Isolation,Identification And Characteristics Analysis Of Spoilage Bacteria In Sichuan Pickle During The Deteriorating Process

Sichuan pickle is the typical fermented vegetable product and its production and sale ranks first in China.But the deteriorated products of Sichuan pickle during the fermentation and storage stages account for 5%~10% of total output and lead to large economic loss every year.The deterioration has become the restriction of the Sichuan pickle industrial development and the essential problem to be solved urgently.The researches involving Sichuan pickle always focused on the microflora changes during the fermentation stage and the spoilage organisms in deteriorated fermented vegetables.The reported spoilage organisms were mainly fungi such as yeast.Therefore,more attentions were paid on the isolation and identification and spoilage bacteria in Sichuan pickle during the deteriorating process in the study.The spoilage characteristics such as lactate utilization,enzyme production and microbial pellicle formation were analyzed.First,physical and chemical properties(including pH,nitrite,total acid,amino-nitrogen and microbial counts)were analyzed and spoilage microorganisms were isolated from different deteriorated stages of Sichuan pickle brine.The deteriorated procedure was divided to three stages: early stage(from day 6 to day 24),mid-stage(from day 25 to day 41)and later stage(from day 42 to day 55).The pickle brine from different stages were diluted and spread on tryptone soya agar(TSA)and 350 clones with different morphology were isolated.Lactate utilization and pH evaluation are the typical spoilage characteristics.Tryptone soya broth(TSB)with lactic acid(4g/L)was used to screen the lactate utilized bacteria.The amounts changes of lactic acid in the TSB were monitored by HPLC and 226 isolates were selected.46 clones from different stages were inoculated into pickle brine(the primary fermentation pickle brine,which was filtrated by 0.22 ?m membrane filter).The results showed that all the46 isolates could utilize lactic acid.According to 16 S rDNA sequencing analysis,Bacillus(5)and Citrobacter(6)were the dominant bacteria in the early stage of the pickle deterioration.Bacillus(3),Providencia(4),Citrobacter(3)and Morganella(2)were the dominant ones on the mid–stage.Morganella(4),Providencia(4)and Enterococcus(2)were the dominant bacteria in the later stage.The lactate utilization and the metabolite of the 46 isolated described above were estimated further.The 46 strains were inoculated into the pickle brine and cultured at 37 ?for 6 days.The results showed that Klebsiella sp.,which was isolated in the early stage of pickle deterioration,utilized lactic acid strongly.The reduction of lactic acid was accounted for 50% of the original amounts.The amount of acetic acid and propionic acid increased and the change rate of was over 140% and 720% respectively.The ratio of lactic acid utilization by C.freundii,S.cohnii subsp.Cohnii and Mo.morganii subsp.Morganii,which were isolated from the middle spoilage stage,were over 10%.The amounts of acetic acid and propionic acid increased and the rate of change was over 350% and 50% respectively.The ratioes of lactic acid utilization by Enterob.aerogenes,B.subtilis and Mi.oxydans,whichwere isolated from the later spoilage stage,were over 6%.The amounts of acetic acid and propionic acid increased and the rates of change were over 200% and 150% respectively.The production of plant cell wall degrading enzymes(PCWDEs)and biofilm formation(commonly known as “sheng hua”)were also typical characteristics of spoilage microorganisms.The PCWDEs production and biofilm formation of the 46 strains were analyzed.The results showed that 46 strains could not produce PCWDEs such as polygalacturonase,pectinase,cellulase,xylanase,amylase,protease and lipase enzymes.The13d-M-Ks-1(Klebsiella sp.)isolated in early spoilage stage,27d-S-Bs-6(B.subtilis)and27d-S-Sc-1(S.cohnii subsp.Cohnii)isolated in the mid-stage,41d-S-Pv-3(P.vermicola)and48d-M-Ks-2(Klebsiella sp.)isolated in the later period formed biofilm strongly.46 bacteria which caused pickle spoilage were isolated during in the different stages of Sichuan pickle brine during the deteriorating process.The characteristics of these spoilage strains including organic acid metabolism,characteristics of producing enzyme and the ability of producing biofilm were analyzed.And that provided a theoretical basis about the rule and prevention during the pickle corruption stage.