Screening Of Lactobacillus Acidophilus From Animal Feces And Preparating Microbial Agent

Lactobacillus acidophilus,an important probiotic in the intestinal tract of animals,has many probiotic properties and is widely used in food,medicine,animal breeding and other fields,and has a very broad market prospect.This research aims to screen Lactobacillus acidophilus from healthy animal intestinal flora that can tolerate the gastrointestinal environment and have potential probiotic properties,and selected a strain of Lactobacillus acidophilus for high-density culture and preparation technology of high activity bacterial agent,which will provide theoretical basis and strain resources for the application in food and feed industry.The specific research results are as follows:(1)Screening and identification of Lactobacillus acidophilus:By using pure-culture method,a total of 12 strains of Lactobacillus acidophilus were screened and identified from the feces of healthy pigs under two feeding modes through molecular biological identification,morphological identification and physiological and biochemical identification.And these strains were stored in the State Key Laboratory of Food Science and Technology of Nanchang University.(2)The probiotic properties of Lactobacillus acidophilus:Compared to other Lactobacillus acidophilus,Lactobacillus acidophilus NCUA065001 had good tolerance to p H 2.5 and 1%bile salts,and its survival rate could reach 80.28%and90.87%respectively.Besides,the survival rate of this strain in the simulated gastrointestinal fluid environment has not significantly decreased,and could better withstand the pressure of the digestive tract environment.The adhesion ability of 12strains of Lactobacillus acidophilus to Caco-2 cells is significantly different.Among them,Lactobacillus acidophilus NCUA065001 has the strongest adhesion ability to Caco-2 cells,which is 5.56%.Lactobacillus acidophilus NCUA065004,NCUA0650013 has the worst adhesion ability to Caco-2 cells,both being 0.09%.The fermentation supernatant and fermentation supernatants adjusted to p H 7.0 of 12 strains of Lactobacillus acidophilus has significant differences in the antibacterial activity to7 common pathogenic bacteria.Some of the fermentation supernatants of Lactobacillus acidophilus had noticeable antimicrobial activities against some pathogens,but it decreased significantly after p H adjustment,while some Lactobacillus acidophilus still had noticeable antimicrobial activities against some pathogens after p H adjustment,the reason for this phenomenon may be was that some Lactobacillus acidophilus produce metabolites other than lactic acid.The fermentation supernatant and fermentation supernatants adjusted to p H 7.0 of NCUA065001 and NCUA0650011 had noticeable antimicrobial activities against some pathogens.In addition,the 12 strains of Lactobacillus acidophilus considered to be safe.Based on the comprehensive function of the strain,NCUA065001 was selected as the target strain for further study.(3)High-density culture of Lactobacillus acidophilus NCUA065001:Based on one-factor experiment,PB experiment,steepest ascent experiment and CCD experiment,the optimal medium of Lactobacillus acidophilus NCUA065001 was determined:maltose 4.94%,peptone fish 3%,bacterial peptone 2.25%,casein peptone1.3%,Mg SO₄·7H₂O 0.29%,anhydrous sodium acetate 0.41%,cysteine 0.08%,triammonium citrate 0.21%,K₂HPO₄ 0.2%,cysteine salt 0.02%.Subsequently,the culture conditions were also optimized and the optimal culture condition were:the culture temperature was 37?,the inoculum amount was 5%,and the initial p H was6.5,which is 4.835 times higher than that of MRS basal medium under normal conditions.The optimization effect was more significant.The high-density fermentation process parameters of Lactobacillus acidophilus NCUA065001 were further optimized,and the best process conditions were determined as follows:nitrogen was introduced,and 20%ammonia water was added to the automatic feedback feeding system to maintain the p H of the fermentation broth at 6.0.(4)Study on the preparation of the bacterial agent of Lactobacillus acidophilus NCUA065001:The optimal freeze-drying process parameters were further determined as follows:centrifugation conditions(8000 r/min and 5 min),protective agent(15%sucrose),pre-freezing time(2 h),the ratio of the bacteria to the freeze-dried protective agent(2:3),and resulting in a 76.79%survival rate of Lactobacillus acidophilus NCUA065001 and 5.2×10¹¹ cfu/g number of viable bacteria.