Studies On Protein Composition Of Soybean Oil Body Concentrate And Its Effect On Properties Of Oil Body Emulsion

Soybean seed cell is mainly filled with oil bodies and protein storage vacuoles,which are the storage organelles for soybean oil and proteins,respectively.Oil body(OB),naturally pre-emulsified soybean oil,is composed of a core of triglycerides matrix covered by one monolayer of phospholipids embedded by OB intrinsic proteins.During the grinding of soybean seeds in aqueous extraction,OBs and extrinsic proteins(soybean storage proteins and bioactive proteins)were released from broken cells,and some extrinsic proteins were bound to OBs.It was found that extraction conditions affected the extrinsic proteins composition of OBs in previous studies,which will affect component and properties of extracted OB concentrate.However,intrinsic and extrinsic protein compositions of soybean OB concentrate,and its effects on properties of OB emulsion have been rarely studied.In this study,protein composition of soybean OB concentrates extracted at different pH were identified;the tendency and mechanism of soybean oleosin hydrolysis induced by endogenous protease were deeply investigated;effects of extrinsic protein composition and oleosin hydrolysis on the physicochemical properties of OB emulsion were systematically analyzed;relationship between de-emulsification of OB emulsion and oleosin hydrolysis induced by endogenous protease was discussed.This study is very meaningful for the application of soybean OB emulsion and aqueous extraction of oil from OB concentrate.The major results are as follows:1.The soybean OB concentrates were extracted at pH 6.8 and 11.0,their protein compositions were identified by electrophoresis and MALDI-TOF/TOF MS;interaction between extrinsic proteins and OBs was also studied.The intrinsic proteins contain caleosin,steroleosin,and three forms of oleosins.Specifically,soybean oleosins contain 24 kDa oleosin(two isoforms,A and B),18 kDa oleosin(3 isoforms)and 16 kDa oleosin(two isoforms).Two caleosins(27 kDa and 29 kDa)and one steroleosin(41 kDa)were also confirmed.The extrinsic proteins,including lipoxygenase,glycinin,β-conglycinin,γ-conglycinin,β-amylase,Bd 30 K and P34,were bound to OBs.However,lectin,KTI and BBI were not bound to soybean OBs.It was found that Bd 30 K and P34 was strongly bound to OBs through the special bond to 24 kDa oleosin isoform A.Other extrinsic proteins were bound to OBs through the weak interactions.2.Soybean pH 6.8-,8.0-,9.5-,and 11.0-OB were extracted at different pHs.In unheated and heated conditions,effects of extrinsic proteins on the physicochemical properties of OB emulsions were examined.In unheated condition,particle size and viscosity gradually decreased,whereas p I and oxidative stability increased with decreasing the extrinsic protein content.In heated condition,coalescence of droplets occurred in OB emulsions with more extrinsic proteins.Protein denaturation and interfacial property of droplets were affected by thermal treatment,which resulted in higher p I of droplets,and higher viscosity and better oxidative stability.The pH 11.0-OB emulsion without extrinsic proteins has good flow behavior,dispersion stability and oxidative stability.3.The tendency of soybean oleosin hydrolysis induced by endogenous protease and its universality in other plant seeds were investigated.At 35°C and pH 6.5,the hydrolysis of 24 kDa and 18 kDa oleosin occurred in soybean pH 6.8-,8.0-,and 9.5-OB,while 16 kDa oleosin and extrinsic proteins were not hydrolyzed;and the hydrolysis of 24 kDa and 18 kDa oleosin did not occurred in pH 11.0-OB.Optimal temperature and p H of the hydrolysis of 24 kDa and 18 kDa oleosin were respectively determined as 35~50°C and pH 6.0~6.5,while ≥60 °C or ≥pH 11.0 could inhibit them.The hydrolysis site of 24 kDa oleosion was the peptide bonds between Lys and Thr.When 24 kDa oleosin was hydrolyzed,6942 Da and 6786 Da were respectively released from 24 kDa oleosin isoform A and isoform B,while others hydrolysis products(about 16 kDa,named HP1 A and HP1B)remained on OBs.P34 and Bd 30 K were linked to HP1 A through special bond,respectively.Remarkably,soybean oleosins did not hydrolyzed without P34 and Bd 30 K.In addition,endogenous protease-induced hydrolysis of oleosins occurred in all selected plant(jicama,sesame,peanut,walnut,sunflower and rapeseed)OB concentrated extracted at pH 6.5.4.Endogenous protease and mechanism of soybean oleosin hydrolysis induced by endogenous protease were deeply investigated.P32 was from P34 by removing a decapeptide at the N-terminus of P34.It was found that P32 was the endogenous protease,inducing hydrolysis of 24 kDa and 18 kDa oleosin.P32 was a special cysteine protease,because E64 could not inhibit proteolytic activity.In addition,1~2 mmol/L DTT could obviously enhance proteolytic activity while NEM and IAA could decrease it.The optimal pH and temperature for hydrolysis of 24 kDa oleosin was 40°C and pH 6.0.After P32 dimers(P32-P32)mixed with 24 kDa oleosin,P32-P32 were firstly dissociated into monomers and bound to 24 kDa oleosin to form P32-24 kDa oleosin through the special bond;then 24 kDa oleosin was gradually cleaved;P32 monomers were changed back to P32 dimers again,which was the mechanism of 24 kDa oleosin hydrolyzed by P32.5.Effect of oleosin hydrolysis induced by endogenous protease on the physicochemical properties of oil body emulsion was examined,and the relationship between de-emulsification of OB emulsion and oleosin hydrolysis induced by endogenous protease was discussed.It was found that the hydrolysis of 24 kDa and 18 kDa oleosin resulted in coalescence and aggregation of droplets,and dispersion stability and oxidative stability of OB emulsion decreased.In addition,de-emulsification of walnut,sesame,and sunflower pH 6.5-OB emulsions occurred at pH 4.0~6.0 and 70~80°C,and 90~98% free oil was released.Oleosin hydrolysis is necessary condition for de-emulsification of OB emulsion.However,de-emulsification was also affected by other conditions,such as the size of OBs.