Effects of Heating Temperature and Cooling Rate on Denaturation of

Chapter 4

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Effects of Heating Temperature and Cooling Rate on Denaturation of Soymilk Protein Makoto Shimoyamada,*,1 Kimiko Tsuzuki,2 Hiroaki Asao,2 and Ryo Yamauchi3 1School

of Food, Agricultural and Environmental Sciences, Miyagi University, 2-2-1 Hatatate, Taihaku, Sendai, Miyagi 982-0215, Japan 2Marusan-ai Co., Ltd., 1 Arashita, Niki-cho, Okazaki, Aichi 444-2193, Japan 3Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan *[email protected]

In order to evaluate the effects of heating and cooling on soymilk protein, raw soymilk, which was squeezed before heating, was heated to 80, 100 or 115°C and then cooled at room temperature or -5°C. The surface SH content of the soymilk proteins decreased with increasing heating temperatures. Furthermore, the SH content of the soymilk that was cooled rapidly at -5°C was higher than that of the soymilk that was cooled slowly at room temperature. The surface hydrophobicity of the soymilk proteins was increased by heating, and the hydrophobicity was slightly higher as a result of cooling at -5°C compared with cooling at room temperature. The raw soymilk was first heated at 115°C (first heating) and then 80°C (second heating). After cooling in an ice bath, decreases in both the SH content and the hydrophobicity of the resulting soymilk were observed as a result of the second heating. This denaturation behavior is different from that of a protein solution prepared from defatted soybeans.

Soymilk is composed of protein suspensions, emulsions consisting of lipids and proteins, and others substances. Soymilk has beneficial qualities that improve human health, for example, lowering of serum lipids and cholesterol (1, 2), © 2010 American Chemical Society In Chemistry, Texture, and Flavor of Soy; Cadwallader, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.


estrogen-like activity (3) and antioxidative activity (4). In Japan, about 200,000 tons of soymilk is consumed each year. Soymilk is prepared from imbibed soybeans by milling, removing insoluble residue and heating. In the case of commercial soymilk, two or more heating steps are included to denature soymilk proteins for easy digestion, to inactivate lipoxygenase and other bioactive components, and to sterilize the soymilk for aseptic filling. It is thought that heating affects the structures and functional properties of proteins. It has been reported that proteins in soymilk form aggregates upon heating and interact differently with lipids (5–7), which affects the physical properties of tofu (8). Regarding the heating and successive cooling and/or freezing of soymilk, Shimoyamada et al. (9) have reported that frozen storage allowed the soymilk to form a gel-like coagulate (freeze-gel). In this process, pre-cooling at -5 °C, which was carried out successively after heat treatment, was essential for the freezegelation of soymilk. Pre-cooling has been divided into two classifications, namely, supercooling and rapid cooling. Small globular ice crystals, which are formed by freezing supercooled soymilk, are effective at maintaining the gel-like structure of frozen and thawed soymilk (10). Through heating, the surface SH content and the surface hydrophobicity of proteins in the soymilk are altered. Rapid cooling increases the SH content and hydrophobicity of the heated soymilk. These results are attributed to the maintained reactivity of the heat-denatured protein molecules (11). These results imply that protein denaturation behavior is affected by cooling as well as heating. In this study, the denaturation behavior of soymilk proteins was monitored after heating and cooling at various temperatures to estimate the effects of cooling rate on the thermal denaturation of soymilk proteins. Two-step heating at first a higher temperature and then a lower temperature can be considered a kind of slow cooling; therefore, the denaturation behavior of the soymilk sample was also monitored after two-step heating at two separate temperatures. In order to estimate the effects of heating and cooling, three heating and two cooling temperatures were selected. At a heating temperature of 80°C, only β-conglycinin is denatured, and glycinin remains native. At 100°C, both β-conglycinin and glycinin are denatured as determined by DSC measurements (12, 13). A temperature of 115°C was selected to consider the sterilization temperature of soymilk. Since a pressure cooker was used, the temperature was limited to 115 °C. Cooling at room temperature is referred to as slow cooling, and cooling at -5°C in a refrigerator is referred to as rapid cooling. In the latter case, the soymilk never froze due to supercooling. The denaturation behavior and precipitation of soymilk proteins were measured after cooling the heated soymilk samples, and the effects of heating temperature and cooling rate were evaluated.

62 In Chemistry, Texture, and Flavor of Soy; Cadwallader, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.

Materials and Methods Preparation of Soymilk and Heat Treatment


Soybeans were soaked in water overnight at 4°C, and milled with water in a blender (6,000 rpm × 5 min, Type R, Teraoka Co., Osaka, Japan). The total volume of imbibed and milling water that was added to 30 g of the dry soybean was 300 mL. The milled soybeans were filtered with 5-ply gauze to afford raw soymilk at less than 60°C. The raw soymilk samples were heated at 80 or 100°C in a water bath, or at 115°C in a pressure cooker. The heated soymilk samples were cooled at -5°C in a refrigerator (rapid cooling) or at room temperature (slow cooling) for 2 h and then put in an ice-water bath (0°C) until the analyses were conducted. Estimation of Precipitate from Heated Soymilk After successive heating and cooling, the soymilk was refrigerated at 4°C for 10 days in a screw-capped test tube and then centrifuged at 1,500 × g for 30 min, and the supernatant was removed by placing the test tubes upside down for half a minute. The weight of the precipitate was used to calculate precipitation rate. Protein Surface SH Content and Surface Hydrophobicity The protein surface SH content was estimated by using 2,2′-dithiobis-(5nitropyridine) (DTNP) (14, 15). The samples were diluted to a volume of 2 mL with 0.1 mol/L phosphate buffer (pH 7.6) and then mixed with 0.5 mL of 5.0 × 10-4 mol/L DTNP ethanol solution. The samples were incubated at 25°C for 20 min, and 2.5 mL of 10% perchloric acid solution was added. The samples were centrifuged at 1,500 × g for 10 min to remove the protein. The resulting supernatant was passed through a 0.45 µm membrane filter, and the filtrate was analyzed by a spectrophotometer (NovaspecII, Pharmacia LKB Biotechnology, Uppsala, Sweden) at 386 nm. The surface hydrophobicity of the soymilk proteins was measured using 8anilino-1-naphtalene sulfonic acid (ANS) (16). The samples were diluted to a volume of 100 µl with 0.01 mol/L phosphate buffer (pH 7.0) and mixed with 20 µl of 8 × 10-3 M ANS solution and 4 mL of the pH 7.0 phosphate buffer. The resulting mixture was analyzed with a fluorescence spectrophotometer (F-2000, Hitachi High-Technologies Co., Tokyo, Japan; excitation: 390 nm; emission: 470 nm).

Results and Discussion Effect of Cooling Rate on Surface SH Content of Soymilk Protein Denatured by Heating Changes in protein surface SH content indicate the making or breaking of disulfide bonds on the surface of protein molecules as well as exposure or concealment of free SH groups. The protein surface SH content of soymilk was determined after heating and cooling (Figure 1). The soymilk sample 63 In Chemistry, Texture, and Flavor of Soy; Cadwallader, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.


measured immediately after heating to 80°C, where the sample was cooled to room temperature in an ice bath, showed that the SH content was almost constant. Soymilk measured after cooling at room temperature for 2 h had a decreased surface SH content. However, rapid cooling of heated soymilk at -5°C decreased the SH content to less than slow cooling at room temperature (Figure 1A). Heating at 80°C was found to denature only β-conglycinin and to maintain the inter-polypeptide disulfide bonds of the glycinin subunits as determined by SDS-PAGE (data not shown). The decreased SH content during cooling possibly depended on structural changes in the denatured β-conglycinin. In the case of heating at 100°C, the surface SH content measured immediately after heating decreased, in contrast to the surface SH content observed after heating at 80°C (Figure 1B). This difference between 80°C and 100°C is thought to arise from denaturation of glycinin. The SH content in the soymilk sample cooled at -5°C for 2 h was between that of the other two, namely, the sample immediately after heating and the sample after 2 h of cooling at room temperature. Furthermore, in the case of heating at 115°C, the SH content initially increased, and then decreased after heating was complete. After 2 h of cooling time, the SH content decreased further as in the case of heating at 100°C (Figure 1C). Cooling at -5 °C suppressed this decrease. By heating at temperatures over 90°C, both β-conglycinin and glycinin are expected to undergo denaturation, dissociation to their constituent subunits, and then aggregation (17). Successive re-association of dissociated subunits occurs at least partially through SH - SS exchange reactions. This behavior results in decreasing SH content. However, it is thought that rapid cooling inhibits disulfide bond formation among heat denatured proteins, resulting in decreased SH content.

Effect of Cooling Rate on Surface Hydrophobicity of Soymilk Proteins Denatured by Heating An increase in protein surface hydrophobicity is thought to indicate thermal unfolding of proteins by estimating the exposure of inner hydrophobic region. In the case of heating at 80°C, surface hydrophobicity increased slightly (Figure 2A). The surface hydrophobicity of the soymilk after 2 h of cooling increased more than that measured immediately after heating; however, there was little difference between cooling temperatures at room temperature and -5°C. The surface hydrophobicity of soymilk heated at 100°C increased significantly. In contrast to heating at 80°C, the hydrophobicity decreased after 2 h of cooling. Cooling at -5°C appeared to maintain higher hydrophobicity than cooling at room temperature (Figure 2B). In case of heating at 115°C, similar results were obtained (Figure 2C). The surface hydrophobicity of rapidly cooled soymilk was between that of the other two, also indicating that cooling at -5°C suppressed the decrease in hydrophobicity after 2 h of cooling.



Figure 1. Effect of cooling rate on surface SH content of heated soymilk. A, Soymilk heated at 80°C; B, heated at 100°C; C, heated at 115°C. ♦, Measured Immediately after heating; ▴, after 2h of cooling at -5C; ▪, after 2h of cooling at room temperature. The protein surface hydrophobicity, which increased upon heating at 100 or 115°C, decreased during cooling for 2 h. This decrease possibly relates to aggregate formation or partial refolding of denatured protein molecules during cooling. Rapid cooling was found to suppress aggregate formation and partial refolding. In our previous work (9, 11), it was reported that rapid cooling of heated soymilk is essential for gel formation in frozen storage, namely, freeze-gelation. Since coagulate formation is thought to be related to precipitate formation, the effect of cooling rate on the precipitation from heated soymilk was investigated (Figure 3). In the case of heating at 80°C, precipitation was higher than that of the soymilk heated at any other temperatures. Furthermore, it was higher than or comparable with that of the raw soymilk. Slow cooling slightly increased precipitation from refrigerated soymilk. Precipitation decreased with increasing heating temperature. In the case of heating at 100 or 115°C, precipitation from soymilk cooled at -5°C was nearly equivalent to that cooled at room temperature.



Figure 2. Effect of cooling rate on surface hydrophobicity of heated soymilk. A, Soymilk heated at 80°C; B, heated at 100°C; C, heated at 115°C. ♦, Measured Immediately after heating; ▴, after 2h of cooling at -5°C; ▪, after 2h of cooling at room temperature.

Figure 3. Precipitation of heated and refrigerated soymilk A, Soymilk heated at 80°C; B, heated at 100°C; C, heated at 115°C. Open bar, Soymilk cooled at -5 °C; Hatched bar, cooled at room temperature.



Figure 4. Precipitation of soymilk heated at two temperatures. Open bar, soymilk heated first at lower temperature and successively heated at 115°C; Hatched bar, heated first at 115°C and at lower temperature. Non, non-heated soymilk. (Reproduced from reference (18). Copyright 2008 The Japanese Society for Food Science and Technology.)

Denaturation Behavior of Soymilk Proteins Heated at Two Successive Heating Temperatures After cooling, the structure of thermally denatured soymilk proteins was altered depending on cooling rates. Regarding the slow cooling as another heating of proteins by residual heat, it is possible for further denaturation to occur, for example, unfolding and/or refolding and aggregating. Thus, we can estimate the effects of two-step heating on the thermal denaturation behavior of the soymilk proteins. The two-step heating was performed as a combination of heating at 115°C in a pressure cooker as a higher temperature and then at 70, 80, 90 or 100°C in a water bath as a lower temperature. Heating first at the higher temperature and then at the lower temperature was carried out in sequence, and the results were compared with those from a reverse sequence, namely two-step heating first at a lower temperature and then at a higher temperature. Precipitate formation was estimated after the two-step heating (Figure 4). The precipitation from refrigerated soymilk increased due to the heating sequence first at 115°C and then at 70 or 80°C. On the other hand, the opposite heating sequence, namely, heating first at 70 or 80°C and then at 115°C, showed considerably lower precipitation. These results showed that combinations and sequences of heating temperatures affect the properties of heated soymilk, such as precipitation. Therefore, heating sequences as well as individual heating temperatures are important factors for controlling the quality of soymilk.



Figure 5. Surface SH content of soymilk heated at two temperatures. Open bar, soymilk heated first at lower temperature and successively heated at 115°C; Hatched bar, heated first at 115°C and at lower temperature; Vertically-striped bar, heated at one temperature. Non, non-heated soymilk. Duplicate measurement was repeated twice. (Reproduced from reference (18). Copyright 2008 The Japanese Society for Food Science and Technology.) The surface SH content of soymilk proteins was measured during two-step heating (Figure 5). The SH content of proteins in the soymilk heated at two different temperatures was lower than that of the soymilk heated at a single temperature. This decrease in the surface SH content is possibly the result of the extended total heating time. Comparing sequences of heating temperatures, there were no significant differences between heating first at a lower temperature and then at a higher temperature, and heating first at a higher temperature and then at a lower temperature. Next, the surface hydrophobicity of the soymilk proteins was measured (Figure 6). When 115°C was the higher temperature in the heating sequence, and 90 or 100°C were the lower temperatures, no significant differences were observed, even if the sequence of the heating temperatures was switched. However, the surface hydrophobicity of soymilk heated first at 90°C and second at 115°C was slightly higher than that heated in the reverse order. All temperatures over 90°C were regarded as being essentially equivalent in terms of the heat-induced unfolding of the soymilk proteins, and no significant difference in surface hydrophobicity was observed. On the other hand, with heating at 70 or 80°C as a lower temperature after initial heating at 115°C, the surface hydrophobicity initially decreased during the second heating step and increased again upon further heating. This decrease during the second heating at 70 or 80°C is attributed to aggregate formation of soymilk protein molecules or partial refolding of the protein molecules that were unfolded during the first heating step. Furthermore, these changes were also thought to relate to the increase in precipitate formation



Figure 6. Surface hydrophobicity of soymilk heated at two temperatures. A, soymilk was heated at 70°C (lower temperature) and 115°C (higher temperature); B, heated at 80°C and 115°C; C, heated at 90°C and 115°C; D, heated at 100°C and 115°C. •, soymilk was heated first at lower temperature (70, 80, 90 and 100°C) and successively at 115°C; ▪, heated first at 115°C and successively at lower temperatures. First heating time was 10 min. Duplicate measurement was repeated twice. (Reproduced from reference (18). Copyright 2008 The Japanese Society for Food Science and Technology.) In conclusion, rapid cooling of heated soymilk suppressed both decreases in the surface SH content and the surface hydrophobicity of soymilk proteins during cooling. Successive heating at two different temperatures affected the denaturation behavior of soymilk proteins, resulting in increasing precipitation of soymilk proteins upon heating at 70 or 80°C as a second heating step after initially heating at 115°C. Since the surface hydrophobicity of the soymilk proteins was initially decreased by the second heating step at 70 or 80°C, this increase in precipitation is possibly related to aggregate formation or partial refolding of the soymilk proteins.

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Effects of Heating Temperature and Cooling Rate on Denaturation of

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