Study On The Action Mechanism Of Endogenous Proteinase In Aqueous Extraction Processing Of Peanut

Peanut is rich in lipid and protein,which exist as oil bodies（OBs）and protein storage vacuoles（PSVs）,respectively.The OB particle is covered by a protein-phospholipid membrane,and the protein is named OB intrinsic protein.During the aqueous extraction processing of peanut,peanut cell was disrupted by grinding,and OBs were released;various proteins,which were named OB extrinsic proteins,were released from disrupted PSVs,and some OB extrinsic proteins were bound to OBs.By filtrating,peanut water extract was obtained.By centrifuging,OB emulsion（crude OBs）and protein water extract were obtained.In our previous study,it was found that the crude OBs contained endogenous proteinases,which hydrolyzed the oleosins（OB intrinsic proteins）.In this study,protein component of crude OBs and the properties of endogenous proteinases were examined;the endogenous proteinases were used to demulsify OB emulsion and to hydrolyze peanut proteins.The main contents and results were as follows:Firstly,the conditions of soaking,grinding,and centrifugation of peanut were examined,and protein component of crude OBs was confirmed.Peanut reached the maximum water absorption ratio of 0.5 g/g peanut,after soaking at room temperature for 9 h or 4^oC for 18 h.By wet grinding,about 88%（w/w）of lipid and 78%（w/w）of protein in peanut kernel were recovered in peanut water extract.The water extract was centrifuged（3000 g for 15 min）to obtain OB emulsion（crude OBs）and protein water extract.The protein component of crude OBs was identified by Tricine-SDS-PAGE,2-D electrophoresis,and MALDI-TOF/TOF MS.It was found that crude OBs not only contained OB intrinsic proteins,but also contained OB extrinsic proteins.The intrinsic proteins of OBs included two isoforms of oleosin（14 kDa and17 kDa）,two isoforms of caleosin（28 kDa and 30 kDa）and one steroleosin（40 kDa）.The OB extrinsic proteins contained lipoxygenase,Ara h 1 allergen（65 kDa）,arachin,Ara h 2allergen（20 kDa）,ferritin,and aspartic proteinase.It was found that aspartic proteinase showed high affinity for OBs,while the other OB extrinsic proteins had weak interaction with OBs.Secondly,the enzymatic properties of endogenous proteinase in crude OBs were investigated.It was found that the aspartic proteinase was the major endogenous proteinase.Optimal pH and temperature of the hydrolysis of 17 kDa oleosin were respectively determined as pH 4.0 and 60^oC,and the proteinase was thermal stable.Metal ions,organic molecules,and peanut varieties all affected the activity of endogenous proteinase.The effects of Na⁺,K⁺and Ca²⁺on the proteinase activity were weak regardless of concentration;Mg²⁺and Zn²⁺at low concentration showed weak effect,but obvious inhibition at high concentration;sorbic acid inhibited proteinase activity,the effects of gallic acid,malic acid,citric acid and phytic acid on the proteinase activity were not obvious,and ascorbic acid could promote proteinase activity;Haihua No.14 had the highest proteinase activity,followed by Haihua No.11,and Luhua showed the lowest activity.Interestingly,the endogenous proteinase not only hydrolyzed OB intrinsic proteins,but also OB extrinsic proteins.At pH4.0 and 60^oC,Ara h 1 and arachin acidic polypeptides（46⁴9 kDa）were quickly hydrolyzed,while arachin basic polypeptides and other proteins were slightly hydrolyzed.Thirdly,the effect of endogenous proteinase on the demulsification of crude OBs was examined.It was found that the hydrolysis of oleosins resulted in the aggregation and coalescence of OBs.Under the synergetic effects of temperature and endogenous proteinase,95%free oil was released.Under the synergism of endogenous proteinase and aqueous extract of peanut skin,87%free oil was released.The demulsification rate was 7%higher than that of papain,3%and 6%lower than that of pepsin and alkaline proteinase,respectively.Finally,the effect of endogenous proteinase on the hydrolysis of protein in protein water extract was studied.The results showed that endogenous proteinase had the highest activity at pH 2.0³.0 and 60^oC.Ara h 1 and arachin acidic polypeptides（46⁴9 kDa）were the most quickly hydrolyzed,arachin basic polypeptides（21.4 kDa）were slightly hydrolyzed.The endogenous proteinase had great potentials to be used in decreasing the immunoreactivity and increasing the digestibility of peanut proteins.This study clarified the protein component in crude OBs,and the properties of peanut endogenous proteinase in crude OBs and protein water extract.So it provides some hints for the economical,environment-friendly and efficient demulsification method of crude OBs,,and the preparation of low immunoreactivity and high digestible protein products.