In vivo utilisation of fructooligosaccharides by sheep faecal bifidobacteria and in vitro antagonistic effects against intestinal pathogens
Li, Y. (2003). In vivo utilisation of fructooligosaccharides by sheep faecal bifidobacteria and in vitro antagonistic effects against intestinal pathogens (Thesis, Doctor of Philosophy (PhD)). The University of Waikato, Hamilton, New Zealand. Retrieved from https://hdl.handle.net/10289/13231
Permanent Research Commons link: https://hdl.handle.net/10289/13231
The application of pro-, pre, and synbiotics has been studied primarily in humans and some other monogastric animals. Very few studies have been made to determine their effects on the ruminant intestinal microflora. This project tested whether or not four commercial food-grade oligo- and polysaccharides (three FOS products and one polysaccharide control, Arabinogalactan) could modify the hindgut microflora of sheep towards a more salutary community in which the health-promoting bacterial groups bifidobacteria and lactobacilli predominate, whereas the potential intestinal pathogens and putrefactive bacteria E. coli and sulphite reducing clostridia are suppressed. A fructo-6-phosphate phosphokatolase (F6PPK) enzyme-based identification protocol was developed and optimised for identifying and large-scale screening of presumptive bifidobacteria isolates from gut contents or faecal samples. An in vivo experiment was then carried out to determine the bifidogenic effect (promotion of bifidobacteria by prebiotics) and the associated antimicrobial effect (suppression of potential pathogens due to the increase in the populations of bifidobacteria) of the four oligo- and polysaccharides on sheep hindgut microflora. Twelve fistulated sheep were managed in a balanced, two Latin square, cross-overdesign experiment, which was run in 5 consecutive periods, with each of 5 treatments (Arabinogalactan, Fibruline, Raftilose, Yacon, and an "acidified saline" carbohydrate-free control) administered to two sheep in each period. Each period consisted of a 1 week of stabilisation to the pelleted diet (no oligo- and polysaccharides), followed by 14 days of daily abomasal supplementation of oligo and polysaccharide/acidified saline, followed by about 12 days of normal pelleted diet. In each period, sheep faecal bifidobacteria, lactobacilli, E. coli/ Enterobacteriaceae, sulphite reducing clostridia, and total anaerobes were enumerated on the day -4 of the "stabilisation" period; days 3 & 9 of the "treatment supplementation" period, and days 15, 16, 19 & 26 after cessation of infusions. Raftilose, Yacon, and Fibruline all exerted significant bifidogenic effects on sheep faecal bifidobacteria after 9 days of daily dosing. Raftilose produced the greatest stimulation of bifidobacteria, reaching counts of approximately 107 CFU/g of faeces. No significant changes in the populations of bifidobacteria were observed in Arabinogalactan-treated sheep. Raftilose and Yacon significantly increased the number of lactobacilli, reaching approximately 107 CFU/g of faeces, after 9 days of daily dosing. With Fibruline, the lactobacilli increase after 9 days of administration was not significant. Arabinogalactan did not elevate the populations of lactobacilli. All four carbohydrate treatments significantly increased the number of total anaerobes to approximately 107 to 108 CFU/g of faeces after 9 days of daily dosing. Supplementation of the test oligo- and polysaccharides had no significant effect on the other determined groups of gut microflora: sulphite reducing clostridia and E. coli/Enterobacteriaceae. There were no significant changes in sheep faecal pH and dry matter content with the four treatments. Further in vitro antagonistic experiments were carried out to determine whether or not the isolated sheep faecal bifidobacteria inhibited the growth of potential intestinal pathogens in fermentation broth containing the bifidogenic FOS. One hundred and seventeen bifidobacterial isolates from sheep faeces, were screened for their capacity to utilise different oligo- and polysaccharides. Eighteen of these, with strong fermentation patterns, were selected for further study. In the first preliminary experiment, the 18 isolates plus 2 reference cultures of bifidobacteria were divided into 5 groups of 4 strains each. The 5 groups of bifidobacteria were compared for their in vitro antagonistic activities against E. coli in Peptone Yeast Extract broth containing Yacon, Raftilose, or Fibruline as primary carbon sources. Two groups, exerting 100% antagonistic effects against E. coli after 48-hour anaerobic co-culture at 37°C, were selected for further examination. The eight individual strains in these two groups were tested individually for their antagonistic activities against E. coli in PY broth containing Yacon or Raftilose. Fibruline was eliminated due to its low antagonistic activity by bifidobacteria. Six isolates, showed 100% inhibitory effects against E. coli, which made them particularly promising for use as probiotics. The pH of the fermented broths showed a clear negative correlation with the rank transformed or angular transformed inhibition rate of E. coli. One bifidobacterial strain, P5-Po4-37, was subsequently investigated for its in vitro antagonistic activity against E. coli by determination of bacterial growth kinetics over 60 hours anaerobic incubation at 37°C in PY broth containing Raftilose or Yacon. In this experiment, two different concentrations of bifidobacteria, 107 to 108 CFU/mL and 103 to 104 CFU/mL, were incubated with 104 to 105 CFU/mL of E. coli. After 30 and 48-60 hours of incubation, the growth of E. coli was completely inhibited by both the higher and lower concentrations of bifidobacteria, respectively. The presence of E. coli did not affect the growth of bifidobacteria. With the higher inoculum level, the populations of bifidobacteria increased by only approximately 1.30 log10 cycles; whereas with the lower inoculum level, the populations of bifidobacteria increased by approximately 5.62 log10 cycles, to attain the same maximum viable counts at approximately 109 CFU/mL. Fermentation products were analysed in Raftilose containing PY fermented broth. The inhibitory activity of strain P5-Po4-37 was associated with the production of acetic and lactic acids. Strain P5-Po4-37 also exerted strong antagonistic activities against Clostridium perfringens, Enterobacter aerogenes, Enterococcus faecalis, Klebsiella pneumoniae, Salmonella Dublin, and Salmonella Menston. These findings indicated that Yacon and Raftilose potentiate an organic acid mediated inhibitory action of bifidobacterial strain P5-Po4-37 against the test potential intestinal pathogens. To date, this demonstration of inhibitory activities has been convincingly made only in in vitro studies. The combination of Yacon or Raftilose and bifidobacterial strain P5-Po4-37 may exert a promising synbiotic effect on sheep hindgut microflora, which will be investigated in vivo in the near future.
The University of Waikato
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