Tolerance Of Clostridium Butyricum And Its In Vitro Probiotic Characteristics Of Broilers

Antibiotics were forbidden gradually all over the world due to the severe effect of veterinary drug residue on human health and the occurrence of drug-resistance strains as a result of antibiotics abuse.The research of green feed additives to replace antibiotics is,therefore,becoming a hotspot in feed field.Clostridium butyricum(C.butyricum),an anaerobic intestinal probiotics,is characteristic of zero vestigial and productivity enhancement on animals compared to antibiotics and chemical synthetic drugs.These unique properties determined that it could be widely used in livestock and poultry diets to increase the economic benefits of the animal husbandry.However,microorganisms,a variety of organisms with different functions,had great differences in their physiological characteristics due to species differences.Therefore,this study was designed to investigate the growth characteristics of a strain of C.butyricum in a particular species,its ability to resist the process of pellet environment during feed processing,and its in vitro probiotic properties.It was looking forward to provide scientific basis for the application of C.butyricum in livestock and poultry production.In the identification test of C.butyricum,its colony morphology was determined by solid plate culture method,and its gas production capacity was determined by liquid culture method.The bacteria was stained by Gram stain to observe bacterial morphology.And its 16 s rDNA was extracted and sequenced to determine its species classification;In the resistance test,it was evaluated the tolerance of C.butyricum during high temperature,high pressure,artificial gastric juice and intestinal digestive enzyme in the trial;In the evaluation of the in vitro probiotic characteristics of C.butyricum,its growth curve and pH change curve were measured by absorption spectrophotometry.The Escherichia coli,Pseudomonas aeruginosa,Staphylococcus aureus,Vibrio parahaemolyticus,and Clostridium perfringens undergoing in vitro antibacterial test in Oxford Cup method were measured with the fermentation supernatant which was dealed with C.butyricum fermentation broth supernatant,row acid and catalase,trypsin,protease K.The volatile fatty acids(VFA)in fermentation broth was determined by gas chromatography(GC).The broiler intestinal mucosal epithelial cells were adopted as adhesion model to conduct the adhesion experiment of C.butyricum.Identification test for C.butyricum: The colony of C.butyricum had a round milky white color and produced a large amount of gas during cultivation.It is the C.butyricum I in Clostridium species;Resistance test of C.butyricum:(1)After treated with simulated feed pellets at a temperatureof 85?for 0,2.5,5,and 7.5 min,the survival rate of C.butyricum decreased significantly(P<0.05).(2)When C.butyricum was treated for 5 min under 0,0.20,0.30,0.40 MPa pressure,its survival rate showed a decreasing trend with increasing pressure,but the difference was not significant(P>0.05).(3)C.butyricum was treated for 48 min in artificial gastric juices(pH 2.00,3.00,and 4.00)and phosphate buffered saline(PBS).Compared with the control group,the survival rate of C.butyricum in each test group was not significantly different(P>0.05).(4)C.butyricum was treated in intestinal digestive enzymes and PBS for 198 min,and the survival rate of the experimental group was significantly lower than that of the control group(P<0.05).The results showed that C.butyricum could tolerate the high temperature and high pressure environment during the processing of broiler feed and had good resistance of gastric juice,but there was low tolerance to intestinal digestive enzymes;Evaluation of in vitro probiotic characteristics of C.butyricum: At 41?,C.butyricum entered the logarithmic phase at 9 h and after 4 h,achieved the stable phase.There was inhibitory effect partly on the five kinds of intestinal pathogens selected in the supernatant.And their inhibition zone diameter was between 10-12 mm.After trypsin and proteinase K treatment,the bacteriostasis disappeared.Studies had shown that organic acids,proteins and/or peptides acted as inhibitors;after reaching the stable phase of C.butyricum liquid culture,the content of acetic acid and butyric acid in the fermentation broth was respectively 3379.59mg/ml and 2609.73mg/ml.Intestinal mucosal epithelial cells of broilers were averagely adhered to 14.1 C.butyricum.Based on the above results,the following conclusions could be drawn: This strain of C.butyricum had normal morphology and physiological characteristics.The C.butyricum had a certain tolerance to high temperature,high pressure treatment and artificial gastric juice.The tolerance of C.butyricum to intestinal digestive enzymes is low.And it could adapt to the temperature and anaerobic environment in the broiler's intestine and proliferate rapidly.Then it could secrete probiotics,such as antibacterial proteins and VFA,and adhere to the surface of the small intestinal epithelial cells in broilers.Based on the above reasons,there would be a wide application prospect and good application effect by this C.butyricum as a feed additive for broilers.