The Study Of Mechanism Of Modori Phenomenon Of Blue Round Scad ISP Protein Isolate And Application

In the present study,proteins isolates were recovered from the muscle of blue round scad(Decapterus maruadsi)using acidic-or alkaline-aided isoelectric solubilization/precipitation(API/KPI).The mechanism of modori phenomenon was investigated.The result showed that API and KPI achieved in recovery yield of 65.0% and 84.6%,much higher than that of protein(MF,myofibrillar protein)recovered by conventional process(54%).API and KPI also possessed the better removals of lipid and ash than total protein(TP).The rheological and texture analysis released that TP,MP and KPI could occurred modori phenomenon when temperature range from 50 to 55?C.And the gelations were observed in temperature higher than 60?C.However,API did not changed significantly.In order to investigate the auto-degradation of proteins,TP,API and KPI were incubated at 50?C.As a result,the myosin heavy chain and tropomyosin were fiercely degraded,especially in the TP and KPI.Compared with TP,proteolytic activity was highly remained in the supernatant of KPI.Using casein as substrate,the endogenous proteinases showed its optimal pH and temperature of 9.0 and 60?C.The inhibitory specificity result showed that proteinase could be effective inhibited by serine proteinase type inhibitor,suggesting the proteinases belong to serine proteinase family.According to above result,a novel serine proteinase was purified from supernatant of KPI to homogeneity by ammoniun sulfate fractionation and a series of chromatographies including DEAE-Sepharose,HiTrap Q-HP,Sephacryl S-200 and Mini Q.The molecular weight of purified proteinase was estimated to be 70 kDa by SDS-PAGE.The fluorogenic substrate analysis showed that proteinase can specifically hydrolyze the peptide with Arg and Lys residue in the carboxyl terminal.Using Boc-Leu-Gly-Arg-MCA as substrate,the optimum temperature and pH of purified proteinase were 50?C and 9.0,respectively.Moreover,the proteinase was strongly inhibited by serine proteinase inhibitors and partially inhibited by pepstatin inhibitors and cathepsin proteinase inhibitors.These results suggested that purified proteinase was serine proteinase,which was probably involved in modori phenomenons of alkaline ISP protein from blue round scads.To clarify the gel formation mechanism of isolated proteins recovered from blue round scad,the physiochemical,conformational and gelation properties of the major protein myosin treated by acidic-(AM)or alkaline-(KM)pH-shifting were investigated and compared with native myosin(NM).Among the samples,AM exhibited the lowest solubility and highest turbidity than NM and KM as the temperature was higher than 40?C,indicating that AM is easily to be denatured and aggregated.Circular dichroism analysis showed that both acidic and alkaline pH treatments led to conformational changes of myosin.When pH was readjusted to neutral,KM recovered its structure similar to NM,while AM failed to refold to its native state.The result of rheology indicated that the G′ of AM did not change obviously during heating.These results strongly suggested that acidic treatment led to an irreversible conformational change in myosin,consequently induced a fierce denaturation and aggregation,and eventually led to a complete loss of gel-forming ability.Alkaline pH-shifting,on the other hand is thus more feasible for protein recovery from blue round scad.To apply the protein isolate of blue round scad on industry,different proportion of soybean protein isolate was added to inhibit the activity of endogenous proteinase.The results showed that 1% soybean protein isolate could effectively inhibit the degradation of myosin heavy chain,and improve the gel strength of surimi.Subsequently,glucono-δ-lactone(GDL)and transglutaminase(TG)were added to KPI to prepare a novel soft surimi product for the elderly and people with chewing and swallowing difficulties.The addition of 2% GDL and 1% TG could promote the gelation of surimi,in which the gel strength of GDL was higher than that of TG group,with higher hardness and whiteness,as well as more compact gel network structure.The results of chemical bonds determination and SDS-PAGE analysis suggested that GDL could improve the hydrophobicity of proteins and promote the formation of disulfide bonds,while TG could promote the cross-linking of myosin to form macromolecular polymers,consequently promoting the formation of protein gelation.