Fabrication And Application Evaluation Of Compound Preparation Of Lactic Acid Bacteria From Tibetan Kefir And Bacillus From Chicken

With the rapid development of the livestock and poultry industry,the abuse of antibiotics is serious,and the problems of bacterial resistance and drug residues have caused serious harm to the sustainable development of the breeding industry and the ecological environment.As a new type of green antibiotic substitute,probiotic preparation has the functions of promoting animal growth,improving body immunity,preventing and controlling pathogenic bacteria infections,etc.Tibetan kefir is a kind of complex of various bacterial phases unique to Tibet.Its rich lactic acid bacteria have the functions of antibacterial,anti-infection and antioxidant activity.Bacillus can secrete digestive enzymes,provide nutrients and improve the body's immunity,and has the advantages of heat resistance and acid resistance.In this experiment,lactic acid bacteria with excellent biological activity and Bacillus with excellent enzyme production ability were separated and screened from Tibetan kefir and chicken manure,respectively.The evaluation test explored the effect of the compound preparation on the growth performance,immune function and intestinal flora of white feather broilers.Provide a theoretical basis for the application of probiotic complex preparations.The main research contents and results are as follows:1.Isolation,identification and biological evaluation of lactic acid bacteria from Tibetan kefirIn order to screen out lactic acid bacteria with excellent biological activity,this experiment used Tibetan kefir as the source of screening strains.Eleven strains of lactic acid bacteria were isolated and selected by the plate culture method.The heat resistance test results show that after heat treatment at 60? for 10 min,the survival rate of 6 strains of lactic acid bacteria is higher than 50%.After 16S rDNA identification,they were all Enterococcus faecium.The results of physical and chemical properties test showed that after 4 hours of culture with artificial gastric juice of pH=3,the survival rate of Y1 was significantly higher than other strains?P<0.05?,which was 52.56%.The surface hydrophobicity and self-polymerization ability of different strains were significantly different?P<0.05?.However,the co-aggregation ability with pathogenic bacteria was not significantly different between the strains?P>0.05?.Among them,Y1 has the highest surface hydrophobicity of 57.77%.Y1 self-polymerization capacity is 35.95%.The results of in vitro antibacterial tests showed that Y1 had higher antibacterial activity than other strains.The above test shows that Y1 Enterococcus faecium has good biological activity,which provides an application basis for the preparation of the next composite preparation.2.Screening,Identification and Biological Evaluation of Bacillus from ChickenIn order to screen out Bacillus with excellent enzyme production performance,this experiment initially screened out 20 strains of Bacillus from chicken manure.Through the protease and amylase production tests,3 strains were screened for enzyme production ability significantly higher than other strains?P<0.05?R5,R12,R16.After 16S rDNA identification,the three strains were all Bacillus amyloliquefaciens.The results of physical and chemical tests showed that the survival rate of R12 was significantly higher than that of R5 and R16 under heat treatment at 85?,90?,and 95? for 10 minutes?P<0.05?.After cultured with artificial gastric juice of pH=3 for 4 h,the survival rate of R12 was the highest,which was 51.49%.The results of in vitro antibacterial tests showed that R12 had the highest antibacterial activity.The above test shows that R12 Bacillus amyloliquefacien has good probiotic properties,which is conducive to the preparation of the next compound preparation.3.Development of probiotic compound preparation and optimization of fermentation conditionsIn order to prepare the optimal probiotic compound preparation,the test used the bacteriostatic activity against the pathogenic bacteria E.coli CMCC44102 and Staphylococcus aureus ATCC25923 as indicators to determine the optimal bacterial concentration compound ratio R12:Y1 to 1:3.On the basis of the most appropriate ratio,the single-factor test was used to optimize the inoculation volume,filling volume and incubation time of the compound preparation,and the optimal fermentation conditions of the composite preparation were determined from the test results:the inoculation volume was 2% and the filling volume When the incubation time is 40% and the culture time is 24 h,the compound has the highest antibacterial activity.4.Evaluation of the application of probiotic compound preparations in broilersThis experiment explores the application effects of probiotic compound preparations by affecting the growth performance,immune function and intestinal flora of white feather broilers The results showed that at 35 days of age,the average body weight of the experimental group ??feeding concentration 1.0×10⁸CFU/mL?and the experimental group ??feeding concentration 1.0×10⁹CFU/mL?compared with the PBS control group,respectively 5.6% and 1.4%.The average body weight of the experimental group ?(feeding concentration 1.0×10¹⁰CFU/mL)decreased by 1.3%;each experimental group had an increased effect on the thymus index and the bursa index compared with the control group,of which groups?and ?,Thymus index of group ? increased by 33.80%,28.73%,15.21%;The bursa index increased by 19.65%,16.72%,and 15.25%,respectively;the experimental groups?and ? can significantly increase the number of cecal lactic acid bacteria and reduce the number of E.coli?P<0.05?;At the same time,the structure of ileum villi in test group ? was more complete than that in control group.The above results indicate that the compound preparation has the effects of promoting the growth of white feather broilers,improving immunity and regulating intestinal flora,and the addition effect of group ? is better.The above test results indicate that this test was conducted by screening strains Y1 Enterococcus faecium and R12 Bacillus amyloliquefacien with excellent biological activity from Tibetan kefir and chicken manure,and made a probiotic compound preparation.The fermentation conditions are optimized to determine the best fermentation conditions.Animal experiments show that the compound preparation can promote the growth of white feather broilers and regulate the intestinal flora.