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Effect of fermentable substrate type and availability on gut microbiota composition and activity

An in vitro cumulative gas production technique was used to assess microbial activity of a complex community, in relation to fermentation of selected energy sources. This was carried out in combination with an in vivo study to examine the effects of two different diets for weaning piglets, so that microbial activities of faeces could be compared from the same animals on the two different diets in the in vivo experiment.

The two diets were: CHO diet (containing added fermentable carbohydrates – including sugar beet pulp (SBP) and wheat starch (WST)), and Control diet without any added fermentable CHOs. Neither diet contained antibiotics nor extra added copper. Twenty-four piglets were selected from 12 litters (2 per litter), weaned at 4 weeks of age (neither creep feeding nor any antibiotic treatment before and during the study), and introduced to one of the two diets. After nine days on the diet, faecal samples were collected from selected animals, and tested for their activity in terms of gas production kinetics, and end products such as volatile fatty acids (VFA), ammonia, and dry matter (DM) disappearance of two test substrates SBP and WST.

From samples taken before and after in vitro fermentation, the bacterial diversity was also analyzed, using denaturing gradient gel electrophoresis (DGGE) analysis of amplified 16S rRNA genes. There were differences both in kinetics and end-products of the substrates. In terms of gas production, significant differences were detected between inocula, though mainly in terms of fermentation kinetics of the two substrates, rather than total gas. For the CHO inoculum, SBP was fermented faster, than for the Control diet animal faeces, while this effect was reversed for WST.

Significantly higher diversity, as measured by DGGE fingerprint analysis, was detected in the microbial community enrichment on SBP as compared to WST at the end of fermentation. The difference between the kinetics of SBP and WST fermentation by faecal microbiota from the CHO diet fed piglets, suggests better adaptation of those piglets to SBP fermentation than to WST fermentation. The WST fermentation differences were more unexpected, though it was realized that a significant amount of starch is known to be fermentable by the small intestinal microbiota. This may have meant that the faecal microbiota was therefore less adapted to fermentation of starch.

It was concluded that the microbial community composition and activity in the GIT may be changed in response to diet, and that this change can be detected in vitro. Secondly, the response of ileal and colonic microbiota of weaning piglets, to dietary addition of 4 fermentable carbohydrates (inulin, lactulose, wheat starch and sugar beet pulp) was analyzed for an animal feeding trial. The same two diets, either enriched in or lacking these fermentable carbohydrates, were fed to piglets for 4 (n=48), and 10 days (n=48), and the lumen associated microbiota was compared using DGGE analysis of amplified 16S rRNA genes.

The results revealed that the addition of fermentable CHOs (= prebiotics) led to a significant enrichment of lactobacilli in the ileum. Furthermore, the bacterial diversity in the ileum and colon of each piglet was measured by assessing the number of DGGE bands and the Shannon index of diversity. A higher number of DGGE bands in the colon (24.2 +/- 5.5) compared to the ileum (9.7 +/- 4.2) was observed in all samples. In addition, at 10 days after weaning, a significantly higher bacterial diversity was detected in the colon of CHO-fed piglets, compared with the control.

Selected samples from the ileum and colon lumen were also investigated using fluorescent in situ hybridization (FISH), and cloning and sequencing of the 16S rRNA. This revealed that Lactobacillus amylovorus- like and L. reuteri- like phylotypes were the most prevalent in the ileum and colon lumen samples of the piglets fed the CHO diet. Newly developed DNA probes targeting these phylotypes allowed their rapid detection and quantification in the ileum and colon by FISH.

These results indicated that the addition of fermentable carbohydrates supports the growth of specific lactobacilli in the ileum and colon of weaning piglets, and increased the bacterial community diversity in the colon. The data therefore supported the hypothesis, that changes of the diet can modulate the composition of the microbiota in the intestine. More specifically, they show that the addition of a careful selection of fermentable carbohydrates can lead to positive changes in the bacterial population in the gut. Furthermore, the last study suggested a significant role of L. amylovorus-like phylotype in the metabolism of fermentable carbohydrates.

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Wageningen University
Hesselink van Suchtelenweg 4
6703CT Wageningen
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