Abstract: Abstract: 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≈1.6 μm) 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 of α-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.