A total of 124 samples were collected from the intestine of broiler chickens, piglet faeces, fermented foods, soils and Chinese herbs. More than 750 strains of aerobic, spore-forming bacteria were isolated from these samples. The inhibitory activity of these spore-forming strains against Escherichia coli K88, E. coli K99, Salmonella typhimurium and Staphylococcus aureus was assessed using a disc plate diffusion assay. The six bacilli with the largest inhibition zones against the four indicator bacteria were chosen and assessed for their resistance to unfavorable conditions within simulated gut environments. The strain Bacillus subtilis MA139 showed full resistance to pH 2, 0.3% bile salts and exhibited the highest antimicrobial activity. Based on these results, B. subtilis MA139 was selected as a potential probiotic and fed to piglets at concentrations of 2.2 x 10(5), 2.2 x 10(6) or 2.2 x 10(7) CFU/g of feed during a 28-day feeding trial. A negative control consisting of the basal diet with no additives and a positive control consisting of the basal diet supplemented with 16 g/ton flavomycin were also included. Ninety piglets between 35 and 40 days old were used in the in vivo animal trials. B. subtilis MA139 enhanced daily gain (P = 0.10) and feed conversion (P = 0.03) compared with the negative control. The performance of pigs fed B. subtilis MA139 supplemented diets did not differ from that of pigs fed the antibiotic diet. There was a significant increase in Lactobacilli cell counts (P = 0.02) and a numerical decrease in E. coli counts (P = 0.05) in the faecal samples of pigs fed B. subtilis MA139 with 2.2 x 10(5) CFU/g of feed. The overall results of this study show that the use of initial co-culture with indicator pathogens, a disc plate diffusion assay and simulated gut environment tolerance tests is one of effective methods of screening Bacillus for probiotic use and that B. subtilis MA139 is a promising alternative to antibiotics for use as a feed additive in piglet diets.
Bacillus has been proposed as a probiotic due to its in vivo effectiveness in the gastrointestinal tract through antimicrobial activities. The present study investigates the effects of Lactobacillus alone or in the presence of Bacillus subtilis MA139 on the inhibition of pathogenic Escherichia coli K88. Mixed cultures were used to predict the possible interactions among these bacteria within the intestinal tract of animals. B. subtilis MA139 was first assayed for its inhibition against E. coli K88 both under shaking and static culture conditions. A co-culture assay was employed under static conditions to test the inhibitory effects of Lactobacillus reuteri on E. coli K88, with or without addition of B. subtilis MA139. The results showed that B. subtilis MA139 had marked inhibition against E. coli K88 under shaking conditions and weak inhibition under static conditions. Lactobacillus alone as well as in combination with B. subtilis MA139 spores exerted strong inhibition against E. coli K88 under static conditions. However, the inhibition by Lactobacillus in combination with B. subilis spores was much higher than that by Lactobacillus alone (P<0.01). B. subtilis MA139 significantly decreased the pH and oxidation-reduction potential values of the co-culture broth compared to that of Lactobacillus alone (P<0.05). The viability of Lactobacillus increased when co-cultured with B. subtilis MA139 because of significantly higher Lactobacillus counts and lower pH values in the broth (P<0.05). The role of Bacillus in the mixed culture models suggests that Bacillus may produce beneficial effects by increasing the viability of lactobacilli and subsequently inhibiting the growth of pathogenic E. coli. Therefore, the combination of Bacillus and Lactobacillus species as a probiotic is recommended.
BackgroundThe beneficial effects of Lactobacillus probiotics in animal production are often strain-related. Different strains from the same species may exert different weight-gain effect on hosts in vivo. Most lactobacilli are selected based on their in vitro activities, and their metabolism and regulation on the intestine based on strain-related characters are largely unexplored. The objective of the present study was to study the in vivo effects of the three lactobacilli on growth performance and to compare the differential effects of the strains on the faecal microbiota and ileum mucosa proteomics of piglets.MethodsThree hundred and sixty piglets were assigned to one of four treatments, which included an antibiotics-treated control and three experimental groups supplemented with the three lactobacilli, L. salivarius G1-1, L. reuteri G8-5 and L. reuteri G22-2, respectively. Piglets were weighed and the feed intake was recorded to compare the growth performance. The faecal lactobacilli and coliform was quantified using quantitative PCR and the faecal microbiota was profiled by denaturing gradient gel electrophoresis (DGGE). The proteomic approach was applied to compare the differential expression of proteins in the ileum mucosa.ResultsNo statistical difference was found among the three Lactobacillus-treated groups in animal growth performance compared with the antibiotics-treated group (P > 0.05). Supplementation of lactobacilli in diets significantly increased the relative 16S rRNA gene copies of Lactobacillus genus on both d 14 and d 28 (P < 0.05)., and the bacterial community profiles based on DGGE from the lactobacilli-treated groups were distinctly different from the antibiotics-treated group (P < 0.05). The ileum mucosa of piglets responded to all Lactobacillus supplementation by producing more newly expressed proteins and the identified proteins were all associated with the functions beneficial for stabilization of cell structure. Besides, some other up-regulated and down-regulated proteins in different Lactobacillus-treated groups showed the expression of proteins were partly strain-related.ConclusionsAll the three lactobacilli in this study show comparable effects to antibiotics on piglets growth performance. The three lactobacilli were found able to modify intestinal microbiota and mucosa proteomics. The regulation of protein expression in the intestinal mucosa are partly associated with the strains administrated in feed.
In order to develop a multi-microbe probiotic preparation of Lactobacillus reuteri G8-5 and Bacillus subtilis MA139 in solid-state fermentation, a series of parameters were optimized sequentially in shake flask culture. The effect of supplementation of B. subtilis MA139 as starters on the viability of L. reuteri G8-5 was also explored. The results showed that the optimized process was as follows: water content, 50 %; initial pH of diluted molasses, 6.5; inocula volume, 2 %; flask dry contents, 30∼35 g/250 g without sterilization; and fermentation time, 2 days. The multi-microbial preparations finally provided the maximum concentration of Lactobacillus of about 9.01 ± 0.15 log CFU/g and spores of Bacillus of about 10.30 ± 0.08 log CFU/g. Compared with pure fermentation of L. reuteri G8-5, significantly high viable cells, low value of pH, and reducing sugar in solid substrates were achieved in mixed fermentation in the presence of B. subtilis MA139 (P < 0.05). Meanwhile, the mixed fermentation showed the significantly higher antimicrobial activity against E. coli K88 (P < 0.05). Based on the overall results, the optimized process enhanced the production of multi-microbe probiotics in solid-state fermentation with low cost. Moreover, the viability of L. reuteri G8-5 could be significantly enhanced in the presence of B. subtilis MA139 in solid-state fermentation, which favored the production of probiotics for animal use.
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