MATERIALS AND METHODS

The main material used in this project was soluble wheat protein (SWP). It was manufactured by the deamidation of wheat gluten. This chapter describes the structure of the gluten proteins and the effect of deamidation on its functional properties. In addition the analytical methods routinely used in the project are described.

The Gluten Proteins and Deamidated Soluble Wheat Protein (SWP)

Gluten Proteins

The storage proteins of wheat are unique because they are also functional proteins. They do not have enzyme activity, but they are the only cereal proteins to form a strong, cohesive dough that will retain gas and produce light baked products. They can be easily isolated by removing starch and albumins/globulins by gently working a dough under a small stream of water. After washing, a rubbery ball is left, which is called gluten. Traditionally, gluten proteins have been classified into four types according to their solubility (Osborne, 1907) as follows:

An alternative classification to that described above has been proposed (Shewry et al., 1986) based on composition and structure rather than solubility (Figure 2.1). Most of the physiologically active proteins (enzymes) are found in the albumin or globulin groups. Nutritionally, the albumins and globulins have a very good amino acid balance. They are relatively high in lysine, tryptophan and methionine (Pomeranz, 1968). The prolamins were among the earliest proteins to be studied, with the first description of wheat gluten being that of Beccari (1745). The prolamins have always been considered to be unique to the seeds of cereals and other grasses and unrelated to other proteins of seeds or other tissues. They have been given different names in different cereals, such as: gliadin in wheat; avenins in oats; zeins in maize; secalins in rye; and hordein in barley. The glutelins are called glutenins in wheat. The gliadins and glutenins are the storage proteins of wheat endosperm and they tend to be rich in asparagine, glutamine, arginine or proline (Abrol et al., 1971; Derbyshire et al., 1976; Kirkman et al., 1982; Larkins, 1981; Spencer and Huggins, 1982) but very low in nutritionally important amino acids lysine, tryptophan and methionine (Appendix A).


Figure 2.1: Classification of wheat gluten proteins based on primary structure relationship and mobility in Gel Electrophoresis.

Cereals are an important protein source and are processed into bread, pasta and noodles, breakfast cereals and fermented drink. For all these applications the quality is determined, to a greater or lesser extent, by the gluten proteins which account for about half of the total grain nitrogen. There are also opportunities to develop novel uses for cereal proteins in both the food and non-food industries. The aim of this project is to study the wheat gluten proteins, particularly the deamidated SWP in relation to their structure and function.

2.1.1.1 Intergenetic Comparisons of Cereal Prolamines

On the basis of information presented by several authors (Hitchcock, 1950; Morris and Sears, 1967; Clayton, 1972; Sakamoto, 1973; Paulis and Wall, 1977; Smith and Lester, 1980), a tree diagram summarizing currently accepted genetic relationships of cereal grains was constructed by Bietz, 1982 (Fig. 2.2).


Figure 2.2: Genetic relationships of cereal grains

Wheat, barley, and rye are classified in the same subfamily (Festucoideae) and tribe (Triticeae) of the grass family (Gramineae), and this close relationship is reflected in the structures of their prolamin storage proteins. Only in wheat, however, do these proteins form a cohesive mass (gluten). Barley and rye are diploids, each with seven pairs of chromosomes, while wheat species are diploid, tetraploid, or hexaploid (Figure 2.3).

Chromosome number in each genome		     	       Genome
						A		B		D
		1				1A		1B		1D
		2				2A		2B		2D
		3				3A		3B		3D
		4				4A		4B		4D
		5				5A		5B		5D
		6				6A		6B		6D
		7				7A		7B		7D

		Figure 2.3: Chromosome structure of wheat.

Total number of chromosome = 42 for hexaploids (used for breadmaking)
		= 28 for tetraploids (Durum wheat used for pasta)
		= 14 for diploids (primitive wheat)
The last two originated from the hybridization of related wild diploid species. Bread wheat (Triticum aestivum) is an allohexaploid with three genomes (each having seven pairs of chromosomes called A, B, and D). The A and D genomes are thought to be derived from a wild diploid wheat (Triticum sp.) and the related wild grass Aegilops squarrosa (T. tauschii), respectively. The origins of the B genome are not known. Durum or pasta wheats (T.durum) are tetraploid, with only the A and B genomes. Because the A, B, and D genomes are derived from related diploid species, they have genes that encode related but not identical proteins. The interactions of proteins encoded by the different genomes are important in determining the precise physical and technological properties of dough produced from pasta and bread wheats. Each chromosome consists of a long arm and a short arm joined by a centromere (Fig.2.4).

Genes coding for the gliadin proteins are located on the short arms of groups 1 and 6 chromosomes (Wrigley and Shepherd, 1973; Brown et al.,1981). The group 1 chromosomes control all the omega-gliadins, most of the gamma-gliadins, and a few of the beta-gliadins, whereas genes on the group 6 chromosomes code for all the alpha-gliadins, most of the beta-gliadins, and some of the gamma-gliadins. The genes coding for gliadin proteins occur as a single complex locus on each of the short arms of groups 1 and 6 chromosomes rather than at two or more loci. The HMW glutenin subunits are coded by genes (loci) on the arms of chromosomes 1A, 1B, and 1D. These loci are designated Glu-A1, Glu-B1, and Glu-D1, respectively. Electrophoretic studies have revealed appreciable polymorphism in the number and mobility of the HMW subunits in different wheat cultivars. That is, the genes on the chromosome 1 long arms show multiple allelism. Based on electrophoretic studies, Payne et al. (1981) proposed that there were two main types of subunits, the x type (of high relative molecular weight, Mr ) and the y type (low Mr). This subdivision has been supported by chemical and genetic evidence.


Genes for HMW Glutenins = Glu-A1, Glu-B1 and Glu-D1 Genes for LMW Glutenins = Glu-A3, Glu-B3 and Glu-D3 Gli-A1, B1 and D1 = all omega gliadin, most of the gamma gliadin and a few of the beta gliadin Gli-A2, B2 and D2 = all alpha gliadin, most of beta gliadin and some of gamma gliadin.

Figure 2.4: Chromosomal location of major protein groups of hexaploid wheats.

A particular subunit is specified by noting its chromosome, followed by its classification as x or y, and finally a number (designating the protein subunit), this number increasing with decreasing Mr (Appendix A). In contrast to the HMW subunits, the LMW glutenin subunits are encoded by genes on the short arms of chromosomes 1A, 1B, and 1D, these loci being designated Glu-A3, Glu-B3, and Glu-D3. The allocation of genes coding for particular proteins has been achieved by use of genetic variants in combination with electrophoresis. Examples of these variants are aneuploids, lines that are deficient in a single chromosome. Much of the pioneering work in this area was conducted by Sears (1969). In simple terms, when a particular chromosome is missing, this may coincide with the disappearance of a certain band or bands in the electrophoretic pattern of the wheat protein, thus identifying the chromosome that carries the gene(s) responsible for the synthesis of the missing protein(s).

Storage proteins are usually synthesized in very large amounts and consequently need to be stored in a highly concentrated form and in a subcellular compartment in which they are separate from other metabolic processes. This is achieved by a combination of specific solubility properties and deposition into protein bodies (1 - 20mm). The protein bodies have been found both in the aleurone cells and the endosperm (Fig.2.5). The solubility properties are determined by the primary structures of the individual proteins and their interactions by non-covalent forces (notably hydrogen bonds and hydrophobic interactions) and by covalent disulphide bonds. Storage proteins are synthesized on the rough endoplasmic reticulum with a signal peptide which directs the nascent polypeptide into the lumen of the endoplasmic reticulum and is itself removed by proteolytic cleavage. The signal sequences of the storage proteins all display the characteristics for transported proteins in other organisms (Von Heijne, 1983) such as length, hydrophobicity, and the presence of an amino acid with a small uncharged side chain prior to the N-terminus of the mature protein. There is some sequence homology between the signal sequences for closely related zeins (Spena et al., 1982). Thus, it appears that the features of this protein segment that have been conserved in evolution are the nature of the amino acids and perhaps the tertiary structure of the signal sequence rather than the actual amino acid sequence.


Fig.2.5. Diagrammatic representation of a cross-section of a seed with endosperm from the inside towards the outside: the large starchy endosperm (S), the aleurone layer (A), nucellus (N), the integument formed by the tegment (T1) made of crushed cells, and the testa (T2) with sclerous cells sometimes covered, in certain species by mucilaginous cells. (From Champagnat et al., 1969).

By analogy with animal systems (Kreil, 1981), the function of signal sequences in plant storage proteins is to facilitate the translocation of storage protein into the lumen of the ER as the first step in intracellulartransport (Fig.2.6). Protein folding and disulfide bond formation are considered to occur within the lumen of the endoplasmic reticulum, and may be assisted by molecular chaperones and by the enzyme protein disulfide isomerase respectively (Freedman, 1989; Gething and Sambrook, 1992). The precise mechanism of intracellular transport of storage proteins from their site of synthesis to their site of deposition are still largely unknown but a two way hypothesis has been proposed by Shewry (1993).


Figure 2.6: Schematic illustration of the signal hypothesis (Blobel, 1977).

Gliadin

The gliadins are divided into four groups, called alpha-, beta-, gamma-, and omega-gliadins, based on their electrophoretic mobility at low pH (Woychick et al., 1961), but more than 30 components can be separated by two-dimensional electrophoretic procedures. The amino acid compositions of the alpha-, beta-, and gamma-gliadins are similar to each other and to that of the whole gliadin fraction (Tatham et al., 1990a). The omega-gliadins contain little or no cysteine or methionine and only small amounts of basic amino acids. All gliadins are monomers with either no disulphide bonds (omega-gliadins) or intrachain disulphide bonds (alpha-, beta-, and gamma-gliadins). Although no complete sequences of omega-gliadins have been determined, Kasarda et al. (1983) purified a number of individual components from bread and pasta wheats and determined their relative molecular weights (Mrs) by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE); and the Mrs fell between 44,000 and 74,000, with most above 50,000. The alpha-, beta-, and gamma-gliadins have lower Mrs, ranging between about 30,000 and 45,000 by SDS-PAGE and by amino acid sequencing (Appendix A). The latter approach has shown that the alpha- and beta-gliadins are closely related, and both are now usually referred to as alpha-type (in contrast to the gamma-type) gliadins. Although there is some overlap, the alpha-type gliadins generally have lower Mrs than gamma-type gliadins (Appendix A).

In dough formation, the gliadins do not become covalently-linked into large elastic networks as the glutenins but act as a ‘plasticiser’, promoting viscous flow and extensibility which are important rheological characteristics of dough. They may interact through hydrophobic interactions and hydrogen bonds. Krejci and Svedberg (1935) used ultracentrifugation to analyse the gliadin fraction extracted with aqueous ethanol. Although the protein was not homogeneous, a principal non-spherical component with a molecular weight of about 34,500 was present. Lamm and Poulsen (1936) using diffusion measurements showed gliadin to have a high degree of asymmetry with a calculated axial ratio of 8:1 whilst Entrikin (1941) using dielectric measurements showed an axial ratio of 13:1. Hydrodynamic studies showed that gliadins had a low intrinsic viscosity, indicating a compact, globular conformation (Taylor and Cluskey, 1962; Wu and Dimler, 1964). Grosskreutz (1961) reported detailed X-ray scattering and electron microscopy studies of two gluten fractions, the first gliadins and the second fraction a major glutenin subfraction called a1-gluten (not the same as alpha-gliadin).

The gliadins appeared to have a doughnut shape, about 340Å in diameter and 90Å thickness, with a central hole of about 100Å in diameter. Grosskreutz (1961) suggested that this shape resulted from the flattening of a hollow, sphere during drying. Kasarda, (1980) divided wheat varieties into two types (Type 1 and Type 2) based on the major features of the gel electrophoretic patterns of their alpha-gliadins. A sub-fraction of the alpha-gliadins of Type 1 wheat variety was named A-gliadin which was found to be encoded by genes on chromosome 6A. The other alpha-gliadins were coded by genes on chromosomes 6A, 6B and 6D (Figure 2.4). At pH 3, the A-gliadins were found to be partially unfolded and dissociated into monomers. As the pH was increased, the molecules became compactly folded and aggregated to form microfibrils (Figure 2.7).


Figure 2.7: Schematic representation of the aggregation of A-gliadin subunits into fibrillar forms (Kasarda, 1980).

At pH 5 and at an ionic strength of 0.005 M, electron microscopy revealed that the aggregates collected by ultracentrifugation had a microfibrillar structure about 80Å in diameter and up to 3000 or 4000Å long (Kasarda et al., 1967). CD and ORD studies of A-gliadin dissolved in 10-5 M HCl at pH 5.0 showed that it was made up of 33-34% alpha-helix (Kasarda et al., 1968). The molecule was found to be more stable than most globular proteins. 65% of the helical structure present at 25°C remained when the temperature was raised to 90°C. The effects of heating were reversible when cooled.

The primary structure of alpha-type gliadins can be divided into five different domains (Kasarda et al., 1984). Domain I consists of non-repetitive N-terminal sequences and of repetitive sequences rich in glutamine, proline and aromatic amino acids. Domain II contains a polyglutamine sequence with a maximum of 18 residues of glutamine. Domains III and V are homologous to the corresponding domains of gamma-type gliadins and low Mr subunits of glutenin. Domain IV, is unique to alpha-type gliadins and is rich in glutamine but poor in proline. Most alpha-type gliadins contain six cysteine residues, located in domain III (four residues) and V (two residues). Because of the monomeric character of alpha-type gliadins, and the absence of free sulphydryl groups, it has been assumed that the cysteine residues are linked by three intramolecular disulphide bonds (Kasarda et al., 1987). On the basis of the sequence homology to g-type gliadins and low Mr subunits, definitive positions for disulphide bridges have been postulated for alpha-type gliadins (Köhler et al., 1993).


Figure 2.8: Proposed disulphide bond structure of alpha-type gliadins (designation of domains to Kasarda et al., 1984, and of cysteine residues according to Köhler et al., 1993).

The gamma-type gliadins are single monomeric proteins with only intra-chain disulphide bonds and are considered to be the ancestral type of S-rich prolamin (Shewry et al., 1986). Complete amino acid sequences of several gamma-gliadins have been deduced from genomic and cDNA sequences (Bartels et al., 1986; Okita et al., 1985; Rafalski, 1986; Scheets et al., 1985). These sequences showed a clear domain structure, with a non-repetitive sequence of 14 residues at the N-terminus, an N-terminal repetitive domain based on a heptapeptide repeat motif (consensus Pro.Gln.Gln.Pro.Phe.Pro.Gln) and a non-repetitive C-terminal domain which contained all the cysteine residues. Structural studies, using circular dichroism and structure prediction, indicated that the two domains adopt different conformations. Whereas the repetitive domain adopts a beta-reverse turn rich conformation, the non-repetitive domain is rich in alpha-helix (Tatham et al., 1990b). Scanning tunnelling microscopy (STM) and small-angle X-ray scattering (SAXS) results indicated that the gamma-gliadins have a compact conformation, with axial ratios of approximately 1.5:1 (Thomson et al., 1992). A SAXS study of an S-poor prolamin, C hordein, with a repetitive motif (consensus Pro.Gln.Gln.Pro.Phe.Pro.Gln.Gln) similar to that of the repetitive domain of the gamma-gliadins (consensus Pro.Gln.Gln.Pro.Phe.Pro.Gln) has been reported (I’Anson et al., 1992). The SAXS data indicated that in solution C hordein behaved as a worm-like chain (an extended conformation) with a high degree of flexibility. The similarity of the repeat motifs would indicate that the N-terminal domain of the g-gliadin may adopt a similar conformation in solution to that of C hordein.

Small deformation oscillatory measurements on gliadin between 50-70°C showed the elastic modulus (G’) to be roughly equal to the viscous modulus (G") in magnitude, but a large increase in G’ was observed at temperatures above 70°C (Madeka and Kokini, 1994). The increase in the elastic component was attributed to crosslinking reactions occurring among gliadin molecules, resulting in the formation of a network structure. The G’ reached a peak at 120°C, and the G" increased to a plateau value in the temperature range 90-110°C. On further heating G" fell to a minimum value at 120°C, whereas G’ was at its maximum, indicating maximum structure build-up. At this point, the aggregation reaction appeared to have been completed and a highly crosslinked network formed. On increasing the temperature even further, a reduction in G’ with a simultaneous peak in G" was observed. This indicated a softening of the crosslinked gliadin at 130°C.

Glutenin

The glutenin proteins make up 35-40% of flour protein and consist of subunits that form large polymers (Mrs above 1 x 106 and possibly exceeding 1 x 107 ) stabilised by interchain disulphide bonds (Field et al., 1983). After reduction, both HMW and LMW subunits were observed by SDS-PAGE. The latter resembled the monomeric alpha-type gliadins in amino acid composition and had Mrs in a similar range. The HMW subunit contain significantly less proline and had an Mrs value from 80,000 to 160,000 by PAGE. Extensive genetical analysis of the HMW subunits by Payne and co-workers (Payne, 1987), established that genes coding for the HMW subunits are located on the long arms of chromosomes 1A, 1B and 1D at complex loci designated Glu-A1, Glu-B1 and Glu-D1 respectively. By contrast, genes for the LMW subunits and the gliadins are located on the short arms of the same chromosomes (designated Gli-A1, Gli-B1 and Gli-D1 respectively). The HMW subunits consist of nonrepetitive domains of 88-104 and 42 residues at the N- and C-termini, respectively, separated by a longer repetitive domain (481-690 residues). Variation in the repetitive domain is responsible for most of the variation in the size of the whole protein, and it is based on random and interspersed repeats of hexapeptide and nonapeptide motifs, with tripeptides also present in x-type subunits only (Appendix A). Structure prediction indicated that the N- and C-terminal domains are predominantly alpha-helical, while the repetitive domains are rich in beta-turns.

Several models for the structure of wheat glutenin have been proposed. One of the earliest molecular models was that of Ewart (1968). He subsequently modified the model in 1972 and 1979. Ewart’s latest model shows one disulphide bond between two adjacent polypeptide chains of glutenins, which consist of linear polymers. Ewart pointed out that the rheological properties of a dough are dependent on the presence of rheologically active disulphide bonds and thiol groups as well as on secondary forces in the concatenations (Ewart, 1979).

Kasarda et al. (1976) proposed an alternative model in which glutenin has only the intrachain disulphide bonds. They suggested that the intrachain disulphide bonds force glutenin molecules into specific conformations that facilitated interaction of adjacent glutenin molecules through non-covalent bonds, thereby causing aggregation.

Khan and Bushuk (1979) proposed a model of functional glutenin complexes that contained both inter- and intrachain disulphide bonds. On the basis of results from SDS-PAGE, they proposed an aggregate of two types of glutenin complexes, I and II. In their model, glutenin I comprised subunits of molecular weight 6.8 x 104 and lower, held together through secondary forces, such as hydrogen bonds and hydrophobic interactions: glutenin II comprised crosslinked subunits of molecular weights above 6.8 x 104, linked by interchain disulphide bonds.

Shewry et al. (1989) proposed a model for HMW (y-type subunit 9), which has at the N-terminus, a non-repetitive sequence of 80-100 residues containing three to five cysteine residues. At the C-terminus, there is another non-repetitive sequence of 42 residues including one cysteine, this region being identical for all subunits (Appendix A). The central region of the polypeptide (490-690) consisted of a number of repeated sequences forming beta-turns. From the dimensions of the subunits as determined by hydrodynamic measurements, the beta-turns were thought to be organised into a loose spiral (Figure 2.9).

When glutenin was heated above 50°C there was a dramatic increase in viscosity and G’ (Schofield et al., 1983), which led to network formation by protein-protein aggregation at temperatures > 80°C. At 90°C, glutenin gelled (crosslinked) through the formation of disulphide bonds, reaching a maximum structure build-up at 135°C, with a maximum G’ value and a minimum G" value. As the temperature increased further, G" increased, reaching a maximum at 150°C, at which point G’ dropped drastically, suggesting softening of the glutenin crosslinked network.


Figure 2.9: Structure of a typical high-molecular weight subunit of glutenin. (Shewry et al., 1989).

Deamidated Gluten

Wheat gluten is available as a by-product of the wheat starch industry and is used in food applications. The insolubility of gluten in aqueous solutions is one of the major limitations for its more extensive use in food processing for example in dairy products. Gluten insolubility is due to the high concentration of nonpolar amino acid residues such as proline and leucine and the polar but non-ionisable residue glutamine, and to the low concentration of ionisable side chains such as lysine, arginine, glutamic acid and aspartic acid. The interactions between glutamine and asparagine side chains through hydrogen bonds play an important role in promoting association of gliadin and glutenin molecules (Beckwith et al., 1963; Krull et al., 1966). Many researchers have developed methods for modifying the solubility and functional properties of gluten. Gluten modification via deamidation can be achieved in two ways, namely chemical deamidation (acid solubilisation) under acidic conditions and high temperature (Wu et al., 1976) or enzymatic treatment (Kato et al., 1987; Bollecker et al., 1990; Popineau and Thebaudin, 1990). Whether chemically or enzymatically induced, the deamidation of gluten proteins resulted in an increased charge density on the protein, causing changes in protein conformation due to electrostatic repulsion. These charge-induced conformational changes resulted in enhanced surface hydrophobicity due to the exposure of hydrophobic residues (Matsudomi et al., 1982). The increased surface hydrophobicity coupled with the presence of more negatively charged polar groups resulted in a modified protein with amphiphilic characteristics which made an ideal surface active agent for use as an emulsifier or foam stabiliser. Even though surface hydrophobicity increased, protein solubility was also enhanced due to decreased protein-protein interactions. Levels of deamidation as low as 2-6% can enhance the functional properties of proteins (Matsudomi et al., 1985; Hamada and Marshall, 1989). Acid deamidation has been reported to leave behind an astringent mouth-feel, although this can be overcome by extraction with alkaline isopropanol and then isopropanol after deamidation (Finley, 1975).

Deamidation is a hydrolytic reaction, similar to the peptide-bond cleavage reaction, which is catalysed by proteases (Jencks, 1969). It is catalysed by acids and bases (nucleophiles), and requires a water molecule (Figure 2.10). The general acid, HA, catalyses the reaction by protonating the amido -NH- leaving group of the Asn side chain. A general base (the conjugate base, A- or hydroxide ion) can attack the carbonyl carbon of the amido group or activate another nucleophile by abstraction of a proton for attack on the amide carbon. The transition state is inferred to be an oxyanion tetrahedral intermediate, whose stabilisation by proton donors increases the rate of the reaction. The order of acid- and base-catalysed steps in Figure 2.10 vary with reaction conditions, particularly pH. The pH of maximum stability of Asn and Gln in peptides is around pH 6.0. Wright and Robinson (1982) showed how specific amino acid side chains are likely to function in catalysing the deamidation of Asn and Gln in peptides and proteins. The Ser and Thr side chains can function as general acid groups, providing a proton to the leaving group or stabilising the transition state. Asp, Glu, and His side chains are all nucleophiles at neutral pH, which can attack the carbonyl carbon of the amide side chain or function as general bases to activate nucleophiles. The gluten proteins lack Asp and do not have enough of His (Appendix A). Lys and Arg, which correlate with high deamidation rates when next to Asn and Gln in sequence , may stabilise the oxyanion intermediate. Gluten proteins lack Lys, although HMW glutenin has one Lys in the middle of Gln residues. However, there are numerous Arg residues next to Gln in HMW glutenins which may influence the reaction rate. The gamma-gliadins and the HMW glutenins, but not the alpha-gliadin and LMW glutenins have, terminal Gln which, may cyclisize with a terminal carboxylate group to form an unstable anhydride which may break down to deamidated glutamate (Wright and Robinson, 1982).

Figure 2.10: General mechanism of deamidation.

In studies on model tetra and penta-peptides of different sequences, a broad range of deamidation rates was observed (Robinson and Rudd, 1974). Several generalisations were made from the sequence dependence of these rate data:

Deamidation in peptides and proteins generally require the participation of a water molecule to go to completion. In peptides, there are minimal obstructions to water access to the labile amide. However, the more stable protein structures may limit access of water to amide groups and so influence the rates of any deamidation reactions. Deamidation rates of Asn and Gln residues on the surface of proteins will not be limited by water access, while those that occur in the interior of proteins may be. Such a limitation will be determined by the static protein structure and by the frequency with which buried Asn and Gln are exposed to solvent during rapid dynamic changes in the structure due to thermal motion.

Soluble wheat protein (SWP)

SWP is the product of the deamidation (20%) of gluten, produced by AMYLUM NV - Belgium. The following properties were provided in the product specification information sheet.

Properties

It is a bland, white creamy low viscous sodium-salt of a soluble wheat protein isolate. By its unique combination of functional properties like emulsifying capacity, gelling, binding and water-retention, the product can be used in several food applications like meat-preparations, soups, sauces, dressings, imitation dairy etc. It can also be combined with other functional proteins like caseinates, soy isolates, and may result in all types of synergies.

Chemical Data

Protein (N x 6.25): 86%; moisture: 5%; fat: 8%; ash: 5%; carbohydrates: 1%; pH 6.5-7.5.

Microbiological Data

Total plate count: max. 5000/gr; Coliforms: absent in 0.1 gr; Moulds: max. 100/gr; Yeasts: max. 100/gr; Salmonella: absent in 25 gr; St. aureus: absent in 1.0 gr.

Solubility

A 1% protein solution was stirred for 30 minutes, the pH adjusted and the solution was centrifuged at 5000g for 20 minutes. The solubility (nitrogen solubility index) was expressed at different pH values in % as:

nitrogen content in the supernatant NSI = ----------------------------------------- total nitrogen content
produced a solubility profile shown in Figure 2.11

Figure 2.11: Solubility profile of SWP

Emulsifying properties

Three characteristics may be distinguished:

Table 2.1: The emulsifying properties at different pH values.
pH EC
g oil/g protein
EA
%
ES
%
547052.9100
657051.7100
762052.5100

The emulsifying capacity, in relation to sodium caseinate, is comparable at pH 7 and higher at pH 5 and 6.

Gelling properties

A 15% protein solution was heated in 200ml cans for 60 minutes. The gel strength, expressed in grams, was measured at room temperature with a Stevens LFRA Texture Analyser (cylindrical plunger 1") to a penetration depth of 40mm at a speed of 2.0mm/sec.

Table 2.2: Effect of temperature and pH on gel strength.
Temperature(°C) pH Gel strength
95645
748
1056180
7260
1206480
7370

SWP (15% - 20% w/v) formed a gel at 115°C. Use of reducing agents such as cysteine and bisodium sulphite (Na2SO3) reduced the gelling temperature to 65°C - 70°C, accompanied by an increase in gel strength.

Viscosity

The solutions were prepared by mixing for 13 minutes (3min. at 2000rpm, 10min. at 1500rpm). 17.25 to 25g of product per 100ml (for resp. 15 and 20%). The pH was adjusted and the viscosity was measured within the first 3 minutes with a Brookfield RVT at 20 rpm.

Table 2.3: Effect of concentration and pH on viscosity.
Concentration pH Viscosity(mPa.s)
15%71000
20%66000
73300
81900



Georges-Louis Friedli, PgDip., MSc., CH(t)., MACN., PhD.
drfriedli@friedli.com

Last Updated September 1996