| Recent years, epidemic disease emergenced on and on, which became the important reason to restrict the benefit of chicken. Especially pathogenic escherichia coli caused diarrhoea of chicken, large areas, serious infectivity, high death rate. At present, feedstuff of chicken added antibiotics additive in second treatment dose to inhabit and kill pathogeny,which brought a series of problems. At first, though it killed pathogeny, it killed normal commensal bacteria of chicken at the same time. It destoried the balance of microorganism; at second, antibiotic resistant pathogeny and variation pathogeny came into being. These compelled breeders to add more antibiotics, use new medican, which brought higher antibiotic resistant pathogeny and intestinal micro envirenment deteriorated further.A great deal of researches indicated that microorganism preparation could adjust the microorganism balance of intestines. It prevented and cured diarrhoea, at the same time, avoided antibiotic resistant. It has more benefits which antibiotics can't compare. It has development potential, so countries of the world proposed and extended, focused attention on it.300 strains of bacillus isolated from feces of healthy youth chicken, in which 152 strains resisted pathogenic escherichia coli and J-4 has the most significant inhibiting effect on pathogenic escherichia coli. Through morphological observation, physiological and biochemical experiments and 16S rDNA gene sequence analysis for identifying its species, it is confirmed that J-4 is Bacillus subtilis.Microorganism preparation excrete polypeptide which have high pathogenic resistance and reduce easily. This test analysed fermentation liquid of J-4. Organic impregnant extraction, salt out, high protein enzyme sensitivity, acid and temperature endurement, were made. It figured out that the pathogenic resistance substance were peptides.Most probiotic bacilli have pathogenic resistance out of body and invalid in intestine. The main problems are that stomach acid, gallbladder salt and intestinal juice killed the bacillus. To get over these disadvantages, detection was made to observe the tolerability of bacterial strains. After taking effect in stomach acid and the pH is 2.0 for 3 hours, the survial rate is 90%; after taking effect in gallbladder salt and the enderance gallbladder salt concentration is 0.3%, after taking effect in intestinal juice for 3 hours, the survial rate is 80%. Results show that, strain J-4 can surve and establish in intestine. The survial rate of J-4 spores is more than 88%after 70 minutes heat in 80℃hot water, so J-4 strain is expected to become probiotic bacillus.To increase the concentration of J-4 fermentation liquid for diarrhoea prevention and cure, we optimized the fermentation condations. Single-factor test and orthogonal experiment methord indicated that the best carbon source was corn flour, the best nitrogen source was earthnut flour and the best inorganic salt was MnSO4·H2O, the optimum fermentation medium and optimum fermentation conditions for concentration production of J-4 strains as following:corn meal in 1%, earthnut flour in 2%, MnSO4·H2O in 0.07%, NaH2PO4·2H2O in 0.2%, Na2HPO4·2H2O in 0.4%. The optimum fermentation conditions were:original pH value was 7.0, volume was 30 mL/250 mL, inoculation amount was 2%, rotate speed was 220 r/min and formentation temperature was 37℃. After optimization, the concentration production of J-4 was improved to 1.83×109 cfu/mL compared with the original 2.23x108 cfu/mL,7.2 times more. Spore production rate was up 95%. It established foundation for further research and application.To test security of strain J-4, the formentation liquid was feed to mice and chicks. We didn't found any pathological changes in viscera and intestine, so strain J-4 was safety Bacillus subtilis subsp.subtilis. To get more information of effect on diarrhoea disease, former of diarrhoea caused by athogenic escherichia coli in mice was made. Formentation liquid of J-4 was feed to cure.The results showed that it could promote the growth of beneficial bacteria such as lactic acid bacteria and it could reduce the growth of harmful bacteria such as escherichia coli. The diarrhoea rate and the mortality rate of J-4 was distinctiy lower than comparison group. The former of diarrhoea caused by athogenic escherichia coli in chicken was made, which received effective prevention and treatment from J-4 stain.Probiotic bacillus J-4 had excellent effect on diarrhoea disease of chicken. It provided new probiotic bacillus to prevent and cure diarrhoea of chicken. |
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