Study On The Quality And Microbial Diversity During The Fermentation Of Large Yellow Croaker With Sweet Rice Wine

With the success of large yellow croaker cultured technology,the yield of large yellow croaker increased year by year,but the process of large yellow croaker is still based on frozen or skimmed frozen.The relatively single process technology has restricted the sale of large yellow croaker.The object was to analyze the variation of nutritional components,volatile substance as well as microbiota structures in the process of fermentation using the techniques of physical and chemical analysis,instrumental analysis as well as molecular biotechniques.Meanwhile,culture-independent technique metagenomics analysis system was also applied to investigate the microbiota variations.The main conclusions are as follows:(1)The variation of nutritional components of large yellow croaker during fermentation were analyzed and evaluated.The changes of moisture content and crude protein content were not obvious,but the contents of crude fat and ash decreased significantly.The contents of water,crude protein,crude fat,ash in fresh fish samples were 74.19%,17.57%,10.43%and 1.00%,while those of the sample collected on the thirty-fifth days were 54.52%,19.05%,13.17%and 2.68%respectively.The main saturated fatty acids were palmitic acid and stearic acid,while the highest levels of monounsaturated fatty acids were palmitoleic acid and oleic acid.The content of EPA and DHA was higher in the polyunsaturated fatty acids,and the content of EPA and DHA was at a higher level in the whole process of fermentation,reaching about 18%.Amino acids ratios of samples collected from different fermenting stages were reasonable and the content of flavor amino acids increased gradually.The minimum ratio of EAA/TAA was more than 38%,exceeding WHO/FAO standard(35.38%).Meanwhile,the minimum ratio of EAA/NEAA was over 70%,higher than the WHO/FAO reference protein pattern proposed standards(60%).(2)The variation of volatile flavor compounds in the process of fermentation were studied.The volatile flavor compounds of large yellow croaker during different periods of fermentation were extracted and identified by headspace solid-phase microextraction-gas chromatography-mass spectrometry.The contributions of volatile compounds to global odor perception were evaluated by a relative odor activity value(ROAV)combined with odor threshold.The results indicated that the key flavor compounds of the muscle sample collected from 0d and 1d were 1-Nonanal,2,3-Pentanedione,octanal,1-Octen-3-ol,heptaldehyde,hexanal,ethyl acetate and 3-Methyl-1-butanol.And the key components of those samples of 12d and 16d fermented days were ethyl acetate,ethyl caprylate,benzaldehyde,2-ethylfuran,hexanal,phenethyl alcohol,phenylacetaldehyde,2,4-heptadienal and 3-methyl-1-butanol.To the 30th and 35fth fermenting days the main flavor components were ethyl acetate,ethyl caprylate,2-decenal,benzaldehyde,2-ethylfuran,hexanal,phenethyl alcohol,phenylacetaldehyde,1-octen-3-ol,2,4-heptadienal and 2-pentylfuran.On the whole,the content of the volatile compounds increased from 51.51%to 84.06%during the fermenting processing.(3)The dominant microbial flora in the process of fermentation were isolated and identified.In total,108 Bacillus amyloliquefaciens,4 Bacillus methylotrophicus,6 Bacillus subtilis,22 Enterococcus faecium,5 Morganella morganii,4 Raoultella ornithinolytica,5 Vibrio spp.,5 Enterobacter aerogenes,3 Shewanella spp.,3 Staphylococcus warneri,3 Arthrobacter spp.,2 Chryseobacterium shigense spp.,2 Pseudomonas spp.,2 Psychrobacter spp.,2 Weissella hellenica,1 Citrobacter freundii,2 Staphylococcus epidermidis,1 Staphylococcus saprophyticus,1 Staphylococcus aureus,1 Wickerhamomyces anomalus,1 Kluyvera cryocrescens and 1 Bacterium lacticum isolates were picked up and purified.There were many kinds of microorganisms in the system,the number of Bacillus,Enterobacter and Staphylococcus were more than the others.In early stage of the fermentation,the number of Staphylococcus was large and caused the content of free fatty acid increased of the fermented meat products.The rapid multiplication of the Weissella hellenica changed the pH and flavors of fermented foods.The pH decreased rapidly and the other microbial species were inhibited.In the latter of this period,only Bacillus and a small amount of lactic acid bacteria remained,and the pH was stable at about 3.90.(4)Metagenomics was used in this study to reveal the variation of the micobiota structure in the fermenting process of large yellow croaker.The variation of bacteria during different periods was analyzed and dominant bacteria in each period were ensured through metagenomics.The results showed that as fermented time went on,the bacterial flora became increasingly single,samples of abundance decreased gradually,the diversity decreased and eveness deteriorated.Gram-positive bacteria were the predominant bacteria of samples in the whole process.In the early fermentation stage,the predominant bacterial flora was composed of Bacillus(45.06%).In the end of this period,the predominant bacteria were Weissella(26.38%)respectively.The predominant fungus in samples has changed a lot during fermentation,from Zopfiella(8.46%)at the beginning to Wickerhamomyces(94.71%)in the middle of process.According to cluster analysis of bacterial samples at genus level,we put these samples into five categories:1)samples fermented with wine and that fermented for 1d;2)samples fermented for 4-8d;3)samples fermented for 16d;4)samples fermented for 30d;5)fresh samples.The results showed that as time went by,the structure of microbial flora in fermented products changed gradually.