The Probiotics And Intestinal Colonization Of Bacillus Coagulans In Common Carp（Cyprinus Carpio L.）

Recently,various diseases of fish are closely related to intensive farming,which has caused the increasing mortality of fish,destruction of aquaculture environment.The use of antibiotics to control pathogens and diseases has been increasing environmental stress and the disruption of intestinal flora in fish.Recently,probiotics have attracted great attention because of it generally safe to fish and environment,which are a potential alternative to antibiotics.First,a strain of bacteria by using the morphological characteristic,16S r RNA gene sequence analysis and phylogenetic tree identification,which was initially identified as Bacillus coagulans.Probiotic characteristics of B.coagulans were test in this experiment,such as the capacity of acid production,antagonistic activity against pathogens,resistance to bile,high temperature and low p H,antibiotic sensitivity.The results showed that:B.coagulans could withstand 90℃,p H of 3.0 and 0.3%concentration of bile salt;B.coagulans was co-culturing with Escherichia coli,Edwardsiella tarda,Aeromonas hydrophila,Staphylococcus aureus could inhibit their growth.Furthermore,B.coagulans was sensitive to streptomycin,chloramphenicol,erythromycin,tetracycline,vancomycin,kanamycin,clindamycin,ampicillin and amoxicillin.To further study the practical function of B.coagulans in aquatic breeding,240 healthy common carp that specifications consistent（32.50±0.41 g）were randomly divided into 4 groups（each treatment was repeated three times）.Four experimental diets were prepared:the control diet with different concentrations of B.coagulans suspension:0（CK）,10~7 CFU/g（A）,10~8 CFU/g（B）,and 10~9 CFU/g（C）respectively.Feed for 30 and 60 days,then indexes were detected to examine the effects of supplementing different concentrations of B.coagulans in common carp on the growth,serum biochemical indexes,intestinal morphology,intestinal microbiome and immune response against A.hydrophila.In addition,this study was to examine the colonization of B.coagulans in intestine of common carp.The following were the mainly results:1.Effect of B.coagulans on the growth,serum biochemical indexes,antioxidant indexes and disease resistance against A.hydrophila of common carp:After 30 days of feeding trial,although the growth performance did not differ among groups,the FBW,WGR and SGR in treatments were all higher than those in the control group,FCR decreased with the concentration of B.coagulans increased.The fish fed in10~9 CFU/g B.coagulans diet could remarkably improve the glucose（GLU）,cholesterol（CHO）and acid phosphatase（ACP）,which could significantly degrade the content of high density lipoprotein cholesterol（HDL-C）（P<0.05）.The activities of catalase（CAT）and total antioxidant（T-AOC）were significantly increased in group C,and the production of malondialdehyde（MDA）was significantly inhibited in experimental groups（P<0.05）.After 60 days of feeding trial,the growth indexes in the experimental groups were all significantly higher than those in the control group,FCR was significantly decreased（P<0.05）.The fish fed 10~7 CFU/g B.coagulans showed the best growth indexes（P<0.05）.The activities of GLU,HDL-C and ACP were significantly promoted in the experimental groups with adding B.coagulans（P<0.05）.The content of CAT and SOD in group C were significantly improved,and significantly degraded MDA in group A and B（P<0.05）.At the end of 60-day feeding,the common carp were injected with 0.1 m L of A.hydrophila（1×10~8 CFU/m L）for a week,then the serum cytokines were detected 24 hours later.The results showed that compared with the control group,the expression levels of pro-inflammatory cytokines interleukin 1β（IL-1β）and interleukin 12（IL-12）in group A and B were significantly upregulated（P<0.05）,and B.coagulans could upregulate the expression levels of anti-inflammatory cytokines interleukin 10（IL-10）and transforming growth factorβ（TGF-β）in the experimental groups（P<0.05）.2.Effect of B.coagulans on intestinal morphology,intestinal digestive enzymes and intestinal microbiome of common carp:As the experimental results showed,after 30 days of feeding,the activities of protease in foregut and hindgut,and lipase in foregut of group B were significantly higher than those in control group,while the activities of amylase in intestine and lipase in midgut and hindgut of group C were significantly higher than those of control group（P<0.05）.At the end of the 60-day culturing experiment,adding B.coagulans to the diet could significantly promote the lipase activity in the intestine of common carp;the protease activity in the intestine of group C was significantly increased（P<0.05）.According to the observation of intestinal morphology,adding B.coagulans to the diet had no significant effect on the integrity of intestinal tissue structure of common carp,but the intestinal microvilli were arranged orderly;the height of midgut and hindgut villi were significantly increased（P<0.05）.In addition,B.coagulans had a significantly promoting effect on the thickness of intestinal muscle layer（P<0.05）.12 samples were sequencing using Illumina Misep^TM PE 300.A total of 528,841 optimized sequences were obtained after filtered out dirty quality sequences.At the phylum level,the relative abundance of Firmicutes increased significantly with the concentration of Bacillus coagulans increased（P<0.05）,while Bacteroidetes disappeared in group C.At the genus level,the relative abundance of Gemmobacter and Bacillus increased significantly compared with the control group（P<0.05）.3.Colonization of B.coagulans in intestinal tract of common carp:the recombinant B.coagulans could be detected by gavaging of 10~9 CFU/m L of common carp in 1-14 days.The recombinant B.coagulans could stay in the intestinal tract of common carp for more than 14 days,and the number of B.coagulans colonized in the hindgut were more than other parts,which was beneficial for B.coagulans to play a better role in healthy feeding of common carp.