Study On Heat-induced Inactiviation Of Bowman-birk Trypsin Inhibitor In Soymilk

Urease,Bowman-Birk inhibitor(BBI),Kunitz trypsin inhibitor(KTI)and soybean agglutinin(SBA)are four kinds of protein anti-nutritional factors in soymilk.Urease,KTI and SBA were inactivated easily.The high resistance to heat of BBI should be related with its conformation which contains 7 disulfide bonds(SS).Due to the complicated soymilk system,this paper studied the process of separation and purification of BBI and the heated-induced BBI inactivation mechanism were studied.Afterwards,the mechanism was studied by simulating soymilk system gradually.According to the complex coacervation of protein-polysacccharide,turbidity curve was made to analyse experimental conditions.It was found that there was a highest productivity at protein/?-carrageenan(CG)=5:1(w/w),pH 3.85.The condition of highly purified BBI-A(>96%)was protein/CG=4:1(w/w),pH 3.75.The purity of BBI concertrate which contained three groups: BBI-A(38.8%),BBI-C(11.1%)and BBI-D(21.9%),were above 70% by two-dimensional electrophoresis?mass spectrum and HPLC.By heating BBI concertrate and BBI-A at 60~100°C,the effect of the component and purity of two samples were studied.The results showed that TIA declined faster than CIA;the structure of BBI destoryed gradually;the broken of SS;the content of amino acid decreased.BBI did not tend to form intermolecular cross-link with another BBI,but slightly with non-BBI proteins.Based on some previous studies and the results the conformational change of BBI was attributed to the ?-elimination reactions.Based on the results above,the inactivation and conformational changes of BBI-A was attributed to the broken of SS.By reducing SS of BBI-A with dithiothreitol(DTT),differnet reducing samples were prepared(9.15%?23.43%?40.02%?62.09%?88.75%?99.01%)to study the changes of inactivation and choformation of BBI-A.The effects of SS reduction on TIA/CIA could be divided into three steps: when one exposed SS was reduced,both TIA and CIA were decreased to about 60%;with increasing reduction of exposed SS,CIA was gradually decreased to 8%,and TIA gradually to 26%;with further reduction of buried SS,CIA was gradually decreased to 2%,and TIA slightly to 24%.Far-UV CD spectra showed that secondary structure of BBI-A was slightly changed,whereas near-UV CD spectra showed that conformation of BBI-A was substantially changed after five exposed SS were reduced,and further reduction of buried SS affected the conformation to some extent.The results from Tricine-SDS-PAGE and C8 column showed the same trend as near-UV CD spectra.The results above supported the hypothesis that hydrophobic collapse of the exposed hydrophobic patches into a regular hydrophobic core was occurred after the reduction of SS in BBI-A.There were reducing sugar(glucose and fructose)in soymilk.This part used the mixture of BBI and reducing sugar to study the inactivation of BBI.The results showed that reducing sugar accelerate the decrease of TIA in contrast to CIA.BBI possessed two independent sites of inhibition,one at Lys 16-Ser 17 against trypsin and the other at Leu 43-Ser 44 against chymotrypsin.According to the structure,Maillard reaction between reducing sugar and Lys 16 contributed to the decrease of TIA.CIA was not affected by Maillard reaction.Interestingly,BBI mixed with reducing sugar without heating,the sample of TIA and CIA increased.After several experiment,there was still the trend.Other experiments were needed to solve the problem.At last,effect of soy protein(SP)on inactivation of BBI during heating process.The results showed SP could accelerate the inactivation of BBI.SP had significant effect on TIA before 1 h,the effect not obvious with heating time prolonging.SP always accelerated decrease of CIA and the effect growing with heating time prolonging.The increasing of BBI concentration would not make inactivating faster.With Tricine-SDS-PAGE profiles,the main reason of inactivation of BBI was thermal aggregation reaction between BBI and SP(little SH/SS exchange reaction).This study optimized the heated-induced inactivation of BBI.Taking samples heated for 3 h for example:(1)The mixture of BBI concentrate and reducing sugar,TIA of BBI decreased 45%.?-elimination and follow-up reactions and reducing sugar contributed to 23% and 22% individually.CIA of BBI decreased 31%.?-elimination and f ollow-up reactions and reducing sugar contributed to 30% and 1% individually.(2)The mixture of BBI concentrate and SP,TIA of BBI decreased 29%.?-elimination and follow-up reactions and SP contributed to 23% and 6% individually.CIA of BBI decreased 49%.?-elimination and follow-up reactions and SP contributed to 30% and 19% individually.