Research On Proteolysis And Flavor Formation Mechanism During Suanyu Fermentation

Suanyu,a traditional fermented aquatic product with unique flavor,is not only nutrition-rich,but also can be stored long.Therefore,Suanyu products have promising prospect in China.Our previous studies showed that fermentation with inoculation of some microorganisms isolated from Suanyu could speed up degradation of protein and improve the flavor quality of Suanyu.However,proteolysis and flavor formation mechanism during Suanyu fermentation are not clear.In this study,key odor compounds and taste compounds were investigated.Changes of proteins,peptides,and free amino acids were studied.Meanwhile,the role of microorganisms in protein degradation and formation of volatile compounds derived from amino acids were discussed.The results are of definite academic significance to further understand the mechanism of microorganisms in protein degradation and aroma formation.In addition,this study would provide an evident theoretical foundation for flavor optimization and product quality stabilization in freshwater fish processing industry.Firstly,free amino acids and small peptides,as taste compouns,were analyzed.Results showed that Asp and Glu had the highest TAV,and they played a synergic effect on the umami taste.27 small peptides,mainly dipeptides and tripeptides,were isolated in WSN of Suanyu.Secondly,volatile compounds were detected by GC-MS in Suanyu from Guizhou and Guangxi.Results showed that the main volatile compounds of Suanyu from Guizhou and Guangxi were mainly terpenoids,phenols,alcohols,and esters successively.Key odor component were 1-octen-3-ol,ethyl acetate,3-methyl-butyl acetate,ethyl hexanoate,nonanal,eucalyptol,and linalool,determined by the comparison of relative odor activity Value(ROAV)of volatile compounds.Proteolytic changes in Suanyu were determined.The p H value gradualy decreased to about 4.5,accompanied with an increasing trend detected in trichloroacetic acid–soluble peptides concentration.A decrease in sarcoplasmic and myofibrillar protein fractions was accompanied by an increase in the insoluble fraction.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)showed most bands of sarcoplasmic,myofibrillar,and insoluble proteins lightened or disappeared due to proteolysis or denaturation.New bands emerged especially in insoluble proteins.In the beginning of fermentation,the main parts were free amino acids(<0.18KDa)and peptids(>10KDa).Fluctuation of molecular weight distribution of the water-soluble nitrogen in Suanyu was found during fermentation,and free amino acid <(0.18KDa)and samll peptids(0.18-0.5 kDa)became the main parts in the end product.Free amino acids analysis showed that nearly all amino acids increased,except Trp,Arg and His.Intense proteolysis in Suanyu brought numerous of small peptides and free amino acids,which contribute to the taste and flavor of Suanyu.With comparison and analysis of electrophoretograms and free amino nitrogen in neutral and acidic model system,the role of endogenous enzymes and microbial enzymes in proteolysis of Suanyu was investigated.In model system without acidification,no significant change was found in the electrophoresis pattern before or after incubation for all samples.Free amino nitrogen of the control showed no significant change,while that of inoculate samples changed obviously.It indicated that endogenous enzymes did not work without acid activation in neutral medium,whereas microbial enzymes could hydrolyze small molecular proteins or peptides.In model system with acidification,significant changes were found in the electrophoresis pattern and free amino nitrogen during the incubation of all samples,but no intergroup difference was discovered.This illustrated that endogenous enzymes played a major role in sarcoplasmic and myofibrillar protein hydrolysis,while the proteolysis performance of microbial enzymes were negligible in acidic medium.Presumably,microorganisms hydrolyzed small molecular proteins and peptides by secreting proteases in early fermentation period before acidification.As fermentation proceeds normally,endogenous enzymes were activated by acids produced by lactic acid bacteria,whereupon a large amount of proteins were hydrolyzed to peptides and amino acids.Hence,acid,endogenous enzymes and microbial enzymes participate together in proteolysis of Suanyu protein.The role of microorganisms in formation of volatile compounds derived from Suanyu proteolysis was investigated by establishing flavor model system.All the strains grew well in the model system.Lactobacillus plantarum 120 and Pediococcus pentosaceus 220 could reduce the system’s acidity through lactic fermentation,activate endogenous enzymes,and release a large number of free amino acids.Staphylococcus xylosus 135 and saccharomyces cerevisiae 152 could not activate endogenous enzymes as a result of their weak acid-producing ability.Production ability of volatile compounds depended on strains.Lactobacillus plantarum 120 and Pediococcus pentosaceus 220 could produce ethanol and acetic acid by heterolactic fermentation,and metabolize Leu and Phe to 3-methyl-1-butanol and phenethyl alcohol.Staphylococcus xylosus 135 had a strong ability to synthesize acetoin,and transfer Leu into 3-methyl-1-butanol.But phenethyl alcohol was not detected.Saccharomyces cerevisiae 152 could generate ethanol and carbon dioxide by alcoholic fermentation,and transfer Leu,Ile,Val,and Phe into 3-methyl-1-butanol,2-methylbutanol,2-methyl propanol,and phenethyl alcohol and phenylcarbinol,separately.No significant increase in the varieties of quantities of volatile compounds of groups with combined inoculation compared with that of single ones was found.Certain compounds decreased or even disappeared,such as ethanol,carbon dioxide,phenethyl alcohol,and so on.But meanwhile,some compounds,which were not found in single inoculation samples,derived from amino acids were detected,such as 2-methyl propanol,3-methyl-1-butyric acid and 3-Methylthiopropanol.The results of principal component analysis indicated that the formation of flavor compounds is related to the strains inoculated.Phenethyl alcohol and 3-methyl-1-butanol were the major products in systems,which production might be closely associated with Lactobacillus plantarum 120,Pediococcus pentosaceus 220 and saccharomyces cerevisiae 152.While Staphylococcus xylosus 135 did not contribute significantly to the formation of flavor compounds derived from amino acid.