Kasetsart J. (Nat. Sci.) 46 : 804 - 811 (2012)

1 Faculty of Agro Industry, King Mongkut’s Institute of Technology Ladkrabang, Bangkok 10520, Thailand.2 Faculty of Home Economics Technology, Rajamangala University of Technology Phra Nakhon, Bangkok 10300, Thailand.* Corresponding author, e-mail: cayongyuth@yahoo.com

Received date : 12/03/12 Accepted date : 30/07/12

Characteristics of Proteins from Fresh and Dried Residues of Soy Milk Production

Yuporn Puechkamut1,* and Woralak Panyathitipong2

ABSTRACT

Fresh soy milk residue (okara) received from a soy milk factory was dried at 60 °C to obtain dried okara. Proteins were extracted from fresh and dried okara at pH 9 and 80 °C. The results found that the greatest recovery of extracted protein from fresh okara was achieved with an extraction time of 2 h. The surface hydrophobicity of the protein extracted from dried okara was higher than that of fresh okara. The sodium dodecyl sulfate (SDS) patterns determined by polyacrylamide gel electrophoresis (PAGE) of okara protein isolates were similar to that of commercial soy protein. The okara protein isolates seemed to have better functionality in their emulsion stability, foam capacity and fat absorption capacity compared to commercial soy protein but not in water absorption capacity. There were no signifi cant differences in the functional properties among proteins extracted from fresh and dried okara. The essential amino acid contents of okara protein isolate were comparable to those of the FAO scoring pattern with the exception of valine, tyrosine and sulfur-containing amino acids.Keywords: soy protein, okara

INTRODUCTION

Soymilk residue (also known as okara) is produced as a byproduct in the manufacturing of soymilk and tofu. Every kilogram of dry beans made into soymilk or tofu generates about 1.1 kg of okara, which contains 76–80% moisture (Liu, 1997). Most okara is sold as animal feed. However, okara contains about 27% protein (dry basis), 10% oil, 42% insoluble fi ber and 12% soluble fi ber (O’Toole, 1999). Okara protein has good nutritional quality and a superior protein effi ciency ratio. The protein can be extracted from okara at alkaline pH; Ma et al. (1997) reported that the protein recovery obtained at pH 9 and 80 °C was

signifi cantly higher than when incubated at 25

°C. With its high moisture content, fresh okara is very easily spoiled by microorganisms. In the factory, okara is usually dried to reduce the low moisture content prior to sale as animal feed. However, the severe heat treatments cause protein denaturation that affects the functionality of the protein. Therefore, in the present study, the effect of drying on the yield and characteristics of the extracted okara protein were studied. The protein isolates were prepared from fresh and dried okara. The physicochemical and functional properties of the extracted proteins were compared with commercial soy protein to assess the potential to use them as a food ingredient.

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MATERIALS AND METHODS

Fresh okara was received from a soymilk factory (Greenspot Co. Ltd, Thailand). The water was removed from the okara by a hydraulic press to obtain 80% (wet basis) moisture content. A commercial soy protein, Supro EX33, was provided by Solae LLC (Thailand). All chemical reagents were analytical reagent grade.

Preparation of dried okara The okara was dried in a tray dryer at 60 °C for 75–80 min to reduce the dried okara to 4–5% (wet basis) moisture content. The dried okara was vacuum packed and kept at 4 °C prior to use in the following experiments.

Extraction of okara protein Proteins were extracted from the fresh and dried okara according to the method of Ma et al. (1997). The okara was mixed with distilled water at a ratio of 1:8, respectively, and the pH was adjusted to 9 by 2 N NaOH. The mixture was stirred at 80 °C for 1–2 h and was then centrifuged at 9,000 rpm for 20 min. The extracted protein in the supernatant was precipitated by adjusting the pH to 4.5 and recovered by centrifugation at 12,000 rpm for 20 min. The precipitated protein was neutralized using 2 N NaOH. The protein isolate was freeze dried and defatted using hexane. The defatted okara protein isolates were kept and applied to the following experiments.

Physicochemical properties The surface hydrophobicity of the protein samples was determined by the fl uorescence probe method using 1-anilino-8-naphathalenesulfonate (Nakai et al., 1996). Polyacrylamide gel electrophoresis (PAGE) in sodium dodecyl sulfate (SDS) was performed with 12% gels according to the method of Laemmli (1970). The sample buffer contained 2% 2-mercaptoethanol. The standard marker was used with the molecular weight ranging from 14,000 to 70,000 Da.

Functional properties The emulsion properties were determined by a turbidimetric method according to Hill (1996). The foam capacity was determined according to Phillips et al. (1987) and the height of the foam was recorded as the foam capacity. The water and fat absorption capacities were determined according to the procedures described by Chan and Ma (1999) and El-Adawy (2000), respectively, and expressed in grams per gram of water and grams of soy bean oil bound per gram of protein, respectively.

Amino acid composition analysis The amino acid contents of the protein were determined according to the procedure described by Spackman et al. (1958). The analyses of the physicochemical and functional properties were performed in triplicate. Analysis of variance and Duncan’s multiple range tests were used to establish the signifi cance of differences among samples at the P ≤ 0.05 level, whereas statistical differences of the amino acid contents in comparison to the commercial soy protein were evaluated for signifi cance by a t-test at the P ≤ 0.05 level.

RESULTS AND DISCUSSIONS

Extraction yield of okara protein isolates Proteins were extracted from fresh and dried okara at pH 9 and the recovery yields were determined (Table 1). The recovery yields of fresh and dried okara with an extraction time of 1 h were not signifi cantly different. However, when the extraction time was increased from 1 to 2 h, the yield increased. Fresh okara with an extraction time of 2 h gave the highest recovery yield. The recovery yields of okara protein isolates from all conditions were about 12% (dry basis). Nevertheless, okara contains about 25% protein on a dry basis (O’Toole, 1999), indicating that the remaining protein fraction (approximately 13% of the okara) was in the residue. This result

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correlated to the results of Ma et al. (1997). During soy milk production, the severe heat treatments and mechanical grinding cause extensive protein denaturation (Zayas, 1997). This may result in low solubility of the protein at an alkaline pH. To improve the effi ciency of the extraction method, other methods, such as chemical modifi cation to enhance the ionic groups, may increase the solubility (Vojdani, 1996). However, the extraction method using an alkaline pH was simple and was not harmful to the protein.

Physicochemical properties of okara protein isolates The 1-anilino-8-naphathalenesulfonate surface hydrophobicity of okara protein isolates is shown in Table 2. In the case of a globular protein like soy protein, the surface hydrophobicity is correlated to the degree of denaturation (Voutsinas et al., 1983; Nakai et al., 1996). When a globular protein is denatured, its compact structure is unfolded, leading to the exposure of buried hydrophobic groups. The surface hydrophobicity of the okara protein isolate was found to increase as the extraction time at 80°C increased. Also, the surface hydrophobicity of protein extracted from

dried okara was higher compared to that of fresh okara. The heat during drying and extraction may have caused the protein structure to unfold which enhanced the binding of the fl uorescence probe to the buried hydrophobic groups resulting in higher hydrophobicity being recorded (Nir et al., 1994). Figure 1 shows the SDS-PAGE patterns of okara protein isolates that were extracted from fresh and dried okara and commercial soy protein. The major bands, according to the estimated molecular weight, correspond to α’-, α- and β-subunits of β-conglycinin (7S globulin) and the acidic polypeptide (AP) and basic polypeptide (BP) of glycinin (11S globulin), similar to those observed by other workers (Sathe et al., 1987; Honig and Wolf, 1991; Toda et al., 2007). The results found that there were no major differences in the electrophoresis patterns among sample proteins. The heat during soy milk production and okara drying may cause soy protein conformational changes, but their major subunits still remain. However, in comparison with the commercial soy protein, the okara protein isolates appeared to have less dense bands, suggesting that lower protein contents were present. This result indicated that

Table 1 Effect of extraction time on mean (± SD) recovery yields of okara protein isolates from fresh and dried okara.

Extraction time Fresh okara Dried okara (h) (g per 100 g, dry basis) (g per 100 g, dry basis) 1 12.0ab±0.5 11.6a±0.5 2 12.9c ±0.4 12.3b±0.3a b c = Mean values with the same superscript letter are not signifi cantly different (P ≤ 0.05).

Table 2 Mean (± SD) surface hydrophobicity of okara protein isolates and commercial soy protein. Protein Surface hydrophobicityProtein from fresh okara (extraction 1 h) 320.74a±15.3Protein from fresh okara (extraction 2 h) 352.43b±20.5Protein from dried okara (extraction 1 h) 356.50b±17.7Protein from dried okara (extraction 2 h) 381.93c±14.3Commercial soy protein (Supro EX33) 344.46b±22.1

a b c = Mean values with the same superscript letter are not signifi cantly different (P ≤ 0.05).

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the purity of the okara protein isolates was less than that of the commercial soy protein leading to the low protein content.

Functional properties of okara protein isolates Emulsifying characteristics are one of the important functional properties of soy protein for the meat processing industry (Liu, 1997). Therefore, the emulsion properties of okara protein isolates were determined in the present study. Table 3 shows the emulsion activity and emulsion

stability of okara protein isolates The emulsion activity of okara protein isolates was similar to that of commercial soy protein. However, the okara protein isolates seemed to have a higher emulsion stability compared to commercial soy protein. The mechanical grinding and heat during soy milk production alter the globular structure of protein (O’Toole, 1999). This may increase the number of polar groups and result in the formation of a strong fi lm around an oil droplet which leads to an improvement in the emulsion stability (Nakai and Li-Chan, 1993).

Figure 1 Polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS-PAGE) patterns of okara protein isolates and commercial soy protein: (A) Fresh okara; (B) Dried okara. (Lane 1: commercial soy protein isolate; Lanes 2 and 3 indicate okara protein isolates from extraction times of 1 and 2 h, respectively; Lane 4: standard weight marker; α’, α and β = α’-, α- and β-subunits of β-conglycinin, respectively; AP and BP = Acidic and basic polypeptide of glycinin, respectively.

Table 3 Mean (± SD) emulsion activity and mean (± SD) emulsion stability of okara protein isolates and commercial soy protein.

Protein Emulsion activityA Emulsion stabilityA

Protein from fresh okara (extraction 1 h) 2.73±0.04 0.13b±0.01Protein from fresh okara (extraction 2 h) 2.72±0.03 0.18b±0.02Protein from dried okara (extraction 1 h) 2.77±0.04 0.20b±0.01Protein from dried okara (extraction 2 h) 2.76±0.06 0.17b±0.03Commercial soy protein (Supro EX33) 2.72±0.05 0.09a±0.02a b c = Mean values with different superscript letters within the same column are signifi cantly different (P ≤ 0.05).A = Absorbance at 500 nm (OD500 × dilution factor).

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The foam capacity and water and fat absorption capacities of okara protein isolates are described in Table 4. All the okara protein samples had a similar foam capacity but their capacities were higher than in commercial soy protein; there was the same relationship with the emulsion stability. Protein in the okara isolates may have appropriate charges which induce a protein-protein interaction and promote the formation of fi lms at the air-water interface, hence increasing foam capacity (Were et al., 1997; Molina and Wagner, 2002). There was no significant difference in the fat absorption capacity among the okara protein samples (Table 4). However, the fat absorption capacity of commercial soy protein was signifi cantly less than those of the okara protein isolates. Chan and Ma (1999) also reported that unmodifi ed okara protein isolate had a higher fat absorption capacity than that of commercial Supro 610. Compared to the commercial soy protein, the protein in the okara isolates may have had a more suitable structure that favored the physical entrapment of oil. In contrast to the other functionalities, the results showed that there was a signifi cant difference in the water absorption capacity among the okara protein samples; fresh okara with an extraction time of 1 h gave the highest water absorption capacity. It seems that more heat during extraction and drying reduced the capacity of the protein in the okara isolates to entrap water. Nevertheless, for other functionalities, the results

found that there were no signifi cant differences among the okara protein samples. In the present study, the temperature used for drying okara was 60 °C. This drying treatment may not have been severe enough to disturb the emulsion properties, foam capacity and fat absorption capacity of the proteins extracted from the dried okara. Moreover, with the exception of water absorption, increasing the extraction time from 1 up to 2 h did not affect the functionalities of the okara protein isolates.

Amino acid composition of okara protein isolate There were no signifi cant differences among most of the functionality parameters of the protein isolates extracted under the various conditions and fresh okara seemed to give a higher recovery yield compared to dried okara. Therefore, in the present study, the protein extracted from fresh okara was chosen for the analysis of the amino acid composition. Table 5 shows the amino acid contents of the protein extracted from the fresh okara. Statistical analysis of differences in the amino acid contents compared to those of a previous study of soy protein isolate (Sarwar et al., 1983) was undertaken. The results showed that most of the amino acid contents of the okara protein isolate were less than those of the soy protein isolate with the exception of cysteine, methionine, threonine, histidine and glycine. When compared to the WHO ⁄ FAO ⁄ UNU (2007) reference pattern of proteins established for adults,

Table 4 Mean (± SD) foam capacity and mean (± SD) water and fat absorption capacities of okara protein isolates and commercial soy protein.

Foam capacity Water absorption Fat absorption Protein (cm) capacity capacity (g per gram) (g per gram)Protein from fresh okara (extraction 1 h) 7.45b±0.15 6.01c±0.09 3.11b±0.14Protein from fresh okara (extraction 2 h) 7.60b±0.22 5.02b±0.11 3.54b±0.14Protein from dried okara (extraction 1 h) 7.33b±0.31 5.63b±0.15 3.05b±0.17Protein from dried okara (extraction 2 h) 7.50b±0.17 4.12a±0.20 3.39b±0.10Commercial soy protein (Supro EX33) 5.90a±0.20 4.75a±0.08 1.07a±0.07a b c = Mean values with different superscript letters within the same column are signifi cantly different (P ≤ 0.05).

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the essential amino acid profi le of the okara protein isolate was comparable with the exception of the sulfur-containing amino acids (cysteine and methionine), valine and tyrosine which were lower in the okara protein isolate. Liu (1997) reported that the limiting amino acid in soy protein is a sulfur-containing amino acid. However, soy protein is rich in lysine, and this amino acid is limited in cereals, which are generally rich in sulfur-containing amino acids (Rao et al., 2002). Hence, the protein form in soy or okara is an ideal protein source for complementing cereal proteins.

CONCLUSION

The results showed that there were no major differences in the SDS-PAGE patterns among proteins extracted from fresh and dried okara and

Table 5 Amino acid contents of mean (± SD) okara protein isolate. Amino acid Okara protein isolateA Soy protein isolateB FAO scoring patternC

(g per16 g N) (g per16 g N) (g per16 g N)Essential Cyst(e)ine 2.03b±0.08 1.19a 2.20 Isoleucine 3.58a±0.11 4.72b 3.00 Leucine 6.42a±0.23 8.51b 5.90 Lysine 4.98a±0.31 6.34b 4.50 Methionine 1.09a±0.14 1.24a 2.20 Phenylalanine 3.90a±0.27 5.62b 3.80 Threonine 5.30b±0.20 3.84a 2.30 Tyrosine 2.82a±0.17 4.04b 3.80 Valine 3.70a±0.20 4.91b 3.90 Histidine 2.60a±0.12 2.54a 1.50Nonessential Alanine 3.74a±0.15 4.37b Arginine 4.18a±0.23 7.87b Aspartic acid 10.42a±0.16 11.80b Glutamic acid 17.26a±0.08 20.70b Glycine 4.42a±0.13 4.22a Proline 4.67a±0.14 5.42b Serine 4.05a±0.19 5.3b

A = Protein from fresh okara; B = Sarwar et al. (1983); C = WHO/FAO/UNU (2007).a b = Mean values with different superscript letters within the same row are signifi cantly different (P ≤ 0.05).

they were similar to that of commercial soy protein. Functional properties such as the emulsion stability, foam capacity and fat absorption capacity of okara protein isolates were better than those of commercial soy protein. Except for the water absorption capacity, the results showed that there were no signifi cant differences in functionalities among the proteins extracted from fresh and dried okara. The essential amino acid profi le of the okara protein isolate was comparable to that of the FAO/WHO standard with the exception of the sulfur-containing amino acids and valine and tyrosine. The study showed that a valuable protein can be produced from the low-value residue from soy milk production. Moreover, these results generally indicate that drying okara at moderate temperature can preserve the functionality of the extracted protein.

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ACKNOWLEDGEMENTS

The research project was supported by a grant from the Thai government. The authors would like to thank Ms. Supichayangkul N. for her valuable support in technical aspects throughout the study.

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