Identification,Hydrolysis Conditions Of Sesame Endogenous Proteases And Its Application In Oil Body Demulsification

Due to its advantages in environmental protection and comprehensive utilization of lipid and protein,aqueous enzymatic extraction has attracted more and more attention.However,the aqueous enzymatic extraction requires the addition of enzyme,which increases the processing cost.The previous research in our laboratory revealed that there were endogenous proteases in floating emulsion(oil body concentrate)obtained by centrifugation of sesame milk,which had potential to replace the commercial protease for enzymatic hydrolysis of sesame oil body concentrate to produce food oil.However,there is no systematic study on the hydrolysis conditions of these endogenous proteases.Based on this,the strategy of this study was as follows: Firstly,the effect of pretreatment before centrifugal separation on the separation of lipids and proteins,then the types of endogenous proteases and their hydrolysis conditions were systematically investigated,and finally the effects of endogenous proteases on the demulsification of oil body concentrate was investigated.Firstly,the process conditions for the preparation and centrifugal separation of sesame milk were explored.Three times of grinding can make more than 90% of the lipids,87% of the protein and 89% of the solids in the sesame into the sesame milk.When the pH of the sesame milk was adjusted to 8.0,80% of lipids and 7% of proteins from sesame milk could be distributed in the oil body concentrate by centrifugation.Secondly,endogenous proteases in oil body concentrate were identified,and their hydrolysis pH and temperature ranges were investigated.The detection results from LC-MS/MS,two-dimensional electrophoresis and MALDI-TOF/TOF-MS showed that there were a variety of endogenous proteases in oil body concentrate,including endopeptidases and exopeptidases: endopeptidases included seven types of aspartic proteases,two types of serine proteases and three types of metalloproteinases;exopeptidases included one type of tripeptidyl peptidase II and two types of serine carboxypeptidases.Relative abundance:aspartic proteases > serine proteases > metalloproteases.Oleosins were hydrolyzed in the range of pH 3.0~9.0,and the fastest rate was at pH 4.0,while non-oleosin proteins could be hydrolyzed in the range of pH 3.0~4.0,and the fastest rate was at pH 3.0.In the range of30~60°C,endogenous proteases could effectively hydrolyze oleosins,and the optimal temperature was 50°C.The broad range of hydrolysis pH was consistent with the presence of many endogenous proteases.Thirdly,the types of endogenous proteases were confirmed by protease inhibitor experiments,LC-MS/MS and free amino acid analysis,and the hydrolysis pH ranges of various endogenous proteases were investigated.The results showed that the endogenous proteases were aspartic proteases,serine proteases(including endopeptidases and exopeptidases)and metalloproteases.The hydrolysis pH range of aspartic proteases was3.0~6.0,and the optimal pH was 4.0;the hydrolysis pH range of serine proteases(endopeptidases: subtilisin-like protease and serine protease SPPA)was 4.0~9.0,and the activity was the strongest at pH 5.0,among which the hydrolysis activity in the acidic pH range was provided by subtilisin-like protease.Serine carboxypeptidases can exert hydrolysis at pH 3.0~9.0,and the activity was the strongest at pH 4.0.Metalloproteases could hydrolyze oleosins in the alkaline pH region.The results were consistent with the above identification results.Fourthly,based on the hydrolysis conditions of the endogenous proteases in the oil body concentrate,the effects of hydrolysis time,solid content,particle size and protein content on oil body demulsification were systematically investigated.The results showed that oil body concentrate with the longer of hydrolysis time were easier to be demulsified,oil body concentrate with larger particle size were easier to be demulsified,and oil body concentrate with lower protein content were also easier to be demulsified.When the solid content was50%,the demulsification ratio was 86% at pH 4.0 and 50℃ for 2 h.Finally,in order to further improve the demulsification ratio,the effect of freeze-thaw assistance on the demulsification ratio was investigated,and the oil quality was evaluated.The results showed that the demulsification ratio of the freeze-thaw treatment(demulsification ratio was about 71%)alone was much lower than that of hydrolysis and then freeze-thaw.After hydrolysis for 2 h and freeze-thaw,the demulsification ratio could reach to94.22%.The sesame oil was clear and transparent,the content of oleic acid and linoleic acid exceeded 86%.The acid value was 0.58 mg/g,the peroxide value was 0.15 mmol/kg,and the contents of vitamin E,sesamin and sesamolin were 75.15 mg/100 g,6.03 mg/g and 4.58 mg/g,respectively.Phospholipid content(dry basis)of by-product reached to 120.08 mg/g.