Analysis Of Spoilage Flora Of Freshwater Fish In Inner Mongolia During Low-temperature Storage And Research On Signal Molecules Of Specific Spoilage Bacterial Populations

China's freshwater fish is large in production and rich in species,In most regions,sales of live fish account for a major portion,and stores under low-temperature preservation conditions.The low processing level leads to the easy corruption of freshwater fish,causing huge economic losses to fish industry and a great waste of resources.Previous studies have shown that the spoilage of freshwater fish is mainly caused by spoilage bacteria.The growth and decay of most Gram-negative spoilage.bacteria are regulated by the self-secreted quorum sensing signal molecule AHLs.In this paper,the common carassius auratus,carp and striped bass from Inner Mongolia were selected as research samples to reveal their quality changes at 4?,and used High-throughput sequencing to explore changes in bacterial status at the point of fresh,fresh end,corruption.We isolated and screened specific spoilage organisms(SSO)from freshwater fish in Inner Mongolia and inoculated fish juice models to simulate corruption.Subsequently,a screen-printed electrochemical sensor using RBL-2H3 as a biometric element was constructed to monitor and evaluate the level of AHLs changes.It provides new and highly efficient technologies and means for the monitoring of corruption of freshwater fish,providing a theoretical basis and foundation for the prevention of corruption of freshwater fish during cryogenic storage.The main methods and results of this paper are as follows:1.Freshwater fish were stored at 4?,and the pH,electrical conductivity,microbial count,TVB-N and QI scores of samples increased significantly(P<0.05)during the cold storage.The corruption of striped bass was fastest,followed by carp,and the carassius auratus was the slowest.The fresh end point of carassius auratus was selected as the 12th day,striped bass and carp were the 8th day,and the corruption point of three freshwater fish were the 16th day.High-throughput sequencing analysis showed that the composition of bacteria at the fresh end poin was more diverse than that at the point of fresh and corrupted.The dominant genus at the fresh point includes:Aeromonadaceae?Ochrobactrum?Shewanella?Acinetobacter?Rhodococcus,etc.Aeromonadaceae?Pseudomonas?Shewanella?Vagococcus?Carnobacterium?Moraxellaceae,etc.is the dominant genus at the fresh end point.The dominant genus at the corruption point are Aeromonadaceae?Pseudomonas?Shewanella?Vagococcus,etc.Throughout the storage process,the proportion of Aeromonadaceae decreased,the proportion of Shewanella and Pseudomonas gradually increased,and Pseudomonas and Shewanella were identified as the dominant spoilage bacteria during the process of cold-storage of freshwater fish in Inner Mongolia.2.From the three kinds corruption samples,the growth test and colony morphology observation combined with physiological and biochemical identification was performed at 40C.A total of 43 candidate strains were isolated and screened.Through 4? growth test and colony morphology observation combined with physiological and biochemical identification,a total of 43 selected strains were isolated.We extract strain DNA for 16S rDNA sequencing.It was found that the candidate strains occupied the main components of Aeromonas,Shewanella,and Pseudomonas.Pseudomonas fluorescens strain L228 and Shewanella baltica OS678 are the SSO of Inner Mongolia freshwater fish in the 4? storage.3.Multi-walled carbon nanotubes(MWNTs)was selected to modify screen-printed electrode(SPCE)with a concentration of 8 mg/mL,scanning electron microscopy and electrochemical characterization showed that the basic resistance of the SPCE modified by MWNTs electrode was greatly reduced,and the sensitivity was improved.The optimal concentration of graphene oxide(GO)in the sodium alginate(NaAlg)gel was 0.1 mg/mL.XRD and FT-IR results show that GO and NaAlg fused well after gel formation.The results of CV,DPV,and EIS tests demonstrated the successful construction of the cell sensor.The resistance of the cells in the concentration range of 1×103 to 1×108 cells/mL linearly increased on the electrode.And the linear equation was y=720.04x+397.35,R2=0.9853,1×107 cells/mL was selected as the optimal cell concentration in follow-up experiment.4.After CCK-8 cell viability assay,Ca2+ fluorescence probe detection and flow cytometry,the results showed that the five AHLs had different degree of damage on RBL-2H3 cells and induced apoptosis.Transmission electron microscopy and scanning electron microscopy showed that AHLs was mainly due to cell membrane damage.The release of cell contents was accompanied by particle detachment.The cells with severe damage were shrunk and even broken,and the nucleus was ablated.An electrochemical sensor was used for the 30C12-HSL and C4-HSL detection.The 3OC12-HSL concentration in the range of 0.1-1.0 ?M,linear relationship between ?Ret and 3OC12-HSL concentration is y=-1530.5x+3789.6,R2=0.9904;C4-HSL concentration is in the range of 0.2?1.0?M,the linear equation is y=-1321.5x+4340.5,R2=0.9755,and the detection limit of both is around 0.1?M.The results show that the electrochemical cell sensor can be used for AHLs level evaluation.5.The Pseudomonas and Shewanella were respectively inserted into the sterile fish juice to simulate spoilage at 4?.With the prolongation of time,the bacterial concentration and TVB-N value gradually increased,and the protein concentration decreased.The change of cell Resistance value showed that the concentration of AHLs increased gradually from 0 to 6 days,and reached the highest level at 6th day,then decreased.The ability of the strain to decay was also decreased,indicating that AHLs regulate the ability of corruption.