Study On The Mechanism Of Histamine Reduction By Lactobacillus Plantarum LPZN19 In Fermented Sausage

Histamine as the most toxic biogenic amine widely exists in fermented food.It was found that the average histamine content in traditional natural fermented sausages in many parts of China was more than 100 mg/kg,which may endanger human health.Studies have shown that lactic acid bacteria inoculation fermentation can reduce biogenic amines in sausage,but its internal mechanism is still unclear.In this study,lactic acid bacteria with strong ability to reduce histamine and strains with strong ability to produce histamine were screened from fermented sausages.The inhibitory mechanism of lactic acid bacteria on histamine-producing bacteria and the effect of lactic acid bacteria on the expression of key genes in the process of histamine synthesis were explored to clarify the inhibitory mechanism of lactic acid bacteria and its metabolites on histamine formation.In addition,the degradation mechanism of histamine by biogenic amine degrading enzymes was elucidated by investigating the degradation process and degradation products of histamine by biogenic amine degrading enzymes.The main research results are as follows:1.Screening and identification of histamine-reducing bacteria and histamine-producing bacteria in fermented sausagesFive strains were isolated from natural fermented sausage samples in western,eastern,northern,central and southern Sichuan,which met the standard of meat product starter and had strong histamine reduction ability.At the same time,five strains with strong histamine production ability were also screened.After identification,among the 5 histamine-reducing strains,No.4,24 and 72 were Enterococcus faecium,No.19 was Lactobacillus plantarum,and No.43 was Lactobacillus pentosus.Among the 5 histamine-producing strains,C1 was Enterobacter carcinogen,C2 and C4 were Enterobacter kobe,C3 was Morganella morganii,and C18 was Proteus mirabilis.The biological characteristics of 5 histamine-reducing strains showed that the growth,acid production and salt and nitrite tolerance of No.19 and No.24strains were significantly stronger than those of the other three strains.In summary,L.plantarum No.19 was selected as histamine-reducing bacteria,named as L.plantarum LPZN19,and P.mirabilis C18 was selected as histamine-producing bacteria,named as P.mirabilis PM18.The above two strains were used for further study.2.Mechanism of Lactobacillus plantarum LPZN19 and its metabolites inhibiting histamine formation by Proteus mirabilis PM18The minimal inhibitory concentration（MIC）and antibacterial curve of LPZN19 cell-free supernatant（LCFS）against PM18 were determined by double dilution method.The cell integrity,cell morphology,expression of histidine decarboxylase（hdc A）gene and histidine/histamine transporter（hdc P）gene,and intracellular and extracellular histidine and histamine contents of PM18 were studied to explore the inhibitory mechanism of LPZN19on histamine formation of PM18.Antibacterial activity and the content of organic acids and fatty acids of the cell-free supernatants of Lactobacillus plantarum LPZN19（LCFS）with different fermentation time was determined by Oxford cup method and GC-MS.Then,we identified and analyzed the metabolites in CFS by GC-MS and multivariate statistical analysis method.Finally,LCFS was isolated by semi-preparative liquid chromatograph,and the antimicrobial metabolites were identified.The MIC of the LCFS was 12.5μl/m L,and the antibacterial curve assay showed an inhibited growth of PM18.The increase of cell wall,cell membrane permeability and potential energy indicated that PM18 cell wall and cell membrane were damaged.The results of scanning electron microscope（SEM）and transmission electron microscope（TEM）revealed that the LCFS disrupted the normal morphology and structure of the cell.Moreover,the difference of intracellular and extracellular histidine and histamine content between the experimental group and the control group showed that the histidine decarboxylation ability and histidine/histamine transport ability of PM18 were weaker than those of the control group,which was consistent with the results of the decrease of the expression of hdc A and hdc P genes and proteins and the histidine decarboxylase activity in the experimental group.The results of antibacterial activity of LCFS at different fermentation time showed that the LCFS at 24 h had the strongest antibacterial activity,which was confirmed by the highest contents of organic acids and fatty acids of LCFS at 24 h.Lactic acid,phenyllactic acid,malic acid,aspartic acid,dodecanoic acid and propionic acid are the main differential metabolites.The LCFS was separated by semi-preparative liquid chromatography to obtain 4 main antibacterial components,including organic acids and fatty acids.In addition,we speculate that there are some amino acids with antibacterial activity in the antibacterial components.Therefore,the mechanism of L.plantarum LPZN19 and its metabolites inhibiting the formation of histamine by P.mirabilis PM18 at the cellular and molecular levels was clarified.3.Whole-genome sequencing and functional annotation of L.plantarum LPZN19The whole genome of LPZN19 was sequenced by Pac Bio RS II sequencing platform.The results showed that the genome included one chromosome and three plasmids,and the genome size was 3,350,978 bp and the GC content was 44.43%.Prokka software was used to predict the coding genes of the assembled genome,and it was found that the genome contained 3301 genes,including 3150 coding sequences（CDS）,68 t RNA,16 r RNA,and 1tm RNA.According to the functional annotation information of the whole genome of LPZN19,the multicopper oxidase（MCO）gene is the only gene found to have histamine degradation function.The gene is located on the chromosome,and the sequence length is1506 bp,and the genome does not contain amino acid decarboxylase gene.In addition,there were no acquired resistance genes and virulence genes in LPZN19 genome,and no genes related to human pathogenicity.Above study identified the genes with histamine degradation function in the LPZN19 genome,and showed that LPZN19 LPZN19 is a safe,reliable probiotic that does not produce biogenic amines and can be used in the processing of fermented meat products.4.Prokaryotic expression of L.plantarum LPZN19 multicopper oxidase（MCO）and its mechanism for histamine degradationProkaryotic expression,induction and purification of MCO were performed by homologous recombination and nickel column affinity chromatography,and its enzymatic properties were explored on this basis.The structure of MCO was identified by circular dichroism spectrometer.On this basis,the induction effect of histamine on the structure of MCO was studied by spectroscopic techniques.Finally,the degradation products of histamine by MCO were identified.The results showed that the purified recombinant MCO showed a single band by SDS-PAGE,and the enzyme activity reached 248.60 U/L.The recovery rate was 33.04%,and the purification multiple was 7,indicating that the purification effect of the enzyme was good.The enzymatic properties showed that the optimum temperature and p H of the enzyme were 50°C and 3.5,respectively.Zn²⁺,Mg²⁺and Cu²⁺activated the enzyme,and 1 mmol/L Cu²⁺increased its activity by 2.3 times.The enzyme activity decreased with the increase of Na Cl concentration.When the concentration was 5%,the enzyme activity could reach 57.71%.The recombinant MCO has high activity for histamine,tyramine,cadaverine and putrescine,indicating that the enzyme has strong degradation ability for the four main biogenic amines.The results showed that the secondary structure of MCO mainly includesβ-sheet and random coil,which contains four Cu²⁺and is a typical metal oxidase.Spectroscopic studies showed that the secondary structure,domain and tertiary structure of MCO changed to a certain extent with the increase of histamine concentration,indicating that MCO interacted with histamine.Degradation products of histamine by MCO were identified as imidazole acetaldehyde,hydrogen peroxide and ammonia.The above results showed that the recombinant MCO had high purity and activity,and had high catalytic activity for for 4 common biogenic amines.In addition,the degradation mechanism of histamine by MCO was clarified,and the degradation products of histamine by MCO were consistent with biogenic amine oxidase and biogenic amine dehydrogenation enzyme.5.Verification of histamine reduction mechanism in fermented sausageTaking fermented sausage inoculated with LPZN19 as the object,the expression levels of hdc A and hdc P genes and proteins in different stages of sausage production were verified,and the degradation products of histamine in fermented sausage were identified.Finally,the histamine content was determined.The results showed that the expression of hdc A and hdc P genes and proteins in fermented sausage showed a downward trend,which was consistent with the down-regulation of hdc A and hdc P genes and proteins under pure culture conditions,indicating that LPZN19 could significantly inhibit the expression of hdc A and hdc P genes and proteins in fermented sausage.The effect of histamine in fermented sausage on MCO produced by LPZN19 was reduced to imidazole acetaldehyde.In addition,the histamine content in inoculated sausage was significantly lower than that in the control group.In summary,the reduction mechanism of histamine in fermented sausage was verified.The results showed that Lactobacillus plantarum LPZN19 could inhibit the growth of Proteus mirabilis PM18 and the expression of hdc A and hdc P and inhibit the formation and accumulation of histamine by producing antibacterial substances and damaging the cell structure of amine-producing bacteria PM18.On the other hand,LPZN19 can produce MCO to degrade histamine to imidazole acetaldehyde,further reducing the content of histamine in fermented sausage...