Screening And Identification Of A Strain Bacillus Amyloliquefaciens From Broilers And Its Feeding Effects On Broilers

This study was conducted to screen a strain of mucosa-associated Bacillus from broilers and subsequently confirm its in vivo effectiveness as a potential probiotic.Test A: Screening and identification of mucosa-associated Bacillus isolates from broilers. An ileum sample(about 1 cm long) was aseptically collected from a healthy broiler, washed with sterile saline(0.85% Na Cl) and soaked in 500 ?l of 0.016%dithiothreitol. After vortexing and centrifugation the supernatant was incubated in a 65?water bath for 45 min to kill any residual vegetative cells, and streaked on a MH agar plate.Then the clonies were scraped into 20 ml MH broth and grown overnight at 37?. 1ml of the culture was treated with 10 ml of simulated gastric fluid and simulated intestinal fluid respectively for 12 h, centrifugated and washed twice with sterile saline, and transferred into Difco sporulation medium(DSM) to promote production of spores. After heat treatment(65? for 45min) 100 ?l of serial DSM culture dilution were streaked on MH agar plates to obtain different Bacillus clonies resistant to both simulated gastric fluid and simulated intestinal fluid. Different clones were picked and cultured in MH broth before storage of the isolates at-80�C in MH broth with 30% glycerol. Agar plate screening methods of LBST and MH containing E.coli K88 were sequentially conducted to screen out the Bacillus isolate with the greatest capacity to produce amylase, protease and antibacterial substances, and finally the screened Bacillus isolate was identified by 16 Sr DNA gene sequence analysis.After the treatments of simulated gastric fluid and simulated intestinal fluid 53 Bacillus clones were obtained, and from those clones one Bacillus strain Y with the greatest capacity to produce amylase, protease and antibacterial substances was selected as the potential Bacillus probiotic, identified as Bacillus amyloliquefaciens by 16 Sr DNA gene sequence analysis.IVText B: The feeding effects of the screened Bacillus isolate on broilers. 600eleven-day-old male yellow broilers were divided into 4 groups with 3 replicates of 50 birds each and fed 4 experimental diets respectively in each of three growth periods(small,11~21 day old; medium, 22~42 day old; large, 43~70 day old). The diets, in mash form,and fresh water were supplied ad libitum. The birds were reared at room temperature and exposed to 24 h per day. For each growth period, the 4 experimental diets were formulated as follows: the basal diet; BSP10, the basal supplemented with the Bacillus spore premix(BSP) at 10 mg/kg(106CFU/g); BSP100, the basal supplemented with BSP at 100 mg/kg(107 CFU/g); BSP1000, the basal supplemented with BSP at 1000 mg/kg(108 CFU/g). At the end of each feeding experiment period, the growth performance was determined.Besides, at the end of the feeding experiment 4 birds from each group with its body weight similar to the average body weight of the group were slaughtered; thereafter, the blood serum of each bird was prepared to determine serum biochemical parameters, cecal digesta was collected to determine microbial indicators, and duodenum, jejunum and ileum were sampled to determine their morphological indices.For the whole growth stages, There significant difference in F/G ratio among groups(P< 0.05), whereas the mortality decreased with that of BSP1000 being significantly lower than the control(P < 0.05). With the BSP dose increasing the F and G increased with those of BSP1000 being significantly higher than the control respectively(P < 0.05), whereas the F/G ratio and mortality decreased with those of BSP1000 being significantly lower than the control respectively(P < 0.05). Whereas the F/G ratio and mortality decreased with those of BSP1000 being significantly lower than the control respectively(P < 0.05).There was no significant differences in serum A/G ratio, ALT and AST among groups(P > 0.05), but with the BSP dose increasing the TP, ALB and GLOB increased with those of BSP1000 being significantly higher than the control(P < 0.05), whereas the BUN, LPS and MDA decreased with those of BSP1000 being significantly lower than the control respectively(P < 0.05).For the duodenum, there was no significant difference in VH, CD or VH/CD(P >0.05); for the jejunum, there was no significant difference in CD(P > 0.05), but with the BSP dose increasing the VH and VH/CD increased with those of BSP1000 being significantly higher than the control(P < 0.05); for the ileum, with the BSP dose increasing the VH and VH/CD increased with those of BSP1000 being significantly higher than the control(P < 0.05), whereas the CD decreased with those of BSP1000 being significantly lower than the control respectively(P < 0.05).With the BSP dose increasing E.coli count(ECC) in the ceacal digesta decreased with that of BSP1000 being significantly lower than the control(P < 0.05), whereas the the lactic acid bacterial count(LABC) and LABC/ECC ratio increased with those of BSP1000 being significantly higher than the control respectively(P < 0.05).Conclusion:1. A strain of mucosa-associated Bacillus amyloliquefaciens Y, resistant to simulated gastric fluid and simulated intestinal fluid, and capable of producing amylase,protease and antibacterial substances, could be isolated from the ileum of healthy broiler.2. The supplementation of Bacillus amyloliquefaciens Y 108 CFU/g could improve the intestinal flora, intestinal villu smorphology and antioxidant capability, and enhanced feed conversion efficiency and feed intake, thus promote growth of broilers.