Study On The Rot-causing Microflora In Oyster Gill And Its Regulation Mechanism For Spoilage

Oyster is a filter-feeding bivalve molluscan shellfish gathered in the gills of a large number of micro-organisms.Mmicro-organisms in oyster gill use the nutrients for proliferation after shucked,and affect the the oyster qualities and shelf life.So far,the role of microflora in oyster spoilage,especially the role of microflora in the oyster gill spoilage was hardly investigated.Therefore,the microbial community structure and its variation,as well as the spoilage nature of the oyster gills were not fully revealled in the gills.The introduction of the DGGE,HPLC,Quantitative PCR and other new technologies into the study of the microbial community structure and variation,qualities changes,and biogenic amine production mechanism were performed in oyster gills.Further study of MAP on the microflora and its biogenic amine production mechanism during spoilage was carried out,in order to provide targeted basis on oyster preservation and scientific guidance for the application of preservation technologies.(1)The microbial isolation and identification for oyster gillsTraditional methods of counting,isolation and identification were used to analyze the microbial community structure and quantity for different seasons and different parts of the oyster.The results showed that the initial aerobic bacteria count(APC)of oyster gills was higher than that at the exocuticle or overall.The APC,Enterobacteriaceae,Pseudomonas and LAB in oyster gill increased during storage at 4 0C,10 ℃ and 20 ℃.Little difference in the microflora structure of oyster gills was found between winter and spring.(2)The effects of different temperatures on the microflora in oyster gillThis article established DGGE analysis method for the oyster gill microorganisms.A stepwise gradient centrifugation phenol-chloroform method was applied to extract the total bacterial genome DNA from oyster gill to analysize the microbial community structure and its variation during storage at 4 0C,10 ℃ and 20℃.The results showed that the DGGE method analysis of bacterial DNA extracted from the gill matix was more direct reflects on the microflora than the total bacterial DNA extracted from the culture medium.The microflora structure in fresh oyster gills were complex and had high diversity;Lactococcus raffinolactis and Enterobacter sp.were the dominant bacteria at 4 ℃ storage,other bacteria species including Pseudomonas sp.,Aeromonas spp.,Lactococcus spp.,Weissella confuse,Enterococcus spp.and Lactobacillus sakei..The L.raffinolactis,L.lactis,Lactobacillus curvatus and L.lactis subsp.Lactis were the dominant bacteria for 10 ℃ storage.Mocroflora structure in oyster gill was different at different refrigerated storage temperatures.Microbial community structure and its variation are complicated during storage period.(3)Biogenic amine production analysis in bacterial strains isolated from oyster gillThe HPLC column derivatization analysis was used to analysize the biogenic amines for culture bacterial strains isolated from the oyster gill at different storage temperatures.The results showed that the HPLC column derivatization method can be effective separation and determination of cadaverine,putrescine,histamine,tyramine and spermidine;Biogenic amine-producing positive genus included Enterobacter iaceae,Aeromonas,Lactobacillus,Lactococcus,Enterococcus,Leuconostoc and Clostridium;The cadaverine,putrescine and tyramine were the main biogenic amine production for bacteria strains isolated from shelf life endpoint of different storage temperatures,but histamine and spermidine were hardly found in these strains.The cadaverine,putrescine and tyramine were the main biogenic amine in oyster gill tissue during storage at different temperatures,and increased in the whole storage period,especially the cadaverine was at highest concentration,but the histamine and spermidine amount were at low level.The biogenic amines in oyster gill during spoilage were regarded as cadaverine,putrescine and tyramine.The changes of these three biogenic amines can evaluate the qualities of oyster gills.(4)Microbial effect on the changes of chilled oyster gill qualities during spoilagePearson Correlation was used to analyze the relation between qualities and microbiology in oyster gills.Correlations among the microbial indices(APC,Enterobacteriaceae,LAB Pseudomonas)and bio-chemical indices(TVBN,TMA,putrescine,cadaverine and tyramine)for refrigerated oyster gill were significant;the APC and TVBN,the LAB and tyramine,Enterobacteriaceae and putrescine,Pseudomonas and putrescine were the highest correlation,and the regression equation were significant.(5)Microbial community structure and variation in oyster gills under MAP storageDGGE and cluster analysis of oyster gill microflora for modified atmosphere packaging(C02:N2(50%:50%);C02:02(70%:30%);C02:02(50%:50%))at 4℃storage was performed.The results showed that the modified atmosphere packaging changed the community structure,inhibit the growth of Enterobacteriaceae and prolong the shelf life of oyster;the major bacteria were almost common in the same MAP condition for the different storage period dispite of the few differences in community structure.For 4 ℃ storage,the dominant bacteria in oyster gills for CO2:N2(50%:50%)and CO2:O2(50%:50%)packaging were Lactobacillus sakei,Lactobacillus curvatus and Lactococcus lactis subsp.Cremoris;Lactobacillus sakei,L.lactis subsp.Cremoris and L.raffinolactis for CO2:O2(70%:30%)。(6)Microbial effect of modified atmosphere packaging on oyster gills and bio-amine production of LABPearson Correlation was used to analyze the relation between qualities and microbiology in oyster gills under MAP.MAP of CO2:O2 inhibited the growth of APC and Enterobacteriaceae,thereby inhibited the accumulation of cadaverine,putrescine and tyramine in the oyster gill,and the TVBN value and TMA value were at lower level throughout the refrigerated storage period than other MAP groups;CO2:N2(50%:50%)packaging significantly inhibited the growth of Enterobacteriaceae,and retarted the increase of TVBN value TMA values,cadaverine,putrescine,APC,and Enterobacteriaceae,and had a great impact on the senses of oyster gill with acidity,but hardly inhibited the growth of LAB,therefore,CO2:N2(50%:50%)packaging can not effectively extend the shelf life.The characteristics of the bogenic amine production of lactic acid bacteria were investigated by HPLC and decarboxylase(TDC)gene amplification methods,and then using quantitative PCR analysize gene expression changes in tdc.HPLC method and the TDC gene amplification method were showed that the biogenic amine production strains isolated from CO2:N2(50%:50%)were more than that from CO2:O2(70%:30%)and CO2:O2(50%:50%)in TVBN end shelf life.Amine-production strain numberes of Lactococcus and Enterococcus was not significant difference,but had a significant lower number than the strains of Lactobacillus;Tyramine production mainly depends on the species of Lactobacillus.Quantitative PCR analysis of tyramine decarboxylase gene expression in the oyster gills under MAP storage.The results showed that CO2:O2(70%:30%)had the lowest copies of the TDC gene for LAB,followed by CO2:O2(50%:50%),and then CO2:N2(50%:50%).MAP of CO2:O2(70%:30%)promoted the growth of Lactobacillus sakei inhibit the growth of Lactobacillus curvatus which was the strong tyramine producing strain.This can maintain the good qualities and prolong the shelf life of oyster.MAP of CO2:N2(50%:50%)induce the growth of LAB with strong TDC gene production,and can not achieve good preservation effect.