Analysis On Pellicle-forming Properties Of Spoilage Microorganisms Under Different Vegetable Fermented Conditions

Fermented vegetable is naturally fermented in relatively closed ponds or altars by a complex microbial system,which is dominated by lactic acid bacteria(introduced by vegetables and excipients).Fermented vegetable is produced and consumed throughout the world.However fermented vegetable is prone to deteriorate because of no sterilization and the complex microbial system.Accoriding to the statistics,the spoilage fermented vegetable in industrial production annually accounts for 10% to 20% of the total processing volume and leads to huge waste and economic losses.Pellicle formation is one of the typical features of spoiled fermentated vegetable.At present,the study on the pellicle-forming microorganism and its characteristics in fermented vegetables and the influence of the fermentation vegetable environment pellicle formation are not yet in-depth.In view of this,the pellicle-forming microorganisms and the pellicle formation characteristics are detected.Furthmore,the key factors affecting the formation of pellicle and the expression of pellicle related genes in model medium are analyzed.Firstly,the pellicle-forming microorganisms are isolated and identified.The samples of spoilage pellicle in fermented vegetable are collected and hanged in sterile saline.After serial dilution,tryptone soy agar(TSA)is used to isolate the pellicle formation microorganisms of dilution.Fifty isolates have been selected randomly from the TSA and inoculated into the fermented brine model medium(TFB).Six isolates which could form pellicle on the surface of the media are identified by 16 S r DNA or 18 S r DNA sequences.The results show that the pellicle-forming isolates are bacteria and identified as Bacillus subtilis,Citrobacter freundii,Staphylococcus cohnii subsp.cohnii,Providencia vermicola,Klebsiella sp.and Enterobacter cloacae,respectively.Secondly,the pellicle formation characteristics of six pellicle-forming microorganisms have been studied in fermented vegetables model medium.Lactobacillus plantarum is used as a microbial agent to ferment 6 kinds of vegetables(Cucumis sativus,Brassica pekinensis,Rupr Brassica juncea,Red Raphanus sativus,Vigna unguiculata and Capsicum annuum),and 18 kinds of model medium are prepared from different stage.It is used to simulate the fermented vegetable environment.The composition of nitrogen content,minerals and reducing sugar in the model medium is analyzed by the Keselidine method,the Graphite furnace-atomic absorption spectrophotometer and the 3,5-dinitrosalicylic acid colorimetric method.Those pellicle-forming microorganisms are inoculated into different model medium and cultured for 15 days.The characteristics of pellicle formation are analyzed.The results show that Bacillus subtilis could form pellicle in 4 fermented vegetables and is the most likely to form pellicle.While Providencia vermicola could only form pellicle in fermented cucumber and is the least likely to form pellicle.Six pellicle-forming microorganisms could form pellicle in cucumber model medium,and no strain could form pellicle in Red Raphanus sativus model medium.it is obvious that,Cucumber is the most likely substrate for the above microorganism to form pellicle,while Red Raphanus sativus is the opposite.Bacillus subtilis was further cultured in cucumber and Raphanus sativus model medium.Bacillus subtilis shows a normal growth trend in cucumber model medium,forms pellicle and rises the value of p H in the culture solution.In Raphanus sativus model medium,the number of Bacillus subtilis continus to decrease from the initial concentration of 4.0 log(CFU/m L),no pellicle is formed,and the value of p H in the culture solution remains unchanged.Thirdly,the effect of fermentation vegetable environment on pellicle formation related gene expression have been analyzed.Numerous studies have shown that the expression of sin I,eps H,eps C,and tas A genes in Bacillus subtilis are positively related to the formation of pellicle,and the sin R gene is negatively.Bacillus subtilis was cultured in four model medium: CVJ,CFVJ,RSVJ,RSFVJ and a control medium(TSB),respectively.Expression of pellicle-forming related genes of Bacillus subtilis is analyzed by the method of 2-??Ct comparative Ct in fluorescence quantitative PCR.The results show that fermented cucumbers could promote the expression of those promoting pellicle-forming genes and inhibit the expression of sin R in Bacillus subtilis.The effect of fermented Red Raphanus sativus is opposite.In summary,Bacillus subtilis,Citrobacter freundii,Coriolus subcordiformis,Providencia,Klebsiella,and Enterobacter cloacae are typical spoilage microorganisms in fermented vegetables.From the cellular level and the molecular level,Bacillus subtilis is the most likely to forming pellicle.Providencia vermicola is the least likely to form pellicle and could only form pellicle in fermented cucumber.Fermented cucumber is the most suitable medium for the growth of pellicle-forming microorganisms and forming pellicle,while fermented Red Raphanus sativus is the least.