Study On The Cause Of Aggregation Of Soy Protein Hydrolysates And Its Inhibition

Enzymatic hydrolysis of soybean protein is one of the main methods to improve structure and function of soybean protein.Hydrolysates with different degrees of hydrolysis(DH)have different functions,limited hydrolysates can effectively improve the emulsifying and foaming properties while extensive hydorlysates have blood pressure and cholesterol lowering effect,antioxidation,antifatigue and other biological functions.However,there is a serious aggregation and precipitation behavior of soybean protein after hydrolysis and inactivation of enzyme,which results in a significant decrease in the yield of soluble hydrolysates,thus seriously affects its economic value.In this subject,the aggregation behavior of soy protein and the main component in soy protein,β-conglycinin(7S)and conglycinin(11S)was studied in detail.The behavior and rule of the aggregation of soy protein with different thermal treatment were also analyzed.How to inhibit the enzymatic aggregation and increase soluble hydrolysates was explored preliminary in order to provide the basis for enzymatic modification of soybean protein in the future.First of all,7S and 11 S globulins,the main components in soy protein isolate are isolated and purified.In this paper,the aggregation behavior of hydrolysates from SPI,7S and 11 S with papain were studied respectively.by means of changes in DH and turbidity,molecular weight distribution and electrophoresis.The results showed that the degradation of 7S was most rapid and complete,while 11 S was relatively slow and SPI was between the two.There are different degree of aggregations in the process of hydrolysis of SPI,7S and 11 S protein.From the perspective of the amount and speed of the formation of aggregates,11 S protein was more dominant.When DH was about 5%,the yield of supernatant for 7S was higher than 11 S after digestion.It was shown that the 7S and SPI protein aggregations was mainly composed of small molecular weight peptides while the aggregates of 11 S protein may contain much larger fragments.The surface hydrophobicity of 7S protein decreased while the surface hydrophobicity of 11 S globulin increased.Tricine-SDS-PAGE also shows that the three aggregates contain certain disulfide linkages,especially in 11 S proteins.All of the above results suggest that the hydrophobic group induced by 11 S and the aggregation of disulfide bonds may be one of the reasons for the decrease of the yield of soluble hydrolysates of soybean protein.Then,the effects of thermal denaturation on the aggregation of SPI,7S and 11 S were studied in detail.The changes of structure of SPI,7S,11 S protein after thermal treatment at 100°C,80°C and 95°C for 5 min and 10 min respectively were characterized by using DSC and reductive electrophoresis.The results showed that 7S and 11 S proteins were partially denatured after thermal treatment at 80°C and 95°C for 5 min,and the denaturation peak was not existed after 10 min treatment.The heated proteins were hydrolyzed by papain,and the enzymatic aggregation was studied by means of the changes in turbidity and molecular weight distribution and electrophoresis.The results showed that hydrolysis of 7S,11 S and SPI proteins after heat treatment were significantly accelerated,the order is 7S10min>7S5min =SPI10min >11S5min >SPI5min >7S0min > SPI0min> 11S0min;the yield of supernatant with 5 min heat treatment is the highest compared with other samples.Molecular weight distribution showed that molecular weight of supernatant of 7S decreased with the heating time increases while SPI tended to be larger molecular weight.Reduction electrophoresis of supernatant and precipitation showed that both the patterns are very similar,suggesting similar composition of supernatant and precipitation and the presence of disulfide bonds in precipitation.The above results showed that the heat treatment leads to the expansion of SPI,7S and 11 S structure,and the hydrolysis rate increased,and the yield is increased.When the thermal denaturation reached a certain extent,the main chain of the protein was broken that would help promote the formation of aggregates.Finally,the category of interaction force involved in aggregation were studied.Different concentrations of denatured reagents such as guanidine hydrochloride,urea and SDS and reducing reagents like β-Me was added into different soy protein aggregates.It can be found that the turbidity of aggregates decreased with the increasing concentration of denaturant,while the turbidity of aggregates in β-Me remained the same.The content of hydrophobic amino acid and sulfhydryl in the aggregates were higher than that in the supernatant.All of these showed that hydrophobic interactions played a major role in aggregation of 11 S and SPI protein and some other noncovalent interactions may also play a supporting role,covalent bond not involved.On the basis of the above,the control method of enzymatic aggregation was proposed.Before inactivation of enzyme,different reagents such as 0.1% Na2SO3 and 0.1% Cys and 0.1% SDS was added to the hydrolysate,The results showed that the 7S,11 S and SPI proteins of yield of supernatant adding 0.1% SDS was increased by 12.57%,10.73% and 18.77% respectively.the addition of 0.1% Cys can obtain the higher content of supernatant than 0.1% Na2SO3 It is suggested that the SPI and 11 S aggregates may contain more hydrophobic interaction force,in addition,there are some disulfide bonds,but the aggregates of 7S is more hydrophobic.