Research On Bio-Diversity Of Microorganisms In Huangshan Stinky Mandarinfish Fermentation

Mandarinfish is a kind of edible freshwater fish which is tender, delicious and nutritious. Traditional stinky Mandarinfish is manufactured from fresh madarinfish and a small amount of spice by short-term spontaneous fermentation at the condition of low-salt (5%) and low-temperature (10-14℃). It is one of the most famous traditional dish in Anhui prvince and note for its uniquely firm but tender texture and special strong odor, so it has a good reputation from all the consumers.In the sduty, stinky Mandarinfish samples were collected from different fermenting stages, and phenotypic and genotypic tests were carried out to all the isolates from the samples. Denaturing Gradient Gel Electrophoresis (DGGE) was used to check the variation of the microbial population during the fermentation. The research results were as follows:In total of 60 lactic acid bacteria were obtained from Huangshan stinky Mandarinfish samples. Firstly, the morphology characters were investigated for general clustering, then the 60 isolates were classified as 18 groups according to their physiology and biochemistry characters. Furtherly, combining the results of RFLP 16S rDNA analysis obtained with HaeⅢ, HhaⅠ, HinfⅠ, TrulⅠ, and the results of (GTG)5-PCR fingerprint analysis, representative strains were selected for 16S rDNA sequencing. Finally, the 60 isolates were identified to 6 genus and 8 species, including Lactococcus lactis, Lc.garvieae, Lc.raffinolactis, Streptococcus parauberis, Lactobacillus sakei, Vagococcus sp., Macrococcus caseolyticus and Enterococcus hermanniensis. The strains isolated from initial fermenting stages belonged to 6 species, in which the predominant LAB were Lb.sakei and S. parauberis. As the fermentation went by, Lb.sakei became predominant lactic acid bacteria after the fourth day, account for 92% of late fermenting stage.In total of 53 Enterobacteriacea were obtained from Huangshan Stinky Mandarinfish samples collected from different fermenting phase. The morphology characters was studied initially, then combining the rusults of RFLP 16S rDNA analysis by AfaⅠ, MspⅠ, HaeⅢ, and the results of ERIC-PCR fingerprint analysis, representative strains were selected for 16S rDNA sequencing. On the whole, the 53 Enterobacteriacea was classied to 3 family,13 genus and 14 species, including: Providencia rettgeri, Morganella organ, Moellerella wisconsensis, Proteus vulgaris, Klebsiella sp., Buttiauxella grestis, Hafnia alvei, Plesiomonas shigelloides, Aeromonas caviae, A. veronii, Acinetobacter junii, Enterobacter sp., Kluyvera ascorbata, Enterobacter ludwigii.According to the data acquired from API 20E experiment, except for the Non-fermentative bacteria Ac. Junii, and Aeromonas sp., all of the other isolates could be identified to species level and showed a good correspondence to the results obtained from genetic identification.The salt-tolerant bacteria obtained from Stinky Mandarinfish exhibited a higher bio-diversity with 44 isolates classified to 9 genus by directed 16S rDNA sequencing. In total,17 Psychrobacter spp.,15 Vibrio spp.,3 Staphylococcus spp.,3 Macrococcus spp.,2 Oceanimonas spp.,1 Marinobacter sp.,1 Arthrobacter sp.,1 Brevibacterium sp. and 1 Marinilactibacillus sp. were detected.In initial stage of fermentation, the salt tolerant bacterial population showed a higher diversity. However, starting from the fermenting day, predominant bacteria appeared and various species took over the places in different fermenting stages. Psychrobacter and Vibrio were the predominant bacteria on the forth fermenting day, and Vibrio became the unique dominant population on the eighth fermenting day of Stinky Mandarinfish final product.The DGGE analysis of 16S rDNA V6-V8 region revealed that Enterococcus sp., Shewanella putrefaciens, Macrococcus caseolyticus, Exiguobacterium acetylicum were dominant strains during the fermentation. Enterococcus existed during the whole fermenting stage with a high abundance. Shewanella putrefaciens was detected in second day samples, and decreased in later fermentation stages until end. M. caseolyticus appeared in fresh Mandarinfish, undetected in second day, and increased from fourth day of fermentation to end. A. veronii, Uncultured Aeromonas sp. and A. salmonicida were only detected in fresh Mandarinfish and second day. Brochothrix thermosphacta and Pseudoalteromonas issachenkonii were detected in fresh Mandarinfis and the fourth day separately.The results of DGGE analysis showed that there were a large number of spoilage microorganisms in the fermentation process. Enterococcus sp. and M. caseolyticus were the common bacteria isolated in fermented milk and meat, but the other isolates had no relation to food fermentation.All the results showed that that there were certain number of fermentation strains which used as starter culture in traditional fermented fish products, but there were also a large number of spoilage microorganisms, pathogens or conditioned pathogen in the fermentation process. The existence of those bacteria may threaten the safety of Stinky Mandarinfish products.The results were helpful for screening industrial starter, controlling the hazards of harmful microbe and improving the quality and safety of stinky Mandarinfish.