Research On Screening, High Cell Density Cultivation And Mixture Of Lactic Acid Bacteria For Pickle Fermentation

Pickle is a traditional fermented food which has gained extreme popularity amongconsumers for its unique flavor and various kinds of health effects. Pickle fermentation isthrough high concentration of salt, using the microbes (mainly lactic acid bacteria) on thesurface of the vegetables to conduct natural fermentation, thus forming its unique color andflavor.Lactic acid bacteria is the main microbes participated in pickle fermentation. Thepreparation of direct vat set (DVS) of lactic acid bacteria starters is the prerequisite ofshortening the production cycle, improving product quality, ensuring product safety andrealizing standardized and large-scale industrial production of pickle. The screening of lacticacid bacteria, the high cell density cultivation and mixed inoculation of different lactic acidbacteria is the key to the preparation of DVS of lactic acid bacteria starters.In this paper, in order to develop a new kind of mixed DVS specific for picklefermentation, we screened lactic acid bacteria from the original culture fluid of various kindsof pickles specific for pickle fermentation, research on the high cell density cultivation andutilization of mixed lactic acid bacteria culture to ferment pickle. The main results are list asfollows:(1) Six strains have been screened from the original culture fluid of various kinds ofpickles, for their fast acid production rate and good nitrite depletion ability. With morphologiccharacter, physiological and biochemical experiment and16S rDNA identification, strain H3,H10, H12, H13were identified as Lactobacillus brevis (L.brevis), strain H8was Lactobacillusbuchneri, and strain H21was Lactobacillus sake.(2) The four selected L.brevis strains have been identified for the fermenting capabilityof cabbage using L.brevis CICC6239as control strain. After24h of fermentation, thefermented cabbage juice of strain H3contains the biomass of1.34×109CFU·mL-1, with pH3.44and titratable acidity of0.52%. The unique flavor compounds of the cabbage juicedetected was the most, indicating the most complete fermentation of cabbage juice among thefive strains.(3) The high cell density cultivation of H3was studied in shaking-flask level for mediumand primary culture condition optimization, and culture condition optimization was conductedin3-L fermentor level, then further carried out a pilot-scale optimization in30-L fermentorlevel. The optimized medium consists of glucose20g·L-1, yeast extract30g·L-1, citric acid1.53g·L-1, sodium citrate18.58g·L-1, VB620g·L-1, MgSO4·7H2O0.58g·L-1, MnSO4·5H2O0.25g·L-1, Tween-801g·L-1; An7%(v·v-1) concentration of inoculum was added to modifiedMRS medium for fed-batch cultivation in a3-L fermentor. The strain was grown at30°C, andthe pH was maintained at6.8by addition of20%(v·v-1) NH4OH. the glucose concentrationwas maintained at20g·L-1during the fermentation process with a500g·L-1glucose solution.A two-step controlled dissolved oxygen strategy was conducted: in0-8h of cultivation, thedissolved oxygen level was maintained20%by coupling the agitation speed and aeration; inthe late logarithmic growth phase (after8h), the aeration volume was set as0.75vvm and stirring speed was set as250r·min-1. The maximum viable cell counts was reached at22hwith the biomass of1.58×1010CFU·mL-1. In30-L fermentor level, the maximum viable cellcounts was reached at24h with the biomass of1.54×1010CFU·mL-1.(4) The effect of L. brevis H3and Lactobacillus plantarum CICC23138as startersindividually or combined on the quality of pickled cabbage was investigated. The cabbagewas fermented at30℃for72h. The viable cell counts, pH, titratable acidity, nitriteconcentration, flavor compounds and sensory analysis were measured. Results showed thatthe combination of H3and CICC23138with the inoculum concentration of1.5%and1.5%had the best effect on pickle fermentation. After72h of fermentation, the viable cell countsreached1.25×109CFU·mL-1, with pH3.13and titratable acidity of0.79%, nitriteconcentration of0.21mg·kg-1. The flavor components was the most and the score of sensoryevaluation was88.6/100.