The Dynamic Change Of Microbial Flora Of Vegetables Fermented With Starters During Spoilage

The researches of fermented vegetables always focused on the microflora changes during fermentation stage and the identification of spoilage organisms.But the dynamic change of microbial flora of fermented vegetables during deteriorated period is rarely studied.The microflora changes during the fermentation and the identification of spoilage prganisms in fermented vegetables are always the important research reseach filed at present.The main reported spoilage fungi were yeasts,because yeast always grew when the oxygen leaked into the fermentation systems.Therefore,the microflora changes during the deteriorated period in fermented vegetables,which were produced by stater Lactobacillus plantarum,were monitored in this study.2 fermented systems,oxygen leak and no oxygen,were carried out during the storage periods.Different microorganisms in feremented vegetables during spoilage stage were enumerated by selective medium.16S rDNA libraries were constructed to investigate the microbial flora at different stage.In order to ensure fidelity of the flora analysis 5 different DNA extraction methods were compared.Purity,concentration of the extracted DNA and 16S rDNA PCR amplification products were evaluated.The results showed that the quality of DNA extracted by soil DNA extraction kit of Fuji biotechnology company met the demand superiorly and was used for further study.Under anaerobic condition,lactate acid bacteria(LAB)(3.73×10~7 cfu/mL)was the main group of total bacteria(4.68×10~7 cfu/mL)when pH from 5.7±0.1 down to 3.4±0.1.During storage,LAB accounted for 53.7%of total bacteria.When the spoilage occured,the total number of bacteria rapidly increased nearly four orders of magnitude,and LAB were unable to be detected.The fungus revived to 6.03×10~5 cfu/mL at late-spoilage stage.During fermentation,L.plantarum dominated the fermented phase,but L.brevis was growing up during the storage time.On day 36,L.brevis(4.57×10~4 cfu/mL)replaced L.plantarum(7.76×10~3 cfu/mL)to be the main LAB.What's more,in non-spoilage samples,L.brevis was always the major one at storage and maintained 10~4 cfu/mL.In spoilage samples,L.plantarum grew to 2.00×10~5 cfu/mL and regained to be the chief one at the beginning of spoilage process(pH 3.4±0.1?4.0±0.1).While pH exceeded 4.5,L.plantarum was undected but other spoilage microorganisms such as Enterobacter and Clostridium grew up rapidly accounting for more than 23%and 18%respectively of total bacteria.Under air leaking condition,biofilm was formed on the surface of the brine.Biofilm is the typical properties of the fermented vegetables that have been spoilage.During the early 15days,pH of samples decreased from 5.5±0.2 to 3.6±0.1 and kept stable with the total bacteria declining from 1.50×10~7cfu/mL(S8)to 7.00×10~5log cfu/m L(S15).With the pH increased from 4.5±0.3 to 6.3±0.3,total bacteria climbed from 3.02×10~6 cfu/mL(S29)to 4.61×10~8cfu/mL(S57).The change of microorganism counts in biofilm was similar with that in brine.However,percentage of LAB in the brine and biofilm was different.In the brine,lactate acid bacteria accounted for 71.11%(S1)at beginning,and lowered from 63.33%(S8)to be undetected.From day 15,the proportion of LAB in biofilm was higher than that in brine.On day 29 and 43,LAB in biofilm was 61.29%(M29)and 30.00%(M43)which were much higher than that in the brine with 36.67%(S29)and 1.7%(S43).Flora of microbe at storage stage was different from spoilage time.During storage and early-spoilage process,L.plantarum and Cit.freundii were dominant.During middle-spoilage process,Ec.malodoratus and Prop.acidipropionici were the major ones.Leu.mesenteroides could be detected on day22,while couldn't on day36.And during late-spoilage process,Providencia rettgeri?Morganella morganii?Shewanella algae?Corynebacterium nuruki emerged,then grew vigorously.Under air leaking or anaerobic conditions,the fermented vegetables would become spoilage at different degree.At early-spoilage stage,L.plantarum grew and decreased to undetected level and the spoilage occured.Spoilage microorganisms began to grow at the same time.Furthermore,air leaking or anaerobic conditions,the composition of bacteria is different.Species of bacteria in air leaking jars(23 species)were much more than that in no air leaking jars(9 species).And the dominate specie is Clostridiumwith no air leaking.The result of this study provided theoretical basis for further analysis of the spoilage in fermented vegetables and for guidance dicision of the spoilage control and prevention.