentrate with

hydrocolloids and β-glucosidase. Journal of Food Science. 2000, 65, 591-595.

8) Wang, H. J.; Murphy, P. A. Isoflavone content in commercial soybean foods.

Journal of Agriculture and Food Chemistry. 1994, 42, 1666-1673.

100

Chapter 5

The aggregation profile of 2S, 7S and 11S coagulated by


101

5.1 ABSTRACT

The aggregation process of the proteins coagulated by GDL was monitored by using

atomic force microscopy (AFM). Solutions of 11S, 7S and 2S proteins after heating

at 100oC for 10 minutes was mixed with glucono-δ-lactone (GDL) and formed

aggregates with different aggregation profiles. When the three protein solutions (11S,

7S and 2S) were mixed with GDL and deposited onto the mica for one, two and four

minutes; 11S proteins formed the largest clusters of aggregates, 2S proteins formed

smaller cluster of aggregates than 11S but bigger cluster of aggregates than 7S and

7S proteins formed the smallest cluster of aggregates. It was also found by turbidity

measurement that when GDL was added to the three protein fractions, the level of

turbidity was in the order of 11S>2S>7S. Both these results showed that when GDL

was added to the three proteins fractions, the speed of aggregations was in the order

of 11S>2S>7S.

102

5.2 INTRODUCTION

Gelation and aggregations are closely related. It has been found that depending on

the conditions, both glycinin (11S) and β-conglycinin (7S) are able to form large

aggregates when heated (Mills et al., 2001). There are various ways to analyze

protein aggregates like particle size distribution, electron microscopy, dynamic

viscoelasticity measurement and spectrophotometric methods (Dybowska and Fujio,

1998). Atomic force microscopy (AFM) is widely used to study the structure of

biological macromolecules (McIntire and Brant, 1999). Its high resolution makes it a

powerful tool to study protein aggregation. AFM is able to image the shape of the

protein particles and aggregates (Morris et al., 2001).

In our work, the aggregation process of the various soy protein fractions coagulated

by GDL was monitored by using atomic force microscopy (AFM). 11S, 7S and 2S

proteins when react with GDL will form gels at a different speed and these

differences could be explained by looking at their respective aggregation profiles. It

was hoped that a greater understanding of the interactions of the different soy protein

fractions would shed more light on the factors that contribute to tofu texture.

103

5.3 MATERIALS AND METHODS

5.3.1 Sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE)

Protein fractions were analyzed using SDS-PAGE in a mini-Protean® 3 cell (Bio-

Rad laboratories, USA). 100 µl of sample (4mg/ml) of sample was mixed with 95 µl

of sample buffer (the sample buffer preparation was attached in Appendix I) with 5µl

of mercaptoethanol was heated for 95 oC for four minutes. Then 10 µl of this sample

mixture was applied to the gel. The concentrations of the stacking and running gels

were 4% and 12% respectively. The gel formulations for the resolving gels and the

stacking gels were attached in Appendix II. Precision Plus protein standards (Bio-

Rad, USA) are used as the molecular weight markers. The stacking, resolving and

electrolysis buffer preparation was attached in Appendix I and the voltage used for

the migration was 125V. After migration, the protein fractions were stained with

0.1% Coomassie Brilliant Blue in water-methanol-acetic acid (4:1:5) and destained

with water-methanol-acetic acid (85:7.5:7.5).

104

5.3.2 Atomic force microscopy (AFM)

The images of the aggregates were obtained by AFM according to the method of

Mills et al. (2001) modified as given below. Protein solutions were heated for 10min

at 100oC and cooled to room temperature. Freshly prepared GDL solution was added

to them such that the resultant mixture contained protein at 4.0% (w/v) and GDL at

0.4% (w/v). A drop of sample solution (30µl) was deposited onto freshly cleaved

mica for 1 and 2 and 4 minutes. Then argon gas was used to blow dry the samples.

The samples were imaged under ambient conditions with a Multimode™ AFM

(Digital Instruments, Veeco Metrology Group; Inc, Santa Barbara, CA) connected to

a Nanoscope® IIIa scanning probe microscope controller (Digital Instruments,

Veeco Metrology Group Inc, Santa Barbara, CA). All images were acquired in

tapping mode.


Turbidity due to aggregation was determined according to the method of Molina and

Wagner (1999) modified as given below. Turbidity was measured using Shimadzu

UV-visible spectrophotometer (Japan). Protein solutions were heated for 10min at

100oC. Freshly prepared GDL solution was added to them such that the resultant

mixture contained protein at 4.0% (w/v) and GDL at 0.4% (w/v). The absorbance of

1ml of mixture in a semi micro cuvette was measured at wavelength of 600nm.

105

A B C


5.4.2 Sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE)

Figure 5.1. SDS-PAGE of the soy protein fractions, lane (A) is 2S protein fraction;

lane (B) is 7S protein fraction; lane (C) is 11S protein fraction.

Figure 5.1 shows the SDS-PAGE patterns of the three soy protein fractions (2S, 7S

and 11S). In lane A, the molecular weight of 2S protein fraction was found to be

Molecular weight (kDa)

250 150 100

75 50 37 25 20 15 10

α

α’

β

acidic

basic

106

about 18 kDa. In lane B, the 7S protein fraction is make up of three subunits – α, α’

and β of molecular weight of 75 to 50 kDa. And in lane C, the 11S protein fraction is

make up of acidic and basic subunits of about 37 kDa and 20 kDa respectively.

The extraction technique for both the 11S and 7S fractions is acid precipitation while

the extraction technique for 2S is salt precipitation. Generally, Polson et al., (2003)

found that the efficiency of acid precipitation technique was higher than salt

precipitation technique. The extraction technique for 2S is salt precipitation instead

of acid precipitation because it was found that 7S was often co-extracted with 2S

during acid precipitation. This could be due to the close pI value of the two fractions

(pI of 7S is 4.8 and pI of 2S is 4.5).

5.4.2 AFM

The aggregation process of 11S with GDL was the fastest. It can be seen clearly

from Figure 5.2 a and 5.2 b that the particles of 11S become a cluster of aggregates

in just a minute upon the addition of GDL. When 11S proteins and GDL were

deposited onto mica for longer period of time, the size of aggregates increased as

shown in Figure 5.2 b-d.

107

The aggregation process of 7S with GDL was the slowest. It can be seen from Figure

5.3 a-d that the cluster of aggregates 7S protein did not change as significantly as the

cluster of aggregates of 11S and 2S when GDL was added. This showed that GDL

needed more time to react with 7S protein as compared to 11S and 2S to cause a

change in the aggregation profile at room temperature for the same length of time.

When 7S proteins and GDL were deposited onto mica for longer period of time, the

number of similar size cluster of aggregates increased.

It can be seen in Figure 5.4 a-d that the size of the aggregates increased as the time

deposited onto mica increased. For one, two and four minutes, the size of the cluster

of aggregates of 2S with GDL is smaller than the cluster of aggregates of 11S with

GDL but is larger than the cluster of aggregates of 7S with GDL. Hence the

aggregation process of 2S with GDL was slower than 11S with GDL but faster than

7S with GDL.

108

(a) (b)

(c) (d)


of 0.4% GDL: (b) 11S & GDL deposited onto mica for 1min. (c) 11S & GDL

deposited onto mica for 2 min (d) 11S & GDL deposited onto mica for 4min. Scan

size: 3µm by 3µm. The colour intensity corresponds to the height of the sample and

thus brighter spots denoted a greater aggregate height.

109

(a) (b)

(c) (d)


of 0.4% GDL: (b) 7S & GDL deposited onto mica for 1min. (c) 7S & GDL deposited

onto mica for 2 min (d) 11S & GDL deposited onto mica for 4min. Scan size: 3µm

by 3µm. The colour intensity corresponds to the height of the sample and thus

brighter spots denoted a greater aggregate height.

110

(a) (b)

(c) (d)


of 0.4% GDL: (b) 2S & GDL deposited onto mica for 1min. (c) 2S & GDL deposited

onto mica for 2 min (d) 11S & GDL deposited onto mica for 4min. Scan size: 3µm

by 3µm. The colour intensity corresponds to the height of the sample and thus

brighter spots denoted a greater aggregate height.

111

Protein solutions of 11S, 7S and 2S heated at 100oC for 10 minutes and without the

addition of GDL were deposited onto mica for the same period of time (1min, 2min

and 4min) to determine that the changes in the size or number cluster of aggregates

as seen in Figure 5.2 to 5.4 were due to the coagulating effect of GDL. It was found

that all the three proteins fractions when deposited onto mica for 1min, 2min and

4min formed aggregates that have similar aggregations size at confidence level of

95% (Table 5.1). This shows that the differences in the aggregates size in Figure 5.2

to Figure 5.4 were due to the effect of GDL.

The images of protein solutions of 11S (Figure 5.2a), 7S (Figure 5.3a) and 2S

(Figure 5.4a) without the addition of GDL, all showed large aggregates instead of

uniform particles or linear fibrous aggregates found by Mills et al., 2001 using 7S

protein solution of 0.005% (w/v). These large aggregates could be due to the effect

of heating the 4% (w/v) protein solution at 100oC for 10mins. Similar ‘macro

aggregates’ have been observed for heating 1% (w/v) solutions of 7S at 100oC for 10

min (Mills et al., 2001). Mills et al., 2001 suggested that the ‘macro aggregates’

maybe conglomerates of the smaller fibrous structures observed at lower protein

concentration.

112

Table 5.1. Average size of protein particles formed when 4 % protein solution heated

at 100oC for 10 minutes was deposited onto mica for 1, 2 and 4 minutes.

Average size of

protein deposited

onto mica for 1min

/ µm

Average size of

protein deposited

onto mica for 2min

/ µm

Average size of

protein deposited

onto mica for 4min

/ µm

11S protein

0.22 ± 0.02 a

0.23 ± 0.03 a

0.21 ± 0.03a

7S protein

0.09 ± 0.02 b

0.09 ± 0.02 b

0.08 ± 0.03 b

2S protein

0.13± 0.03 c

0.12 ± 0.04 c

0.14 ± 0.04 c

a denotes average size of 11S was not significantly different at P<0.05. b denotes

average size of 7S are not significant different at P<0.05. c denotes average size of

2S are not significant different at P<0.05. Single factor ANOVA was carried out.

113


Molina and Wagner, 1999 have found that turbidity (A600 nm) can be used to estimate

the degree of protein aggregation. When GDL was added to the three proteins

fractions, it was found from the turbidity graph (A600nm against time) that as time

increased, the A600nm increased and thus the level of turbidity increased which in turn

meant that the degree of aggregation also increased; thus the turbidity graph can be

used to determine the speed of aggregation. From Figure 5.5, the level of turbidity

increased with time as more aggregates were formed and reached a maximum when

homogenous gel was formed. The order of turbidity was in the order of 11S>2S>7S.

These results suggested that the speed of aggregation of the proteins were in the

same order of 11S>2S>7S.

Figure 5.5. Turbidity measurement of 11S, 7S and 2S protein solutions.

Turbidity measurement

0

0.5

1

1.5

2

0 5 10 15 20 25

Time (ks)

A 60

0 nm 11S

2S7S

114

5.5 CONCLUSION

Aggregation profile of soy protein could be used to explain the difference in the

gelation of protein with GDL. The aggregation profile of 11S, 7S and 2S proteins

with GDL at room temperature (Figures 5.2 to 5.5) showed that 11S proteins formed

large aggregates fastest followed by 2S and than 7S. Faster aggregations could lead

to faster gelation which was reported by Khim et al. (1995) who used rhemoter to

determine the gelation speed of 7S and 11S. They reported that 11S formed faster

gelation with GDL. Thus from our results, 2S should form faster gels with GDL as

compared to 7S with GDL but slower gels when compared to 11S with GDL. The

presence of 2S will affect the coagulation of soy protein with GDL. 2S although have

the least percentage composition in soy protein as compared to 7S and 11S will play

a significant role during the gelation process.

ACKNOWLEDGEMENT

This research was funded by National University of Singapore Academic research

grant #R-143-000-179-112. Special thanks to Mr. Darius Oh Hup Aik, Mr. Zhou

Xue Dong and Ms. Dai Xue Ni for their technical support in the operation of AFM.

115

REFERENCE

1. Doe, E. Gels and gelling of globular proteins. Trends in Food Science and

technology. 1993, 4,1-5.

2. Dybowska, B. E.; Fujio, Y. Optical analysis of glucono-δ-lactone induced by soy

protein gelation. Journal of Food Engineering.1998, 36, 123-133.

3. Fukushima, D. Recent progress of soybean protein foods: chemistry, technology,

and nitrition. Food Review International. 1991, 7,323-351.

4. Kohyama, K.; Murata, M.; Tani, F.; Sano, Y.; Doi, E. Effects of protein

composition on gelation of mixtures containing soybean 7S and 11S globulins.

Bioscience Biotechnology Biochemistry. 1995, 59, 240-245.

5. Kohyama, K.; Nishinari, K. Rheological studies on the gelation process of



6. Lakemond, C. M. M.; de Jongh, H. H .J.; Paques, M.; Vliet, T. V.; Gruppen, H.;

Voragen, A. G. J. Gelation of soy glycinin; influence of pH and ionic strength on

network structure in relation to protein conformation. Food Hydrocolloids. 2003,

17, 365-377.

7. McIntire, T. M.; Brant, D. A. Imaging of carrageenan macrocycles and amylase

using noncontact atomic force microscopy. International Journal of Biological

macromolecules. 1999, 26, 303-310.

116

8. Mills, E. N. C.; Huang, L.; Noel, T. R.; Gunning, P.; Morris, V. J. Formation of

thermally induced aggregates of the soya globulin β-conglycinin. Biochimica et

Biophysica Acta. 2001, 1547, 339-350.

9. Molina, M. I.; Wagner, J. R. The effects of diavalent cations in the presence of

phosphate, cirate and chloride on the aggregation of soy protein isolate. Food

Research International. 1999, 32,135-143.

10. Morris, V. J.; Mackie, A. R.; Wilde, P. J.; Kirby, A. R.; Mills, E. C. N., Gunning,

A. P. Atomic force microscopy as a tool for interpreting the rheology of food

biopolymers at molecular level. Lebensm. Wiss. Technology. 2001, 34, 3-10.

11. Nagano, T.; Hirotsuka, M.; Mori, H.; Kohyama, K.; Nishinari, K. Dynamic


agricultural food chemistry. 1992, 40, 941-944.

12. Polson, C.; Sarkar, P.; Incledon, B.; Raguvaran, V.; Grant, R. Optimization of

protein precipitation based upon effectiveness of protein removal and ionization

effect in liquid chromatography-tandem mass spectrometry. Journal of

Chromatography B. 2003, 785, 263-275.

13. Rao, A. G.; Rao, M. S. A method for isolation of 2S, 7S and 11S proteins of

soybean. Preparative Biochemistry. 1977, 7, 89-101.

14. Tay, S.L.; Perera, C. O. Effects of glucono-δ-lactone on 7S, 11S proteins and

their protein mixture. Journal of food Science. 2004, 69(3), FEP 139-143.

117

Chapter 6

The functional and structural properties of 2S soy protein in

relation to other molecular protein fractions

118

6.1 Abstract

The purpose of this investigation is to develop a better understanding of the

structure-function relationship of the 2S fraction of soy protein that has not been

considered in earnest by the research community. Defatted soy flour was used to

extract the three major fractions of the protein (2S, 7S and 11S). It was found that 2S

exhibits better foaming and emulsification properties than the remaining two

molecular fractions. Work was extended to structural properties, which were

monitored using spectrophotometry, atomic force microscopy (AFM), scanning

electron microscopy (SEM), small-deformation dynamic oscillation on shear, and

large-deformation compression testing. An experimental protocol utilising glucono

delta lactone (GDL), GDL with N-ethylmaleimide (NEM), or GDL with urea was

capable of identifying the nature of molecular interactions responsible for gelation.

Surprisingly, it was found that in the initial stages of structure formation, 2S fared

better than 7S, with 11S exhibiting the highest rates of aggregation. Given time,

however, 7S produced a firmer network with a better water holding capacity than

that of 2S. Physical interactions, as opposed to disulphide bridging, were found to be

largely responsible for the changing functionality of the molecular fractions

throughout the experimentation from the formation of a vestigial structure to that of a

mature gel.

119

6.2 INTRODUCTION

Soy protein is increasingly being used as a functional ingredient in an effort to

imitate desirable organoleptic properties in processed food products (Nunes et al.,

2003). It is advantageous that among legumes, soybean has a high protein content

containing three major molecular fractions (11S, 7S and 2S). The molecular weight

of these three fractions are approximate 300, 180 and 18 kDa, with the percentage

content being 41.9, 34 and 15%, respectively (Fukushima, 1991). Through the years,

research in structure-function relationships has been carried out mainly on 11S and

7S.

Regarding the interfacial studies, 11S was found to be a rather poor foaming and

emulsifying agent due to the difficulty in adsorbing at the air-water interface (Rickert

et al., 2004). This was attributed to low surface hydrophobicity, low chain flexibility

and the high molecular weight of the protein (Wagner and Guéguen, 1995).

Furthermore, Utsumi et al. (2002) modified 11S at a molecular level thus designing

recombinant systems where the extent of hydrophobicity of the C-terminal region

would determine largely the emulsifying properties. In the case of 7S, the extension

region found in subunits α and α’ possessed the required functionality for good

solubility and emulsification.

Glucono delta lactone (GDL) induced gelation has been extensively studied, and

Kohyama and Nishinari (1993) reported that the breaking stress of the 7S gel was

120

smaller than for 11S. Similarly, the rate of gelation for the 7S was lower than 11S. In

addition, the low isoelectric point of 7S maintained a relatively high pH value in the

gel, but the protein possessed lower cohesiveness, gumminess and lightness than the

11S preparation at 4% solids (Kohyama et al., 1995; Tay and Perera, 2004).

Scanning electron microscopy (SEM) showed that 11S produced a coarse network

with pore size of 2-3 µm whereas 7S exhibited a finer structure of about 0.5 µm pore

size (Kohyama et al., 1995). Puppo and Añón (1998) reported that acidic gels of the

two molecular fractions are stabilized by noncovalent bonds, with the contribution of

disulfide bridging to network stability becoming prominent only in an alkaline

environment. Finally, Mills et al. (2001) argued that precursors of a three-

dimensional structure in the form of insoluble macroaggregates can be captured

during thermal denaturation of a dilute solution of 7S (1% solids).

The scarcity of studies dealing with the relationship between structure and functional

properties of 2S may be two fold: Clearly, the well-known allergenicity of the

albumin family of 2S found in Brazilian nuts did not help to raise an interest in the

physicochemical properties of the material (Nordlee et al., 1996). Thus, several food

allergens associated with the protein fraction of 2S obtained from various natural

sources (sunflower seed, sesame seed, soybean, etc.) have been reported, for

example, the Kunitz trypsin inhibitors in (Moroz et al., 1980). According to Lin et al.,

however, the extent of allergenicity of the 2S fraction from soybean has not been, as

yet, rigorously documented (Lin et al., 2004). Another reason is the composition of

the soy protein, as 2S is the smallest of the three major fractions (Fukushima, 1991).

121

Putting the above considerations aside, Burnett et al. reported on the emulsification

properties of two variants of 2S from sunflower seed (Burnett et al., 2002).

Formulations of 11S with 2S from sesame flour were tried as a substitute for

traditional uses of dairy and soybean proteins in beverages and were met with an

acceptable response from a hedonic sensory evaluation panel (Lopez et al., 2003).

Sorgentini and Wagner (2002) found that whey soy protein which is made up of a

mixture of 2S and 7S fractions exhibited good foaming capacity within a broad pH

range from pH 2 to 10. The current investigation aims to provide further insights into

the foaming, emulsification, aggregation and gelation properties of the 2S soy

protein, and discusses this profile with the corresponding behaviour of the more

established molecular counterparts.

122

6.3 MATERIALS AND METHODS

6.3.1 Measurements of Foaming and Emulsification

Foaming properties were determined as follows: Air, at a flow rate of 90 cm3 / min.,

was introduced into 6 ml of 0.1% protein solution in a graduated column of 10 mm

diameter with a coarse glass frit at the bottom. Bubbling was continued for 140 s.

The volume of liquid incorporated to the foam was determined by measuring the

volume in the liquid remained in the column (Molina and Wagner, 2002).

The emulsifying properties of the samples were determined as follows: The

emulsions, 1.0 ml of corn oil and 3.0 ml of the protein solution (0.2%) were prepared

in 0.035 M phosphate buffer at pH 7.6 and were homogenized using Ultra turrax®

T18 homogenizer (IKA® Works, Wilmington, NC). Homogenization was carried out

at 20°C for 1 min at 18,000 rpm. 50 µl of the emulsion were taken from the bottom

of the container at different time intervals and diluted with 5 ml of a 0.1% sodium

dodecylsulfate solution. The absorbance was determined at 500 nm (Pearce and

Kinsella, 1978). The surface hydrophobicity of the protein sample was estimated by

the method of Rawel et al. (2002).

123

6.3.2 Formation of Aggregates

Protein preparations were heated for 10 min at 100oC and then cooled to 25oC to

form a homogeneous dispersion. Aggregation would occur following addition of a

freshly prepared GDL solution to the system, with the resultant mixture containing

protein at 4.0% (w/v) and GDL at 0.4% (w/v). In order to block the chemical

interaction in the aggregates, 10 mM N-ethylmaleimide (NEM) were added to the

cooled sample an hour prior to the addition of GDL. Physical interactions in the

aggregates were inhibited by adding urea (6M) to the system in accordance with the

procedure described for NEM.

6.3.3 Turbidity and Images of the Aggregates.

Aggregation during the GDL acidification was determined by measuring the

turbidity of 1 ml of the soy sample in a semi micro cuvette as the absorbance at 600

nm and 25oC using a UV-visible spectrophotometer (Shimadzu model UV 1601,

Kyoto, Japan) (Molina and Wagner, 1999).

Images of the vestigial structure formation were recorded using atomic force

microscopy (AFM) (Veeco Metrology Group Inc, Santa Barbara, CA) (Tay et al.,

2005). A drop of sample solution (30 µl) was deposited onto freshly cleaved mica for

124

4 minutes. Then argon gas was used to blow dry the samples. These were imaged

under ambient conditions with a Multimode™ AFM connected to a Nanoscope® IIIa

scanning probe microscope controller. All images were acquired in tapping mode

using Veeco Nanoprobe™ tips (Veeco Metrology Group Inc, Santa Barbara, CA).

6.3.4 Scanning Electron Microscopy (SEM)

Single 2S, 7S and 11S gels containing 4.0% (w/v) protein and 0.4% (w/v) GDL were

left to age at room temperature (25oC) for 24 hours prior to experimentation. Next,

samples were deep frozen in liquid nitrogen and freeze dried. Materials were fixed to

a stub and coated with platinum using Sputter Coater (JEOL model JFC-1300, Tokyo,

Japan). Networks were observed using SEM, which operated at an accelerating

voltage of 15kV (JEOL model JSM-5600LV, Tokyo, Japan).

125

6.3.5 Rheological Measurements

Similar gels to those used for SEM and WHC analyses were subjected to large-

deformation compression testing. Work focused on the hardness of the gels which

was determined by compressing cylindrical disks of 15 mm diameter and 10 mm

height to 80% of their original height (Tsoga et al., 1999). A 35 mm diameter probe

in a TA.XT2i texture analyzer (Stable Microsystems, Godalming, Surrey, UK) was

employed and the compression rate was 0.1 mm/s. Temperature was ambient.

Small-deformation mechanical measurements on shear were performed with the

Advanced Rheometrics Expansion System (ARES), which is a controlled strain

rheometer (Rheometric Scientific, Piscataway, NJ). A parallel-plate geometry of 50

mm diameter and 1 mm gap was used. Samples were prepared as described in

“Formation of Aggregates”, loaded onto the platen of the rheometer, and their

exposed edges were covered with a silicone fluid (50 cs) to minimize water loss.

These were subjected to an isothermal run (25°C) for up to 24 hr at the frequency of

1 rad/s. The time sweep was followed by a frequency sweep between 0.1 and 100

rad/s. In both cases, a strain within the linear viscoelastic region was used. The

experimental protocol provides readings of the storage modulus (G'; elastic

component of the network), loss modulus (G"; viscous component), and a measure

of the ‘phase lag’ δ (tan δ = G" / G') of the relative liquid-like and solid-like

character of the material (Kasapis, 2004).

126


6.4.1 Foaming and Emulsification Properties

The air and water phases in foams form a thermodynamically unstable system due to

unfavorable interactions at the interface, and the different densities of the two

domains. Figure 6.1a reproduces the profiles of foam formation and subsequent

destabilization in our soy fractions following air injection to solutions for up to 140 s.

To the left of the dotted line, the curve indicates foam formation. The process

involves rapid diffusion of the protein to the air/water interface, adsorption, and

reorientation to form a film leading to reduction in the interfacial tension (German

and Phillips, 1994). In this context, stable foam formation relates to an increasing

volume of water being incorporated into the biphasic system. On the right side of the

dotted line, the drop in the profile of volume development as a function of time

reflects the destabilization of the foam, which will eventually collapse due to liquid

drainage.

127

Figure 6.1a

Figure 6.1b

Figure 6.1. The interfacial behaviour of the three soy protein fractions, i.e., 11S, 7S

and 2S, as demonstrated for (a) the foaming and (b) the emulsifying

properties.

Foaming properties

01234567

0 50 100 150 200 250 300

Time (s)

Volu

me

(ml)

11S7S2S

Emulsifying properties

0

0.2

0.4

0.6

0.8

0 50 100 150 200 250 300

Time (s)

A 50

0nm 11S

7S2S

128

The maximum volume of water (Vmax) incorporated in the foam of 2S, 7S and 11S is

6.1, 4.4 and 2.5 ml, respectively. Thus 2S is able to intake the highest amount of

water, a result that argues for the protein forming the best adsorption medium in the

air/water interface. The relatively poor foaming performance of 11S is highlighted

since the Vmax value is obtained early within the air injection period (80 s in Figure

4.1a). The stability of the foam beyond air injection is also of interest and, in the

present context, this is defined as the time of half-drainage of water that was

incorporated in the foam after 140 s (t1/2) (Molina and Wagner, 2002). As for the

foam creation, 11S exhibits the least stability, with the value of t1/2 being 60 s, as

compared for the 2S and 7S air/water structures (t1/2 ~ 160 s).

It has been demonstrated that the higher the hydrophobicity of a protein fraction, the

more stable the film that forms at the air/water interface (Sorgentini and Wagner,

2002). Furthermore, the gradient of the fluorescence intensity vs. protein

concentration can be used as an index of surface hydrophobicity (Ho) (Rickert et al.,

2004; Wu et al., 1999). Following this approach, this experimental work found that

11S exhibits the least hydrophobic character (Ho = 388) among the soya fractions (Ho

for 2S is 483, and 446 for 7S) at neutral pH. Wu et al. (1999) also reported that 7S is

more hydrophobic than 11S at pH 7.0. The hydrophobicity index of the protein

fractions can rationalise the stable film formation by 2S and 7S at the air/water

interface.

129

Next, the emulsification properties of the soy protein fractions were examined. This

was assessed from the turbidity of the emulsion, since reduction of the oil/water

interface tension and suspension of the oil droplets by the continuous aqueous phase

leads to an increase in turbidity (Diftis and Kiosseoglou, 2003). In Figure 6.1b, the

absorbance values, which indicate the turbidity level of the emulsions formed by the

2S, 7S, and 11S at time zero, are 0.72, 0.51 and 0.35, respectively. Once more, 2S

was found to possess better functional properties (stabilization of the oil droplets

against coalescence this time round), as compared to the higher molecular weight

counterparts. The fraction corresponds to the least percentage composition of the

soybean protein, but possesses attractive foaming and emulsification characteristics

that render weight to potential applications as a single functional ingredient or a

tailor-made addition to a protein composite.

6.4.2 Aggregation of the Molecular Fractions

Soy protein is a texture modifier and, as such, the phase viscosity increases through

the gradual build up of aggregates which may lead eventually to the formation of a

continuous gelled phase. The remaining part of this discussion focuses primarily on

the development of structure in our molecular fractions and its rationalisation. A

starting point to consider is the change in pH with time due to addition of GDL.

Figure 6.2 reproduces the gradual increase in the acidity of the three protein fractions

following addition of the acidifier, which hydrates slowly to form gluconic acid.

130

Upon standing for 30 ks, a noticeable drop in pH values is recorded ranging from 5.3

to 4.5 for the 7S and 11S gels, respectively. Clearly, 7S exhibits the best buffering

capacity against the added protons and this information will prove to be useful in the

examination of the effect of generated acidity on structure formation.

Figure 6.2. pH variation as a function of time following GDL addition at ambient

temperature for the three soy protein fractions.

pH of the soy fractions and GDL

4

5

6

7

0 5 10 15 20 25 30

Time (ks)

pH

11S7S2S

131

Both atomic force microscopy and spectrophotometry are particularly suited for the

capture of vestigial structure in the time domain (Tay et al., 2005). In Figure 6.3a,

the pink tracks, which contrast strongly with the dark brown background, represent

domains of an intense aggregate built up. This takes place within 4 minutes of adding

GDL to the solution of 11S at ambient temperature. It appears that the fraction

exhibits rapid kinetics of aggregation. In spite of the addition of GDL, the

aggregation process of 7S is slow and cluster formation remains rather sporadic

within the time frame of experimental observation (Figure 6.3b). 2S forms

intermediate size structures, which are imaged as white discontinuous inclusions in

the viscous suspension (Figure 6.3c). Clearly, the rate of the aggregation process is

governed by the extent of acidification discussed in Figure 6.2, with the most acidic

environment of 11S and 2S being inducive to the development of a proteinacious

structure in the absence of thermal treatment.

132

Figure 6.3a Figure 6.3b

Figure 6.3c

Figure 6.3. Atomic force microscopy images of the three types of soy protein

aggregates: (a) 11S, (b) 7S, and (c) 2S following addition of 0.4% GDL and

deposition onto mica for 4 min (scan size: 3µm by 3µm).

133

Semi-quantitative confirmation of the relative patterns in structure development can

be obtained from turbidity measurements at ambient temperature. Increase in

turbidity, owing to build up of aggregation, is reflected in the values of absorbance

(A), which presently are recorded at 600 nm to eliminate potential chromophoric

complications from molecular groups at lower wavelengths. There is an early

increment in readings for 11S which reach a plateau at 2.5 ks (Figure 6.4a). This is

followed by 2S and 7S achieving constant levels at 15 and 24 ks, respectively. Thus

the degree of protein aggregation unveiled in the micrographs of the preceding

paragraph is consistent with the order of turbidity development using

spectrophotometry.

Figure 6.4a

Turbidity of soy protein fraction with GDL

0

0.5

1

1.5

2

0 5 10 15 20 25 30Time (ks)

A 60

0nm

11S7S2S

134

Figure 6.4b

Figure 6.4c


three types of soy protein aggregates following GDL addition: (a) overall

profile, (b) in the presence of urea, and (c) in the presence of NEM.

Effects of urea on the turbidity of soy protein fractions with GDL

0

0.5

1

1.5

2

0 5 10 15 20 25 30

Time (ks)

A 60

0nm 11S

7S2S

Effects of NEM on the turbidity of soy protein fraction with GDL

0

0.5

1

1.5

2

0 5 10 15 20 25 30

Time (ks)

A 60

0nm 11S

7S2S

135

The isoelectric point (pI) of 11S is 6.4, followed by 7S (4.8) and 2S (4.5) (Dreauet et

al., 1994). Based solely on the effect of pI and for the same pH value, the extent of

aggregation of the three molecular fractions following GDL addition should be in the

order of 11S > 7S ~ 2S. However, the actual buffering capacity was found to create

the distinct sequence of 11S > 2S > 7S, with the 2S exhibiting three-dimensional

formations earlier than expected. Clearly, simple pI considerations are not adequate

to rationalize the experimental trend, and factors such as the nature and extent of

intermolecular forces responsible for structure development should now be

considered.

The prevailing type of interactions in soy protein was probed with urea and N-

ethylmaleimide (NEM). The former is well known to destabilize forces of a

secondary nature, i.e., hydrophobic, electrostatic, and hydrogen bonding (Chronakis

et al., 1995). As shown in Figure 6.4b, addition of urea has an adverse effect on the

kinetics of vestigial structure formation for all fractions, but the aggregation profile

of 11S recovers fully during the isothermal run. In the case of 7S and 2S, there is an

irreversible loss in molecular functionality, with the values of absorbance leveling

off well below those recorded in the absence of the reagent. Urea preserves chemical

interactions in the system and, in Figure 6.4b, these create the following order of

structure formation: 11S > 2S~7S. However, this outcome is not congruent with the

overall response observed in Figure 6.4a.

136

N-ethylmaleimide, on the other hand, is used to block chemical interactions in the

nature of the thiol groups (Alting et al., 2003). Gratifyingly, NEM addition to soy

preparations in Figure 6.4c yields aggregation rates which are effectively identical to

those first recorded in Figure 6.4a. This result argues convincingly for the absence of

thiol-group contributions to structure at the vestigial stage. Physical interactions

survive in the presence of NEM and create the descending order of 11S > 2S > 7S

also noted in the absence of reagents, thus rendering the effect of pI and thiol groups

secondary in early aggregation.

6.4.3 Formation of Three-Dimensional Structures Upon Ageing

A preliminary exploration of the structural features of soy preparations included the

water holding capacity (WHC) and rapid compression testing. In doing so, samples

were left to age for 80 ks at ambient temperature. WHC examination at the end of the

standing period yielded the following results: 11S gel (70.1%), 7S (64.4%), and 2S

(42.2%). Compression testing focused on the force required to fracture the gel

(hardness) thus producing values of 0.77, 0.42 and 0.14 N with decreasing molecular

weight distribution of the macromolecules in the soy network. It is clear, at this point,

that the gel hardness and WHC properties exhibit a common pattern of functionality

according to the order: 11S > 7S > 2S. These basic features of the soy network are

not related directly to the kinetics of aggregate formation earlier examined, at which

the 2S exhibited accelerated functionality.

137

Further confirmation of the incongruity between the overall trend in the aggregation

profile and the final rate of gelation in the soy protein fractions is presented by

means of Scanning Electron Microscopy (SEM). Figure 6.5a illustrates a well-

developed, “honeycomb’ structure obtained for the mature 11S gel at ambient

temperature following addition of GDL. By comparison, the three dimensional

arrangement of 7S appears to include a high number of cavities with a broad size

distribution (Figure 6.5b). The imperfections in the organization of the layers of 7S

should be responsible for the formation of soft gels with WHC properties inferior to

those of 11S. Both 11S and 7S, however, form networks with contour-length pores

that are able to trap water molecules effectively in comparison with the 2S gel. Thus,

extensive structural defects are noted in the three-dimensional image of the 2S

network resulting in a poor water holding capacity and the softest intermolecular

assembly (Figure 6.5c).

138

Figure 6.5a Figure 6.5b

Figure 6.5c

Figure 6.5. Electron microscopy images of the three types of soy protein gels: (a)

11S, (b) 7S, and (c) 2S (magnification: 1500×).

139

The unexpected deterioration in the functionality of the aged 2S gel needs also to be

explained. To understand the gelation process, once more, we employed the interplay

between the formation of chemical gels, where crosslinking is covalent, and physical

gels, which contain non-covalent crosslinks. Figure 6.6a reproduces the development

of turbidity recorded between 30 and 80 ks, following GDL addition, and at the

remaining experimental conditions described earlier for shorter time periods of

observation (0 to 30 ks in Figure 6.4). For all fractions, a plateau is obtained at a

single reading of absorbance, an outcome which signifies the formation of an

“infinite” network of molecules, that is macroscopic in size, so that its turbidity

characteristics exceed the maximum reading capacity of the signal detector of the

instrument.

Regarding the addition of chemical reagents, a single spectroscopic response

identical to that in Figure 6.6a is recorded for the three soy fractions in the presence

of NEM (results not shown here). However, the behaviour of our systems changes

considerably once urea eliminates the physical contributions to network stability.

Thus in Figure 6.6b, 11S cross-links appear to be affected the least by this

environment but the 7S network is severely disrupted. The molecular structure of the

2S is rather different to that of 7S and involves some contributions from thiol groups,

with the overall profile of the underlying “gel state” in the presence of urea being

11S > 2S > 7S. These results are, of course, at variance with the WHC, textural and

structural properties reported in this section, thus emphasizing the importance of

physical associations in the development of the mature 7S network.

140

Figure 6.6a

Figure 6.6b


three types of soy protein gels following GDL addition: (a) overall profile,

and (b) in the presence of urea.

Effects of urea on the turbidity of soy protein fractions with GDL

0

0.5

1

1.5

2

30 40 50 60 70 80Time (ks)

A 60

0nm 11S

7S2S

Turbidity of soy protein fraction with GDL

0

0.5

1

1.5

2

30 40 50 60 70 80

Time (ks)

A 60

0nm 11S

7S2S

141

Complementary evidence that the morphology of our networks is quite distinct from

chemical gels and that physical forces predominate in the long distance scales of

molecular interactions is probed by rheological, particularly viscoelastic,

experiments (Tabilo-Munizaga and Barbosa-Canovas, 2005). This approach is

invaluable in the context of the present investigation for making measurements on

aged gels that are not subject to “upper bound” limitations in readings seen for

spectroscopy in Figure 6.6a. Thus, gel cure experiments of the soy preparations were

carried out in real time using small-deformation oscillatory rheometry. The pre-

gelled system (in the fluid state) was loaded on to a parallel plate assembly at

ambient temperature, and one of the plates was oscillated at a fixed frequency and

fixed maximum strain. As the gel begins to “set up”, readings of G´ and G" are

recorded for 80 ks to match the timescale of observation employed in spectroscopy

experiments.

Figure 6.7 reproduces the characteristic time profile of gelation obtained for the three

soy fractions at the concentration of 4% and in the presence of GDL. To avoid clutter,

only the G' trace is illustrated. Materials exhibit a pasty viscoelasticity with a solid-

like response, i.e., values of the storage modulus predominate over those of the loss

modulus, with the damping factor, tan δ, being less than 0.1. There is a rapid

development in the elastic component of the 11S network, which achieves an

apparent equilibrium modulus of ≈ 1 kPa relatively early within the time run (5 ks).

The cold-setting behaviour of 7S is considerably slower but, overall, it forms a

142

sigmoidal modulus-time profile indicative of co-operative gelation. In contrast, 2S

exhibits a progressive increase in the values of G' which appear to level off at about

60 ks. The values of G' of the 2S network are substantially lower than for the two

remaining counterparts, never exceeding 50 Pa (log G' = 1.7). Finally, addition of

NEM produced a mechanical response qualitatively similar to that of Figure 6.7,

whereas in the presence of urea, aggregate formation was unable to create a

sufficient volume of intermolecular interactions for the formation of cohesive three-

dimensional structures within the experimental timescale of observation (results not

shown).

Figure 6.7. Time course of G' for the three soy fractions at 25°C, frequency of 1 rad/s,

and strain of 0.1%.

Time course of G'

-1.5

-0.5

0.5

1.5

2.5

3.5

0 10 20 30 40 50 60

Time (ks)

log

( G' /

Pa

) 11S

7S

2S

143

6.5 CONCLUSION

The present paper explores the functional and structural features of 2S soy protein

and then compares them with those of the higher molecular-weight counterparts (11S

and 7S). It is arguable that based on the superior interfacial properties of 2S,

inclusion of the molecular fraction in a variety of food products may improve their

foaming and emulsification performance. Addition of GDL results in levels of

acidity that do not follow the conventional pattern dictated by the isoelectric point of

the three molecular fractions. Thus, GDL strengthened 2S systems exhibit

unexpected signs of an early aggregation owing to non-covalent molecular

interactions. However, further curing of the material at ambient temperatures results

in soft gels with an inferior three-dimensional structure and a water holding capacity

in relation to the remaining two proteinaceous fractions. Throughout the course of

experimentation, the importance of these physical forces emerges again and again,

i.e., from the slow kinetics of the vestigial structure formation in 7S, as compared

with 2S, to the development of a firm network in the cured 11S gel.

144

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149

Chapter 7

Conclusions and future studies

150

7.1 Conclusions

In this thesis, the functional and structural properties of soy protein fractions (11S,

7S and 2S) were studied.

The two major soy protein fractions, 7S and 11S, and the mixtures were reacted with

acid and salt coagulants to study the acid-induced and salt-induced gelation. The

mixtures of 11S:7S when reacted with acid coagulants, GDL possess an interesting

interplay of gelation properties based on polymeric composition and time of thermal

treatment. Mixtures of 11S:7S when reacted with GDL produced quantifiable

gelation behavior based on the premise that higher levels of 7S in the composite

would require longer times of thermal treatment to achieve comparable

physicochemical properties. The mixtures of 11S:7S when reacted with salt

coagulants will result in different types of curd formation. Based on these differences

we will be able to determine the coagulating power of the salt coagulants. The

coagulating powers of various salts was in the order of CaCl2 > MgCl2 > CaSO4 >

MgSO4.


benefits, since isoflavones are lost during aqueous extraction of soy protein fractions.

Thus it was important to improve the isoflavone content of the protein during

extraction. κ-Carrageenan when added to soy flour was found to improve the

retention of isoflavones during the extraction of 11S. κ-Carrageenan not only is able

151

to enhance the isoflavone constituent in 11S but also able to modify the functional

properties of 11S. κ-Carrageenan when added to soy flour to extract 11S caused 11S

to have better foaming properties and improved gelling properties of calcium

sulphate and magnesium sulphate gels.

Through the years, research in structure-function relationships has been carried out

mainly on 11S and 7S, and limited studies is carried out on 2S. The functional and

structural features of 2S soy protein were studied and compared with that of the

higher molecular-weight counterparts (11S and 7S). It was found that although, the

2S corresponds to the least percentage composition in soy protein as compared with

7S and 11S, it plays a significant role in the foaming, emulsifying and aggregation

process of the soy proteins. Thus based on the superior interfacial properties of 2S,

inclusion of the molecular fraction in a variety of food products may improve their

foaming and emulsification performance.

The aggregation process is governed by the extent of acidification in the acid-

induced gelation. It was found that the order of the acidity of the soy protein

fractions gel was the same as the order of the aggregation process of the three protein

fractions. The order of the aggregation process was 11S > 2S > 7S. It was found that

of pI and physical interactions were responsible for the 11S protein fraction having

the fastest aggregation rate, while physical interactions was responsible for the

aggregation process of 2S which is faster than 7S.

152

The aggregation profile of soy protein fractions cannot be used to explain the

difference in the rate of gelation of protein fractions with GDL. It was found that the

rapid aggregation of the 2S does not correspond to firm gels with improved water

holding capacity. The 2S soy fraction formed soft gels with an inferior three-

dimensional structure and a water holding capacity as compared with the other two

protein fractions. Throughout the course of experimentation, the importance of these

physical forces emerges again and again. It was found that in the initial stages of

structure formation, 2S exhibiting higher rates of aggregation than 7S. Given time,

however, 7S produced a firmer network with a better water holding capacity than

that of 2S. Physical interactions, as opposed to disulphide bridging, were found to be

largely responsible for the changing functionality of the molecular fractions.

In conclusion, this thesis develops understanding of the physico-chemical properties

of single (11S, 7S and 2S) and mixed soy protein fractions (11S and 7S), and in

particular the 2S fraction that has not been considered in earnest by the research

community. The investigation on the molecular interactions of the protein fractions

was carried out in order to understand the basis of the functionality and the various

modes of interactions of the fractions that occurred especially in the aggregation and

acid induced gelation conditions. The information obtained from the studies on the

functional properties of each of the protein fractions should facilitate inclusion of

these mixtures as a single functional ingredient or as a tailor-made addition to a

protein composite.

153

7.2 Future studies

In this project, the functional and the structural properties of individual protein

fractions (2S, 7S and 11S) and the mixture of the two major protein fractions (7S and

11S) were studied. In order to further understand the basis of the soy protein

fractions on the gelation and structural properties, more studies about the protein-

protein interaction of the minor protein fraction (2S) with the major protein fractions

(7S and 11S) are need to be carried out in the near future. Studies on 2S mixtures

with 11S and / or 7S will assist in the identification of general patterns of behaviour

in biphasic gels, with the view of facilitating the inclusion of these mixtures in

industrial product formulations

The minimal concentration required for protein to form gel and the phase separation

of the mixed protein systems of the 2S and 11S or 2S and 7S could be obtained from

the graph of the semilog plot of the storage modulus (G’) against the concentration

of the protein. Information regarding the gel strength of the mixed protein system

could be investigated using large-deformation compression testing and small-

deformation mechanical measurements, and networks of the mixed protein gel could

be investigated with SEM.

154

Appendix I. Buffer solutions preparation for SDS-PAGE

1. The sample buffer was prepared by mixing all the reagents as below.

Reagents

Volume / ml

Deionized water

3.55

0.5M Tris-HCl, pH 6.8

1.25

Glycerol

2.5

10% (w/v) SDS

2.0

0.5% (w/v) bromophenol blue

0.2

2. The stacking buffer was prepared as follows: 6g of Tris base was dissolved in

60ml of deionized water. The pH was adjusted to pH 6.8 with 6 N HCl. 0.4 g of

sodium dodecyl sulfate (SDS) was dissolved in the solution and the total volume was

topped up to 100ml with deionized water.

155

3. The resolving buffer was prepared as follows: 18.15g of Tris base was dissolved

in 80ml of deionized water. The pH was adjusted to pH 8.8 with 6 N HCl. 0.4 g of

sodium dodecyl sulfate (SDS) was dissolved in the solution and the total volume was

topped up to 100ml with deionized water.

4. Electrolysis buffer was made up of 3.03g of Tris base, 14.4 glycine and 1g of

sodium dodecyl sulfate (SDS). The pH of the solution is approximately pH 8.3.

156

Appendix II. Gel formulation for SDS-PAGE

1. Seperating Gel

Reagents

Volume / ml

Deionized water

5.25

Seperating buffer

3.75

30% acrylamide / bis

6.0

The monomer solution was prepared by mixing all the reagents above. Prior to

pouring the gel to cast, 10 µl of TEMED and 50 µl of ammonium persulfate (10%)

were added and the mixture were swirl gently to initial polymerization.

157

2. Stacking Gel

Reagents

Volume / ml

Deionized water

1.25

Seperating buffer

3.05

30% acrylamide / bis

0.65

The monomer solution was prepared by mixing all the reagents above. Prior to

pouring the gel to cast, 5 µl of TEMED and 25 µl of ammonium persulfate (10%)

were added and the mixture were swirl gently to initial polymerization.

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