Development And Application Of Complex Probiotics From Pigs

In recent years antibiotics are used as additives to be widely added to domestic animals diets.Which are play an important part in diseases prevention and cure and promoting growth.There are a munber of questions to be studied in every detail,such as:drug-residues and bacterial resistance.After many practice and research,probiotics are a safty and nontoxic replacer of antibiotics.Bacillus licheniformis and Enterococcus faecalis were isolated from the contents of the rectum of the healthy pigs,these bacterias hold the stabilization of intestinal microflora.Bacillus licheniformis and Enterococcus faecalis can not only inhibit the growth of pathogenic bacteria,but also produce nutritious substances.So Bacillus licheniformis and Enterococcus faecalis can be added to domestic animals diets.Acorrding to the common screeing methods for probiotics,Bacillus licheniformis strain B1 and Enterococcus faecalis strain NA14 were isolated that have the probiotic characteristics,the aim of this study is the research of biological property,bacteriocin and application of strain B1 and NA14.the tests show that these two strains can be increasing weight and enhancing immunity of piglets.1.Screening,Identification and Biological characteristics of Bacillus licheniformis and Enterococcus faecalis from the healthy pigs3 Bacillus and 18 Enterococcus from the contents of the rectum of the healthy piglets were isolated in this study.In antibacterial test Bacillus strain B1 and Enterococcus strains NA1,NA3,NA4,NA10,NA14,NA18 had evident inhibitoty action on Escherichia coli O157,Staphylococcus aureus and salmonella enteritidis C78-1 that were used as indicator strains.The strain B1 was calassified as Bacillus licheniformis and The strains NA4 and NA14 were calassified as Enterococcus faecalis according to the results of morphology observation,physiology biochemical test and the analysis of 16SrRNA suquences.Future studies about the biological characteristics of there stains have shown that the strains have a strong tolerance to artificial gastric juice,artifical intestinal juice,cholate and bile.The strain B1 can withstand artificial gastric juice for 2h and artifical intestinal juice for 2.5h,the survival rate of the strain would maintain 100%when it was treated with 0.3%cholate for 12h and 90 ℃ for 15min.The number of the strains NA4 and NA14 had reduced by an order of magnitude when they were processed with artificial gastric juice for 2h,but the survival rate of the strains would maintain 100%when they were treated with artifical intestinal juice.Strain NA14 is the better tolerance to cholate than NA4.There two strains have a strong tolerance to 60 ℃.In antibiotic susceptibility test the strains B1 and NA14 were sensitive to many kinds of common antibiotics,only there two strains have a tolerance to penicillin,chlortetracycline and bacillosporin B,which indicated that the strains didn’t have resistance of antibiotic,it could’t level transfer among microorganisms in animals.So the strains can be used with some antibiotic.The adherence rate of the strains B1 and NA14 to IPEC-J2 cell were 0.88%and 8.7%,the result show that Enterococcus faecalis have a strong adhesive ability to cell.2.Purifying and Identification of antibiotic substances produced by the two strainsThe fermented liquor of the strains B1 and NA14 showed broad-spectrum antibacterial action.they display strong antibacterial activity to all bacterium in this test.Antibiotic substances in supernatant of culture medium were purified by ammonium sulfate precipitation method.The B1 bacteriocin had a favorable ability of bile stability and acid base stability,its antibacterial activity didn’t change after being treated at 100℃ for 30min and pH 6-9.In addition to protease K,the bacteriocin could resistant common proteases.The molecular weight of the bacteriocin was between 3000-5000Da by Ultrafiltration device.The highest resistant temperature and the best pH of the NA14 bacteriocin were 50℃ and between 6-7 respestively.Only it also was sensitive to protease K.The molecular weight of the NA14 bacteriocin was between 5000-10000Da.At last,the bacteriocins of strains B1 and NA14 were detected by PCR,the results show that NA14 can product enterocin A.3.Optimization of fermentation process of bacteaial strains B1 and NA14On the basis of the fermentation basal medium,the fermentation condition and medium component of the strains B1 and NA14 were optimized.The optimal cultural condition of strain B1 were the initial pH of 6.5,bacterial load of 8%,cultural temperature of 37℃ and cultural speed of 190r/min.the best cultural component:glucose(5%),beef extract(20g/L),CaC12(10 g/L)and MgS04(5 g/L).The best optimal cultural condition of strain NA14:the initial pH of 7.0,bacterial load of 6%,cultural temperature of 37℃ and cultural speed of 180r/min.the best cultural component:sucrose(1%),beef extract(20g/L),CaC12(lg/L)and MgS04(1 g/L)4.Appilication of the probioticsIn the study,one hundred and sixty weaned piglets of three-way cross-bred[Duroc×(Landrace X Large White)]which were average 30 day-old were chosed and assigned to four groups:fed with basic diet added chlortetracycline(75mg/kg),basic diet added chlortetracycline and Bacillus licheniformis(75mg/kg and 2×1011CFU/g),basic diet added chlortetracycline and Enterococcus faecalis(75mg/kg and 2×1011 CFU/g)and basic diet added chlortetracycline,Bacillus licheniformis and Enterococcus faecalis(75mg/kg,1.5×1011 CFU/g and 5×1010CFU/g).The total testing time was 30 day.The results indicated that the probiotic groups could increase the daily weight of piglets and cut down the ratio of feed conversion and diarrhea.The probiotics could inhibit the growth of E.coli and promote the production of immunity,The complex-Probiotic-Preparation was better than the single-Probiotic-Preparation by comprehensive evaluation.