Research On The Multi-strains Fermentation Process And Characteristic Flavor Substances Of Zhejiang Rosy Vinegar

Zhejiang rosy vinegar is a traditional condiment with long history and popular with consumers among southeast areas of China.Zhejiang rosy vinegar is made of indica rice and the natural fermentation microorganism comes from surrounding air and straw vat cover.The fermentation process of rosy vinegar is a trilateral process,the production of reducing sugar,the production of alcohol and the production of acidity are carried out at the same step.However,fermentation process of rosy vinegar is affected due to seasonal temperature and environmental conditions.Several production and processing steps,such as nesting and vat turning,are mostly done by manual operation.Therefore,the quality of rosy vinegar is difficult to reach consistent,and the yield and scale are limited.In this study,the effects of multi-strains fermentation on the quality of rosy vinegar were evaluated,including Aspergillus oryzae,Aspergillus niger,Monascus purpureus and Rhizopus oryzae.The characteristics of enzyme activity of traditional rosy vinegar during saccharification process were used as a model design to obtain the mixing ratio of each strain,which were performed the new saccharification process of factory koji-making instead of traditional saccharification process.The difference of quality and yield between multi-strains and traditional fermented rosy vinegar were compared.Further,high-throughput sequencing and metabolomics were applied to reveal the key metabolites and pathways of quality and flavor during the fermentation and aging stages.The main results are as follows:（1）Among the four fermented strains,A.oryzae had the highestα-amylase activity of 1631U/g,and R.oryzae had the highestβ-amylase and acid protease activities of 1522 U/g and 1867U/g,respectively.The enzyme activities of traditional rosy vinegar in the saccharification stage were characterized as follows:α-amylase:β-amylase:acid protease ratio is close to 1:1:2.Based on the characteristics of the enzyme system in the saccharification process of traditional rosy vinegar,the ratios of A.oryzae:A.niger:M.purpureus:R.oryzae were obtained as 5:9:1:0（group I）,12:0:2:1（group II）and 9:4:1:1（group III）.（2）The above mixed strains of three ratios were applied to the fermentation production,and natural fermented rosy vinegar was used as the control group.During the initial 10 days,the fermented mash of vinegar could all be stratified in about ten days of liquid fermentation,and the acetate film could grow normally during the fermentation process.The content of reducing sugar in groupsⅠ,ⅡandⅢwas 21.14,13.21 and 20.13 g/100m L at the beginning of fermentation,which was significantly higher than that of control group 2.03 g/100m L（P<0.05）,followed decreased to 0.31,0.12 and 0.24 g/100m L after five days of fermentation,and then gradually increased to 0.4,0.26 and 0.73 g/100m L at the end of fermentation.The content of alcohol in groups control,Ⅰ,ⅡandⅢreached to 4.9,7.95,8.6 and 8.35%（v/v）at about 10 days of fermentation,respectively,and then consumed undetectable gradually.The acidity continued to rise in the fermentation process,which arrived to 5.95,6.02,6.97 and 6.93 g/100 ml at the end of fermentation in groups control,I,II and III.The yield of rosy vinegar produced by different strains was calculated,reached to 4.77,5.55 and 5.84,respectively.GroupⅡandⅢwas significantly higher than the control group at 4.98（P<0.05）.（3）The flavor substances in rosy vinegar samples were determined by HPLC and HS-SPME-GC-MS.Eight organic acids were detected in all three groups of multi-strains fermented and traditional rosy vinegar,and the organic acid contents of groups I,II and III reached 51.51,54.45 and 71.312 mg/L,respectively,which were higher than the traditional control group of44.89 mg/L.Among them,the contents of lactic acid,acetic acid,pyroglutamic acid and succinic acid increased significantly.Compared with the volatile content of 4245.07 mg/L in the traditional rosy vinegar,the total volatile content of the multi-strains fermented rosy vinegar increased significantly,reaching 9557.02,9548.90 and 8641.14 mg/L.The contents of the main flavoring substances including phenylethyl alcohol,ethyl acetate,isoamyl acetate and phenylethyl acetate,etc.in multi-strains fermented rosy vinegar were significantly increased.The result of sensory evaluation showed that the basic quality and flavor of rosy vinegar produced by groupⅠ,ⅡandⅢwere slightly improved in ester aroma and vinegar aroma,but decreased in sweetness.The vinegar scores of groupⅠ,ⅡandⅢwere groupⅠ:19.68,groupⅡ:20.31,groupⅢ:22.5,compared to 20.05 of control group.Thus,rosy vinegar fermented with A.oryzae:A.niger:M.purpureus:R.oryzae as 9:4:1:1（groupⅢ）has higher flavor and quality.（4）The formation of characteristic flavors in the fermentation and aging stages of rosy vinegar were explored based on multi-omics techniques.The fermentation process of rosy vinegar is a trilateral process involving in various microbial activities.The microbial count showed that the yeast exhibited the highest population of 6.59 log CFU/m L around 10 days of fermentation,and then gradually died out.High-throughput sequencing further investigated the microbial changes in the fermentation process,showing that the rosy vinegar had high species abundance at the beginning of fermentation.The abundance of lactic acid bacteria and acetic acid bacteria reached more than 90%as the fermentation proceeded,dominating the fermentation process of rosy vinegar.As the fermentation process prolonged,the concentration of total volatiles increased from 4077.23 mg/L in the middle of fermentation to 6269.57 mg/L at the end of fermentation.The content of alcohols and acids increased significantly in the early fermentation stage,but decreased in the late stage of fermentation and aging process due to consumed as precursors.The total volatile compounds of rosy vinegar were 6059.18,7063.04and 7585.96 mg/L after one,three and five years of aging,respectively.Esters,aldehydes and ketones and heterocyclic substances were produced by esterification or Maillard reaction.The contents of ethyl acetate,phenethyl acetate,acetyl methyl and tetramethylpyrazine were greatly increased,promoting rosy vinegar with richer aroma.Moreover,the changes of non-volatile flavor substances of rosy vinegar in fermentation and aging stages were further evaluated based on the analysis of UHPLC-GC/MS.The results showed that the main substances were carbohydrates,fatty acids,amino acids and other similar derivatives.With VIP>1,P-value<0.001（t-test）,and Log₂FC>3 or<0.33,308 differential metabolites were screened during fermentation,and organic oxygen compounds,lipids and lipid-like molecules were metabolized by Acetobacter and Lactobacillus to produce organic acids,amino acids and derivatives.402differential metabolites were screened during aging,amino acids,including phenylpropanoids and polyketides,nucleosides,nucleotides and other substances,which could be produced by chemical reactions or Maillard reactions.All of these substances are mainly involved in 67metabolic pathways,in which the biosynthesis of valine,leucine and isoleucine being the most relevant pathways in the fermentation process,the biosynthesis of arginine and the metabolism of D-glutamine and D-glutamate being the main pathways in the aging process.In conclusion,the multi-strains fermentation process can successfully ferment rosy vinegar,which is suggested that the multi-strains fermentation process can be used in the actual production of rosy vinegar to expand the yield and maintain the traditional quality and flavor.Alcohols,acids and esters are produced by the metabolism of lactic acid bacteria and acetic acid bacteria during the fermentation stage of rosy vinegar.The metabolic pathways of amino acids in the aging stage promote the formation of various amino acid substances,which provide a rich taste to rosy vinegar.The above results suggesting that the aging stage has an important influence on the quality of rose vinegar.