Texture-Structure Relationships in Textured Soy Protein. V. Influence of pH and Protein Acylation on Extrusion Texturization

Can. Insr. Food Sci. Technol. J. Vol. 15, No. 4, pp. 294-301, 1982 Pergamon Press Ltd. Printed in Canada.

Texture-Structure Relationships in Textured Soy Protein. V. Influence of pH and Protein Acylation on Extrusion Texturization R.W. Simonsky and D.W. Stanley Department of Food Science University of Guelph Guelph, Ontario NIG 2Wl

Abstract

portance de la solubilite dans la formation de la texture. La microscopie electronique par balayage a revele que les extrudats acyles furent moins cellulaires et plus denses que le materiel temoin. La fraction soluble des proteines acylees montra une mobilite electrophoretique plus grande a la suite de la perte des groupements amines cationiques.

Data were gathered to further support the hypothesis that formation of texture, measured in terms of shear values and sensory responses, during the thermal extrusion of soy protein involves protein-protein interactions based on NH" groups. It was shown that ninhydrin, a reagent capable of irreversibly binding with free amino groups, significantly inhibited texture formation. When texturization was studied as a function of pH adjustment (- 4-10), maximum Warner-Bratzler shear values and sensory response were found to occur close to pH 8, where amino groups are unprotonated and more reactive. At the extremes of the pH range extrudates lost structural integrity, were more soluble, and an examination of their microstructure showed that the interconnected longitudinal vacuoles separated by thick cell walls normally seen were replaced by a denser globoid system. Soy protein isolate was acylated using either acetic or succinic anhydride. Both treatments resulted in a significant decrease in extrudate texture but increases in extrudate solubility; the latter finding tends to discount the importance of solubility in texture formation. Scanning electron microscopy revealed that the acylated extrudates exhibited less cellularity and were denser than the control materials. The soluble fraction of acylated proteins demonstrated increased electrophoretic mobility as a result of the loss of the cationic amino groups.

Introduction Perhaps the most imaginative use of soy protein for human food is in the production of textured products such as meat analogs and meat extenders. Projected sales for textured soy proteins, however, have proven to be over optimistic (Sair, 1981). Although economic, consumer and regulatory factors have had a major effect on this situation, the failure to fully understand the extrusion process has retarded technological progress and limited product development. The major quality attribute of extruded soy proteins is texture, but the way in which heat, pressure and shear combine during this process to reorganize protein molecules into a desirable meat-like structure is not understood and, thus, only empirical knowledge is available to extrusion processors. We have previously attempted an investigation of this area and the objective of the present research was to gain a further understanding of extrusion texturization of soy proteins by varying the nature of the feed material.

Resume Les donnees presentees corroborent I'hypothese que la formation de la texture, mesuree en termes d'indices de cisaillement et d'evaluation sensorielle, au eaurs de I' extrusion thermique de proteines de soja implique des interactions proteine-proteine basees sur les groupements NH". 11 a ete demontre que la ninhydrine, un reactif capable de creer des liaisons irreversibles avec les groupements amines libres, interfere significativement avec la formation de la texture. L'etude de la texturisation en fonction du pH (4-10) a montre que les indices de cisaillement Warner-Bratzler et les reponses sensorielles maxima se sont situes aux environs de pH 8, alors que les groupements amines sont sans charge positive et plus reactifs. Aux extremes de la marge de pH les extrudats perdirent leur integrite structurale, furent plus solubles, et I'examen de leur microstructure mit en evidence un systeme globolde plus dense 11 la place des vacuoles longitudinales interreliees separees par des parois cellulaires epaisses que I'on observe normalement. De I'isolat de proteine de soja fut acyle avec de I'anhydride acetique ou de I'anhydride succinique. L'un ou I'autre des traitements ont cause une diminution significative de la texture de I'extrudat mais une augmentation de sa solubilite.· Cette demiere observation tend 11 rabaisser I'im-

Materials and Methods Soybean Meal Defatted soybean meal was obtained commercially (Victory Soy Mills, Toronto, Ont.), ground to pass a 0.61 mm screen and blended. The meal contained 9.73% moisture and 50.1% protein (db, N x 5.71). Soybean Isolate A soybean isolate was used (Promine D, Central Soya Co., Inc., Chicago, Ill.) that had a moisture content of 5.82% and a protein content of 87.3% (db, N x 5.71).

0315-5463/82/040294-08$3.00/0 Copyright © 1982 Canadian Institute of Food Science and Technology

294

Preparation of Material for Extrusion Soy proteins were rehydrated to 27% moisture using a Hobart Kitchen Aid mixer (K5-A). The rehydrated material was allowed to equilibrate overnight at 4°C.

Extrusion A laboratory scale thermal extruder (C. W. Brabender Co., South Hackensack, N. J., Model 2003) was used to produce texturized protein materials. Details of its construction, instrumentation and operation were reported previously (Burgess and Stanley, 1976; Maurice et a/., 1976; Maurice and Stanley, 1978). A 4:1 compression screw was employed when extruding soy grits and a 2: I compression screw was used for soy isolate and acylated protein. Raw material was gravity fed into the extruder using a stainless steel hopper. Pressure was monitored by a Bourdon-type gauge inserted directly into the extruder barrel. Extrusion parameters (Table I) were selected on the basis of previous work to yield optimum texture.

Texture Measurement The texture of extrudates was measured with a modified Warner-Bratzler shear using a deformation rate of 8.1 cm/min. Maximum peak height (maximum force) resulting from the shearing of one strand of extruded material by one blade was recorded; thirty replicates were tested per treatment. This was found to be sufficient to correct for much of the variation produced by differences in strand diameter. Sensory analysis was performed using a fifteen member untrained panel that was asked to judge tactile properties of extrudates by pulling or bending individual strands. Panelists rated samples by placing a vertical mark on a 15 cm unstructured scale ranging from "lacking cohesiveness" to "extremely cohesive." Each treatment was evaluated twice by the panel. Both measuring methods used have been found to accurately monitor changes in the texture of extruded soy protein (Maurice et a/., 1976).

Preparation of Samples

is summarized in Figure I. A control sample was exposed to the procedure excepting the addition of the acetic anhydride or succinic anhydride acylating agents. Extent of acylation was measured using the ninhydrin method of Franzen and Kinsella (1976). The difference in absorbancies (580 nm) between the control and acylated protein was expressed as the percentage of NH~ groups blocked.

Chemical and Physical Evaluation Solubility of proteins was determined by extracting material ground to pass a .36 mm sieve mixed in a ratio of 2.0 g protein to 100 mL solution for I h at room temperature .. The resulting slurry was centrifuged for 30 min at 15°C (16,300 x g). The supernatant was filtered through Whatman #42 paper prior to protein analysis. Protein quantification was accomplished using the biuret method for clear liquids or the Kjeldahl method for all other samples. Polyacrylamide gel electrophoresis was carried out using a Canalco unit (Canalco Instrument Co., Rockville, Md.). Protein was extracted from feed material and extrudates by running buffer (Tris-glycine, pH 9.3) for I h and the extract filtered and/or centrifuged. Exact volumes (10-30 MoL) were loaded onto 7.5% polyacrylamide gels cast in glass tubes and run at a current of 5 mA/gel. Following separation, gels were stained for 2 h using aniline black and subsequently destained in acetic acid. The resulting gels were scanned in a Joyce 10% PROTEIN SLURRY

~

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ALKALINE PROTEIN SLURRY

~

Add acylaling agent

ALKALINE ACYLATED PROTEIN SLURRY Dilute with 37.8'C (100'F) H20 Centrifuge for 30 min

SUPERNATANT

A series of soybean meal samples varying in pH was prepared by adding different volumes of 12 N HCI or 50% NaOH to the rehydration water. pH of the meal and corresponding extrudates was measured using a Fisher Accumet 140 pH meter (Fisher Scientific, Toronto, ant.) on a slurry obtained by agitating 5.0 g dry material with 100 mL distilled water for 15 min. With the exception of the two highest pH levels (9.48, 10.53 before extrusion; 8.98,9.93 after extrusion) there was little effect (± .20 pH units) of extrusion on pH. Salt was added to a control sample (.56 M NaCI/kg soybean meal) to account for any ionic effects caused by the addition of acid or base. Acylated soy protein isolate was prepared using the method of Melnychyn and Stapley (1973). This procedure

SOLIDS

Adjust pH 10 4.0

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SUPERNATANT

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NEUTRAL ACYLATED PROTEIN

lyophilize

DRIED ACYLATED PROTEIN

Fig. 1. Method used to acylate soy proteins.

Table 1. Parameters used in the extrusion of soy proteins. Temperature CC) Screw speed Material Soy meal Soy isolate and acylated isolate

(rpm)

Rear heater

Centre heater

60 80

96 96

Can. Inst. Food Sci. Tu·hnoi. J. Vol. 15. No. 4. 1982

Die heater

Product

172

176

154

164

176 170

Simonsky and Stanley/295

Loebl Chromoscan densitometer (Joyce, Loebl and Co., Ltd., Gateshead, England) equipped with 620 nm and 2-00 filters. Scanning electron microscopy was performed on vacuum dried gold/palladium coated samples in an Etec Autoscan microscope (Etec Corp., Hayward, Calif.).

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Results and Discussion

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Previous results (Burgess and Stanley, 1976) indicated that the addition of citric acid or ninhydrin to soybean meal significantly reduced the Warner-Bratzler shear force values of the subsequent extrudates. It was hypothesized that these results were due to an inhibition of intermolecular amide bond formation since these compounds competed for possible amino and carboxyl binding sites. In the preliminary stages of the present work, several amino acids and ninhydrin were again evaluated as extrusion additives. As before, shear force values were lowered by the presence of such additions as 0.7 M aspartic acid or cysteine and saturated ninhydrin solution when used as the rehydration media for soy meal. For example, rehydrating soy meal in saturated ninhydrin reduced Wamer-Bratzler shear values of the resulting extrudate by 41 % and a similar experiment with soy isolate resulted in a 30% decrease (both significant at P~ .05). Subsequent analysis revealed, however, that the two amino acids, but not ninhydrin, also significantly reduced the pH of the extrudate. Thus, it became of importance to differentiate these two effects. Varying the pH of soybean meal through the addition of either HCl or NaOH produced profound changes in both sensory and instrumental measurements of texture (Figures 2 and 3). While these two methods correlated highly (r = .86; P~.OI), Wamer-Bratzler shear force values could not be obtained for extrudates produced at the three lowest pH values since the individual strands were too small to be evaluated. In general, highest shear force and sensory values were produced at about pH 8 and descended on both sides of this optimum. Harper (1979) found that adjusting the pH of soy meal downwards to 5.5 with acid salts or dilute acids will increase the doughiness of the extrudate, but increasing the pH to 8.5 with base produces a more tender, less chewy product that rehydrates more rapidly; NaCl at levels up to 3% were reportedly useful in increasing the firmness of the rehydrated texturized material and enhancing pH adjustment effects. In the present work, NaCl was found to produce extrudates with higher shear force values. The macroscopic appearance of the extrudate (Figure 4) exhibited a loss of structural integrity at the extremes of the pH scale. Appearance correlated well with shear force and sensory data; longer, thicker strands gave higher instrumental values than shorter, less cohesive ones. It was noted that lowering the pH of the material produced extruder surging as previously indicated by Atkinson (1970) while output using alkaline soybean meal was more even. Adding NaCl to soybean meal resulted in a denser extrudate of shorter strand length. Hermansson (1973) reported that minimum solubility of soy proteins occurs in the presence of salt, perhaps due to the formation of ionic bonds within and between protein molecules 296/Simonsky and Stanley

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leading to the formation of aggregates. This may produce a denser extrudate. Scanning electron micrographs of the extruded samples (Figure 5) show the control extrudate to consist of familiar interconnected longitudinal vacuoles (Maurice et al., 1976; Stanley and deMan, 1978) separated by thick cell walls. Highly acidified and alkaline extrudates lost this cellular appearance which was replaced by a dense globoid system; the addition of NaCI produced a similar lack of cellularity. The extrusion of moderately alkaline soybean meal (pH 7.11, 7.89 and 7.99), in contrast, yielded extrudates with increased cellularity and thicker cell walls. Both the macro and microscopic views of extrudate structure serve to emphasize the important relation between structure and texture in the extruded products. 1. !t1.\'1. Can. Sci. Techllol. Aliment. Vol. 15, No. 4, 1982

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Fig. 4. Influence of pH adjustment on macrostructural integrity of extruded soy protein. Top - acidic: A - 6.78 (control); B - 6.57 (NaCI); C - 4.28; D - 4.70; E - 5.40; F - 5.71; G - 6.28. Bottom - basic: A - 6.78 (control); B - 7.11; C - 7.89; D - 7.99; E - 9.48; F - 10.53.

These findings warrant the conclusion that texture and structure of soybean extrudate are altered as a result of changes in pH. One explanation of this effect is pH induced changes in protein solubility. Thus, in the review Can. Inst. Food Sei. Technol. J. Vo!. 15. No. 4, 1982

of Kinsella (1978), it is suggested that lower solubility and increased susceptibility to thermal denaturation at extreme pH values may lead to more rapid aggregation in the extruder, a more dense protein network, and subSimonsky and Stanley/297

Fig. 5. Influence of pH adjustment on microstructure of extruded soy protein. Left - acidic: A - 6.78 (control); B - 4.28; C - 4.70; D - 5.40; E - 5.71; F - 6.28. Right - basic: A - 6.59 (NaCI); B - 7.11; C - 7.89; D - 7.99; E - 9.48; F - 10.53.

sequent reduction in the size of entrapped moisture droplets and resulting vacuoles. This, in turn, increases product density and retards rehydration. However, if a comparison is made between soy protein solubility data from Smith and Circle (1938) and extrudate texture as a function of pH, it will be seen that while between pH values of 4 and 8, both solubility and texture increase with pH; above this level protein solubility does not decrease but extrudate texture is markedly reduced. Extrudate solubilTable 2. Protein extractability of pH adjusted soy extrudates. pH prior to extrusion 4.28 4.70 5.41 5.71 6.28 6.78 (control) 7.11 7.89 7.99 9.48 10.53

Protein extracted (%)1 14.90a 13.21b 12.50b,c 12.33b,c,d 12.29c,d 12.82b,c 12.39b,c,d 12.14c,d 11.54d I I.76d 15.55a

'Protein extractable by 0.5 M PO, buffer, pH 7.0, for I h at room temperature (2.0g/IOO mL), average of duplicates. Means bearing similar letters are not significantly different (Duncan's Multiple Range Test, P"".05).

298/Simonsky and Stanley

ity (x) was measured (Table 2) and highest solubilities were found at the pH extremes where shear values (y) were lowest. This effect was statistically significant with r (x,y) = -.72, P";;.05. These data also suggest that partial protein hydrolysis may have occurred. Thus, protein solubility is probably not a major factor in texture formation. In reality, both high and low nitrogen solubility index (NSI) soy meals have been reported to produce better texture during extrusion (Smith, 1975; Salazar de Buckle et al., 1977; Harper, 1979) which suggests the importance of other parameters. A second explanation of the influence of pH on thermal extrusion of soy protein could be its effect on the rate and/or extent of protein-protein interactions in texture formation. A slightly alkaline pH resulted in the highest texture values, and those interactions favoured in this range may be important in the texturization phenomenon. Means and Feeney (1971) indicate that both the reactivity of the NH 2 groups and oxidative disulphide bonding are promoted at slightly alkaline pH. Electrophoresis patterns were examined (Figure 6) to investigate the influence of pH adjustment on soy proteins. It can be seen that thermal extrusion results in the appearance of several faster moving protein bands, indicating the presence of smaller molecular weight proteins in the soluble fraction. Cumming et al. (1973) reported J. Inst. Can. Sci. TechnoJ. Aliment. Vol. 15, No. 4, 1982

similar results in extruded soybean meal. It was found that while neither NaCl nor alkali produced extrudates with electrophoretic patterns different from the control, the addition of acid led to a gradual disappearance of medium and low molecular weight proteins. Since the decrease in texture of acidic extrudates closely parallels this reduction it may be that proteins of sufficient molecular size are required for texture formation. Lundgren (1949) stated that an average molecular weight in the range of 10,000 to 50,000 daltons is required for the formation of synthetic molecular fibres, and hence texture. It is difficult on the basis of electrophoretic data to make inferences concerning texture enhancing protein-protein interactions, since these reactions are accompanied by a loss in solubility that prevents their examination by this method. Direct evidence concerning the fate of specific protein reactive groups requires the use of perturbing agents. In order to evaluate the importance of free NH t groups in the thermal extrusion of soy protein, acetic and succinic anhydrides were used as acylating agents. Preliminary experiments showed that using 7.0 g acylating reagent per 100 g protein yielded maximum (>85%) blocking of free amino groups in the production of the large quantities of feed material required for extrusion texturization. Properties of the final modified proteins are shown in Table 3. This table also gives the results of texture analysis of acylated soy protein by both sensory and instrumental methods. Acylation results in a significant reduction in extrudate shear values; these data are consistent with the hypothesis that free NH t groups are important during thermal extrusion of soy protein. Kuo et al. (1979) found significant differences between the texture of untreated and succinylated soy proteins that had been texturized using a nonextrusion "Village Texturizer." Also shown in Table 3 are solubilities of the various extruded protein samples. While, as demonstrated in the pH experiments, thermal extrusion greatly reduces protein solubility, acetylation and succinylation significantly increased solubility over that of the control. Meyer and Williams (1977) reported that acylation of soy proteins enhanced their solubility and led to a greater resistance to aggregation. These data tend to further discount the explanation of texture formation solely on the basis of solubility differences. Scanning electron micrographs of extrudates from soy protein isolate and acylated soy protein (Figure 7) revealed that extrusion of acylated proteins resulted in a denser product with fewer air cells

pH 6.28

6

6 pH 5.41

pH 4.28

Fig. 6. Influence of pH adjustment on electrophoretic patterns of extruded soy protein.

than those made from the control materials. Good texture formation, then, seems dependent upon the appearance of cellularity in the structure. The influence of acylation on soluble protein electrophoretic mobility can be observed both before and after extrusion (Figure 8). Peak I on the densitometer tracings was chosen to demonstrate this effect on proteins prior to extrusion. Rf values of 0.14, 0.21 and 0.27 were calculated for this peak in control, acetylated and succinylated samples; the peak directly preceding Peak I probably represents larger proteins that entered the gel matrix but did not migrate appreciably. Succinylated and acetylated proteins moved more rapidly in the gel than the control material, with the former modification outdistancing the latter. This is expected since acetylation replaces ammonium cations by neutral acetyl groups, whereas succinylation converts cationic amino groups to anionic succinate residues. This successive increase in net negative charge results in greater affinity for the cathode and increased electrophoretic mobility. In both cases the cation group

Table 3. Propenies of acylated soy proteins. Propeny % NH, groups blocked

Percent yield (db) Pre-extrusion solubility' (%) Sensory score, transformed" Wamer-Bratzler shear (kg) Post-extrusion solubility' (%)

Control'

Acetylated

0 81.62 81.78c 5.OOa 9.58a 3.25c

88.43 87.90 90.32b 2. 94b 6.21b 8.53b

Succinylated

Soy isolate

87.77 93.83 92.15a 3.59b 6.46b 18.66a

39.60d 3.80b 6.31b 4.38c

I Soy isolate subjected to all acylation steps excepting addition of acylating reagent. 'Protein extracted by water in 1 h at room temperature (2.0 g/lOO mL), average of duplicates. "Cm from control plus 5.0.

Means within a row bearing similar letters are not significantly different (Duncan's Multiple Range Test, P";;.05). Can. Inst. Food Sei. Teehnol. J. VD\. 15. No. 4. 1982

Simonsky and Stanley/299

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would no longer be available to form intermolecular amide bonds with carbonyls, our previous hypothesis for texture formation in extruded soy protein (Burgess and Stanley, 1976). Extrusion initially results in the formation of lower molecular weight proteins, presumably through subunit dissociation (Cumming et al., 1973). While this general pattern holds with modified proteins, there was a relative increase in larger and/or slower migrating soluble proteins and nonmigrating proteins in succinylated extrudates compared to the other samples. It would seem that succinylation inhibits protein dissociation or interaction of dissociated subunits. This observation reinforces the need of a mechanism to analyze insoluble extrudates, about which very little is known. The results obtained cannot rule out the possibility of groups other than NH~ being involved in the texturization process, however, blocking them was shown to reduce texture. These data, along with the inhibitory effect of ninhydrin, and the finding that shear and sensory values are enhanced at a mildly alkaline pH, at which NH~ groups are reactive, strengthens the hypothesis that proteinprotein interactions through amino groups are an important factor in soy protein texturization phenomenon. 300/Simonsky and Stanley

Note Added Rhee et al. (1981) have very recently published results of experiments similar to those undertaken in this research. They also examined the influence of pH, protein modification and addition on extrusion texturization. Because the two research groups did not obtain comparable data or make similar conclusions, it is of importance to examine the studies carefully. These authors' observations in the following areas apply directly to the present work: (I) Effect of pH. The influence of pH on thermal extrusion was studied over the range of 5.3-9.0. Texture measurements of rehydrated material showed a significant decrease with increasing pH. This is in sharp contrast to other data presented in this paper, where textural parameters (instrumental and sensory), macro and microstructure, electrophoretic data and protein solubilities, all lead to the conclusion that, under the conditions of the experiment, maximum texturization occurs around pH 8. It is of interest to note that Rhee's data seems to indicate that increasing pH enhances the ability of the resulting extruded soy proteins to interact with water, whereas the present study shows a drop in protein extractability with increasing pH. J. Insf. Can. Sci. Technol. Alimellt. Vol. 15. No. 4, 1982

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(2) Effect of NaCI. Rhee and coworkers found that salt added at I% and 2% significantly hindered texture formation, but in the present work, a level slightly over 3% enhanced texture formation. (3) Effect of protein modification and additives. A decrease was noted by Rhee in the rheological properties of nonextrusion textured soy flour that had been succinylated, while the solubility of the material increased. The magnitude of these changes are similar to those shown for thermally extruded soy protein in the presently reported work. The conclusions drawn from the data were, however, totally different in that the authors deduced that even though succinylation inhibits texture formation, the occurrence of any texture discounts a role for intermolecular amide bonds in this process. Subsequently, evidence was given for a role of disulphide bonding in texture formation based on the addition of disulphide reducing agents (cysteine-HCl and Na~SO:;) increasing texture and a disulphide bond enhancer (KIO:I ) reducing texture. Work done in conjunction with the present study indicated a different role for cysteine-HCl; when this amino acid was added at a rate of .7 M to the rehydration media, Wamer-Bratzler shear values were reduced by 60% (P~.05). However, measurement of the extrudate revealed that the pH was reduced significantly (1.33 units). From subsequent pH experiments, it was concluded that textural changes observed as a result of amino acid addition may be due solely to pH effects. In any case, if disulphide bonding is important in extrusion one might expect disulphide reducing agents to weaken textural bonds, while oxidizing agents would enhance them. Although the opposite was observed, Rhee et al. conclude that their data "further proves that the change of disulphide linkages is an essential reaction for texture formation. " Call. Insl. Food Sci. Technol. J. Vol. 15, No. 4, 1982

This research was supported in part by the Natural Sciences and Engineering Research Council and the Ontario Ministry of Agriculture and Food.

References

Acetylated Protein

0

The reader will, no doubt, find it difficult to reconcile these disparate results. It should be obvious that the mechanism by which proteins associate during thermal extrusion is not yet scientifically established and, until it is known, predictive control and manipulation of extrusion processes will remain elusive.

Atkinson, W.T 1970. Meat-like protein food products. V.S. Patent No. 3,488,770. Burgess, L.D. and Stanley, D.W. 1976. A possible mechanism for thermal extrusion of soybean protein. Can. Ins!. Food Sci. Technol. J. 9:231. Cumming, D.B., Stanley, D. W. and deMan, J.M. 1973. Fate of water soluble protein during thermoplastic extrusion. J. Food Sci. 38:320. Franzen, K.L. and Kinsella, J.E. 1976. Functional properties of succinylated and acetylated soy protein. J. Agric. Food Chem. 24:788. Harper, J.M. 1979. Food extrusion. CRC Cri!. Rev. Food Sci. Nutr. 11:155. Hermansson, A.M. 1973. Determination of functional properties of protein foods. In: Proteins in Human Nutrition. J.W.G. Porter and B.A. Rolls (Eds.). Academic Press Inc., New York, NY. Kinsella, J.E. 1978. Texturized proteins: Fabrication, flavoring and nutrition. CRC Cri!. Rev. Food Sci. Nutr. 10:147. Kuo, C.M., Kazenzadeh, M. and Rhee, K.C. 1979. Texturization properties of succinylated soy flour. Paper 219. Institute of Food Technologists Annual Meeting, S!. Louis, MO. Lundgren, H.P. 1949. Synthetic fibers made from proteins. Adv. Protein Chem. 5:305. Maurice, TJ., Burgess, L.D. and Stanley, D.W. 1976. Texture-structure relationships in texturized soy protein. Ill. Textural evaluation of extruded products. Can. Ins!. Food Sci. Technol. J. 9:173. Maurice, TJ. and Stanley, D. W. 1978. Texture-structure relationships in texturized soy protein. IV. Influence of processing variables on extrusion texturization. Can. Ins!. Food Sci. Technol. J. 11: I. Means, G.E. and Feeney, R.E. 1971. Chemical Modification of Proteins. Holden-Day, Toronto, ON. Melnychyn, P. and Stapley, R.B. 1973. Acylated protein for coffee whitener formulations. V.S. Patent No. 3,764,711. Meyer, E.W. and Williams, L.D. 1977. Chemical modification of soy proteins. In: Food Proteins Improvement Through Chemical and Enzymatic Modification. R.E. Feeney and J.R. Whitaker (Eds.). American Chemical Society, Washington, DC. Rhee, K.C., Kuo, C.M. and Lucas, E.W. 1981. Texturization. In: Protein Functionality in Foods. J.P. Cherry (Ed.). American Chemical Society, Washington, DC. Sair, R.A. 1981. Marketing plant proteins in Europe. In: Utilization of Protein Resources. D. W. Stanley, E.D. Murray and D.H. Lees (Eds.). Food and Nutrition Press, Inc., Westport, CT Salazar de Buckle, T, Zapata, L.E., Mercedes de Sanoval, A., BenGera, I. and Shomer, I. 1977. Relationship between structure and texture of extruded soy and cottonseed flour products. J. Food Quality 1:253. Smith, A.K. and Circle, S.J. 1938. Peptization of soybean proteins. Extraction of nitrogenous constituents from oil-free meal by acids and gases with and without added salts. Ind. Eng. Chem. 30:1414. Smith,O.B. 1975. Extrusion and forming: Creating new foods. Food Eng.47:48. Stanley, D. W. and deMan, J.M. 1978. Structural and mechanical properties of textured proteins. J. Texture Stud. 9:59. Accepted May 6, 1982

Simonsky and Stanley/30l