微射流均质预处理提高大豆分离蛋白酶解效率及酶解产物乳化性能

Microfluidization pretreatment improving enzymatic hydrolysis of soy isolated protein and emulsifying properties of hydrolysates

为了探讨微射流均质预处理对大豆分离蛋白酶解效率及酶解产物乳化性能的影响，该文研究比较了微射流均质预处理前后大豆分离蛋白酶解产物的理化性质（水解度、亚基组成、蛋白溶解性、表面疏水性和分子量分布）和乳化性能（通过测定分析样品乳状液的平均粒径和微观结构评估样品的乳化性能）的变化。研究表明：大豆分离蛋白经过微射流均质预处理后采用木瓜蛋白酶水解，其酶解产物（水解度为1.7%）与对照大豆分离蛋白和未经预处理的酶解产物相比，在较低浓度下（30 g/L）制备出粒径细小的稳定乳状液（体积平均粒径≈1.6μm）。微射流均质预处理提高了大豆分离蛋白中α-7S和A-11S亚基的酶解敏感性，使酶解产物在水解度1.3%～1.7%范围内蛋白溶解性显著增加（P〈0.05），同时保持较高的表面疏水性值，与未经预处理的酶解产物相比形成了更多具有界面活性的可溶性多肽（分子量主要分布在11.3 kDa左右），在乳化过程中可有效防止乳液滴间发生桥联絮凝。因此微射流均质预处理是一种辅助提高大豆蛋白酶解效率和酶解产物乳化性能行之有效的方法。研究结果可为大豆蛋白深加工蛋白乳化剂提供理论和方法参考。

The current popularity of soy proteins, due to their low cost and high nutritional value, continues to drive soy research and commercial development of new food products. However, the poor emulsifying properties have limited the application of soy proteins in emulsion-based food products. Modification of soy proteins by enzymatic hydrolysis for improved functionalities is a well-accepted and safe method, but soy proteins are resistant to enzymatic hydrolysis due to their compact structures that protect many of the peptides bonds. Recently, it has been reported that microfluidization treatment can disrupt the quaternary and tertiary structure of globular proteins and break up protein aggregates, which may cause the exposure of more cleavage sites. However, limited information is available concerning the effects of microfluidization pretreatment on the enzymatic hydrolysis pattern of soy proteins or on the emulsifying properties of its hydrolysates. Hence, the objective of this work was to study the effects of combining microfluidization pretreatment and controlled enzymatic hydrolysis using Papain on the emulsifying properties of soy protein isolates （SPI）. Microfluidizaiton pretreated SPI （MSPI） was prepared using a microfluidizer at a specific pressure level of 120 MPa. Papain was used as protease for the preparation of SPI hydrolysates （SPIH） and MSPI hydrolysates （MSPIH）. Oil-in-water emulsions （20% v/v sunflower oil, 20 g/L protein sample, pH=7.0） were formed by SPIH and MSPIH with various DH （degree of hydrolysis）. And the analysis of mean droplet sizes and microstructures of these emulsions showed that emulsions formed by control SPI and SPIH were unstable to bridging flocculation, suggesting their poor emulsifying capability. This findings may be explained by the fact that there were insufficient soluble protein with high surface activity existed in control SPI and SPIH. In contrast, some MSPIH （DH was 1.3%-1.7%） showed that the emulsifying capability and emulsions stabilization against bridging flocculation were markedly improved. Compared with control SPI and SPIH （0.6% DH）, MSPIH （1.7% DH） was capable of producing a stable fine emulsion （d43≈1.6μm） at a lower concentration （30 g/L）, suggesting its better emulsifying capability. Composition of subunits, protein solubility （NSI）, surface hydrophobicity （H0） and molecular weight distribution of SPIH and MSPIH have been measured in order to study the underpinning mechanisms of improving emulsifying capability for SPIH and MSPIH. The analysis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles of different hydrolysates showed that microfluidization pretreatment could significantly improve the enzymatic accessibility of subunits ofα-7S and A-11S in SPI that were resistant to Papain hydrolysis. As a result, compared with SPIH, MSPIH showed a stronger increase in protein solubility and a more moderate change in surface hydrophobicity. Therefore, when DH was 1.3%-1.7%, MSPIH not only had greatly increased protein solubility （NSI was 51.3%-58.2%）, but also retained sufficiently high surface hydrophobicity （H0 was 1021.7-1614.2）. These finding suggested that after microfluidization pretreatment, more soluble polypeptides with high surface activity were produced during Papain hydrolysis in MSPIH, which may be the main cause for the increase in their emulsifying capability. Moreover, compared with control SPI and SPIH （0.6% DH）, MSPIH （1.7% DH） showed a smaller molecular weight distribution mainly around 11.3 kDa, and probably had a better flexibility of protein structure, which benefited for the adsorption and unfolding of protein molecule at the oil-water interface and for preventing the bridging flocculation of droplets. In summary, this study demonstrates that modified soy proteins can be an excellent emulsifying agent for food and other applications. It also demonstrates that combining microfluidization pretreatment and controlled enzymatic hydrolysis can be an effective way for the functionality modification of globular proteins.