Screening Of Lactobacillus Acidophilus In Vitro And Study On High Density Fermentation Of Excellent Strain

Probiotic Lactobacillus acidophilus has the beneficial functions of enhancing immunity,relieving lactose intolerance and treating diarrhea.However,due to its slow growth rate,low cell density and complex nutrient requirements,the industrial application range is limited.Therefore,screening Lactobacillus acidophilus with good probiotic function and carrying out high-density culture are the key steps and technical bottlenecks for its application in food dietary supplements and microecological preparations.This article takes Lactobacillus acidophilus as the research object,the tolerance of artificial gastrointestinal juice and bile salt of the strain was determined.The strain with strong tolerance was screened for subsequent experiments.Then,by optimizing its static culture conditions,medium composition and high-density fermentation process,the biomass of the strain was significantly increased.Finally,combined with transcriptomics to study the gene expression and metabolic pathways of Lactobacillus acidophilus in the high-density fermentation process.The experimental results are as follows:(1)The results showed that the survival rate of Lactobacillus acidophilus IMAU81186 was 85.76%in p H 2.5 artificial gastric juice(3h),and the survival rate of Lactobacillus acidophilus IMAU81186 in artificial intestinal juice are 85.77%(4h)and33.69%(8h)respectively.The bile salt tolerance of IMAU81186 was also strong,and the bile salt delay time was 0.39h.(2)The static culture conditions of Lactobacillus acidophilus IMAU81186 were as follows:initial p H 6.5,temperature 35?,inoculum volume 3%.The optimal medium for Lactobacillus acidophilus IMAU81186 was glucose 30.18 g/L,soybean peptone 37.35g/L,fish peptone 18.68 g/L,sodium citrate 2.46 g/L,sodium acetate 6.125 g/L,K₂HPO₄2.46 g/L,Mg SO₄·7H₂O 0.4 g/L,Mn SO₄·5H₂O 0.04 g/L,serine 0.01 g/L,uracil 0.3g/L.After optimization,the number of viable cells reached(2.14±0.24)×10⁹ CFU/m L,which was 2.74 times higher than that before optimization((7.82±0.28)×10⁸ CFU/m L)CFU/m L.The optimal fermentation conditions of Lactobacillus acidophilus IMAU81186were as follows:constant p H 5.5,20%ammonia.When Lactobacillus acidophilus IMAU81186 was cultured in 5L full-automatic mechanical stirring fermentor,the growth rate,logarithmic phase and cell density were significantly increased.The number of viable cells reached(5.5±0.43)×10⁹CFU/m L which was 7.03 times higher than that before optimization((7.82±0.28)×10⁸ CFU/m L).(3)In this thesis,the gene expression of Lactobacillus acidophilus in different growth stages was analyzed by transcriptomics.Based on the difference of expression quantity of samples,the differentially expressed genes in each growth stage were selected and analyzed.The results showed that the key metabolic genes of glycolytic pathway,such as hexokinase(glk),phosphofructokinase(pfk)and pyruvate kinase(pyk),had higher expression levels at each growth stage,and their expression levels were significantly increased at logarithmic stage,and decreased to varying degrees at stable stage.In nucleotide metabolism,the de novo synthesis of purine nucleotide metabolism is mainly in logarithmic phase.Furthermore,it was found that the gene of pyrimidine nucleotide metabolism showed an upward trend in the whole growth period,which continuously provided energy for the growth and reproduction of the strain.