Physicochemical and functional property changes in soy protein isolates stored under high relative humidity and temperature (2025)

Keywords: Soy protein isolate, Storage, Water activity, High temperature, Functional property

Materials

Soy protein isolate (SPI) ADM974 was provided by Archer Daniels Midland Company (Decatur, IL, USA). The protein isolate had 4.8%±0.32 moisture content, 92.00%±0.33 crude protein, 0.01%±0.01 crude fat, and 3.35%±0.01 ash. In order to obtain samples with the desired aw, 300g of the original SPI with an aw of 0.25 (SPI-0.25) was sprayed evenly with 30.6g water (12.6% final moisture content) and mixed well. The moistened SPI was then sealed in the pouch and stored at 5°C for several days to assure that moisture equilibrium occurred throughout the sample, thus obtaining an SPI having an aw of 0.75 (SPI-0.75). The water activity of SPI-0.75 was then determined with a water activity and moisture meter (Hygrolab 2, Rotronic AG, Bassersdorf, Switzerland). Measurements were done in triplicate at 25°C.

Storage experiment

Samples (300g) of SPI-0.25 and SPI-0.75 were separately packaged in sealed laminated pouches (30×20cm, Ny+LLDPE). The pouches were incubated separately at 25 and 45°C in different incubators for 224 d and removed every 28 d. SPI samples were examined with respect to color, gel hardness, emulsifying stability index, foaming properties, viscosity, nitrogen solubility index, and surface hydrophobicity.

Color analysis

The color of the SPI samples was measured by the modified method of Shih et al. (2009). The Hunter color of the SPI samples was determined with a colorimeter (Nippon Denshoku Kogyo Co., Tokyo, Japan). The reported values are the averaged values of four duplicate tests for each sample. Results were expressed as tri-stimulus values (L: lightness [0=black, 100=white], a: -a=greenness, +a=redness, and b: -b=blueness, +b=yellowness) of the Hunter color scale. The instrument was set at the reflectance mode. A numerical total color difference (ΔE) was calculated as

$$\mathrm{\Delta E}={\left[{\left(\mathrm{L}-\mathrm{Lo}\right)}^2+{\left(\mathrm{a}-\mathrm{ao}\right)}^2+{\left(\mathrm{b}–\mathrm{bo}\right)}^2\right]}^{1/2}$$

where Lo, ao, and bo are the Lab values of reference samples, which herein are the control sample’s Lab values.

Gel hardness

Gels were prepared according to Martins and Netto (2006) with some modifications. Suspensions (pH7.0) containing 100ml of 20% protein in a 100ml beaker were stirred for 10min. The beakers were sealed, heated at 90°C for 15min in a water bath, and then cooled to 25°C. Samples were then stored for 24h in a refrigerator at 6°C before being analyzed for texture. Gel texture profiles were determined using a TA.XT-2 Texture Analyser (Stable Microsystems, Surrey, UK). The analyses were carried out at 25°C. Gels were compressed twice to 60%. The operating apparatus was a cylindrical probe having a 10mm diameter (p/10), test speed was 1.0mm/s, and the applied force was 25kg. Hardness, defined according to Bourne (1982), was measured.

Emulsifying stability index

The emulsifying properties of SPI samples were determined by the turbidometric method of Guo et al. (2007). To prepare the emulsions, 20ml of pure soybean oil and 20ml of each sample solution (0.5% protein) in 0.1M phosphate buffer (pH7.0) were mixed together in a 100ml beaker at 25°C and homogenized with a Polytron homogenizer (Model PT 1200E, equipped with a high-foam PT-DA 1205/2EC-E generator) (Kinematica AG, Luzern, Switzerland) at maximum speed (~20,000rpm). The emulsion was immediately transferred into a 15ml plastic beaker (dia. 3.0cm). Aliquots of freshly prepared emulsion (50ml) were taken 0.5cm from the bottom of the beaker and dispersed into 5ml of 0.1% sodium dodecyl sulfate (SDS) solution. Absorbance was measured at 500nm against a 0.1% SDS solution blank in a spectrophotometer. Emulsions were kept undisturbed for 10min at 25°C and then 50ml aliquots were taken 0.5cm from the bottom of the beaker and dispersed into 5ml of 0.1% SDS solution. Absorbance was measured at 500nm as previously described. Emulsion stability was expressed as an index, ESI (%), of the homogenized samples, defined as

$$\mathrm{ESI}=\left(\mathrm{A}10/\mathrm{A}0\right)\times 100\%$$

where A10 and A0 represent the absorbance at 500nm after 10min and time zero, respectively.

Foaming properties

The foaming properties [foaming capacity (FC), foam maximum density (FMD) and foam drainage stability (FS)] of the samples were evaluated by the high speed agitation method described by Agyare et al. (2009). First, 0.4g SPI sample was dissolved in 20ml of distilled water. The sample was then introduced into a 100ml plastic measuring cylinder and blended with a Polytron homogenizer (Model PT 1200E equipped with a high-foam PT-DA 1205/2EC-E generator) (Kinematica AG, Luzern, Switzerland) at maximum speed (~20,000rpm). The total volume of foam generated was measured immediately and FC was computed according to Motoi et al. (2004). FC was used to calculate FMD using the formula of Ru’ız-Henestrosa et al. (2007a). Foam was allowed to stand undisturbed for 10min and the volume of liquid that was then drained from the foam was used as the indicator of foam drainage stability (FS) (Mimouni et al. 1999).

Viscosity

The viscosity of SPI was measured in 20% (w/w) dispersions in distilled water. Viscosity was measured at 25°C in a Brookfield Viscometer RVT using a Helipath stand, 93-spindle series, at 10rpm. Values are expressed as centipoises (cp).

Nitrogen solubility index

The NSI of SPI samples was measured by method modified from Pinto et al. (2005). Five grams of each sample were dispersed in 200ml of water by stirring in a water bath at 30°C for 120min, then centrifuged at 400g for 10min. Total and soluble nitrogen (supernatant) were determined with the micro-Kjeldahl method (AOAC 2000) in triplicate using a conversion factor of 6.25. The correlation between total and soluble nitrogen fractions allows the calculation of solubility with the formula

$$\mathrm{NSI}\left(\%\right)=\left(\mathrm{NS}/\mathrm{NT}\right)\times 100\%$$

where NS is soluble nitrogen and NT is total nitrogen.

Surface hydrophobicity (RSo)

The hydrophobicity of SPI was determined with a hydrophobicity fluorescence probe ANS (8-anilino-1-naphthalene sulfonic acid) according to Zhang et al. (2005). Samples of SPI were diluted to 1mg/ml in 0.01M phosphate buffer at pH7.0 and serially diluted with the same buffer to obtain protein concentrations of 0.00625–0.1mg/ml. Then, 10μl of ANS (8.0mM in 0.1M phosphate buffer, pH7) was added to 2ml of the sample. Fluorescence intensity (FI) was measured with a Varioskan Flash spectral scanning multimode reader (Thermo Fisher Scientific, Waltham, MA) at wavelengths of 390nm (excitation) and 515nm (emission). The original SPI sample was used as the control. The degree of hydrophobicity was expressed as a ratio of the fluorescence intensity of the treated sample to that of the control. The initial slope of FI versus protein concentration was used as an index for protein hydrophobicity.

Statistical analysis

Data were analyzed by an analysis of variance (ANOVA) using a general linear model. Duncan’s multiple range test was used to determine the differences among samples. Significant levels were defined as probabilities of ≤0.05. All processing treatments were done in triplicate.

Color analysis

The color analysis results are presented in Tables 1. The L value of the original SPI was 89.05. The range of L values that changed during storage were 89.04–89.37 for SPI-0.25 and 89.04–89.56 for SPI-0.75, significantly higher than SPI-0.25 (data not shown). The a values in the samples that were stored at 45°C increased 12% (SPI-0.25) to 100% (SPI-0.75) (Table 1). In contrast, samples stored under high aw exhibited 46% decreases in a values at 25°C and 100% increases at 45°C. The b values were increased by 0% (SPI-0.25, 25°C) to 18% (SPI-0.75, 45°C). The ΔE values increased 69% (SPI-0.25) to 254% (SPI-0.75) in samples stored at 45°C.

Color changed significantly even after 28 d at 25°C and low water activity. The results indicate that aw, temperature, and storage time all have significant effects on the Maillard reaction, although aw had a more pronounced effect on the Maillard reaction than temperature or storage time. The general tendency for browning to increase with increasing aw was also observed by Hsu and Fennema (1989); Kehrberg and Johnson (1975), and Labuza and Saltmarch (1981). Davies et al. (1998) studied SPI stored at different aw and temperatures and with or without glucose and found that in the absence of glucose only soy protein underwent browning. Otherwise, the rate of browning increased with increasing temperature and aw.

Gel hardness

Protein gels were composed of three-dimensional matrices or intertwined networks partially associated with polypeptides having entrapped water. The ability of proteins to form gels and provide structures for holding water, flavor, sugar, and other constituents of food are useful in food applications and developing new products, providing an added dimension to protein functionality. Table 2 shows the changes in SPI gel hardness under different treatments and storage times. Our results showed a decrease of almost 50% in hardness of all of samples stored for 224 d, although hardness values changed significantly after only 28 d of storage in the high aw groups.

Surface properties

Foaming properties

There were no significantly different of all the foaming capacity of the samples, and the range were from 15 to 16. There was a decrease of approximately 50%~61% in the foam maximum density (FMD) in all samples stored for 224 d, but this occurred in SPI-0.75 samples after only 28 d at 45°C (Table 4). Since the interfacial behavior of proteins depends on their physical, chemical, and conformational properties (size, shape, amino acid composition, etc.), the composition of SPI might changed which affect the foam maximum density (FMD). Ru’ız-Henestrosa et al. (2007b) analyzed the effect of different condition on foam properties of soy globulins (7S and 11S at 0.1wt%) and found the 7S globulin had higher foam maximum density (FMD) then 11S globulin at pH7 and I 0.05M.

There was a decrease of 43%- 50% in the foam stability (FS) in all samples stored for 224 d except for SPI-0.75 at 45°C, which decreased to 16% after 28 d of storage and then increased to 92% (Table 4). The FS of SPI-0.75 stored at 45°C decreased from 70 to 16 after 28 d of storage and then increased to 92 after 112 d. This is an unusual result for which we have no explanation. Our results are in contrast to the finding of Hsu and Fennema (1989) that the effects of aw, storage temperature, and storage time on FS of WPC are all significant and that FS improved during three months of storage. This change was most dramatic at 40°C. Furthermore, FS values after six months were inversely related to aw (as they were initially).

Viscosity

The decrease in viscosity changed during storage by 8% (25°C) to 27% (45°C) for SPI-0.25 and 81% (25°C) to 96% (45°C) for SPI-0.75, significantly higher than SPI-0.25 (Table 5). Viscosity decreased rapidly from 70.27 cp to 3.80 cp for SPI-0.75 after 28 d of storage at 45°C. This means that viscosity decreased as aw increased. Therefore, the effect of aw on viscosity is more significant than temperature.

There may also have been an interaction between temperature and aw because the greatest decrease was in the viscosity of the SPI-0.75 sample stored at 45°C.

Nitrogen solubility index

Generally, higher water activity values resulted in greater reductions in nitrogen solubility index (NSI) values (Table 6). The decrease in the NSI of isolates stored at 25°C occurred gradually, showing a solubility loss of only 37% for the SPI-0.25 after 224 d of storage. The reduction was more pronounced during the first 28days of storage for samples stored at 45°C, reaching 66% for SPI-0.25 and 81% for SPI-0.75, but variation after this period was small. In other words, higher aw values result in lower NSI values, and therefore the effects of aw on NSI are greater than temperature.

Our results show that SPI quality decreases gradually during long periods of storage, in an intensity dependent on the conditions, mainly temperature and relative humidity. We found that storage at 45°C led to a significant decrease in NSI for all tested samples (including SPI and defatted soy flours), similar to the results of Pinto et al. (2005) but to a higher degree than for SPI. Our results indicate that prolonged storage at higher temperatures and aw may cause protein denaturation, and as a consequence protein insolubilization, which can limit product utilization.

Surface hydrophobicity

Generally,surface hydrophobicity (RSo) increased as water activity increased (Table 7). The increase in RSo of the isolates stored at 25°C occurred gradually, showing an increase from 0% to 5.23% for SPI-0.25 after 224 d of storage. This increase was more pronounced during the first 28 d of storage at 45°C, being 0%-2.61% for SPI-0.25 and to 4.76% for SPI-0.75. It continued increasing significantly until the end of storage. Hydrophobicity is one of the most important structure- related factors influencing the functional properties of proteins, and surface hydrophobicity is significantly correlated with protein gelation properties (Hua et al. 2005). The denaturation of proteins is known to expose hydrophobic groups and thus increase surface hydrophobicity Ibrahim et al. (1993) reported that dehydrated whey proteins stored at 80°C for 7 d underwent changes in surface hydrophobicity and conformation.

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