Structural Changes of Soymilk Proteins during Heating and Cooling

Chapter 22

Structural Changes of Soymilk Proteins during Heating and Cooling, and Freeze-Gelation of Soymilk

Downloaded by UNIV OF ARIZONA on July 27, 2012 | http://pubs.acs.org Publication Date: December 1, 2003 | doi: 10.1021/bk-2004-0871.ch022

Makoto Shimoyamada, Kayoko Tômatsu, Satomi Oku, Wataru Koseki, and Kenji Watanabe Faculty of Agriculture, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan

The heated soymilk could be converted to a gel-like coagulate by freezing. In thisfreeze-gelation,cooling of heated soymilk at -5°C before freezing (precooling) was very important. The structural changes of soymilk proteins and interactions among them during heating and cooling was thus evaluated. Surface SH contents and surface hydrophobicities of soymik proteins, which increased by heating of soymilk, decreased to a lesser extent by cooling at-5°Cthan by cooling at room temperature. Precooling was shown to suppress refolding of thermally denatured proteins. Further, considering the effects of some additives such as sodium dodecylsulfate, 2-mercaptoethanol and sucrose, the inteaction among proteins and the freeze-gelation mechanism were discussed.

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© 2004 American Chemical Society In Nutraceutical Beverages; Shahidi, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.


291 A large number of people drink soymilk for its health benefits, especially in Japan, where its consumption has gradually increased over the past several years. Soymilk is an emulsion consisting mainly of soybean proteins and lipids, which nutritionally and functionally have excellent qualities. It is utilized as not only beverage itself, but also, for its ingredients. Asians including Japanese have been consuming tofu, soybean curd, yuba, and sheets of dried soymilk skin among others. As for the gel derived from soy protein, heat induced gel (1,2) and tofu gel (3-6) are well characterized, and there are too many reports in the literature during the past half century. Further, the soy protein dispersion or paste have been reported to form gel-like coagulates by heating and freezing (7-9), or heating and refrigeration (10). Here soymilk was used as a sample instead of soy protein dispersion; it was converted to gel-like coagulate by heating and freezing (//). Raw soymilk was heated for 3 min in an autoclave, then immediately precooled in a freezer at -5°C for 2 h. The cooled soymilk was put into afreezerat 20°C and stored for 14 days. The frozen soymilk was thawed in a warm water bath. Soymilkfreeze-gelis similar to soft tofu or yogurt (Figure 1). This freezegel is processed without the addition of coagulants such as calcium sulfate, magnesium chloride, glucono-ô-lactone, etc., thus differing from traditional tofu gel (3-6). The significance of heating and cooling steps of soymilk was of interest in order to clarify the structural changes of proteins in the soymilk during heating and cooling, including precooling step, in terms of the soymilk freezegelation.

Figure 1. Soymilk freeze-gel

These structural changes of protein molecules are thought to be important for the interactions among the proteins. Interactions among protein molecules in aggregation have been widely investigated. Disulfide, hydrophobic and electrostatic interactions are considered important for heat-induced gelation (2J2). In thefrozeninsolubilization of soybean protein solutions, disulfide bond

In Nutraceutical Beverages; Shahidi, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.


292 formation and exchange were reported to be important intermolecular interactions (8), while for cold gel formation, the hydrophobic interactions were more important (70). Thus, the interactions among protein molecules during freeze-gelation, in which the precooling step is considered to be very important for uniform texture of the freeze-gel (11,13) was studied. The precooling step allows the formation of spherical ice crystals, which were found to be distributed uniformly infrozensoymilk duringfreeze-storage(75). Attempts were also made to clarify the significance offreeze-storageas affected by pre-cooling in the protein structures of soymilk. For this, the free thiol content and the surface hydrophobicity of protein were estimated during precooling. The effect of precooling on the structure of heated soybean protein during cooling is discussed herein. Thefreeze-gelationmechanism of soybean, especially, the interactions among the protein molecules, were also studied.

Structural Changes in Soymilk Proteins During Heating, Cooling and Freezing Generally, soymilk was prepared by sucessively heating and squeezing soybean which was ground with water. However, we prepared raw soymilk under mild heating condition below 70°C, where main proteins in soybean are considered to maintain their native structures. The raw soymilk and the heated soymilk, prepared by heating of the raw soymilk at 100°C, were frozen in a freezer (-20°C) for 7 days. Appearances of products after thawing in warm water bath (30°C) are shown in Figure 2. The heated soymilk precipitated by freezing and thawing, but the raw soymilk did not. These data suggest that heating process is essential for frozen precipitation of soymilk, namely thermal denaturation of soymilk proteins would leadfrozenprecipitation of proteins and other constituents. Then the effect offrozenstorage time of heated soymilk on the precipitation after freeze-storage was estimated. At the beginning, the precipitation increased with the increase of the frozen time and reached plateau for about 14 days offrozenstorage at -20°C. Thefrozenstorage period affected a little the precipitation of soymilk, but precipitation increased with the heating time and reached plateau for 3 min of heating of soymilk (data not shown). Raw soymilk was reported to formfreeze-gelby sucessive heating (110°C, 3 min), precooling (-5°C, 2 h) and frozen storage (-20°C, 14 days) (77). Behavior of precipitation of soymilk described above is almost consistent with the previously reported data. Further, the precooling was reported to be essential for the formation offreeze-gel(77). So, the thermal denaturation behaviors of soymilk proteins during heating and cooling was estimated. At first, surface SH contents of soymilk proteins were measured using


293 90

• 70


1

50 0

7

14

21

28

Frozen storage (days) Figure 2. Freezing and thawing treatment of heated or unheated soymilk. 2,2'-dithiobis-(5-nitropyridine) (DTNP) (14,15). Soymilk was adequately diluted with a 0.1 M, pH 7.6 phosphate buffer. An aliquot (2 mL) was mixed with 5x10" M DTNP in ethanol (0.5 mL) and incubated for 20 min at room temperature. The resulting solution was mixed with a 10 % perchloric acid solution (2.5 mL) and centrifuged for 10 min at 1,500 xg. The supernatant was filtered and its absorbance read at 386 nm. Surface SH increased by heating and reached a maximum after 1 min of heating (Figure 3), but then gradually decreased with the increase of heating time. These data show that thermal denaturation increased surface SH groups by breakikng S-S bonds and exposing interior SH groups by unfolding of the protein molecules. Further heat treatment may accelerate the exchange and reformation of S-S bond and decomposition of SH group to H S or other compounds, and result in a decrease of the surface SH. Therefore, the SH content decreased to about two third of the value immediately after heating by cooling at room temperature for 2 h. However, it maintained almost equal level to that after heating, when it was left at -5°C. These data show that rapid cooling of soymilk inhibits reforming of S-S bonds and/or coverings of the SH groups by refolding of the protein molecules. These suggest that the cooling of the heated soymilk at -5°C maintains the surface SH, namely potent reactivity by the S-S formation to be higher. The surface hydrophobicities of the proteins in soymilk was subsequently evaluated. The surface hydrophobicity of protein was measured using l-(anilino) naphthalene-8-sulfonate (ANS) (16). A properly diluted sample (0.1 mL) was 4

2



294

Heating time (min) Figure 3. Surface SHof soymilk protein after heating and cooling 3

mixed with 0.01 M phosphate buffer (pH 7.0, 4 mL) and 8*10 M ANS solution (0.02 mL). The mixture was excited at 390 nm and the relative fluorescence intensity was measured at 470 nm in afluorescencespectrophotometer (F-2000, Hitachi, Ltd., Tokyo, Japan). As a result (Figure 4), the surface hydrophobicity of soymilk increased by heating, however the value was almost maintained from 0.5 to 3 min of heating. Unfolding of protein molecule is genarally considered to allow interior hydrophobic region of the protein to 600 r

0

1 2 Heating time (min)

3

Figure 4. Surface hydrophobicity of soymilk protein after heating and cooling (Reproducedfromreference 17) (Reproduced from reference 17. Copyright 2000 American Chemical Society.)



295 expose outside, so increase of surface hydrophobicity was responsible for partial unfolding of soymilk proteins. The hydrophobicity was slightly but significantly higher by cooling at -5°C than at room temperature. Because of the lipids and other constituents, the measurements of hydrophobicity in soymilk may contain some inaccuracies. Even given these restrictions, the data coincided well with the result from surface hydrophobicity in APP solution (data not shown). The surface hydrophobicity, which increased with heat treatment, might maintain at a relatively higher level by rapid cooling to the supercooling state. The precooling of heated soymilk at -5°C suppressed the decrease in surface thiol content and surface hydrophobicity of soybean proteins, which had increased during the heating step. This result might also show that precooling suppressed the refolding of the protein molecules, which had been partially destroyed or unfolded by heat treatment. High levels of surface thiol and surface hydrophobic areas were required to form three-dimensional networks during frozen storage. In earlier work (75), the significance of the precooling step was reported to form uniform, fine ice crystals. The high level retention of surface thiol and hydrophobicity is considered to be another significant aspect of the precooling for freeze-gelation. In order to evaluate the secondary structures of protein molecules during heating and precooling of soymilk. Fourier transform - infrared spectra (FT-IR spectra) were recorded by a System2000 (Perkin Elmer Co.) using Horizontal ATR method. As a result (Figure 5), IR spectra of soymilk showed the amide band I which is composed of the absorptions derivedfromamide bonds (-CO-

Figure 5. IR spectra of soymilk after heating and cooling


296 NH-) and show the proportions of secondary structures, namely α-helix, β-sheet, β-turn and non- ordered structure. The amide band I of raw soymilk had a peak at about 1650 cm" which shifted in heated soymilk to lowfrequency(~1630 cm' *), showed the increase of β-sheet-like or extended structures (18) by heat denaturation. These data may show that soymilk protein molecules were partially and slightly unfolded by heat treatment although they generally maintained their steric structures. During cooling of soymilk at room temperature, the amide band I was almost back to the native state, so the protein moelcules were considered to be almost refolded. However, the shifted band was maintained during cooling at -5°C. Heat denatured proteins were refolded during cooling, but cannot refold by cooling at -5°C because rapid cooling decreased the velocity of conformational changes of proteins. From these data, soymilk proteins were partially denatured by heating at 110°C, changed their secondary structures and exposed interior SHs and hydrophobic regions, and resulted in an increase of the reactivity to other molecules. Further, cooling of thermally denatured protein lead to partial refolding but rapid cooling (precooling at -5°C) caused slow refolding to be slower and maintained the reactivity between protein molecules at relatively high levels.


1

Interactions Among Protein Molecules in Soymilk Freeze-gel and Freeze-gelation Mechanism In order to estimate the involvement of S-S bonds, hydrophobic interactions and hydrogen bonds, the effects of some additives such as sodium sulfite, sodium dodecylsulfate (SDS) and sucrose on the formation of soymilk freeze-gel were evaluated. Figure 6 shows the effect of sodium sulfite which reduces the intra- and intermolecular S-S bonds of protein molecules. Raw soymilk was mixed with sodium sulfite and then heated, precooled and frozen (//). After 2 weeks, the frozen samples were thawed in water at 30°C. In comparison with the control sample (Figure 1), the addition of 0.1% sodium sulfite partially suppressed gelation and led to a heterogeneous precipitation (Figure 6a). Increased sodium sulfite (0.5%) completely inhibited coagulation of soymilk (Figure 6b). These data are cosistent with those reported by Hashizume (9), indicating that the formation of intermolecular disulfide bonds was important for the insolubilization of soybean protein during frozen storage. Hydrophobic interactions were also considered to be important among protein molecules. The addition of SDS led to a partially collapsed surface on the freeze-gel at 0.1% (Figure 7a) and to heterogeneous precipitation at 0.5% (Figure 7b).



297

Figure 6. Effect ofsodium sulfite on freeze-gelation of soymilk (Reproduced from reference 17. Copyright 2000 American Chemical Society.) A, 0.1% ofsodium sulfite added; B, 0.5% added.

Figure 7. Effect ofsodium dodecylsulfate (SDS) on freeze-gelation ofsoymilk (Reproducedfromreference 17. Copyright 2000 American Chemical Society.) A, 0.1% of SDS added; B 0.5% added y

Apparently, a greater concentration of SDS than sodium sulfite was needed to inhibit the formation of the freeze-gel. It is considered that soybean protein subunits, which were dissociated during the heating step (2), were associated with other subunit through intermolecular disulfide bonds or hydrophobic interactions. The resulting aggregates then interacted with other aggregates to form insoluble coagulates. Differing from the above data, the addition of a small amount of sucrose allowed the freeze-gel to be stabler and smoother (data not shown). However, further addition of sucrose lowered the stability of the gel.



298 This unique effect of sucrose may show that sucrose affects not only hydrogen bonds among proteins, but also hydrophobic interactions. The above results showed that disulfide, hydrophobic and hydrophilic interactions are important in the formation of gel texture. Proteins in the soymilk were partially denatured and unfolded by heat treatment. In this state, some of the SH groups and hydrophobic regions were considered to be exposed on the surface of the protein molecules. Then, if the heated soymilk was left at room temperature, the denatured proteins were partially refolded and surface reactivity, which included SH and S-S exchanges and hydrophobic and hydrophilic interactions, lowered. However, if the soymilk was put into a freezer controlled at -5°C, the refolding and S-S reformation of the denatured proteins were delayed and the surface reactivity retained at a relatively high level. When the precooled soymilk was frozen, the protein molecules were freeze-concentrated by forming ice crystals and closely interacted with one another through S-S and hydrogen bonds and hydrophobic interactions. During the frozen storage, interactions among proteins were strengthened and allowed the gel to be more stable. Sucrose showed some different effects on thefreeze-gelation,and the effect depended on the amount added to soymilk. Gekko et al. (19) reported that polyols like sorbitol stabilized the protein structure but destabilized the gel structure. The same authors speculated that the thermal stabilization of proteins can be responsible for the strengthening of intramolecular hydrophobic interaction. A small amount of sucrose addition enhanced the hydrophobic interactions among protein molecules resulting in stable gel formation by improving the association or entanglement of the peptide chains. However, a large amount of sucrose was considered to enhance hydrophilic interactions between proteins and water as well as the hydrophobic interactions, and these interaction to form destabilized cross-links through the hydrogen bonds among proteins.

References 1 2 3 4 5 6

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Watanabe, T.; Nakayama, Ο.; Iwasaki, Ν. Nippon Shokuhin Kogyo Gakkaishi 1963, 10, 163-166 (in Japanese). 8 Hashizume, K.; Kakiuchi, K.; Koyama, E.; Watanabe, T. Agric. Biol. Chem. 1971, 35, 449-459. 9 Hashizume, K. Nippon Shokuhin Kogyo Gakkaishi 1979, 26, 450-459 (in Japanese). 10 Soeda, T. Nippon Shokuhin Kogyo Gakkaishi 1994, 41, 670-675 (in Japanese). 11 Shimoyamada, M.; Tômatsu, K.; Watanabe, K. J. Sci. Food Agric. 1999, 79, 253-256. 12 German, B.; Damodaran, S.; Kinsella, J. E. J. Agric. Food Chem. 1982, 30, 807-811. 13 Shimoyamada, M.; Tômatsu, Κ.; Watanabe, Κ. Food Sci. Technol. Res. 1999, 5, 284-288. 14 Grassetti, D. R.; Murray, Jr. J. F. J. Chromatogr. 1969, 41, 121-123. 15 Obata, Α.; Matsuura, M.; Fukushima, D. Nippon Shokuhin Kogyo Gakkaishi 1989, 36, 707-711 (in Japanese). 16 Hayakawa, S.; Nakai, S. J. Food Sci. 1985, 50, 486-491 17 Shimoyamada, M . ; Tômatsu, K.; Oku, S.; Watanabe, K. J. Agric. Food Chem. 2000, 48, 2775-2779. 18 Surewicz, W. K.; Mantsch, Η. H.; Chapman, D.; Biochemistry 1993, 32, 389-394. 19 Gekko, K.; Li, X.; Makino, S. Biosci. Biotechnol. Biochem. 1999, 63, 22082211.


Structural Changes of Soymilk Proteins during Heating and Cooling

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