| The long-term use of antibiotics and zinc oxide (ZnO) in the diet can lead to severe outcomes, including the induction of antimicrobial resistance, food safety hazards and environmental pollution. Consequently, recent research has been focused on the development of alternatives to antibiotics and high does Zinc Oxide useage to maintain swine health and performance. In the current study, Clostridium butyricum ZJU-F1 (C. butyricum ZJU-F1), which could produce efficient antimicrobial protein, was isolated and characterized from piglets’cecal contents. Subsequently, its probiotic characteristics, intestinal growth kinetics, and acute toxicity was studied in vitro. At last, the protective effect of CBP22, an antimicrobial peptide produced by C. butyricum ZJU-F1, on the immunity and intestinal barrier function in mice challenged with LPS was discussed. After then, the effects and mechanisms of C. butyricum ZJU-F1 replacing antibiotics and reduce the high-does of Zinc Oxide was studied in weaning piglets. Furthermore, the effects of C. butyricum ZJU-F1 on immunity and mechanisms was researched in vitro.1. R & D of C. butyricum.This study was conducted to separate and screen C.butyricum from piglets’cecal contents, and to study its biological characteristics in vitro. C.butyricum strains were isolated by anaerobic cultivation and selected by Cavity AGAR diffusion method, and identified by physiological and biochemical experiments. Six strains of C. butyricum were isolatedthree strains of which had significantly inhibitory effects on E.coli K88, S.aureus ATCC25923. C. butyricum ZJU-F1 was identified to have the strongest antimicrobial activities. This strain was tolerant against low pH, high temperature, cholate and resistant to six antibiotics. The C.butyricun was conserved in the China General Microbiological Culture Collection Center.The probiotic function was compared in vitro between C.butyricum ZJU-F1 and five other C. butyricun strains. Enzyme activities were measured by ELISA kits, volatile fatty acids were measured by headspace-gas chromatography, and the Vitamin B3 was detectd by UPLC. Results showed that C.butyricum ZJU-F1 had the highest ability to produce digestive enzyme, and the amylase and protease actives were up to 2515.43 U/mL and 93.72 U/mL. C.butyricum ZJU-F1 also produced volatile fatty acids, such as Acetic acid and butyric acid, which were reached to 2.29 g/L and 1.67g/L, respectively. And the C.butyricun also produce high concentration of Vitamin D3, which up to 175.95μg/L. C.butyricum ZJU-F 1 have the best comprehensive probiotic function, and it could be a potential alternativefor antibiotics and high does ZnO.The gut growth kinetics of C, butyricum ZJU-F 1 in mice was also studied. C. butyricum ZJU-Fl adhered to each intestinal segments of mice, but the colonization did not last for a long time in vivo. The number of spores reached the maximum on day 1 in different intestinal segments, and then quickly decreased, and excreted on day 7. C. butyricum ZJU-F 1 less adhesion in the jejunum. Ueum, cecum and colon had the highest number of bacterial colonization.Safety evaluation of C. butyricum ZJU-F 1:The acute oral maximum-tolerated dose of C.butyricum ZJU-F1(1.2 × 109 CFU/g) were greater than 20.0g/Kg in mice, and C.butyricum ZJU-F1 was classified as non-toxic substance. The study indicated that C. butyricum ZJU-F 1 could potentially be a safe and green feed additive.2. Effects of antimicrobial peptides secreted by C. butyricum on intestinal inflammation in mouse induced by LPSSeperation, purification and Characterization of antimicrobial peptide from C. butyricum: After ammonium sulfate precipitation, affinity chromatography, ation-exchange chromatography and RP-HPLC, CBP22 was derived from C. butyricum ZJU-F 1. CBP22 is a linear peptide, which composed of 22 amino acids, with molecular weight of 2264.6 Da. The isoelectric point is 8.64. CBP22 showed MIC of 32 μg/mL and 64 μg/mL toward E. coli K88 and S. aureus ATCC 25923, respectively.The effect of CBP22 on the intestinal barrier function and immune function in mice: On the basis of previous separation, purification, and identification of C. butyricum peptide CBP22, this study investigated the effect of CBP22 on the intestinal barrier function and immune function in mice induced by LPS intraperitoneally. Intestinal barrier functionIntestinal permeability Mice injected with LPS showed significantly higher intestinal permeability compared with control group. CBP22 pretreatment significantly prevented permeability increase induced by LPS, which were similar to control mice.Intestinal morphology LPS treatment promoted jejunual epithelial cell shedding, and further led to severe damage to villus apex. Pretreatment with 4 mg/Kg or 8 mg/Kg CBP22 significantly reduced LPS-induced villus apex damage. Compared with mice in control group, mice treated with LPS showed significantly shortened villus (p<0.05), which was markedly prevented by CBP22 pretreatments (p<0.05), and the beneficial effect of 8 mg/Kg CBP22 tended to be better than 4 mg/Kg (p>0.05). When the mice were treated with both LPS and CBP22,no significant change in crypt depth were observed when compared with control group (p>0.05). These results indicated that intraperitoneally injection of CBP22 could significantly reduce LPS-induced jejunal villus damage.Cell apoptosis Compared with control group, mice treated with CBP22 only didn’t bring any significant change to the apoptosis index of jejunal epithelial cell (p>0.05), but LPS treatment induced significantly severe apoptosis (p>0.05). Pretreatment with 4 mg/Kg and 8 mg/Kg CBP22 markedly prevented LPS-induced apoptosis by 82.65% (p<0.05) and 86.81% (p<0.05) respectively, which showed no significant difference with control group (p>0.05). These results indicated that pretreatment with CBP22 could significantly prevent LPS-induced intestinal epithelial cell apoptosis.TJs expression Compared with control group, treatments with CBP22 only promoted the gene expression of ZO-1 (p<0.05), and mice treated with LPS showed significantly decreased ZO-1 and ZO-2 expression (p>0.05). Mice pretreated with CBP22 significantly protected mice from LPS induced decreased expression of ZO-1 and ZO-2 (p<0.05), which were: similar to control group. Immune functionSerum immunoglobulin Compared with control group, mice treated with CBP22 only showed decreased serum IgG concentration (p>0.05). LPS treatment significantly elevated serum IgG, IgA and IgM level (p<0.05). Pretreatments with CBP22 significantly prevented the increase of serum IgG, IgA and IgM concentration (p<0.05), which was similar to control group (p>0.05). Serum cytokines LPS treatment induced a significant increase in serum TNF-α concentration compared with control group (p<0.05), which was significantly reduced by CBP22 pretreatments (p<0.05, compared with LPS group).SIgA concentration in ileum The sIgA concentration in ileum was significantly higher in LPS treated mice compared with control group, which was markedly prevented by CBP22 pretreatments (p<0.05, compared with LPS group). TNF-a expression in ileum LPS treatment significantly increased the gene expression of TNF-a in jejunum compared with control group (p<0.05), which was markedly prevented by CBP22 pretreatments (p<0.05), showing no significant difference with control group (p>0.05).3. Effects of C.butyricum ZJU-F1 and in combination with Bacillus licheniformis on the growth performance, digest function and immunity of weaning pigletsOn the basis of the in vitro health promoting function and safety study of C. butyricum ZJU-Fl, This study was conducted to evaluate the effects of C. butyricum ZJU-F1 and mix probiotics of C. butyricum ZJU-F1 and Bacillus licheniformis (B. licheniformis) on the growth performance, digestion function and intestinal barrier function of weanling piglets, and to investigate whether Cbutyricum ZJU-F1 could replace antibiotics and high does of ZnO in the diets of weaning piglets. A total of 150 [(Landracex Yorkshire) xDuroc] piglets weaned at 23±2 d were randomly divided into the following 5 treatment with 30 piglets per group: Control A (basal diet + antibiotics + 2250 mg Zn/Kg from ZnO), Control B (basal diet + antibiotics + 1125 mg Zn/Kg from ZnO), BL (basal diet + 1125 mg Zn/Kg from ZnO + 109 CFU/Kg B. licheniformis), CB-F1 (basal diet + 1125 mg Zn/Kg from ZnO+108 CFU/Kg C. butyricum ZJU-F1), and CB (basal diet + 1125 mg Zn/Kg from ZnO + 108 CFU/Kg C. butyricum ZJU-F1 + 109 CFU/Kg B. licheniformis). Feed intake and the incidence of diarrhea were recorded daily. On day 14, 6 piglets were randomly chosen from each group and sacrificed for sample collection.Growth performance and Diarrhea rate Compared with Control A group, pigs in the Control B group showed decreased growth performance and elevated diarrhea rate. The addition of B. licheniformis (BL group), C. butyricum ZJU-F1 (CB-F1 group), or the mix of B. licheniformis and C. butyricum ZJU-F1 (CB group) increased the ADG, and decreased the FCR and diarrhea rate compared with Control B group, and the effects in CB-F1 and CB groups reached significant levels (p<0.05), without significant effect on ADFI. These results indicated that B. licheniformis, C. butyricum ZJU-F1, or the mixed probiotics could replace antibiotics with the addition of 1125 mg/Kg ZnO.Digestive function B. licheniformis, C. butyricum ZJU-F1 or the mixed probiotics improved the activity of amylase and lipase in duodenum and jejunum, and the effects on duodenal amylase activity and jejunal lipase were significant (p<0.05), without significant effect on pancreatic digestive enzymatic activity. The addition of B. licheniformis, C. butyricum ZJU-F1 or the mixed probiotics also promoted the apparent digestibility of dry matter, crude protein, calcium, and phosphorus by varying degrees, and these effects of CB-F1 and CB groups were significant (p<0.05). These results indicated that these probiotics could secret digestive enzymes to improve the apparent digestibility of nutrients. Intestian barrier functionIntestinal permeability Compared with control B group, the serum D-lactate concentration and DAO activity were significantly decreased in BL, CB-F1 and CB groups. Compared with control A group, the serum D-lactate concentration and DAO activity in BL and CB-F1 groups showed no significant difference (p>0.05), but the DAO activity in CB group was significantly lower (p<0.05). These results indicated that C. butyricum ZJU-F1 and the mix could relieve weaning induced increase in intestinal permeability.Intestinal morphology Compared with Control B group, the addition of C. butyricum ZJU-F1 or the mixed probiotics significantly elevated villus height and villus: crypt ratio in duodenum, jejunum and ileum. Compared with control A group, the villus:crypt ratio in CB group was significant elevated (p<0.05). The results of duodenal microstructure didn’t show any organelle abnormality, with the epithelial cells well connected. The order of microvilli of each group was CB>CB-F1>Control A>BL>Control B. These results indicated that B. licheniformis, C. butyricum ZJU-F1, and the mixed probiotics could improve intestinal morphology, which may promote nutrients absorption and growth performance.Tight junctions (TJs) expression In duodenum, the gene expression of ZO-1, ZO-2, Claudin-1 and Occludin, were higher than control B group, and the gene expression of ZO-2 and Occludin of CB group were significantly higher than control A group (p<0.05). In jejunum, the gene expression of ZO-1 and ZO-2, and the protein expression of ZO-1 and occludin in CB-F1 and CB groups were significantly higher than control A group (p<0.05). In ileum, the gene expression of ZO-1, ZO-2, Claudin-1 and Occludin, and protein expression of ZO-1, Claudin-1, and Occludin in CB-F1 and CB groups were significantly higher than control B group (p<0.05). The gene expression of ZO-1, ZO-2, Claudin-1, Occludin and protein expression of ZO-1 and Claudin-1 were significantly higher than control A group (p<0.05). The results of cell experiment showed that C. butyricum ZJU-F1 significantly elevated the gene and protein expression of Occludin, and tend to improve ZO-1 and Claudin-1. But inactivated C. butyricum ZJU-F1 failed to bring any significant difference to TJ expression. C. butyricum M588 significantly increased the gene expression of ZO-1 and protein expression of Occludin and Claudin-1. These results indicated that C. butyricum ZJU-F1 could promote TJs gene and protein expression, and further strengthen barrier function.Intestinal mucus expression Compared with control B group, the gene expression of MUC1, MUC4, and MUC20 of duodenum, jejunum, and ileum of CB-F1 and CB groups were significantly elevated (p<0.05). Compared with control A group, the gene expression of MUC1 and MUC4 in duodenum, MUC1, MUC3, and MUC20 in jejunum, and MUC1 and MUC4 in ileum were significantly elevated (p<0.05). These results indicated that C. butyricum ZJU-F1 and the mixed probiotics could promote the expression of mucin, which could enhance the barrier function.Intestinal microflora The addition of C. butyricum ZJU-F1, or the mixed probiotics elevated the proportion of Firmicutes, with the proportion of Bacteroides decreased. Compared with Control A and Control B, the proportions of Lactobacillus were elevated in BL,CB-F1, and CB groups, with the effect in BL group significant (p<0.05). Compared with Control B group, the butyrate concentration in cecum content of BL group was significantly elevated (p<0.05), but the concentrations of acetic acid, propionic acid, and butyrate in cecum content of CB-F1 and CB group were significantly elevated (p<0.05). Compared with control A group, the butyrate concentration in cecum content of CB group was significantly elevated, and concentration of total acid showed an elevated tendency. These results indicated that the addition of C. butyricum ZJU-Fl, or the mixed probiotics could promote the optimization of intestinal microflora, elevate the concentration of SCFA, which may further improve the intestinal microbiota barrier.4. Effects of C. butyricum ZJU-F1 on immune functions and the mechanismsOn the basis of the study of the beneficial effects of C. butyricum ZJU-F 1 on piglets growth performance, serum indexes, intestinal morphology, and microflora, this study investigated the effects of C. butyricum ZJU-F1 on the immune function. Immune functionSystemic immune function The serum concentrations of IgA, C3, and C4 in CB-F1 and CB groups were higher than control A and control B groups. The addition of B. licheniformis, C. butyricum ZJU-F 1, or the mixed probiotics significantly reduced the serum level of pro-inflammatory cytokines (p<0.05), IL-6, TNF-a and IFN-y, which were similar to the control A group (p>0.05). The addition of C. butyricum ZJU-Fl, or the mixed probiotics significantly improved the gene expression of PR-39, PG1-5, PMAP23 in the bone marrow, and Hepcidin in the liver, and PR-39 in the thymus compared with control A and B groups. These results indicated that C. butyricum ZJU-F1, and the mixed probiotics could improve serum level of immunoglobulins and complements, decrease pro-inflammatory cytokine concentrations, and promote antimicrobial peptides expression to strengthen immune function.Intestinal immune function Compared with control A group, the gene expression of TNF-a, IL-6, IL-8, and IL-10 were significantly higher in CB-F1 group (p<0.05), and the gene expression of IL-1β, TNF-a, IL-6, and IL-8 were significantly higher in CB group (p<0.05). Compared with control B group, the sIgA concentration in BL, CB-F1, and CB groups were elevated, with CB-F1 and CB reaching significant level (p<0.05), which were similar to the concentration of control A group (p>0.05). The gene expression of pBDs and PR-39 of CB-F1 and CB groups were significantly higher than control B and control A groups (p<0.05). These results indicated that C. butyricum ZJU-F1, and the mixed probiotics could promote the expression of cytokines, and antimicrobial peptides, and secretion of sIgA to improve intestinal mucosal immune function.Adhension activity The adherence rate of C. butyricum ZJU-F1 to pig intestinal epithelial cell line IPEC-J2 was 8.38%, which was higher than C. butyricum M588, but significantly lower than E. coli K88 (p<0.05). The release rate of C. butyricum ZJU-Fl after adhension was 1.23%, which was significantly lower than E. coli K88 (p<0.05). Co-cultured C. butyricum and E. coli K88 with IPEC-J2 cells showed that C. butyricum ZJU-F1 and C. butyricum M588 both significantly inhibited the adhension of E. coli K88 to IPEC-J2 cells. The exclude and remove experiments revealed that C. butyricum ZJU-F1 decreased the adherence rate of E. coli K88 to 3.87%and 7.05% respectively, which was similar to C. butyricum M588. These results indicated that C. butyricum ZJU-F1 could inhibit the adherence of pathogens to intestinal epithelial cells.Cell experiment C. butyricum ZJU-F1 and C. butyricum M588 both stimulated TLR-2 gene expression, and further induced the expression of MyD88 and activation of NF-κB in IPEC-J2 cells, and inactivated C. butyricum also possessed similar effects. They also induced the expression TNF-α, IL-1β, IL-8, IL-4, and IL-10 (p<0.05). Inactivated C. butyricum tended to induce the expression of TNF-a, IL-1β, and IL-6, without any significant effect on IL-10 and IL-4. These results indicated that C. butyricum could induce the expression of TNF-a, IL-1β, IL-4, and IL-10 to enhance mucosal immune function through TLR-2/MyD88 signaling pathway.To sum up, the current study successfully isolate and develop a noval probiotics with antimicrobial peptide producing property, i.e., Clostridium butyricum ZJU-F1. The antimicrobial peptide CBP22 produced by C. butyricum ZJU-F1 could ameliorate LPS induced intestinal damge in mice. Furthermore, C. butyricum ZJU-Fl could improve growth performance, gut barrier functions and immune functions in weaning piglets. Our results indicated that C. butyricum ZJU-Fl could replace the use of antibiotics and high does of zinc oxide in the diets of piglets. |
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