Study On The Effect Of Milk-clotting Enzyme From Quambalaria Cyanescens On Soy Protein

Quambalaria cyanescens is a milk-coltting enzyme?MCE?producing strain which was isolated and purified from red yeast rice,the MCE has excellent curd activity,It's essentially an aspartic protease?APs?and the curd activity can be reach at610.52SU/mL,it is a kind of microbe rennin strain with great development potential.Using relevant technical means and experimental methods to explore the effect of MCE purified from Quambalaria cyanescens on the aggregation characteristics of soybean protein and soymilk/milk mixing curd could improve the comprehensive utilization of soybean and dairy resources,and provides theoretical basis and scientific guidance for the intensive processing of soybean protein industry in China.?1?The MCE producing from Quambalaria cyanescens were crude separation by centrifuge and microfiltration,then the crude enzyme solution was enriched and concentrated by pall ultrafiltration system though 30kDa?100kDa ultrafiltration membrane.The DEAE cellulose 52 anion exchange columns and G-75 glucan gel column were used to isolated and purified the MCE producing form Quambalaria cyanescens.The activity of MCE rennin was up to 373.7 IMCU/mg,the activity of proteolysis was up to 373.7 IMCU/mg,and the enzyme activity recovery was up to31.5%.The purification multiple was 5.7,and the purity was above 95%verified by SDS-PAGE.?2?The purified MCE was using to treat?-CN,the peptides after enzymatic hydrolysis were N-terminal sequencing by RPLC-ESI-MS/MS,the result was shown that the major peptides after enzymatic hydrolysis are FFSDKIAKYIPI QYVLSRYPSY,FFSDKIAKYIPIQYVLSRYPSYG,FFSDKIAKYIPIQYVLS RYPSYGLN,PTTMARHPHPHLSFMAIPPKKN,after search and comparison through National Center for Biotechnology Information library,the main restriction site of MCE on?-CN were Arg₆-Phe₇ and Tyr₂₈-Gly₂₉,it also showed that the MCE get amino acid hydrolysis preference for glycine?Gly?,tyrosine?Tyr?and glutamine?Gln?.?3?The purified MCE was treated with soybean isolation protein,the SPI degree of hydrolysis and the turbidity of enzymatic hydrolysis system increased with the increase of enzyme treatment time,the enzyme treatment were lead the change of composition and molecular weight distribution of SPI,the subunit of SPI was dissociated and the SPI was hydrolyzed into components with lower molecular weight,SDS-PAGE electrophoresis analysis showed that 7S component had a higher degree of dissociation than 11S component.?4?The present study revealed that MCE from Quambalaria cyanescens could induce the aggregation of SPI.The absolute Zeta???potential of SPI were decline and the average particle size of SPI were changed during the process of SPI aggregation.MCE treatment induced the molecular unfolding of SPI with the changes of secondary structure,result in the change of thermodynamic properties of SPI;During the period,the content of sulfhydryl group in SPI decreased,and some inter-molecular disulfide bonds were formed through SH/SS reaction;the result of intrinsic fluorescence emission spectra,Surface hydrophobicity and circular dichroism shown that enzyme treatment leads to the change of relative conformation of SPI molecules,and the exposure of hydrophobic groups and hydrophobic amino acids inside the protein molecules,result in the increase of surface hydrophobicity of SPI molecules,this led to stronger and more extensive hydrophobic interactions between SPI molecules,the enzyme treatment led to the increase of?-helix content in the secondary structure of soybean protein with higher aggregation tendency and the decrease of?-sheet content which was important for the natural stable spherical conformation.Combining with the study on the forces in the process of SPI aggregation,it was shown that the non-covalent and covalent interactions in the MCE treatment process jointly induced the aggregation behavior of SPI,and the non-covalent forces dominated by hydrophobic interaction were the main driving force of the soybean protease-induced aggregation process.?5?The enzyme induced aggregation process of SPI and the microstructure of SPI aggregates were determined by confocal laser?LSCM?and atomic force microscope?AFM?,the result shown that the MCE treatment was failing to produce enough inter-molecular covalent cross-linking to form a continuous 3D gel structure,instead,the SPI were formed the loose,discontinuous and irregular aggregates through non-covalent forces,and the aggregation degree of SPI and size of SPI aggregates were increased with the increase of enzyme treatment time.Ca²⁺can stabilize the structure of enzyme and accelerate the process of enzymatic hydrolysis.?6?The phase behavior and gel structure fractal analysis and microstructure morphology of mixing soybean milk/milk curd system were studied.Under suitable raw material group distribution ratio and curd conditions,theSoybean milk/milk could form the stable,dense,weakly linked mixed gel system,and its microstructure is similar to that of enzymatic milk curd.The elastic modulus?G'?of the mixed curd is higher than that of the viscous modulus?G'?,this was shown that solids characteristics of curd are more dominant than that of viscosity,indicating that curd has good linear viscoelasticity.Mixed curd has a higher D_f value than milk curd,this result were showing that mixed curd has more dense and compact three-dimensional network structure and excellent texture.