2.1.3 Advantages of the reverse micelle extraction
Reverse micelle has a variety of advantages such as enormous interfacial area, thermodynamically stable and optically transparent, low cost due to the recovery of surfactant and nonpolar solvents, ease of scale-up and simple control of the reaction variables (Sereti V, 2014). Most importantly, due to the similarity of its aqueous cores to the physiological environment, the reverse micelles could prevent the denaturation of encapsulated biomolecules (Bu, 2014). A recent study has demonstrated that the reverse micelle method can better prevent breakage of the natural molecular structure of proteins compared to the traditional alkali solution–acid precipitation method (ASAPM) (Yao et al., 2021). Specifically, 11S globulin extracted by ASAPM had a higher β-fold content compared to 11S globulin isolated by reverse micelles containing more hydrophobic amino acids and fewer sulfur-containing amino acids. As a result, the surface hydrophobicity of 11S globulins obtained by ASAPM was increased. In addition, the 11S globulins separated using reverse micelles were more resistant to high temperatures. Similarly, Du (Du et al., 2020b) reported that more β-sheets but less turn structure was observed in the 7S globulin extracted by reverse micelle, indicating that the native folded structure of protein could be protected by the reverse micelle environment and 7S globulin formed a more compact conformation. It is well known that the functional properties of proteins are influenced by their structure. The low denaturation temperature, the poor thermal stability, and the strong hydrophobic interaction of 7S globulin prepared by the reverse micelle method affect its gelation process. Therefore, an improvement in the quality of thermally induced gelatin of 7S globulin was observed in the reverse micelle environment (Du et al., 2020b). As an advanced soybean protein extraction method, reverse micelles can not only separates and purifies soy protein but also improves the functionality, nutritional properties, and flavour of soy protein and reduces undesirable beany flavor. Zhao et al (Zhao et al., 2018b) concluded that the protein oil absorption capacity, solubility index, emulsification capacity, and stability as well as foaming capacity obtained by AOT reverse micelles were significantly higher than those obtained by alkali extraction isoelectric precipitation (AEIP). Soy is an essential source of amino acids (Zarkadas, 2007). Current research has shown that AOT reverse micelle extracted soy protein is a superior source of protein nutrition suitable for human consumption. For 11S globulins, the total amino acid content of the AOT reverse micelle extract was increased by 5.98% compared to the amino acid composition of the aqueous buffer extract, but the content of 7S globulins was similar. For both 7S and 11S globulins, the major amino acid content in the aqueous buffer solution was lower than that in the AOT reverse micelles (Zhao et al., 2011a).
2.2 Enzyme-assisted extraction
Enzyme-assisted extraction uses water and protease to extract protein from soybeans and is considered an alternative extraction method to alkaline extraction which involves pollution (Campbell KA, 2011). As a mild extraction method, enzyme-assisted techniques minimize side reactions (Sari et al., 2013). Enzyme-assisted extractions are considered environmentally friendly technologies as they offer a green chemistry possibility for the food industry looking for cleaner routes. Recent studies on enzyme-assisted extraction have shown that it offers faster extraction rates, higher recoveries, less solvent use, and lower energy consumption than non-enzymatic methods and therefore represents a potential alternative to traditional solvent extraction methods (Vergara-Barber, 2015. ). Compared to alkaline extraction, the addition of enzymes results in a reduction in protein size due to protein hydrolysis. As a result, proteins are more easily extracted. In addition, the use of enzymes can also be used to lower the processing pH, thus avoiding severe conditions of protein denaturation (Sari et al., 2013). Enzyme-assisted countercurrent extraction significantly increased the protein yield compared to alkaline extraction and acid precipitation. The protein had a larger molecular weight distribution, reduce flavor volatiles, higher thermal stability, and surface hydrophobicity as evidenced by the denaturation temperature and enthalpy change of the protein (Wei et al., 2017). Under alkaline pH conditions, 80% of soybean meal protein is extracted without the addition of enzymes, while the addition of enzymes increases the protein extraction yield of soybean meal to 90% (Sari et al., 2013). Many studies have shown that enzyme-assisted extraction has been used to enhance the nutritional value and alter the structural properties of proteins (Lu, 2016). The process of enzymatic hydrolysis of soy proteins has obtained many peptides in cancer prevention, anti-hypertension, and reducing blood cholesterol (Hoa N T, 2014, ). Compared to natural SPI, SPI prepared by enzyme-assisted treatment has higher hydrophobic amino acid, surface hydrophobicity, and interfacial adsorption properties. This is due to the formation of small soluble aggregates accompanied by protein unfolding. In addition, the significant improvement in emulsification capacity and physical stability of the emulsions may be related to the higher surface protein loading. These results provide a viable route for the production of nutrient-enhanced soy proteins with excellent emulsification properties for application in the food industry as novel functional ingredients (Lu et al., 2016).