Analysis Of Low Temperature Characteristic Spoilage Bacteria And Its Signal Molecule Detection In Freshwater Fish In Northeast China

Bacterial cells trigger Particular gene expression in a cell-density-dependent Manner by producing,secreting molecules that accumulate in the environment in proportion to cell densiy when the density reaches a threshold level,a phenomenon termed ’quorum sensing(QS)’.N-acyl-homoserine lactones is signal molecules in Gram-negative bacteria.In this paper,we choose the content of AHLs produced by characteristic spoilage bacteria during the process of fish rot as the research objects.PCR-DGGE were used to filter out the characteristic spoilage bacteria when the fish reached the corrupt point under low temperature condition.To determine the mechanism of AHLs in inducing mast cell apoptosis by fluorescent staining,FCM and TEM.The model of mast cell was successful established by sodium alginate and oxidized grapheme that combined with the electrochemical sensing technology to check three kinds of AHLs and draw the standard curve.Simulation of pork corruption process,the corrupt fish in the AHLs were detected.Which provides a new theory and basis for AHLs detection in the process of freshwater fish spoilage,and provides a new,fast and accurate detection method for AHLs routine detection.The main research methods and results are as follows:1.We choose three freshwater fishes from DongBei-bighead,crucian and carp as the research objects.PCR-DGGE were used to examine diversity of bacteria in freshwater fishes during cold storage at 4 □.Extract total DNA of the three freshwater fish samples at freshness point and Spoiling point.Population fingerprints of the predominant microbiota were generated by DGGE analysis of universal V3 16SrDNA PCR amplicons,and distinct bands from DGGE were sequenced.Results showed that the bands in DGGE fingerprints changed significantly.At freshness point,the microflora of freshwater fish in DongBei was dominated by Aeromonas,Pseudowmonas,Flavobacterium,Shewanella,Acinetobacter and Lactococcus piscium;Aeromonas,Shewanella,Cetobacterium and Morganella were SSO at spoiling point.2.The effects of three AHLs on mast cells were determined by fluorescence microscopy,flow cytometry and transmission electron microscopy(TEM).The results of fluorescence microscopy showed that three kinds of AHLs had some regulation on mast cells,and apoptosis was observed.Flow cytometry showed that the apoptotic rate of mast cells in AHLs was about 17～20%,30C12-HSL showed the strongest effect.TEM images also showed that three AHLs could degranulate mast cells and ablate the nuclei.3.The graphene oxide was mixed with sodium alginate to form a hydrogel.The compatibility was measured by FT-IR and XRD,and the mast cells were fixed in hydrogel to establish the mast cell model.RBL-2H3 mast cells were used as the sensing medium.Mast cell sensors were used to detect the cells with different cell numbers.The optimal cell numbers were selected,and then the different concentrations of three kinds of AHLs were detected.The linear relationship between the impedance value and AHLs concentration was established by using electrochemical impedance as an index.The results of FT-IR and XRD showed that the optimized graphene and sodium alginate had good compatibility,and the mast cells were successfully immobilized in the hydrogel.The optimum proportion of GO in the hydrogel was 10%by electrochemical sensing,and the hydrogel had good electrical conductivity and plasticity.The linear relationship between the number of cells and the impedance value was found by electrochemical sensor.The linear equation was y=309.636x + 354.711,R2 = 0.99481.Mast cells were stimulated with different concentrations of 30C12-HSL.The higher the concentration of 30C12-HSL,the smaller the impedance value is,and the linear relationship is linear in a certain concentration range.The linear equation is y =-1339.252x + 3580.906,R2 = 0.9948.But when mast cells were stimulated by C4-HSL and C6-HSL,the relationship between concentration and impedance is nonlinear.4.Pseudomonas sp.and Shewanella putrefaciens were selected.The AHLs were extracted at different times.The results showed that the concentration of AHLs in the two strains increased gradually,the Pseudomonas reached the maximum on the 8th day and the Shewanella sp.Reached the maximum at the 6th day,and then gradually becoming steady.