Low Cost Culture,Preservation And Preliminary Application Study Of Lactobacillus Bulgaricus

Lactic acid bacteria is the most commonly used in microecological preparations.It has many beneficial effects on aquaculture,such as improving water quality,regulating microecological balance and improving body immunity.However,the cost of the medium used in the production of lactic acid bacteria is high,and the preservation method such as spray drying method and vacuum freeze drying method has the disadvantages of high equipment cost and complex process control,which is not conducive to the industrial production of lactic acid bacteria.In this study,we studied the high density culture and preservation of lactic acid bacteria,aiming at reducing the production and storage cost of lactic acid bacteria,improving the number of viable counts,and promoting the widespread use of lactic acid bacteria in aquaculture.The medium composition of Lactobacillus bulgaricus 1.2902 was optimized in a 250 mL shake flask,the optimized medium was as follows:glucose 30 g/L,soybean meal 28 g/L,corn flour 14 g/L,whey powder 28 g/L,K₂HPO₄ 3.0 g/L,triammonium citrate 2.5 g/L,sodium acetate5 g/L,Tween-80 1.25 mL/L,MgSO₄·7H₂O 0.2 g/L,MnSO₄·4H₂O 0.0625 g/L,after incubation for 36 hours,the viable counts in the fermentation broth was 5.376×10⁹ CFU/mL,and the cost of the optimized medium was reduced by 4000 yuan/t compared with the MRS medium;the optimum culture conditions were as follows:temperature 37?,initial pH7.1,inoculation amount 4%?v/v?,standing culture,after the culture was incubated for 36 hours under the above culture conditions,the viable counts in the fermentation broth was 6.07×10⁹ CFU/mL,which was significantly higher than that of the MRS medium 5.8×10⁸ CFU/mL.The results of high-density fermentation of Lactobacillus bulgaricus1.2902 in 5 L fermentor show that when the constant pH was 6.5,the ventilation volume was 2 L/min,the stirring speed of the impeller was 200 r/min,and 20%sodium carbonate was used as the neutralizing agent,the viable counts in the fermentor was the highest,after incubation for 30 hours,the viable counts was 1.48×10¹00 CFU/mL,which was significantly higher than that of 6.07×10⁹ CFU/mL in shake flask,and the culture time was shortened by 6 hours.Microbial liquid preservation process research show that under the condition of 4?,the centrifugal rotation rate was 4000 r/min and the centrifugal time was 20 min,the centrifugal yield was the highest,reaching 92.37%;and 0.01 mol/L PBS solution was used as the protective medium,the survival rate of the cells was 14.37%after one week of storage at 4?;in the protective medium solution of 0.01 mol/L PBS,2.02%trehalose,1.72%sodium ascorbate and1.06%sodium glutamate were used as the protective agent,after 4 weeks of storage at 4?,the survival rate of the cells reached 30.89%,which was 29.64%higher than that of the original solution.Add different concentrations of Lactobacillus bulgaricus 1.2902 to feed tilapia for 60 days,the results showed that:compared with the control group,the weight gain rate and specific growth ratio,the number of lactic acid bacteria and the activity of lipase,amylase,trypsin in the intestine,the activity of trypsin in hepatopancreas,the activity of acid phosphatase,alkaline phosphatase,lysozyme,superoxide dismutase,malondialdehyde and catalase in serum were increased in different degrees,while the feed conversion ratio,the number of vibriages in the intestine,the total number of bacteria were reduced to varying degrees.Therefore,the addition of Lactobacillus bulgaricus 1.2902 in feed can promote the growth and digestion,enhance immunity of tilapia.