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Soy Isolate Doughs. III. Protein and Lipid Binding During Dough Mixing
Can. Inst. Food Sci. Technol. J. Vo\. 15, No. 4, pp. 302-306, 1982 Pergamon Press Ltd. Printed in Canada.
Functionality of Soy Proteins in Wheat Flour/Soy Isolate Doughs. Ill. Protein and Lipid Binding During Dough Mixing S.S. Chen I and V.F. Rasper Department of Food Science University of Guelph Guelph, Ontario NIG 2WI
Abstract
seches 11 froid fut 11 peu pres du meme ordre de grandeur que celle observee entre les produits traites aux basses temperatures (cryodessication ou pulverisation) et ceux traites aux hautes temperatures (sechage sur rouleaux). Meme si I'addition d'agents tensio-actifs influen~a I'ampleur des aptitudes 11 I'extraction, le modele relatif aux divers isolats est demeure pratiquement le meme.
The changes in nitrogen and lipid extractabilities during mixing of soy protein supplemented wheat flour doughs were studied using water or alkali extracted protein isolates in the form of isoelectric protein or protei nates subjected to different drying techniques. The studies were performed on blends at 8% supplementation level in the absence or presence of a surfactant (SSL, Tween 60, Span 65). Nitrogen solubility of the supplemented doughs increased during their mixing to maximum consistency by approximately 11% and 23% when 0.05 M acetic acid and phosphate-NaCI buffer (pH 7.6, JL = 0.5) were used, respectively. This increase was considerably higher than that measured with unsupplemented dough. Free lipid extractability of supplemented doughs was reduced in average by 36% compared to 62% reduction in unsupplemented control dough. Among the freeze-dried isolates, the strongest tendency to affect the studied extractabilities was displayed by Ca proteinates. The difference between the effect of these proteinates and other freeze-dried isolates was about the same magnitude as that observed between the lower heat status (freeze- or spray dried) and higher heat status (drum dried) material. Though the addition of surfactants influenced the measured extractabilities, the response pattern with respect to the type of isolate remained practically unchanged.
Introduction The adverse effect of high protein nonwheat products on dough quality and the role of lipid related surfactants in its counteraction have been the subject of numerous studies. In an attempt of better understanding of the mechanisms involved, many studies focused on the changes in the solubility of protein fractions and lipid binding during mixing and baking of supplemented doughs in the presence of different surfactants (Hoseney et al., 1970; Wherli and Pomeranz, 1970; Aidoo and Tsen, 1973a,b; Chung and Tsen, 1975a,b,c,d; Chung and Tsen, 1977; De Stefanis et al., 1977; Chung et al., 1981). Though various models of complexes formed in the dough during its development were proposed in order to explain the interactions between the wheat flour components, protein supplement and surfactant, the nature of these interactions has not been fully elucidated (Hoseney et al., 1970; Krog, 1971; Kim and Robinson, 1979; Chung et aI., 198 I). The present study is part of research work on wheat flour doughs supplemented with soy proteins prepared from defatted soy grits using different preparative treatments. The preparation of the protein isolates and their chemical and physico-chemical properties, as well as their effects on the rheological and baking qualities of the supplemented doughs, were reported earlier (Chen and Rasper, 1982a,b). This paper reports on results of tests in which the relationship between the type of iso-
Resume En partant d' isolats proteiques extraits 11 I'eau ou 11 I' alcali sous la forme de proteine isoelectrique ou de proteinates seches par differentes techniques, on a etudie les changements dans I'aptitude 11 I'extraction de I'azote et des lipides au cours du malaxage des pates de proteines de soja enrichies de farine de ble. Les etudes ont porte sur des melanges enrichis au niveau de 8% avec ou sans agent tensio-actif (SSL, Tween 60, Span 65). La solubilite de I'azote des pates enrichies a augmente au cours du malaxage jusqu'1I consistance maximum par environ 11% et 23% respectivement avec le tampon 11 I'acide acetique 0,05 M et avec le tampon au phosphate-NaCI (pH 7,6, JL = 0,5). Celle augmentation fut considerablement plus forte que celle obtenue avec la pate non enrichie. L'aptitude 11 I'extraction des lipides Iibres a diminue de 36% en moyenne par rapport 11 62% pour la pate temoin non enrichie. De tous les isolats seches 11 froid, ce sont les proteinates de calcium qui ont manifeste la plus forte tendance 11 affecter les aptitudes 11 I'extraction. La difference entre I'effet de ces proteinates et celui des autres isolats 'Present address: Riverdale Frozen Foods, Niagara Falls, Ontario L21 6S6.
Copyright
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0315-5463/82/040302-05$3.00/0 1982 Canadian Institute of Food Science and Technology
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late, as determined by the preparative technique, and the changes in nitrogen and lipid extractabilities were evaluated. The tests were performed in the absence or presence of three different surfactants.
Materials and Methods Preparation of the Protein Isolates Soy protein isolates were prepared under laboratory conditions using different extraction media (water and alkali), different treatment in the separation of the isolated proteins (isoelectric precipitation and neutralization to form Ca, K and Na proteinates), and drying (freeze-, spray and drum drying). The average protein content was 81.3 :±: 0.6% on dry solids basis (Chen and Rasper, 1982a). Preparation of Supplemented Doughs The doughs were prepared from blends in which 8% commercial bread wheat flour (14.88% protein) was replaced by an equal quantity of soy isolate (replacement on dry solids basis). The doughs were mixed to a maximum consistency (500 BU) in the 50 g stainless steel mixing bowl of a Brabender Farinograph (30:±: 1°C) using the constant flour weight method (AACC, 1969). On reaching the maximum consistency, the doughs were immediately frozen in liquid nitrogen, freeze-dried and pulverized to pass through mesh No. 60. Surfactants used in the study included anionic sodium stearoyl2-lactylate (SSL), nonionic lipophilic sodium tristearate (Span 65) and non ionic hydrophilic polysorbate 60 (Tween 60). The first one was supplied by Semmons-Taylor Co. Ltd., Montreal, Que.; the other two were products of Atlas Chemical Industries Canada Ltd., Brantford, ant. All surfactants were used in solid form at 1% level, flour basis. They were added to wheat flour or wheat flour/soy isolate blend prior to the addition of water.
Nitrogen Extractability (NE) For NE tests, dilute acetic acid (0.05 M) and potassium phosphate-NaCl buffer (pH 7.6, J1- == 0.5) were used as extractants. The extraction was carried out three times at 25°C under constant stirring of the slurry with a magnetic stirrer for a period of 30 min, using a I :20 solids to solvent ratio. After each extraction, the slurry was centrifuged for 20 min at 12,100 g (Sorvall centrifuge, Type SS-34 head). Nitrogen content in an aliquot of the extract (I or 2 mL) was determined by the microKjeldahl method (AOAC, 1980). Free and Bound Lipid Extractability Free Iipids in the lyophilized doughs were determined by exhaustive extraction (12 h) in a Soxhlet extraction apparatus with petroleum ether (bp 35-60°C). The air dried residue was further extracted with water saturated n-butanol (WSB) as described by Pomeranz et al. (1966) with the following modifications: a 5 g sample was treated consecutively with three 50 mL portions of WSB for 4, 4 and 2 min in a Sorvall blender (Sorvall Omnimixer 17105, Dupont Instruments, Newton, Conn.) at a speed setting of 5. The rest of the procedure was as described by the authors.
Results and Discussion NE by 0.05 M Acetic Acid Under the conditions used, the solvent extracted 61.9% of the nitrogen of the wheat flour and an average 9.3% of the nitrogen of the protein isolates. Thus, the amount of acid soluble soy protein in the supplemented blends represented only about 7% of that from wheat flour. Assuming that the extractabilities remain the same for the components in the blends, the calculated average value of the acid soluble protein fraction would be approximately 45% of the total protein. From comparison of this calculated value with the experimental data for
Table I. Nitrogen extractability of wheat flour/soy protein isolate doughs (lyophilized) in the absence and presence of surfactant (I % on wheat flour basis). I Extractant: 0.05 M acetic acid. Nitrogen extractability (%)' Surfactant Type of isolate in mixture
None
SSL
Tween 60
Span 65
Mean
None
65.7
57.5
64.3
66.5
63.5
Water extracted isolates::I Isoelectric protein Ca proteinate K proteinate Na proteinate Mean
51.2a 43.7 54.9 52.8a 50.7
41.5b 31.4d 35.3a 35.la 35.8
49.7a 46.7b 52.0a 52.0a 50.1
49.7a 46.8b 47.6b 49.3a 48.4
48.0a 42.2b 47.5a 47.3a
Alkali extracted isolates::I Isoelectric protein Ca proteinate K proteinate Na proteinate Mean
50.8a 47.0b 49.8ab 51.0a 49.7
40.3b 33.3 35.2a 37.0c 36.4
49.0a 42.3 46.9b 44.7b 45.7
49.5a 43.9 49.2 48.2ab 47.7
47.4a 41.6b 45.3c 45.2c
Na proteinate (spray dried) Na proteinate (drum dried)
51.3a 51.5::'
36.2c 32.5
52. la 38.0
50.0a 38. J
47.4a 40.0b
'All data are averages of four replicates (overall standard deviation 0.8%). Data in vertical columns (except of means) not followed by the same letter are significantly different (P<0.05) using Duncan's multiple range test. 'Extractable nitrogen as percentage of the total nitrogen content in lyophilized dough. "Freeze-dried isolates, unless otherwise stated. Can. Inst. Food Sci. Technof, 1. Vol. 15, No. 4, 1982
Chen and Rasper/303
lyophilized doughs, the changes in NE during dough mixing became evident (Table I). The increase in acid soluble protein during mixing of the unsupplemented control dough to 65.7% of the total protein was in agreement with earlier reported data (Tsen, 1967; Tanaka and Bushuk, 1973). With supplemented doughs, this increase was considerably lower. Although there were only small differences found between doughs with differently treated isolates, it may be noted that doughs containing Ca proteinate, both water and alkali extracted, gave NE values distinctly lower than the rest of the test material. From comparing the solubility data of the individual isolates at the given pH (Chen and Rasper, 1982a), it may be concluded that the lower NE values of the Ca proteinate doughs were a reflection of some interactions during dough mixing, rather than solely of a lower solubility of this particular protein supplement. No significant differences were found between doughs supplemented with either water or alkali extracted isolates. As shown earlier, the anionic SSL significantly decreases protein extractability during mixing of wheat doughs (Chung and Tsen, 1975b). This decrease can be attributed to the well documented ability of wheat proteins to bind ionic surfactants (Hoseney et al., 1970; Simmonds and Wrigley, 1972; Fullington, 1974; Chung et al., 1981). Data in Table I show this decrease for both unsupplemented and supplemented doughs. On the other hand, the means for doughs treated with the nonionic surfactants did not indicate any significant changes in NE due to the addition of the surfactant. None of the three surfactants changed the pattern of the NE values with respect to their response to the type of isolate in the blend. The limited involvement of the actual acid solubility of the soy protein isolates in the discussed tests was further evidenced by results on doughs supplemented with Na proteinates of different heat status. In spite of a markedly lower solubility of drum dried proteinate in the dilute acetic acid (1.6% compared with 9.8% and 11.3% for freeze-dried and spray dried proteinates, respectively),
the nitrogen extractability of dough supplemented with this isolate was found about the same as that of doughs containing any of the two other Na proteinates. There was, however, a noticeable difference in the response of these doughs to the presence of the surfactant. Regardless of the type, the addition of a surfactant to dough supplemented with drum dried proteinate reduced its acid soluble protein fraction by a degree exceeding that observed with the two other doughs. NE by Phosphate Buffer (pH 7.6) Table 2 summarizes results of extractions performed with potassium phosphate-NaCI buffer (pH 7.6, M = 0.5). Because of a considerable variation in the nitrogen extractability of the test isolates at pH 7.6 (Chen and Rasper, 1982a), the NE values for the lyophilized doughs were calculated as a percentage of the total extractable nitrogen in each individual composite blend prior to its mixing into dough. The values for the blends ranged from 17.5-18.6% of the total nitrogen. The NE value of wheat flour protein was only 15.7%, obviously because of the higher isoelectric region of wheat gluten proteins (pH 6-9). Unlike acetic acid soluble proteins, the phosphate buffer extractable fraction did not show any significant quantitative changes during mixing of the unsupplemented dough. This observation was in contrast to results obtained with the supplemented blends in which considerable increase in NE took place during dough mixing. These changes appeared to be influenced by the type of protein isolate. In comparing the water and alkali extracted isolates, a lesser degree of solubilization was noticed with doughs supplemented with the latter. Doughs containing Ca proteinate gave consistently the lowest values. As in testing acetic acid soluble fraction, the addition of surfactant did not change the overall pattern of the NE values with respect to their response to the type of isolate. However, it enhanced the extractability, most noticeably in the case of SSL and Tween 60.
Table 2. Nitrogen extractability of wheat flour/soy protein isolate doughs (lyophilized) in the absence and presence of surfactant (J % on wheat flour basis).' Extractant: potassium phosphate-NaCI buffer (pH 7.6, J.t = 0.5). Nitrogen extractability (%)" Surfactant Type of isolate in mixture
None
SSL
Span 65
Mean
None
14.3
15.7
16.8
14.9
15.4
Water extracted isolates:" Isoelectric protein Ca proteinate K proteinate Na proteinate Mean
23.7a 20.5c 25.2 22.5a 23.0
25.5b 23.6d 27.3a 28.la 26.1
24.5abc 22.4 25.6c 29.2 25.4
20.9a 18.7b 24.8c 23.3d 21.9
23.6a 21.3b 25.7c 25.8c 24.0
Alkali extracted isolates:" Isoelectric protein Ca proteinate Na proteinate K proteinate Mean
21.9b 19.9 2I.9b 21.3bc 21.3
25.0b 24.0d 26.3c 26.4d 25.4
24.2a 22.4 23.3d 23.6bd 23.4
20.7d 18.6b 25.4c 22.9d 21.9
23.0a 21.3b 24.3a 23.6a
Tween 60
'All data are averages of four replicates (overall standard deviation 0.6%). Data in venical columns (except of means) not followed by the same letter are significantly different (P<0.05) using Duncan's multiple range test. 'Extractable nitrogen as percentage of the total nitrogen content in lyophilized dough. "Freeze-dried isolates.
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J. Insr. Can. Sci. Technol. Aliment. Vol. 15, No. 4, 1982
Lipid Extractability Because the distribution of free and bound lipid varied with the type of protein isolate (Table 3), the changes during mixing of the supplemented doughs were evaluated by comparing the data for each individual blend prior to its mixing into dough with those determined analytically in corresponding lyophilized dough (Table 4). Binding of lipids during dough mixing has been a well recognized phenomenon (Chiu and Pomeranz, 1966; Chiu et al., 1968; Mann and Morrison, 1974; Chung and Tsen, 1975b,d; Chung and Tsen, 1977). An approximately 60% decrease in free lipid content measured for the unsupplemented dough agreed well with the literature data. A noticeably lesser degree of reduction, accounting for approximately 40% of the values calculated for the blends, was found with the supplemented doughs. Although the type of isolate present in the dough had only a marginal effect, it could be seen that a consistently higher degree of lipid binding took place in doughs supplemented with either isoelectric protein or Ca proteinate, regardless of the type of extraction medium
used in their preparation. In comparing the effect of heat status on the evaluated property, a distinctly higher lipid binding was observed in doughs supplemented with the higher heat status (drum dried) Na proteinate than in those which contained the lower heat status (freeze- or spray dried) supplement. The results became somewhat less conclusive in the presence of the surfactants. Evidently, a great error was introduced into the comparison of the calculated and experimental values by a relatively high solubility of the surfactants in the solvent used. Extractability of pure surfactant in petroleum ether under the conditions of test was determined as 61.8 ± 3.6%, 65.3 ± 4.7% and 98.6 ± 4.4% for SSL, Tween 60 and Span 65, respectively (the data are averages ± standard deviation of six separate determinations). Although these solubilities were taken into account in computing the lipid values for the individual blends, the analytical procedure applied did not allow separation of the changes due to dough mixing in the extractability of lipids from those in the extractability of the surfactant. Differences among surfactants
Table 3. Free and bound lipids of test isolates. (All data on dry solids basis.)' Free lipids (%)
Bound Iipids (%)
Total (%)
0.97 0.68 0.58
6.95 7.66 8.01 7.84
7.92 8.34 8.45 8.42
Alkali extracted isolates:' Isoelectric protein Ca proteinate K proteinate Na proteinate
1.61 0.67 0.39 0.63
6.79 7.68 8.64 8.29
8.40 8.35 9.05 8.92
Na proteinate (spray dried) Na proteinate (drum dried)
0.37 0.48
7.37 2.00
7.72 2.48
Isolate Water extracted isolates:' Isoelectric protein Ca proteinate K proteinate Na proteinate
0.44
'All data are averages of four replicates. 'Freeze-dried isolates, unless otherwise stated.
Table 4. Changes in the distribution of free and bound Iipids during mixing of wheat flour/soy protein isolate doughs in the absence and presence of surfactant (I % on flour basis). I Change in bound Iipids (%)'
Change in free lipids (%)' None
SSL
Tween 60
Span 65
None
SSL
Tween 60
Span 65
None
-62
-11
- 5
-14
+79
+36
-22
+63
Water extracted isolates:" Isoelectric protein Ca proteinate K proteinate Na proteinate Mean
-42 -41 -33 -37 -38
-11 -31 -22 -14 -20
0 - 3 -16 + 4 - 4
-10 -13 -12 -10 -11
+35 +21 +11 +16 +21
+16 +18 +11 +12 +14
-10 -21 - 4 -15 -13
+30 +20 +24 +24 +25
Alkali extracted isolates:" Isoelectric protein Ca proteinate K proteinate Na proteinate Mean
-37 -38 -32 -32 -34
-34 -45 -31 -32 -36
-26 -25 -28 - 7
-26
-22
-19 - 9 -16
+34 +25 +18 +21 +22
+14 +23 +21 +20 +20
-14 -18 - 8 -10 -13
+24 +26 +23 +24 +24
Na proteinate (spray dried) Na proteinate (drum dried)
-30 -42
-28 -38
-12 -16
-10 -16
+23 +67
+27 +42
+89 +85
+20 +59
Type of isolate in mixture
-10
'All data are averages of four replicates. 'Change during mixing expressed as percentage of the calculated lipid value for the blend. "Freeze-dried isolates, unless otherwise stated. Can. Insf. Food Sci. Technol. J, Vol. 15, No. 4, 1982
Chen and Rasper/305
in their binding abilities to components of dough have been well recognized. In spite of all these shortcomings of the testing procedure, data in Table 4 confirmed the earlier reported displacement of some of the bound lipids in fully developed doughs by nonionic surfactants. This displacement may be reflected in an increase in the free lipid fraction of the system (Chung and Tsen, 1977). Such was the case with doughs containing either Tween 60 or Span 65. Though free lipids were reduced during the mixing of these two doughs, the degree of this reduction did not reach such magnitude as in the presence of ionic SSL. More experimental data would have to be collected in order to conclude that the somewhat higher reduction in free lipid content in SSL doughs supplemented with Ca proteinate (both water and alkali extracted) was in any relationship to its baking quality. It may be of interest to note that this particular isolate/ surfactant combination gave rise to loaves which received the most favourable evaluation in the earlier discussed baking tests (Chen and Rasper, 1982b).
Conclusion Because of the isoelectric point of soy protein in the acidic region (pH 4-5), nitrogen solubility tests on lyophilized soy protein supplemented doughs performed with dilute acetic acid revealed only a limited involvement of the supplementing protein. Nevertheless, an indication of interactions affecting the measured property was noticed, especially in the presence of Ca proteinate. Further evidence for these interactions was provided by extractability tests at close to normal reaction. Unlike extractions with dilute acetic acid, extractions with phosphate buffer (pH 7.6) not only confirmed a lower degree of solubilization of protein during mixing of Ca proteinate doughs, but revealed measurable and consistent differences between doughs supplemented with either water or alkali extracted isolates. The results on lipid extractability as affected by the type of protein isolate in the supplemented dough were less conclusive. The response of this property to the type of material added to wheat flour appeared to be more surfactant than protein isolate related. It was, however, noted that the less soluble isolates (isoelectric protein, Ca proteinate) tended to enhance the binding of free lipids to a greater degree than the more soluble alkali metal (K, Na) proteinates. The difference in free lipid reduction between the two mentioned groups of isolates was approximately the same magnitude as that resulting from different heat status of the test Na proteinates.
Acknowledgements This work was part of a research project supported jointly by the Natural Sciences and Engineering Research Council of Canada and the Ontario Ministry of Agriculture and Food. This financial assistance is greatly appreciated.
306/Chen and Rasper
References AACC. 1969. Approved Methods. Method 54-21. American Association of Cereal Chemists, S!. Paul, MN. Aidoo, E.S. and Tsen, C.C. 1973a. Surfactant-protein interactions in model systems. Cereal Sci. Today 18:301. Aidoo, E.S. and Tsen, c.c. 1973b. Influence of surfactants or soy proteins on the extractability, gel filtration, and disc electrophoretic patterns of wheat proteins. Cereal Sci. Today 18:302. AOAC. 1980. Official Methods of Analysis. Method 47.021-47.023. Association of Official Analytical Chemists, Washington, DC. Chen, S.S. and Rasper, V.F 1982a. Functionality of soy proteins in wheat flour/soy isolate doughs. I. Characterization of isolates prepared using different isolation techniques. Can. Ins!. Food Sci. Technol. J. 15(3):203. Chen, S.S. and Rasper, V.F 1982b. Functionality of soy proteins in wheat flour/soy isolate doughs. 11. Rheological properties and bread making potential. Can. Ins!. Food Sci. Technol. J. 15(3):21 I. Chiu, S.M. and Pomeranz, Y. 1966. Changes in extractability of lipids during breadmaking. J. Food Sci. 31:753. Chiu, S.M., Pomeranz, Y., Shogren, M.1. and Finney, K.F 1968. Lipid binding in wheat flours varying in breadmaking potential. Food Technol. 22: 1157. Chung, O.K. and Tsen, c.c. 1975a. Distribution of lipid binding in acid soluble protein components as affected by dough mixing and surfactants. Cereal Chem. 52:823. Chung, O.K. and Tsen, c.c. 1975b. Changes in lipid binding and protein extractability during dough mixing in the presence of surfactants. Cereal Chem. 52:549. Chung, O.K. and Tsen, C.C. 1975c. Functional properties of surfactants in breadmaking. I. Roles of surfactants in relation to flour constituents in dough system. Cereal Chem. 52:832. Chung, O.K. and Tsen, C.c. 1975d. Changes in lipid binding and distribution during dough mixing. Cereal Chem. 52:533. Chung, O.K. and Tsen, C.C. 1977. Functional properties of surfactants in breadmaking. 11. Composition of lipids associated with doughs containing various levels of surfactants. Cereal Chem. 54:857. Chung, O.K., Tsen, c.c. and Robinson, R.J. 1981. Functional properties of surfactants in breadmaking. Ill. Effect of surfactants and soy flour on lipid binding in breads. Cereal Chem. 58:220. De Stefanis, V.A., Ponte, J.G., Jr., Chung, FH. and Ruzza, N.A. 1977. A binding of crumb softeners and dough strengtheners during breadmaking. Cereal Chem. 54:13. Fullington, J.G. 1974. A protein from wheat flour that binds calcium and stearoyl-2-lactylate ions. Cereal Chem. 51 :250. Hoseney, R.C., Finney, K.F and Pomeranz, Y. 1970. Functional (breadmaking) and biochemical properties of wheat flour components. VI. Gliadin-lipid-glutenin interaction in wheat gluten. Cereal Chem. 47:135. Krog, N. 1971. Amylose complexing effect of food grade emulsifiers. Staerke 23:206. Kim, Y.J. and Robinson, R.J. 1979. Effect of surfactants on starch in a model system. Staerke 31:293. Mann, D.L. and Morrison, W.R. 1974. Changes in wheat lipids during mixing and resting of flour-water doughs. J. Sci. Food Agric. 25: 1109. Pomeranz, Y., Chung, O.K. and Robinson, R.J. 1966. The lipid composition of wheat flour varying widely in breadmaking potentialities. J. Am. Oil Chem. Soc. 43:45. Simmonds, D.H. and Wrigley, C.M. 1972. The effect of lipid on the solubility and molecular weight range of wheat gluten and storage proteins. Cereal Chem. 49:317. Tanaka, K. and Bushuk, W. 1973. Changes in flour proteins during dough mixing. Ill. Analytical results and mechanisms. Cereal Chem. 50:605. Tsen, c.c. 1967. Changes in flour proteins during dough mixing. Cereal Chem. 44:308. Wherli, H.P. and Pomeranz, Y. 1970. A note on the interaction between glycolipids and wheat flour macromolecules. Cereal Chem. 47: 160. Accepted May 11, 1982
J. InSf. Can. Se;. Technol. Aliment. Vcl. 15, No. 4, 1982
Functionality of Soy Proteins in Wheat Flour/Soy Isolate Doughs. Ill. Protein and Lipid Binding During Dough Mixing S.S. Chen I and V.F. Rasper Department of Food Science University of Guelph Guelph, Ontario NIG 2WI
Abstract
seches 11 froid fut 11 peu pres du meme ordre de grandeur que celle observee entre les produits traites aux basses temperatures (cryodessication ou pulverisation) et ceux traites aux hautes temperatures (sechage sur rouleaux). Meme si I'addition d'agents tensio-actifs influen~a I'ampleur des aptitudes 11 I'extraction, le modele relatif aux divers isolats est demeure pratiquement le meme.
The changes in nitrogen and lipid extractabilities during mixing of soy protein supplemented wheat flour doughs were studied using water or alkali extracted protein isolates in the form of isoelectric protein or protei nates subjected to different drying techniques. The studies were performed on blends at 8% supplementation level in the absence or presence of a surfactant (SSL, Tween 60, Span 65). Nitrogen solubility of the supplemented doughs increased during their mixing to maximum consistency by approximately 11% and 23% when 0.05 M acetic acid and phosphate-NaCI buffer (pH 7.6, JL = 0.5) were used, respectively. This increase was considerably higher than that measured with unsupplemented dough. Free lipid extractability of supplemented doughs was reduced in average by 36% compared to 62% reduction in unsupplemented control dough. Among the freeze-dried isolates, the strongest tendency to affect the studied extractabilities was displayed by Ca proteinates. The difference between the effect of these proteinates and other freeze-dried isolates was about the same magnitude as that observed between the lower heat status (freeze- or spray dried) and higher heat status (drum dried) material. Though the addition of surfactants influenced the measured extractabilities, the response pattern with respect to the type of isolate remained practically unchanged.
Introduction The adverse effect of high protein nonwheat products on dough quality and the role of lipid related surfactants in its counteraction have been the subject of numerous studies. In an attempt of better understanding of the mechanisms involved, many studies focused on the changes in the solubility of protein fractions and lipid binding during mixing and baking of supplemented doughs in the presence of different surfactants (Hoseney et al., 1970; Wherli and Pomeranz, 1970; Aidoo and Tsen, 1973a,b; Chung and Tsen, 1975a,b,c,d; Chung and Tsen, 1977; De Stefanis et al., 1977; Chung et al., 1981). Though various models of complexes formed in the dough during its development were proposed in order to explain the interactions between the wheat flour components, protein supplement and surfactant, the nature of these interactions has not been fully elucidated (Hoseney et al., 1970; Krog, 1971; Kim and Robinson, 1979; Chung et aI., 198 I). The present study is part of research work on wheat flour doughs supplemented with soy proteins prepared from defatted soy grits using different preparative treatments. The preparation of the protein isolates and their chemical and physico-chemical properties, as well as their effects on the rheological and baking qualities of the supplemented doughs, were reported earlier (Chen and Rasper, 1982a,b). This paper reports on results of tests in which the relationship between the type of iso-
Resume En partant d' isolats proteiques extraits 11 I'eau ou 11 I' alcali sous la forme de proteine isoelectrique ou de proteinates seches par differentes techniques, on a etudie les changements dans I'aptitude 11 I'extraction de I'azote et des lipides au cours du malaxage des pates de proteines de soja enrichies de farine de ble. Les etudes ont porte sur des melanges enrichis au niveau de 8% avec ou sans agent tensio-actif (SSL, Tween 60, Span 65). La solubilite de I'azote des pates enrichies a augmente au cours du malaxage jusqu'1I consistance maximum par environ 11% et 23% respectivement avec le tampon 11 I'acide acetique 0,05 M et avec le tampon au phosphate-NaCI (pH 7,6, JL = 0,5). Celle augmentation fut considerablement plus forte que celle obtenue avec la pate non enrichie. L'aptitude 11 I'extraction des lipides Iibres a diminue de 36% en moyenne par rapport 11 62% pour la pate temoin non enrichie. De tous les isolats seches 11 froid, ce sont les proteinates de calcium qui ont manifeste la plus forte tendance 11 affecter les aptitudes 11 I'extraction. La difference entre I'effet de ces proteinates et celui des autres isolats 'Present address: Riverdale Frozen Foods, Niagara Falls, Ontario L21 6S6.
Copyright
©
0315-5463/82/040302-05$3.00/0 1982 Canadian Institute of Food Science and Technology
302
late, as determined by the preparative technique, and the changes in nitrogen and lipid extractabilities were evaluated. The tests were performed in the absence or presence of three different surfactants.
Materials and Methods Preparation of the Protein Isolates Soy protein isolates were prepared under laboratory conditions using different extraction media (water and alkali), different treatment in the separation of the isolated proteins (isoelectric precipitation and neutralization to form Ca, K and Na proteinates), and drying (freeze-, spray and drum drying). The average protein content was 81.3 :±: 0.6% on dry solids basis (Chen and Rasper, 1982a). Preparation of Supplemented Doughs The doughs were prepared from blends in which 8% commercial bread wheat flour (14.88% protein) was replaced by an equal quantity of soy isolate (replacement on dry solids basis). The doughs were mixed to a maximum consistency (500 BU) in the 50 g stainless steel mixing bowl of a Brabender Farinograph (30:±: 1°C) using the constant flour weight method (AACC, 1969). On reaching the maximum consistency, the doughs were immediately frozen in liquid nitrogen, freeze-dried and pulverized to pass through mesh No. 60. Surfactants used in the study included anionic sodium stearoyl2-lactylate (SSL), nonionic lipophilic sodium tristearate (Span 65) and non ionic hydrophilic polysorbate 60 (Tween 60). The first one was supplied by Semmons-Taylor Co. Ltd., Montreal, Que.; the other two were products of Atlas Chemical Industries Canada Ltd., Brantford, ant. All surfactants were used in solid form at 1% level, flour basis. They were added to wheat flour or wheat flour/soy isolate blend prior to the addition of water.
Nitrogen Extractability (NE) For NE tests, dilute acetic acid (0.05 M) and potassium phosphate-NaCl buffer (pH 7.6, J1- == 0.5) were used as extractants. The extraction was carried out three times at 25°C under constant stirring of the slurry with a magnetic stirrer for a period of 30 min, using a I :20 solids to solvent ratio. After each extraction, the slurry was centrifuged for 20 min at 12,100 g (Sorvall centrifuge, Type SS-34 head). Nitrogen content in an aliquot of the extract (I or 2 mL) was determined by the microKjeldahl method (AOAC, 1980). Free and Bound Lipid Extractability Free Iipids in the lyophilized doughs were determined by exhaustive extraction (12 h) in a Soxhlet extraction apparatus with petroleum ether (bp 35-60°C). The air dried residue was further extracted with water saturated n-butanol (WSB) as described by Pomeranz et al. (1966) with the following modifications: a 5 g sample was treated consecutively with three 50 mL portions of WSB for 4, 4 and 2 min in a Sorvall blender (Sorvall Omnimixer 17105, Dupont Instruments, Newton, Conn.) at a speed setting of 5. The rest of the procedure was as described by the authors.
Results and Discussion NE by 0.05 M Acetic Acid Under the conditions used, the solvent extracted 61.9% of the nitrogen of the wheat flour and an average 9.3% of the nitrogen of the protein isolates. Thus, the amount of acid soluble soy protein in the supplemented blends represented only about 7% of that from wheat flour. Assuming that the extractabilities remain the same for the components in the blends, the calculated average value of the acid soluble protein fraction would be approximately 45% of the total protein. From comparison of this calculated value with the experimental data for
Table I. Nitrogen extractability of wheat flour/soy protein isolate doughs (lyophilized) in the absence and presence of surfactant (I % on wheat flour basis). I Extractant: 0.05 M acetic acid. Nitrogen extractability (%)' Surfactant Type of isolate in mixture
None
SSL
Tween 60
Span 65
Mean
None
65.7
57.5
64.3
66.5
63.5
Water extracted isolates::I Isoelectric protein Ca proteinate K proteinate Na proteinate Mean
51.2a 43.7 54.9 52.8a 50.7
41.5b 31.4d 35.3a 35.la 35.8
49.7a 46.7b 52.0a 52.0a 50.1
49.7a 46.8b 47.6b 49.3a 48.4
48.0a 42.2b 47.5a 47.3a
Alkali extracted isolates::I Isoelectric protein Ca proteinate K proteinate Na proteinate Mean
50.8a 47.0b 49.8ab 51.0a 49.7
40.3b 33.3 35.2a 37.0c 36.4
49.0a 42.3 46.9b 44.7b 45.7
49.5a 43.9 49.2 48.2ab 47.7
47.4a 41.6b 45.3c 45.2c
Na proteinate (spray dried) Na proteinate (drum dried)
51.3a 51.5::'
36.2c 32.5
52. la 38.0
50.0a 38. J
47.4a 40.0b
'All data are averages of four replicates (overall standard deviation 0.8%). Data in vertical columns (except of means) not followed by the same letter are significantly different (P<0.05) using Duncan's multiple range test. 'Extractable nitrogen as percentage of the total nitrogen content in lyophilized dough. "Freeze-dried isolates, unless otherwise stated. Can. Inst. Food Sci. Technof, 1. Vol. 15, No. 4, 1982
Chen and Rasper/303
lyophilized doughs, the changes in NE during dough mixing became evident (Table I). The increase in acid soluble protein during mixing of the unsupplemented control dough to 65.7% of the total protein was in agreement with earlier reported data (Tsen, 1967; Tanaka and Bushuk, 1973). With supplemented doughs, this increase was considerably lower. Although there were only small differences found between doughs with differently treated isolates, it may be noted that doughs containing Ca proteinate, both water and alkali extracted, gave NE values distinctly lower than the rest of the test material. From comparing the solubility data of the individual isolates at the given pH (Chen and Rasper, 1982a), it may be concluded that the lower NE values of the Ca proteinate doughs were a reflection of some interactions during dough mixing, rather than solely of a lower solubility of this particular protein supplement. No significant differences were found between doughs supplemented with either water or alkali extracted isolates. As shown earlier, the anionic SSL significantly decreases protein extractability during mixing of wheat doughs (Chung and Tsen, 1975b). This decrease can be attributed to the well documented ability of wheat proteins to bind ionic surfactants (Hoseney et al., 1970; Simmonds and Wrigley, 1972; Fullington, 1974; Chung et al., 1981). Data in Table I show this decrease for both unsupplemented and supplemented doughs. On the other hand, the means for doughs treated with the nonionic surfactants did not indicate any significant changes in NE due to the addition of the surfactant. None of the three surfactants changed the pattern of the NE values with respect to their response to the type of isolate in the blend. The limited involvement of the actual acid solubility of the soy protein isolates in the discussed tests was further evidenced by results on doughs supplemented with Na proteinates of different heat status. In spite of a markedly lower solubility of drum dried proteinate in the dilute acetic acid (1.6% compared with 9.8% and 11.3% for freeze-dried and spray dried proteinates, respectively),
the nitrogen extractability of dough supplemented with this isolate was found about the same as that of doughs containing any of the two other Na proteinates. There was, however, a noticeable difference in the response of these doughs to the presence of the surfactant. Regardless of the type, the addition of a surfactant to dough supplemented with drum dried proteinate reduced its acid soluble protein fraction by a degree exceeding that observed with the two other doughs. NE by Phosphate Buffer (pH 7.6) Table 2 summarizes results of extractions performed with potassium phosphate-NaCI buffer (pH 7.6, M = 0.5). Because of a considerable variation in the nitrogen extractability of the test isolates at pH 7.6 (Chen and Rasper, 1982a), the NE values for the lyophilized doughs were calculated as a percentage of the total extractable nitrogen in each individual composite blend prior to its mixing into dough. The values for the blends ranged from 17.5-18.6% of the total nitrogen. The NE value of wheat flour protein was only 15.7%, obviously because of the higher isoelectric region of wheat gluten proteins (pH 6-9). Unlike acetic acid soluble proteins, the phosphate buffer extractable fraction did not show any significant quantitative changes during mixing of the unsupplemented dough. This observation was in contrast to results obtained with the supplemented blends in which considerable increase in NE took place during dough mixing. These changes appeared to be influenced by the type of protein isolate. In comparing the water and alkali extracted isolates, a lesser degree of solubilization was noticed with doughs supplemented with the latter. Doughs containing Ca proteinate gave consistently the lowest values. As in testing acetic acid soluble fraction, the addition of surfactant did not change the overall pattern of the NE values with respect to their response to the type of isolate. However, it enhanced the extractability, most noticeably in the case of SSL and Tween 60.
Table 2. Nitrogen extractability of wheat flour/soy protein isolate doughs (lyophilized) in the absence and presence of surfactant (J % on wheat flour basis).' Extractant: potassium phosphate-NaCI buffer (pH 7.6, J.t = 0.5). Nitrogen extractability (%)" Surfactant Type of isolate in mixture
None
SSL
Span 65
Mean
None
14.3
15.7
16.8
14.9
15.4
Water extracted isolates:" Isoelectric protein Ca proteinate K proteinate Na proteinate Mean
23.7a 20.5c 25.2 22.5a 23.0
25.5b 23.6d 27.3a 28.la 26.1
24.5abc 22.4 25.6c 29.2 25.4
20.9a 18.7b 24.8c 23.3d 21.9
23.6a 21.3b 25.7c 25.8c 24.0
Alkali extracted isolates:" Isoelectric protein Ca proteinate Na proteinate K proteinate Mean
21.9b 19.9 2I.9b 21.3bc 21.3
25.0b 24.0d 26.3c 26.4d 25.4
24.2a 22.4 23.3d 23.6bd 23.4
20.7d 18.6b 25.4c 22.9d 21.9
23.0a 21.3b 24.3a 23.6a
Tween 60
'All data are averages of four replicates (overall standard deviation 0.6%). Data in venical columns (except of means) not followed by the same letter are significantly different (P<0.05) using Duncan's multiple range test. 'Extractable nitrogen as percentage of the total nitrogen content in lyophilized dough. "Freeze-dried isolates.
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Lipid Extractability Because the distribution of free and bound lipid varied with the type of protein isolate (Table 3), the changes during mixing of the supplemented doughs were evaluated by comparing the data for each individual blend prior to its mixing into dough with those determined analytically in corresponding lyophilized dough (Table 4). Binding of lipids during dough mixing has been a well recognized phenomenon (Chiu and Pomeranz, 1966; Chiu et al., 1968; Mann and Morrison, 1974; Chung and Tsen, 1975b,d; Chung and Tsen, 1977). An approximately 60% decrease in free lipid content measured for the unsupplemented dough agreed well with the literature data. A noticeably lesser degree of reduction, accounting for approximately 40% of the values calculated for the blends, was found with the supplemented doughs. Although the type of isolate present in the dough had only a marginal effect, it could be seen that a consistently higher degree of lipid binding took place in doughs supplemented with either isoelectric protein or Ca proteinate, regardless of the type of extraction medium
used in their preparation. In comparing the effect of heat status on the evaluated property, a distinctly higher lipid binding was observed in doughs supplemented with the higher heat status (drum dried) Na proteinate than in those which contained the lower heat status (freeze- or spray dried) supplement. The results became somewhat less conclusive in the presence of the surfactants. Evidently, a great error was introduced into the comparison of the calculated and experimental values by a relatively high solubility of the surfactants in the solvent used. Extractability of pure surfactant in petroleum ether under the conditions of test was determined as 61.8 ± 3.6%, 65.3 ± 4.7% and 98.6 ± 4.4% for SSL, Tween 60 and Span 65, respectively (the data are averages ± standard deviation of six separate determinations). Although these solubilities were taken into account in computing the lipid values for the individual blends, the analytical procedure applied did not allow separation of the changes due to dough mixing in the extractability of lipids from those in the extractability of the surfactant. Differences among surfactants
Table 3. Free and bound lipids of test isolates. (All data on dry solids basis.)' Free lipids (%)
Bound Iipids (%)
Total (%)
0.97 0.68 0.58
6.95 7.66 8.01 7.84
7.92 8.34 8.45 8.42
Alkali extracted isolates:' Isoelectric protein Ca proteinate K proteinate Na proteinate
1.61 0.67 0.39 0.63
6.79 7.68 8.64 8.29
8.40 8.35 9.05 8.92
Na proteinate (spray dried) Na proteinate (drum dried)
0.37 0.48
7.37 2.00
7.72 2.48
Isolate Water extracted isolates:' Isoelectric protein Ca proteinate K proteinate Na proteinate
0.44
'All data are averages of four replicates. 'Freeze-dried isolates, unless otherwise stated.
Table 4. Changes in the distribution of free and bound Iipids during mixing of wheat flour/soy protein isolate doughs in the absence and presence of surfactant (I % on flour basis). I Change in bound Iipids (%)'
Change in free lipids (%)' None
SSL
Tween 60
Span 65
None
SSL
Tween 60
Span 65
None
-62
-11
- 5
-14
+79
+36
-22
+63
Water extracted isolates:" Isoelectric protein Ca proteinate K proteinate Na proteinate Mean
-42 -41 -33 -37 -38
-11 -31 -22 -14 -20
0 - 3 -16 + 4 - 4
-10 -13 -12 -10 -11
+35 +21 +11 +16 +21
+16 +18 +11 +12 +14
-10 -21 - 4 -15 -13
+30 +20 +24 +24 +25
Alkali extracted isolates:" Isoelectric protein Ca proteinate K proteinate Na proteinate Mean
-37 -38 -32 -32 -34
-34 -45 -31 -32 -36
-26 -25 -28 - 7
-26
-22
-19 - 9 -16
+34 +25 +18 +21 +22
+14 +23 +21 +20 +20
-14 -18 - 8 -10 -13
+24 +26 +23 +24 +24
Na proteinate (spray dried) Na proteinate (drum dried)
-30 -42
-28 -38
-12 -16
-10 -16
+23 +67
+27 +42
+89 +85
+20 +59
Type of isolate in mixture
-10
'All data are averages of four replicates. 'Change during mixing expressed as percentage of the calculated lipid value for the blend. "Freeze-dried isolates, unless otherwise stated. Can. Insf. Food Sci. Technol. J, Vol. 15, No. 4, 1982
Chen and Rasper/305
in their binding abilities to components of dough have been well recognized. In spite of all these shortcomings of the testing procedure, data in Table 4 confirmed the earlier reported displacement of some of the bound lipids in fully developed doughs by nonionic surfactants. This displacement may be reflected in an increase in the free lipid fraction of the system (Chung and Tsen, 1977). Such was the case with doughs containing either Tween 60 or Span 65. Though free lipids were reduced during the mixing of these two doughs, the degree of this reduction did not reach such magnitude as in the presence of ionic SSL. More experimental data would have to be collected in order to conclude that the somewhat higher reduction in free lipid content in SSL doughs supplemented with Ca proteinate (both water and alkali extracted) was in any relationship to its baking quality. It may be of interest to note that this particular isolate/ surfactant combination gave rise to loaves which received the most favourable evaluation in the earlier discussed baking tests (Chen and Rasper, 1982b).
Conclusion Because of the isoelectric point of soy protein in the acidic region (pH 4-5), nitrogen solubility tests on lyophilized soy protein supplemented doughs performed with dilute acetic acid revealed only a limited involvement of the supplementing protein. Nevertheless, an indication of interactions affecting the measured property was noticed, especially in the presence of Ca proteinate. Further evidence for these interactions was provided by extractability tests at close to normal reaction. Unlike extractions with dilute acetic acid, extractions with phosphate buffer (pH 7.6) not only confirmed a lower degree of solubilization of protein during mixing of Ca proteinate doughs, but revealed measurable and consistent differences between doughs supplemented with either water or alkali extracted isolates. The results on lipid extractability as affected by the type of protein isolate in the supplemented dough were less conclusive. The response of this property to the type of material added to wheat flour appeared to be more surfactant than protein isolate related. It was, however, noted that the less soluble isolates (isoelectric protein, Ca proteinate) tended to enhance the binding of free lipids to a greater degree than the more soluble alkali metal (K, Na) proteinates. The difference in free lipid reduction between the two mentioned groups of isolates was approximately the same magnitude as that resulting from different heat status of the test Na proteinates.
Acknowledgements This work was part of a research project supported jointly by the Natural Sciences and Engineering Research Council of Canada and the Ontario Ministry of Agriculture and Food. This financial assistance is greatly appreciated.
306/Chen and Rasper
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J. InSf. Can. Se;. Technol. Aliment. Vcl. 15, No. 4, 1982