Preparation,Physicochemical Properties And Polyphenol Inactivation Mechanism Of Soybean Trypsin Inhibitor

Soybean trypsin inhibitor(STI)is a primary antinutrient in soybean.However,a growing body of evidence suggests that STI can play a considerable role in the prevention of several diseases including inflammation and cancer.Moreover,it also has a certain effect on the treatment of diabetes and regulation of insulin level.Thus,STI has potential application value and market prospects in the medical field,it is a valuable issue to develop a low-cost and scale method to recover STI from soy whey.On the other hand,to improve the nutritional value of soy products,it is necessary to remove or inactivate trypsin inhibitor in the food industry.These approaches are based largely on the heat treatment.However,heat treatments do not completely inactive all inhibitors,moreover,autoclaving and other extreme conditions applied to inactivate inhibitors may disrupt some vital essential amino acids.So there is a need for economic and green technique as alternative to heat treatments for inactivating trypsin inhibitor.Therefore,to achieve the high-value utilization of soybean processing by-products,both low-cost and scale methods were developed to recover STI from soy whey,and the physicochemical properties of KTI were evaluated as compared with commercial KTI.A biological inactivation method was developed to inactive KTI by EGCG,the mechanism of inactivation of KTI was also analyzed.The main results are as follows:(1)A simple and low-cost Na2SO4 salting-out method was developed to recover soybean Kunitz trypsin inhibitor(STI)from soy whey wastewater.The stability of temperature and pH as well as the secondary structure of STI obtained by this method were studied.The adsorption characteristics and viscoelastic properties of STI at the gas-water interface were evaluated by dynamic dropping analysis combined with the oscillation technique.The relationship between the interface properties and the properties of foam was also analyzed.The results indicated that the optimal conditions of extraction was adding 9%(w/w)sodium sulfate mass to 13%(w/v)solid content of soy whey(pH 4.0),under this condition,the yield of STI was 2.13% with protein content of 88.0%,the trypsin inhibitory activity of STI was 2135.00 TIU/mg.The physicochemical and interfacial properties of STI were found to be essentially the same as commercial KTI(SigmaT9128).(2)Ultrafiltration combined with expanded bed adsorption was an innovative way to separate trypsin inhibitor from soybean whey wastewater.The effects of different resin,pH value and different desorbent concentration on the adsorption performance were investigated.The results showed that Amberlite XAD7 HP had the best adsorption effect on soybean trypsin inhibitor,and the extraction rate was 2.11% by controlling the injection volume of 300 mL,flow rate 5 mL/min and pH value 4 to obtain high purity trypsin inhibitor.The physicochemical properties and interfacial properties of trypsin inhibitors obtained by bed method have been found to be essentially the same as KTI Sigma T9128.(3)The inactivation effect of epigallocatechin gallate(EGCG)on KTI was evaluated.The inactivation mechanism was studied by using stopped-flow,fluorescence quenching,FTIR,thermodynamic(ITC)and molecular docking.The results indicated that EGCG can effectively inactivate KTI,when EGCG/KTI(w/w)is 40,the inhibition rate reached the maximum value of 75.30%.EGCG binds KTI via both hydrophobic and hydrophilic interactions with an association constant of 6.62 × 105 M-1 to form a 1:1 complex.Molecular docking showed that the participation of amino acids includes three amino acid residues(Asn13,Pro72,and Trp117)near the reactive site of KTI,which may prevent KTI from contacting trypsin and hence inactivate KTI.