Optimization Of High-density And Large-scale Fermentation For Lactic Acid Bacteria

As serious concerns had been paid to the abuse of antibiotics,probiotics are becoming more and more widely proposed and used in animal feed,human food and pharmaceutical industries.As a typical microbial resources,lactic acid bacteria have attracted more and more attention.High-density and large-scale fermentation of lactic acid bacteria is to enhance the bacteria density by using certain technical means and special cultivation devices.Its goal is to achieve significantly higher bacterial density than those by the ordinary culture process,and ultimately,to increases the yield of bacterial cells by large-scale fermentation.In the producing industies,the cell density is a key indicator for the fermentation products of lactic acid bacteria.In this study,two lactic acid bacteria of Lactobacillus acidophilus and Lactococcus lactis(subsp.lactis)were selected as test strains,the culture media and conditions were screened and selected with the consideration of their effects on cell proliferation.And then,the conditions of high-density fermentation and composition of protective agents against freeze-drying for lactic acid bacteria were also optimized.The results of this study were as following:(1)Morphological characteristics of two strains of lactic acid bacteriaLactococcus lactis can form a clear white colony on MRS medium,with a diameter of about 1 mm.The opaquec olonies are with round and neat edges,and the surfaces are smooth and wrinkle-free.On the agar plate of MRS medium with addition of CaCO3,the surrounding of the colonies is transparent and forming hydrolysis circle due to acid production.The bacterial cells were spherical and did not form a chain by the observation under the microscope.Lactobacillus acidophilus can also form clear colonies on MRS medium.The colonies were round,white,convex,smooth surface,and clear edges with a diameter of about 1 mm.On the agar plate of MRS medium with addition of CaCO3,a transparent hydrolysis circle was observed around the colonies.Under the microscope,the bacterial cells were in the shape of short rods.Both strains of lactic acid bacteria were Gram-Positive by Gram staining.(2)Optimization of incubation conditions for lactic acid bacteria in laboratory scaleThe culture conditions for the two lactic acid strains were studied in laboratory experiments.The results indicated that inoculation volume(2-7%)did not significantly affected final viable count at the optimal conditions of the whey powder based medium,incubation temperature of 37 ?,and initial pH of 6.5.The maximum viable count of 1.92±0.15×108 CFU/mL was observed after incubation time of 28h.The optimal culture conditions for Lactobacillus acidophilus were determined as incubation temperature of 37?,the initial pH of 6-6.5,and the inoculation volume between 5%and 7%.The maximum viable count of Lactobacillus acidophilus growed in whey powder medium for 32 hours reached 1.53±0.15×108 CFU/mL.(3)Optimization for high-density and large-scale fermentation processWith the increase of the whey powder addition,the dry cell weight of lactic acid bacteria obtained by centrifugation increases continuously.When the whey powder content increased to 60%,the dry cell weight(DCW/L)increased to 13.21 g/L.With the increase of whey powder addition,the dry weight of the lactic acid bacteria in the fermentor can be significantly increased.In terms of the rate of bacterial powder vs raw material,it increased to 23.42%when the whey powder content increased to 60%for Lactobacillus acidophilus.4)Optimization on the protective agent against freeze-drying(lyoprotectant)By addition of 10%skim milk powder + 10%glucose or 10%skim milk powder + 10%trehalose to the bacterial cells obtained by centrifugation,a relative high recovery rate of bacterial cells was achieved.Four kinds of protective agents with remarkable protective properties against freeze-drying(lyoprotectant)were found by further screening and four of them were used in further experiments.The orthogonal experiments,with four factors/agents and three levels,were performed to opitimize the composition of protective agent against freeze-drying for Lactococcus lactis.The results showed that the influence of these four factors on the cell recovery rate after freeze-drying was significant and ranked as glucose>MnSO4>skimmed milk powder>trehalose.All these four factors significantly affected the cell recovery rates and the final experimental results.Through orthogonal experiments,a combination of these four agents was identified with strong protective ability against freeze-dried.And the combination was composed with addition of 90 g/kg glucose,90 g/kg trehalose,60 g/kg MnSO4,and 30g/kg skimmed milk powder to the cells produced.The freeze-dried bacterial powder should maintain at a low temperature during storage.The two kinds of bacterial powders prepared in this experiment were sealed and stored at-20? for 4 weeks,and the survival rate could still exceed 30%.(5)Resistance experiments for lactic acid bacteria against artifical gastroenteric environmentBoth lactic acid bacterial powders showed a certain resistance to artifical gastroenteric environment.The survival rate of Lactococcus lactis was 9.3%after digestion for 3h in aritificial gastric juice and was 8.6%after digestion for 4h in artificial intestinal juice.Based on the number of live bacterial cells,there were still more than 108 CFU/g of viable cells after the 7h digestion in the artifical gastroenteric environments.The strain of Lactobacillus acidophilus were more resistant to aritificial gastric juice than Lactococcus lactis.After 3h digestion in artificial gastric juice,the visible cells of Lactobacillus acidophilus was decreased from 3.64 × 1010 CFU/g to 5.9 X 109 CFU/g,and the survival rate was 16.3%.After 4h digestion in artificial intestinal juice,the survival rate of Lactobacillus acidophilus was 2.3%.The viable cells of Lactobacillus acidophilus could still exceed 108 CFU/g after 7h digestion in the artifical gastroenteric environments.