| Daocai is a kind of fermented vegetable with local characteristics.In recent years,the problem of biogenic amine accumulation in fermented vegetables has attracted public attention.If it is not properly regulated,it will inevitably affect the long-term development of Daocai.In this paper,Taking the Daocai in different production stages of a food processing factory in Zhenyuan County,Guizhou Province as the research object,this paper studies the biogenic amine producing bacteria and degrading bacteria attached to the products,and selects excellent strains that can produce biogenic amines and degrade high concentration biogenic amines,so as to ensure the gain effect of trace biogenic amines on the body and avoid the damage of high concentration biogenic amines to the human body.Firstly,the amine-producing and amine-reducing abilities of the microorganisms isolated from the production samples were measured,and the strains with relatively complete amine-reducing species and higher amine-reducing rates were identified to explore the amine-reducing characteristics of the target strains;secondly,the third generation sequencing technology of pacbio was used to sequence and analyze the strain,and according to the sequencing results to complete the functional annotation and mine target genes;finally,the amine reduction mechanism of the strain was explored by bioinformatics tools and heterologous expression technology.This study not only enriches the types of biogenic amine-degrading bacteria in Zhenyuan Daocai and traditional fermented vegetables,but also provides theoretical support for the amine-degrading mechanism of biogenic amine-degrading bacteria,and provides new technologies and data basis for the healthy development of traditional fermented foods.The main conclusions of this study are as follows:1.42 strains of bacteria were isolated and purified from Daocai samples at different production stages.32 strains with biogenic amine production capacity were screened by plate color development method,and then the amine reduction capacity of each strain was quantitatively determined by HPLC technology.Finally,three strains LS1-002-014,LS1-003-016 and FJ5-002-005 with wide range and strong amine reduction capacity were screened.They were identified as Bacillus amyloliquefaciens,Bacillus subtilis and Bacillus subtilis by morphology and molecular biology.Among them,strains LS1-002-014 and LS1-003-016 could degrade 7 kinds of biogenic amines,and the degradation ability of spermidine was the strongest,and the degradation rates was43.95%and 34.41%,respectively.Strain FJ5-002-005 had obvious degradation effect on tyramine,and the degradation rate was up to 59.40%.However,this strain lacked the ability to degrade histamine and also had amine-producing performance.Compared with the blank group,the amount of histamine production increases by 4.59%.2.Through the amine reduction curve and growth curve,it was found that the biomass of strains LS1-002-014 and FJ5-002-005 reached the lowest value on the 4th day,but the amine reduction rate reached the highest value,while the amine reduction rate of strain LS1-003-016 reached the maximum on the 3rd day.All three strains showed a reduction in biomass and an early stabilisation and decline phase in mixed biomine culture,which was related to the lack of nitrogen source in the medium.A single biogenic amine had a certain inhibitory effect on the growth of the three strains,and the inhibitory effect of tryptamine and spermidine was the most obvious.High concentrations of nitrite could promote the degradation of tryptamine,but considering the harm of nitrite to the human body,itt would not be included in the culture conditions.Strains LS1-002-014 and FJ5-002-005 had the most obvious amine reducing effect when the content of NaCl was 0%at 37℃.However,strain LS1-003-016 had strong amine reducing ability when the content of NaCl was 0%at 30℃.3.Bacillus amyloliquefaciens LS1-002-014 had antibiotic resistance and contains two antibiotic resistance genes:clnA and vmlR,which could resist streptomycin antibiotics,oxazolidinone antibiotics,phenol antibiotics and other antibiotics by changing the target of antibiotics.There were many genes related to the metabolism of biogenic amine precursors and biogenic amine degradation genes in the genome of this strain,such as speA for decarboxylation of arginine to putrescine,speE for decomposition of putrescine,tryptophan transporter trpP,histidine kinase lnrJ,etc.4.Bacillus amyloliquefaciens LS1-002-014 had spermidine synthase（speE）,diamine-acetyltransferase（speG）,arginase（arg）,acetaldehyde and other key amine-degrading enzymes in putrescine and spermidine metabolic pathways Dehydrogenase（ALDH）.Putrescine was converted to spermidine by spermidine synthase（speE）,and then metabolized to lipids by acetaldehyde dehydrogenase（ALDH）;or putrescine was metabolized to lipids by diamine-acetyltransferase（speG）,acetaldehyde Hydrogenase（ALDH）,and finally was completed metabolized by the butyrate metabolic pathway.5.Spermidine synthase speE-1 was composed of 276 amino acids,with a molecular weight of 31425.94 Da and a molecular formula of C1437H2210N352O422S8,which was similar with 1iy9.1.A in the PDB database and the similarity was 95.27%.The enzyme had strong hydrophilicity and belonged to easily soluble protein.The protein contained no transmembrane region,no signal peptide,and existed in the cytoplasm.The enzyme activity was 2.22 U/L,and the highest degradation effect on putrescine was 34.53%.6.Aldehyde dehydrogenase ALDH was divided into ALDH-1 and ALDH-2.ALDH-1 was consisted of 495 amino acids,the molecular weight was 53995.45 Da,the molecular formula was C2411H3770N632O734S20,which was similar with 5gt6.1.A in the PDB database and the similarity was 62.63%.The enzyme was a hydrophilic,soluble protein without transmembrane region.The signal peptide existed in the cytoplasm;the enzyme activity was 9.31 U/L,and the highest can degrade 33.59%spermidine.ALDH-2 was an isomeric protein of ALDH-1 with a molecular weight of about 54 kDa,and the enzymatic activity was 7.78 U/L,which can degrade spermidine up to 38.67%. |
