Screening And Application Of A Beneficial Bacillus From Pigs

The series consequences of antibiotics abuse cause panic in the society. To find a safty and nontoxic replacer is imperative. Probiotics is just the good choice. After many years practice and research, Bacillus spp. is benefit to the growth and development of animals. Bacillus spp. can not only inhibit the growth of pathogenic bacteria, but also produce nutritious substances, and is one of the most potential sources of probiotics.Acorrding to the general screening methods for probiotics and criterion for Bacillus, B. licheniformis strain B36 was isolated. The characterization of this stain as a probiotic and the applied characteristic were studied, then the fermentation product technics was optimized. Its obvious effect on increasing weight and enhancing immunity were proved with piglet-feeding experiments. The results in detail were as follows:1. 160 Bacillus strains were isolated from the dejecta of the healthy and fertile pigs. The strain B36 had evident antistatic ability to the frequent enterpathogenic microorganism in the cultivation industry of domestic animal such as Staphylococcus aureus, Escherichia coli 0139 and Salmonella enteritidis. The strain B36 was calassified as B. licheniformis according to the results of some physiology biochemical tests and the analysis of 16S rDNA sequences. Further research indicated that the strain possess fortis tolerance to the artificial gastric juice, artificial intestinal juice and bile. In addition, it could play an important part in the intestinal tract of animals. At last, this stain possess some heat resistance to some extent, the survival rate of germ would maintain 100% when it was treated below 80℃. However, it would reduce to 5.5% at 100℃for 15 minutes.Besides, the strain B36 was sensitive to many kinds of antibiotics. Only bacillosporin B and ginebatin displayed weaker inhibited-effect ion for the B36 in the 24 tested-antibiotics, which indicated that the strain B36 didn't include drug resistance plasmids and it could't lead to the level transfer of the antibiotic resistance among the microorganisms in the intestinal tract of animals.2. By studing on the physico-chemical characteristic of antibiotic substances purified in the strain B36, it found that B. licheniformis B36 produced a kind of micro-molecular bacteriocin just like proteins, which showed broad-spectrum antibacterial action. Not only did the bacteriocin display strong antibacterial activity to Gram-positive bacterium, but also reflected antibacterial activity to some Gram-negative bacterium. However, it didn't have inhibition-affection to the eukarya such as yeast and mycetes. But this bacteriocin had a favorable ability of thermal stability and acid base stability; it could maintain 100% action after being treated 30 min at 100℃. In the condition of 115℃, 15 min and 121℃, 30 min, the action of the bacteriocin would lose 75% and 100% respectively. The action of the bacteriocin would have no change between the pH 4-10 and it would not loss any activity treated by the organic solvent as well. At last, the bacteriocin could resistant many common proteases except for the protease K, whose action would decrease 75% when treated by protease K. In the follow step, the bacteriocin was researched further with the methods of ammonium sulfate precipitation and gel filtration chromatography Superdex? G-75 analysis, and the result of mass spectrography analysis showed that the molecular weight of bacteriocin was 1422 Da.3. The optimal cultural composition of solid state fermentation for the stain B. licheniformis B36 was obtained through the single element experiment and orthogonal design. B. licheniformis B36 grown for 48 h at 37℃in solid state fermentation; a maximum of 1.73×10¹¹ spores/g dry substrate were obtained using rice straw powder (300 g/kg) and wheat bran (700 g/kg) supplemented with glucose (40 g/kg), peptone (20 g/kg), yeast extract (20 g/kg), KH₂PO₄ (10 g/kg) and CaO (5 g/kg) with an initial moisture content of 65 %.According to the optimal conditions as above, a new and practical type of sack bioreactor (Φ120×800 mm) was invented and used to the pilot scale production of solid state fermentation for the B. licheniformis B36, whose yield of spore could achieve 1.2×10¹¹ CFU/g .Compared with the shallow pan bioreactor, the sack bioreactor could save more culture space and have higher efficacy of production.4. Through the Plackett-Burman design of experiment, various factors was evaluted evaluated, which might influence the spore-production of B. licheniformis B36 in liquid fermentation and finally it was selected the main internal agent effecting the producing of internal agent. Base on the Plackett-Burman measurement result, through the response-contour method to estimate and optimize the major influence factors, a quadratic polynomial mathematical model could be derived. After the significance test of this model with some statistical methods, it could be concluded that this model indicates how those influence factors effect the spore yield for the fermentation of B. licheniformis B36. After taking the derivation of this quadratic polynomial and solving the inverse matrix, the optimal composition of the culture was obtained as follow: amylum maydis 46.6 g/L, peptone 3.8 g/L, legume 21.3 g/L (NH₄)₂SO₄ 1.9 g/L and CaCO₃ 2.4 g/L. The predictive yield of spore production was Y=8.9×10⁹ CFU/mL. The final yield of spore reached 9×10⁹ CFU/mL using the optimize composition of culture and the spores yield in 50 L fermenter reached 1.3×10¹⁰ CFU/ml. 5. In this test, one hundred and twenty hybridization- weaning piglets which were 21±3 day's old were chosed and divided into five groups randomly. The affection of different level B. licheniformis praeparatum addtive to the performance trait of piglets was studied. The results indicated that the addition of B. licheniformis praeparatum could increase the daily weight of piglets significantly and cut down the ratio of feed conversion. Meanwhile, it could prevent piglets from diarrhea and elevate the survival rate of piglets as well as improve surroundings of breeding.