Efficient production of freeze-thaw fractionated soymilk by thawing at high temperatures

Abstract

Raw soymilk separates into two layers upon freeze-thawing. We investigated the effects of high-temperature thawing (50–100 °C) on the freeze-thaw fractionation of soymilk. After freezing, the samples thawed at high temperatures separated into two layers with similar fractions as observed in samples thawed at low temperatures (10 °C). As the thawing temperature increased, the separation time of the samples thawed at high temperatures decreased by less than half, when compared to those thawed at low temperatures. The ratio of the weight of the upper layer to the total weight decreased as the thawing temperature increased. The 7S and 11S proteins in soymilk could be fractionated through high-temperature thawing, similar to that through low-temperature thawing. Protein denaturation did not occur upon fractionation at higher temperatures. These results suggest that raw soymilk can be fractionated even through high-temperature thawing and may contribute to the efficient production of freeze-thaw fractionated soymilk.

Introduction

Soymilk, a nutritious, plant-based drink that is consumed worldwide(i), is a turbid liquid that is produced by extracting soybeans with water. In East Asian countries, soymilk is an important source of tofu curd (soybean curd). Moreover, soymilk is often utilized as a substitute for cow's milk to make desserts such as pudding, ice cream, and cake. In addition, soymilk has recently been used to produce hot pot soup, yogurt, and cheese-like foods (Nishiyama et al., 2013).

Soymilk is used for various purposes, and its applications can be increased if its properties (i.e., the raw material) can be altered. Raw or unheated soymilk can be separated into two layers through freeze-thawing; both layers have different physicochemical properties, such as the protein and lipid contents and the ratio of 7S (mainly β-conglycinin) to 11S (mostly glycinin) globulins (Morita and Yokoi, 2011). Tofu curd made using the 7S globulin-rich fraction (upper layer) is soft and smooth, whereas tofu curd made using the 11S globulin-rich fraction (lower layer) is hard and coarse. We previously reported a novel dessert-like product made from the upper layer and a novel sausage-like product made from the lower layer (Morita, 2013; Morita, 2018). Sensory evaluations have revealed that these products are tastier than those made from whole soymilk.

Foods with novel textures can be developed by using adequately processed soymilk. However, processing techniques that involve freeze-thawing require extended freezing and thawing durations. Freeze-thaw fractionation of raw soymilk is possible even with rapid freezing (Morita et al., 2015). Nevertheless, only raw or unheated soymilk has been subjected to freeze-thaw fractionation, and the effects of thawing temperature on the properties of soymilk have not been elucidated. Thawing at an elevated temperature could potentially result in a reduction in the thawing time, thereby facilitating the efficient production of freeze-thaw fractionated soymilk.

To investigate this hypothesis, this study aimed to elucidate the effects of thawing temperature on freeze-thaw fractionated soymilk and examine the properties of the resulting soymilk.

Materials and Methods

Soymilk preparation  Soymilk was manually prepared from commercially available soybeans (Glycine max (L.) Merr.). Briefly, soybeans were washed and soaked in distilled water at 20 °C for 16 h. The swollen soybeans were then drained and ground into a homogenate with a volume of distilled water equivalent to six times the dry weight of the soybeans. Raw soymilk was obtained by filtering the homogenate through an absorbent cotton sheet to remove the okara (soybean residue). Heated soymilk was prepared by heating raw soymilk in boiling water at a controlled temperature of 90 °C. After reaching the desired temperature, the temperature of the sample was held constant for 2 min and then cooled rapidly in ice-cold water until a temperature of 20 °C was attained.

Freeze-thawing of soymilk  Raw soymilk was aliquoted in 40 mL portions into individual 50 mL plastic tubes (φ30 mm; liquid height: 85 mm) at room temperature. Next, the soymilk samples were frozen at −30 °C for 7 d and then thawed at 10, 50, 60, 70, 80, 90, or 100 °C. The starting thawing temperature for each treatment was −30 °C. Thawing was performed in a thermostatic chamber (10 °C; M-130, TIETECH Co., Ltd., Nagoya, Japan; 50–100 °C; SG400, Yamato Scientific Co., Ltd., Tokyo, Japan). A temperature recorder (TR-52i, T&D Corp., Ltd., Matsumoto, Japan) was placed inside the samples to monitor the temperature during thawing.

Characterization of separated soymilk layers  After freeze-thawing, the separation of the soymilk into two layers was observed visually; the separation state of the soymilk was observed every 5 minutes, and the separation time was defined as the time when the upper layer became uniform with no suspended solids, clearly separated into two layers, and the ratio of the upper layer became almost constant. The supernatant (upper layer) was carefully collected through decantation, the precipitate (lower layer) was excluded, and the wet weight of the upper layer was determined. The ratios of the weights of the upper and lower layers to the total weight of the sample were determined. The solid contents of the soymilk samples were measured using a digital refractometer (PAL-1, Atago Co., Tokyo, Japan).

In addition, freeze-thawed soymilk samples were agitated, centrifuged at 3 000 × g for 10 min, and the supernatant was decanted. The resulting precipitate was weighed to estimate the precipitation rate.

Soymilk protein analysis  Soymilk protein fractions were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Laemmli, 1970) and the protein bands were stained with Coomassie Brilliant Blue R-250. The intensities of the stained protein bands in the gel were determined using CS Analyzer image analysis software (ATTO Co., Ltd., Tokyo, Japan).

Fourier-transform infrared spectroscopy (FTIR)  The absorption spectra of the soymilk samples at 4 cm⁻¹ resolution were determined with 16 scans of each sample in the wave number range of 4000–800 cm⁻¹ using an FTIR spectrometer equipped with a ZnSe crystal cell ATR (FT/IR-4700, JASCO Co., Tokyo, Japan).

Results and Discussion

Effects of thawing temperature on the freeze-thaw fractionation of soymilk  As shown in Fig. 1, the thawing period was inversely proportional to the thawing temperature; the higher the thawing temperature, the shorter the period until the soymilk temperature increased above 0 °C. After freezing, the soymilk samples thawed at high temperatures (50–100 °C) separated into two layers, with fractions similar to those thawed at a low temperature (10 °C; Fig. 2). The passage time through the maximum ice crystal formation zone (−5–0 °C) and the soymilk separation time decreased proportionally as the thawing temperature increased (Table 1). The soymilk samples thawed at 50 °C separated in less than half the time than those thawed at 10 °C, and those thawed at 100 °C separated in less than one-fifth of the time than those thawed at 10 °C. These results showed that higher thawing temperatures allowed for faster separation of soymilk, thereby resulting in a more efficient freeze-thaw fractionation of soymilk.

Fig. 1.

Effect of thawing temperature on the thawing period of frozen raw soymilk.

Fig. 2.

Effects of thawing temperature on the freeze-thaw fractionation of soymilk. (A) Thawing at 90 °C and (B) thawing at 10 °C. White arrows indicate the separation between the upper and lower layers in each sample.

Table 1.Separation time and temperature of raw soymilk thawed at different temperatures.

Thawing temperature (°C)	Passing time from −5 to 0 °C (min)	Time from start of thawing to exceeding 0° (min)	Time from start of thawing to separation (min)	Time from exceeding 0°C to separation (min)	Separation temperature (°C)
10	150	170	230	60	10.9
50	49	59	90	31	24.6
60	41	50	75	25	29.3
70	35	43	65	22	32.4
80	29	37	55	18	33.3
90	26	33	45	12	30.2
100	21	28	40	12	30.4

Conversely, the temperature at which separation was observed was approximately 30 °C, irrespective of the thawing temperature when the soymilk was thawed above 60 °C (Table 1). Therefore, an increase in the thawing temperature resulted in a decrease in the time required to attain a temperature of 30 °C and a reduction in the time from when the temperature of the soymilk exceeded 0 °C to the separation. Furthermore, a strong correlation was observed between the thawing temperature and the time from thawing (exceeding 0 °C) to separation (Fig. 3). Thus, a temperature of 30 °C has notable implications for separation when thawing above 60 °C.

Fig. 3.

Relationship between thawing temperature and time from exceeding 0 °C to separation.

The ratio of the weight of upper layer to the total weight decreased as the thawing temperature increased (Table 2). In contrast, the solid contents of soymilk were greater in the lower layer than in the upper layer. However, the solid contents of both the upper and lower layers remained consistent across all thawing temperatures. The solids content of soymilk is typically 9.2 %, and its composition is primarily 3.6 % protein, 2.0 % lipid, and 3.1 % carbohydrate (MEXT, 2015). In raw soymilk, some proteins interact with lipid emulsions to form lipid/protein complexes (Ono et al., 1991; Guo et al., 1997). The formation of a lower layer during the freeze-thawing of raw soymilk has been attributed to the aggregation of these complexes through disulfide and/or hydrophobic interactions among proteins that have been concentrated during freezing (Morita and Shimoyamada, 2013). Thus, the separation of raw soymilk into two layers by freeze-thawing is due to the spontaneous sedimentation of these aggregates over time. In the case of thawing at 100 °C, the entire soymilk undergoes rapid thawing, resulting in a short sedimentation period of aggregates. Conversely, in the case of thawing at 10 °C, the soymilk is slowly thawed while maintaining a low temperature, resulting in a long sedimentation period of aggregates. We therefore postulated that the decreased ratio of the upper layer was due to the decrease in the time required until separation due to higher thawing temperatures.

Table 2.Effects of thawing temperature on the weight ratios and solid contents of the upper and lower layers of soymilk after freeze-thaw fractionation.

Thawing temperature (°C)	Weight ratio (%)		Solid content (Brix)
	Upper	Lower	Upper	Lower
10	28.7 ± 1.0	71.3	8.5 ± 0.0	16.4 ± 0.2
50	26.8 ± 2.4	73.2	8.8 ± 0.2	16.4 ± 0.3
60	24.2 ± 1.3	75.8	8.6 ± 0.1	16.5 ± 0.1
70	22.8 ± 2.0	77.2	8.9 ± 0.3	16.5 ± 0.1
80	20.9 ± 2.0	79.1	8.8 ± 0.3	16.3 ± 0.2
90	18.5 ± 2.2	81.5	8.8 ± 0.3	16.3 ± 0.1
100	16.0 ± 2.4	84.0	8.9 ± 0.4	16.3 ± 0.2

Upper, upper layer; Lower, lower layer.

Values are means ± standard deviation (n = 3).

Effects of thawing temperature on the protein composition of freeze-thaw fractionated soymilk  In the SDS-PAGE analysis of the proteins from the upper soymilk layer, the protein bands corresponding to 7S globulins (α′, α, and β) were more intense than those corresponding to 11S globulins (acidic and basic) at all tested thawing temperatures (Fig. 4). SDS-PAGE analysis of the proteins from the lower soymilk layer showed that the acidic and basic polypeptide bands derived from 11S globulins were more intense than those of the subunits derived from 7S globulins at all tested thawing temperatures (Fig. 5). Image analysis showed that the 11S/7S ratio of raw soymilk (without freeze-thawing) was 1.1, whereas that of the upper layer was 0.4–0.6 at all tested thawing temperatures and that of the lower layer was 1.4–1.6 (Table 3). Consistent with the results of our previous study (Morita and Yokoi, 2011; Morita et al., 2015), these results suggest that the proteins in soymilk can be fractionated through high-temperature thawing, similar to those in fractionation through low-temperature thawing.

Fig. 4.

SDS-PAGE pattern of the upper layers of soymilk thawed at different temperatures after freezing.

1, Molecular mass marker; 2, Raw soymilk; 3, 10 °C; 4, 50 °C; 5, 60 °C; 6, 70 °C; 7, 80 °C; 8, 90 °C; and 9, 100 °C. α′, α′-subunit of β-conglycinin; α, α-subunit of β-conglycinin; β, β-subunit of β-conglycinin; acidic, acidic polypeptides of glycinin; basic, basic polypeptides of glycinin.

Fig. 5.

SDS-PAGE pattern of the lower layers of soymilk thawed at different temperatures after freezing.

1, Molecular mass marker; 2, Raw soymilk; 3, 10 °C; 4, 50 °C; 5, 60 °C; 6, 70 °C; 7, 80 °C; 8, 90 °C; and 9, 100 °C. α′, α′-subunit of β-conglycinin; α, α-subunit of β-conglycinin; β, β-subunit of β-conglycinin; acidic, acidic polypeptides of glycinin; basic, basic polypeptides of glycinin.

Table 3.Effects of thawing temperature on the 11S/7S ratios of the upper and lower layers of soymilk after freeze-thaw fractionation.

Thawing temperature (°C)	11S/7S ratio
	Upper	Lower
10	0.5	1.6
50	0.4	1.6
60	0.5	1.5
70	0.5	1.5
80	0.5	1.5
90	0.6	1.4
100	0.6	1.4

Upper, upper layer; Lower, lower layer.

Effects of thawing temperature on the denaturation soymilk proteins  Although the amount of precipitate (centrifuged at 3 000 × g for 10 min) remained unchanged at all tested thawing temperatures, it increased in the heated soymilk (Fig. 6). Shimoyamada et al. (2008, 2014) reported that the precipitation of soymilk increased after heat treatment at 70–80 °C. The increase in precipitation is associated with the denaturation and insolubilization of proteins, particularly that of β-conglycinin. Since the freeze-thaw fractionated soymilk obtained in this study can be redispersed and does not form precipitates again when agitated (Morita and Yokoi, 2011), centrifugation after high-temperature thawing can be used as an indicator to evaluate the degree of denaturation. However, since the amount of precipitation in this study did not increase after thawing at high temperatures, we presumed that the denaturation and insolubilization of proteins did not occur.

Fig. 6.

Precipitate in soymilk samples thawed at different temperatures after freezing.

Raw, raw soymilk; Heated, heated soymilk. Data presented as means ± standard error of triplicate experiments.

The results of FTIR analysis are shown in Fig. 7. No significant changes were observed in the absorption spectra of the proteins following thawing at 100 °C. In contrast, the absorption values of heated soymilk exhibited distinct differences in the spectral range of amide I (around 1675–1600 cm⁻¹) and amide II (around 1575–1525 cm⁻¹). Heat denaturation of soybean protein is known to result in notable changes in the amide I and amide II regions (Samadi and Yu, 2011; Mizutani et al., 2019). The shapes of the amide I band in soymilk thawed at 100 °C were almost identical to those in raw soymilk, which was distinctly different from those in heated soymilk. These results indicated that the influence of heat denaturation is minimal even at a thawing temperature of 100 °C. In contrast, a slight difference in amide II was observed between soymilk thawed at 100 °C and raw soymilk; however, the difference was considered negligible and did not seem to impact the process of freeze-thaw fractionation.

Fig. 7.

FTIR spectra of soymilk samples thawed at 100 °C.

Raw, raw soymilk (thin line); Heated, heated soymilk (thick line); 100 °C upper, the upper layer of soymilk thawed at 100 °C (dotted line); 100 °C lower, the lower layer of soymilk thawed at 100 °C (dashed line).

The heated dispersed soy protein can be coagulated through a freeze-thaw process (Hashizume et al., 1971; Shimoyamada et al., 1999). We previously reported that heating soymilk at 80 °C before freezing completely inhibits the freeze-thaw fractionation of soymilk (Morita et al., 2017). Therefore, we speculated that freeze-thaw fractionation does not occur in heat-denatured soymilk. Soymilk contains 7S (mainly ß-conglycinin) and 11S (primarily glycinin) globulins; 7S globulins undergo a conformational change between 65 °C and 75 °C and undergo complete denaturation at 75 °C. In contrast, 11S globulins begin to denature at 80 °C and are completely denatured at 91 °C (Hermansson, 1986; Iwabuchi et al., 1991; Sorgentini et al., 1995; Nagano et al., 1995). In this study, separation occurred at approximately 30 °C (Table 1); thus, we concluded that denaturation did not occur as the denaturation temperature was not attained. Consequently, soymilk fractionation was observed at all thawing temperatures.

Since soymilk thaws from the surface towards the center, the temperature of the surface was likely higher than that of the center, thereby leading to partial denaturation of the proteins. Temperature recorders were placed at the center and inner side of the plastic tubes, and the temperature at the surface was measured and compared to that at the center (Fig. 8). During thawing at 100 °C, although the temperature increased faster at the surface than at the center, the temperature at the surface of the tube was 42.1 °C when the separation occurred (30.4 °C, Table 1), which was also below the denaturation temperature. However, a limitation of our study is that we used a small volume of soymilk (40 mL). It is possible that partial denaturation could occur if the volume or freezing method is modified. Therefore, additional research using larger soymilk volumes and other freezing methods is required.

Fig. 8.

Difference in the temperatures between the center and surface of frozen raw soymilk thawed at 100 °C.

Furthermore, in the high-temperature thawing, the boundary of the separation became almost indistinguishable when the soymilk was maintained at a constant temperature after separation (data not shown). It is conceivable that the once separated lower layer dissociated the lipid/protein complexes as a result of heat denaturation. Therefore, after separation, it is necessary to remove immediately the precipitate that is formed or transfer it to a low temperature.

In addition, we used unheated soymilk for freeze-thaw fractionation; therefore, when soymilk is thawed at room temperature (approximately 20–40 °C), microbial spoilage should be considered. Thus, from a hygienic standpoint, refrigeration or thawing at a high temperature is preferable.

In summary, within the conditions of this study, soymilk can be fractionated even through high-temperature thawing. These results could contribute to the efficient production of freeze-thaw fractionated soymilk. Furthermore, the data obtained in this study may be useful in the utilization of this freeze-thaw fractionation method across diverse production sites.

Acknowledgements  The authors thank Rika Kobayashi from the University of Shizuoka for her valuable advice on the use of FTIR.

Conflict of interest  There are no conflicts of interest to declare.

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