| Weizmannia coagulans is a facultative anaerobic gram-positive bacteria,which can decompose sugars to produce L-lactic acid which is harmless to human body under anaerobic conditions.it is a homotype fermentative bacteria and has inhibitory effect on common intestinal harmful bacteria.Compared with general lactic acid bacteria,Weizmannia coagulans has stronger heat resistance,higher culture temperature,easier processing and preservation,and is not easy to infect bacteria.The spores produced by it are highly resistant to stress and have strong characteristics of high temperature resistance,acid resistance and bile salt resistance.Because the spores are resistant to high temperature and easy to be processed and preserved,it has better economic benefits as a microecological preparation.In this study,several strains of Bacillus were isolated from the natural fermentation of Artemisia argyi,and a strain of Bacillus was screened by bacteriostatic test.The species of the probiotic Bacillus was identified by morphological,physiological and biochemical tests and 16S r DNA sequence homology analysis,and it was identified as Weizmannia coagulans(Y1).Then the biological characteristics of Weizmannia coagulans were studied from the aspects of growth performance,acid production,acid and alkali resistance,bacteriostasis,temperature tolerance and drug sensitivity,and then the sporulation conditions of Weizmannia coagulans were optimized from two aspects of culture conditions and culture medium formulation.the optimized formula was used to produce spores and corn meal was used as the carrier to make bacterial preparations.Finally,the probiotic benefits of Weizmannia coagulans preparation were evaluated by animal experiments.The results are as follows:1)isolation and identification of Weizmannia coagulans.A total of 8 strains of Bacillus were isolated from Artemisia argyi,of which 3 strains had inhibitory effect on intestinal pathogenic bacteria.According to the results of physiological and biochemical characteristics and 16S r DNA molecular identification,Y1 was Weizmannia coagulans,T1was Bacillus tequilensis,D1 was Bacillus sonorensis.Among them,Y1 had the best bacteriostatic activity,and Y1 was selected as the experimental strain in the subsequent experiment.according to the results of the growth performance test,Y1 reached the logarithmic growth period after 12 hours,and the living bacteria count reached 2.12×10~9cfu/m L after 24 hours.The results of acid tolerance test showed that the living bacteria count could still reach 10~6 cfu/m L after 24 hours of anaerobic culture under the acid condition of p H3,which proved that it had good acid resistance.According to the results of temperature tolerance test,Weizmannia coagulans can grow at 30℃to 60℃,and grow best at45℃.According to the results of acid production test,after 24 hours of anaerobic culture,p H decreased to 4.5.the results of drug sensitivity test showed that Weizmannia coagulans Y1showed strong drug resistance to enrofloxacin and sulfamethoxazole.2)Optimization of sporulation conditions of Bacillus coagulans.After optimizing the sporulation conditions of Bacillus coagulans,the optimum sporulation conditions were obtained as follows:temperature 45℃,initial medium p H 7,shaking speed 180r/min,inoculum volume 5%,300m L conical flask culture medium 50m L,culture time 5 days.The best medium for sporulation was glucose 5g/L,wheat bran 5g/L and soybean meal 10g/L Magneto Na Cl 5g/L.The final spore yield can reach 6.13×10~8cfu/m L,and the number of spores of condensed Bacillus coagulans powder preparation can reach 1.56×10~9cfu/g after being concentrated and dried with corn meal as carrier.3)Animal test of Weizmannia coagulans.Growth performance test:group A was the control group,fed with common food and intragastric administration of PBS buffer 0.2m L every day.Group B was fermented feed group,fed with Weizmannia coagulans preparation fermented grain and intragastric administration of PBS buffer 0.2m L every day.Group C was fed with common grain and Weizmannia coagulans suspension(10~8cfu/m L)0.2m L every day.The data were collected and analyzed after 21 days of feeding.The results showed that the weight gain rate of the fermented feed group was significantly higher than that of the control group and the bacteria group,and the ratio of feed to meat was lower than that of the control group,indicating that the fermented feed prepared with Weizmannia coagulans could increase the body weight of mice and reduce the ratio of feed to meat.Salmonella challenge test:after 21 days of feeding,each mouse was fed with Salmonella suspension(10~9cfu/m L)0.2m L.The results showed that in the first day after salmonella challenge,the overall body weight of the control group decreased,while that of the fermented feed group and the bacterial solution intragastric administration group increased.The body weight of the control group was significantly different from that of the fermented feed group and the bacterial solution intragastric administration group(P<0.05),and the feed-meat ratio of the control group was higher than that of the fermented feed group and the bacterial liquid gastric perfusion group.The results showed that both fermented feed and suspension of clotting bacillus could prevent the weight loss and feed/meat ratio of mice caused by salmonella. |
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