| Bacillus pumilus SE5 is a probiotic isolated from intestine of fast growing grouper(Epinephelus coioides),its cell wall components(whole cell wall,whole peptidoglycan,lipoteichoic acid and cell wall protein)were extracted to explore the effects on growth performance,digestion enzymes activities,immune function and intestinal microbiota of grouper(Epinephelus coioides).The results are presented as follows:1.The extraction and analysis of cell wall components of B.pumilus SE5,including whole cell wal,whole peptidoglycan,lipoteichoic acid and cell wall protein.Peptidoglycan was extracted by suspended with 10%TCA and boiled for 1 hour,then removed the lipid by chloroform methanol mixture,digested by typsase and lyophilized.The extracted peptidoglycan is light brown solid,insoluble and its yield is 16.83%.The extraction was authenticated whole peptidoglycan by infrared spectrum,transmission electron microscope and lysozyme analysis.Its total sugar content is 40.18%,the content of glucosamine is 36.87%,the protein content is 48.10%,the amino acid profiles are basically preserved compared with SE5 and the molecular weight is about 10 kDa.Lipoteichoic acid was extracted by the mixture solution of n-butanol and water,subjected to hydrophob ic interaction chromatography on octyl-Sepharose CL-4B.Then the peak content solution of phosphorus was collected and lyophilized.The extraction is yellow solid,soluble,5.31%yield and authenticated lipoteichoic acid by infrared spectrum with the characteristics of glycerol phosphate,amide and glycosyl group.Whole cell wall was extracted by suspended with lysate and ultrasonic in ice bath,centrifugated and precipitate was lyophilized.Its total yield is 62.17%,the total sugar content is41.36%,the glucosamine content is 13.61%,the protein content is 40.07%,and the amino acid profiles are basically consistent with SE5.Cell wall protein was extracted by suspended with mixture lysate and ultrasonical disrupted.Then centrifugated,the supernatant was collected,dialyzed for 48 h and lyophilized.The protein yield is 13.29%,the protein content is 89.4%,and the molecular weight ranges between 10-15 kDa and one more band located at 35 kDa.2.Effects of cell wall components of B.pumilus SE5 on growth performance,digestion enzymes activities,serum immune enzymes activities and head-kindey immune related genes expression in grouper(Epinephelus coioides).840 juvenile fish(average initial body weight 7.85±0.21 g)were randomly divided into 7groups with 4 replicates and feed the corresponding diets for 9 weeks.Basal diet was formulated with soybean meal,fish meal,fish oil and flour,and other six experimental diets were added with1.0×10~8 cfu/g of live,heat-inactivated probiotic SE5 or cell wall components(whole cell wall,whole peptidoglycan,lipoteichoic acid,and cell wall protein)extracted from the same dose of probiotic,respectively.The results showed that FBW,WGR and SGR improved significantly in peptidoglycan group and lipoteichoic acid group(P<0.05)and fish fed peptidoglycan containing diets showed highest values.In addition,the lowest FCR and highest PER were observed in peptidoglycan group(P<0.05).Dietary administration of peptidoglycan group and lipoteichoic acid group improved the activities of lipase,amylase and trypsin significantly(P<0.05).Dietary administration of peptidoglycan and lipoteichoic acid improved the activities of ACP,AKP and T-SOD significantly(P<0.05).In addition,fish fed diets contain peptidoglycan and lipoteichoic acid promoted the complement C3 and IgM levels significantly(P<0.05).Peptidoglycan group and lipoteichoic acid group had significantly augmented genes expressions of TLR2,NOD2,IL-8,IgM,Hepcidin-1,Epinecidin-1 and?-defensin in the head-kindey of grouper(P<0.05).In conclusion,peptidoglycan or lipoteichoic acid extracted from B.pumilus SE5 could improve the growth performance,digestive enzymes activities,specific and nonspecific immunity of juvenile grouper.3.Effects of cell wall components of B.pumilus SE5 on intestinal morphology,microbiota and immune related genes expression of grouper(Epinephelus coioides).This charpter was aimed to study the e ffects of these components on intestinal biopsy,microbiota and immune related genes expression,including TLR1,TLR2,TLR3,TLR5,NOD2,MyD88,IL-8,Hepcidin-1,?-defensin,Epinecidin-1,IgM,C-type Lectin and HSP90.The results showed that the duplicature height of the live bacteria group,the cell wall protein group,the lipoteichoic acid group,the peptidoglycan group and the cell wall group was significantly higher than that of the control(P<0.05),and the muscularis thickness in each experimental group was higher than that of the control(P<0.05).The ACE and Shannon inde xes in lipoteichoic acid group and the peptidoglycan group were higher significantly than the control(P<0.05).The abundances of class Bacilli and genus Lactobacillus in lipoteichoic acid group and peptidoglycan group were higher(P<0.05),while genus Vibrio lower than the control(P<0.05).This suggested that the key components(lipoteichoic acid and peptidoglycan)of SE5 could modulate the intestinal micriobita.The relative expressions of TLR1,TLR2,TLR3,TLR5,NOD2,MyD88,IL-8,IgM,C-type Lectin,Hepcidin-1,Epinecidin-1 and?-defensin were significantly higher in the lipoteichoic acid group and the peptidoglycan group than those in the control(P<0.05).In conclusion,peptidoglycan and lipoteichoic acid extracted from B.pumilus SE5 could be recognised by TLRS and NOD2 in the intestine of grouper,mediate the downstream signaling pathway,lead to the expressions of antibiotic peptides and IgM and modulate the intestinal micriobita. |
PDF Full Text Request