| Research was done to screening and identify the strain from healthy rabbit cecum which could have a strong inhibition effect of diarrhea pathogens, such as Escherichia coli, and to preliminarily investigate the feeding effects of the strain. With E. coli Salmonella and Shigella as the indicator bacteria, screening and rescreening were done to select the strain with the strongest inhibition of E. coli. Tolerances of the strain for bile salts, gastrointestinal environment and high temperature environment were investigated. And the sensitivity of the probiotic strain against antibiotics and the safety of the strain were evaluated. The specie was identified through morphological observation, physiological and biochemical tests and 16S rDNA sequence analysis. Stain Tu-569 that was screened out from healthy rabbit cecum, with stronger inhibition of E. coli, Salmonella and Shigella, was identified as Bacillus velezensis. Results showed that strain Tu-569 had characteristics of good temperature tolerance, tolerance of 0.4% bile salts, normal growth and reproduction in gastrointestinal environment, sensitivy to streptomycin sulfate, oxytetracycline and gentamicin sulfate in medium dose, tolerance to diaveridine, furazolidone and sulfaguanidine in medium dose and safety for use in young rabbit, which showed its potential as feed additives to feed young rabbits.Single factor test of medium conditions in antibacterial substances production from strain Tu-569 were done to select suitable types of carbon sources, nitrogen sources and inorganic salts, and the medium composition was determined by the orthogonal test. Also, the orthogonal test of fermentation conditions in antibacterial substances production from strain Tu-569 was done to determine suitable temperature, bottling amount, rotation speed, inoculum size and fermentation time. The best medium and fermentation conditions for strain Tu-569 production of antibacterial substances was tound to be as follows: 1.5% soluble starch, 1% peptone, 0.07% MnSO4·H2O, 0.05% MgSO4·7H2O, initial pH 5.0, 2% of the seed medium inoculated to 50 ml/250 ml triangle bottle, 30℃incubation for 24 h. Under these condition, the antibacterial circle area of the fermentation liquid of Tu-569 was up to 322.0 mm2. Through the optimization of fermentation conditions, the best medium and fermentation conditions for strain Tu-569 to produce antibacterial substances were obtained. The production of antibacterial substances had been significantly improved.Research was done with various treatments, such as salting out with ammonium sulfate, extraction with different organic solvent, heating, different pH environment, proteases, ions, protein protective and denaturant agents, to analyze properties of extracellular antibacterial product from probiotic strain Tu-569. The extracellular antibacterial product was a polypeptide with a molecular weight of about 3.5-8 kD. The majority of the antibacterial polypeptide was salted out by 60% ammonium sulfate. The antibacterial polypeptide showed a good stability with heat, but could be degraded by proteases. The antibacterial polypeptide could be concentrated from butanol extraction without loss of antibacterial activity. The antibacterial activity of antibacterial polypeptide could be enhanced by Mn2+, but would be reduced in lower pH (pH 3) or higher pH (pH 11) buffer solutions. When surfactants orβ-mercaptoethanol was present in buffer solutions, antimicrobial peptides would show complete loss of the antibacterial activity. The peptides antibacterial activity against E. coli in 1mg of crude protein was equivalent to 2μg of gentamicin sulfate, 10μg of streptomycin sulfate or 50μg of oxytetracycline. Antimicrobial peptides of Tu-569 strain had a high and stable antibacterial activity against Escherichia coli, which is useful in applications of animal husbandry, aquaculture, food and other fields.The purification method of extracellular antibacterial peptide from the fermentation broth of Tu-569 strain was investigated. Fermentation was done under the optimal fermentation conditions producing antibacterial peptides of strain Tu-569. 23 L of fermentation broth which was rich in antibacterial peptides was obtained. Fermentation broth was centrifuged to obtain the supernatant which was treated by ammonium sulfate salt precipitation with the final concentration of ammonium sulfate at 60%. Dialysis was done after ammonium sulfate salt precipitation and antibacterial activity were determined. By DEAE-Sepharose Fast Flow ion-exchange chromatography, SDS-PAGE and other methods, components with strong antibacterial activity were purified. Results showed that elution peak 3 was getted using DEAE-Sepharose Fast Flow ion-exchange column eluting with 0.3mol/L KCl at pH 7.2. It was confirmed by Tricine SDS-PAGE that elution peak 3 was composed of the main component with relative molecular mass of approximately 7.8 kDa and minor constituents with the molecular weight of higher than 20.1 kDa.Bacillus velezensis strain Tu-569 fermentation broth was fed to young rabbits by adding it in different rations to the feed.The growth performance, diarrhea rate and death rate were determined by the small-scale feeding experiment. Fresh rabbit feces were respectively sampled at 7d, 14d, 21d and 28d to determine activities of digestive enzymes, such as cellulase, amylase, lipase and protease. The amount of Bacillus, lactic acid bacteria, yeast and E. coli in rabbit feces samples were determined using traditional microbiological methods by different selective media. And total DNA in feces were extracted to make intestinal colonies polymorphism fingerprinting by ERIC-PCR. The diversity index of flora in the feces was analyzed using Quantityone 4.6.2. Blood indicators were determined by collecting blood from ear vein after feeding for 28 days. The young rabbits were dissected, and changes in weight, color and texture of organs, such as heart, liver, spleen, stomach, kidney and thymus, was observerd to determine the health state of young rabbits. Results showed that there was no difference between different groups in ear length, ear width, length and bust. Compared with the control group, there was no difference in feed intake but the highest feed conversion rate was seen by adding 15mL/100g strain Tu-569 fermentation broth in the diet of young rabbits. Diarrhea and resultant death could be effectively prevented by adding 15mL/100g or 25mL/100g strain Tu-569 fermentation broth in the dietary of young rabbits. Compared with the control group, there was significant difference in indexs, such as leukocyte, erythrocyte, blood erythrocytes, lymphocyte percentage and monocytes percentage, and no significant difference in other indexs by adding strain Tu-569 fermentation broth in the diet of young rabbits. There was significant increase in weight of spleen and thymus by adding 15mL/100g strain Tu-569 fermentation broth in the diet of young rabbits, which could affect the immune function of young rabbits. The time required for the flora balance could be shortened and the amount of Bacillus, yeast and lactobacillus could be increased by adding strain Tu-569 fermentation broth in the dietary of young rabbits. Microbial flora diversity would appear earlier in the rabbit intestinal by adding 15mL/100g strain Tu-569 fermentation broth in the dietary of young rabbits. Digestive enzyme activities in young rabbits intestinal would be increased by adding 15mL/100g strain Tu-569 fermentation broth in the dietary of young rabbits, which could promote the digestion and absorption of nutrients. These studies had shown that strainTu-569 has good applications potential as an agent for the prevention and control of young rabbits diarrhea and improving the production performance of young rabbits. |
PDF Full Text Request