Study On The Applications Of Seed Endogenous Proteases In Soybean Processing

Proteases can be used to improve the nutritional,functional and sensory qualities of proteins.In the previous studies,it has been confirmed that endogenous proteases can play important physiological roles in soybean germination and growth,such as providing raw materials for biosynthesis and providing nitrogen sources.However,it is unclear whether these endogenous proteases will play a role in soybean processing.Based on the effects of proteases on modifying the nutrition and function of proteins,this paper used the soymilk system to investigate the composition,hydrolysis conditions and hydrolysis activity of endogenous proteases(endopeptidases and exopeptidases),and based on this,to examine the roles and advantages of endogenous proteases in the preparation of new types of soymilk,and the nutrition and function of traditional acid-soaked soybean seeds were evaluated on the viewpoint of the hydrolysis of soybean proteins by endogenous proteases.The main research contents and results are as follows:Firstly,LC-MS/MS analysis was used to determine the endopeptidase,exopeptidase and inhibitors in the soymilk.The results showed that 29 proteases and 24 protease inhibitors were identified in the soymilk.The proteases included five aspartic endopeptidases,five serine endopeptidases(including four subtilisin-like proteases),two cysteine endopeptidases,one matrix metalloproteinase,one prolyl endopeptidases,two oligopeptidases,ten aminopeptidases(including one tripeptidyl peptidase 2)and three carboxypeptidases.By comparing with the previous related studies,it was found that the prolyl endopeptidase,oligopeptidases,puromycin sensitive aminopeptidase,leucine aminopeptidases,aminopeptidase M1,aminopeptidase P1 and lysosomal Pro-X carboxypeptidase were not ever reported,and the others were reported.There were 13 trypsin inhibitors,four BBIs,two serpin-ZX,one subtilisin inhibitor and four cysteine protease inhibitors.Amongst them,the serpin-ZX and subtilisin inhibitor were not reported in previous studies.In addition,one glutamate decarboxylase was also detected in the soymilk system.Secondly,the Tricine-SDS-PAGE,protease inhibitor experiment,TCA-NSI and free amino acid analyses were used to investigate the hydrolysis conditions,proteolytic activity and thermal stability of soybean endogenous proteases.The Tricine-SDS-PAGE analysis showed that the aspartic endopeptidases in the soybean milk system could significantly hydrolyze ?-conglycinin and 11 S globulin at p H 2.5-p H 3.0 and 35°C-45°C;P34 probable thiol protease(P34)hydrolyzed oil body intrinsic proteins at p H 5.0-p H 9.0,and showed better proteolytic activity at p H 6.0-p H 7.0.The free amino acid analysis showed that the exopeptidases could exert activity at p H 4.0-p H 9.0,and peaked at p H 6.0 and 45°C;the glutamate decarboxylase showed catalytic activity at p H 5.0-p H 7.0,and also peaked at p H 6.0 and 45°C.By incubation at p H 6.0 and 45°C for 6 h,the contents of free amino acids and ?-aminobutyric acid in soymilk doubled.The TCA-NSI results showed that the endopeptidases and exopeptidases could exert synergistic activity at p H 3.0-p H 6.0 and 25°C-65°C,and peaked at p H 5.0 and 55°C.The hydrolyzed peptides identified by LC-MS/MS showed that the hydrolyzed peptides were greatly hydrolyzed from trypsin inhibitors and lipoxygenase at p H 5.0 and 55°C,but the ones from ?-conglycinin 11 S globulin were a few.Then,this thesis examined the applications of endogenous proteases in soybean processing:(1)The application of P34 for the defatting of soymilk.This section further confirmed that the hydrolysis of oil body intrinsic proteins by P34 was optimally occurred at p H 6.5 and 40°C.After the oil body intrinsic proteins were hydrolyzed,the physical stability of the oil bodies was weakened.The thermal treatment would promote the coalescence of P34-hydrolyzed oil bodies into larger oil droplets,which could be easily floated by centrifugation.The results showed that after the soy milk was hydrolyzed at p H 6.5 and 40°C for 1 h,then heated in a boiling water bath for 20 min,and then centrifuged at 60°C(3000g,15 min),41.45% of the oil bodies in the soymilk could be floated.With increasing temperature during centrifugation,more and more oil bodies were separated,which could reach to 47.37% at 90°C.At this time,approximately97% of proteins were still remained in soymilk.(2)The roles of endogenous proteases in the processing of acid-soaked soybean seeds.The results showed that 0.8% citric acid solution could soak out the most protein components.In this condition of soaking soybean seeds for 32 h at 25°C,the protein components soaked out accounted for 15.47% of the total proteins in soybean seeds.In them,the free amino acids soaked out were 1768.0 mg/100 g soybean seeds,and the ?-aminobutyric acid was 114.0mg/100 g soybean seeds.Finally,the proteolytic activity of sesame endogenous proteases on soybean proteins was examined.The results showed that the sesame endogenous proteases could efficiently hydrolyze soybean proteins,especially heat-denatured ones.The proteases exerted considerably high activity at p H 4.0-p H 5.5 and 40?-60?,and optimal at 5.0 and 50?.The TCA-NSI value reached to 35.02% by 6 h incubation in the optimal condition.