Exogenous lactobacilli mitigate microbial changes associated with grain fermentation (corn, oats, and wheat) by equine fecal microflora ex vivo

Authors: HARLOW, B - University Of Kentucky; LAWRENCE, L - University Of Kentucky; HARRIS, P - Waltham Centre For Pet Nutrition; Aiken, Glen; Flythe, Michael

Submitted to: PLOS ONE
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/2/2017
Publication Date: 3/30/2017
Citation: Harlow, B.E., Lawrence, L.M., Harris, P.A., Aiken, G.E., Flythe, M.D. 2017. Exogenous lactobacilli mitigate microbial changes associated with grain fermentation (corn, oats, and wheat) by equine fecal microflora ex vivo. PLoS One. https://doi.org/10.1371/journal.pone.0174059.

Interpretive Summary: Horses that are performing athletically sometimes need more calories than they can get from forage alone. Cereal grains (like corn, oats and wheat) have starch, which provides additional calories. However, if too much of these grains are fed, not all of the starch will be digested and absorbed by the horse. The starch that makes it through to the hindgut can be fermented into acids by the hindgut bacteria. Too much fermentation acid production can cause metabolic problems for horses. We previously found that feeding different types of starch to horses resulted in the growth of different species of starch-utilizing bacteria. In particular, the bacteria of the genus Lactobacillus was associated with less fermentation acid production and fewer total starch-utilizing bacteria. These observations led to the hypothesis that there was competition between different species of starch-utilizing bacteria. We brought horse feces into the laboratory and extracted the bacteria. The equine bacteria were permitted to ferment ground grain and make fermentation acids. The fermentation could be altered by adding extra lactobacilli (any one of three species of lactobacilli or a mixture of all three). In general, the exogenous lactobacilli decreased acidification, decreased the total number of starch-utilizing bacteria and increased the number of some types of beneficial bacteria. Furthermore, the lactobacilli did not have to be alive to have these effects. Living bacteria or "probiotics" are fed to horses and other animals for many reasons. However, it sounds odd to propose ameliorating the problems associated with starch fermentation by adding starch-fermenting bacteria. Nevertheless, lactobacilli interfered with starch fermentation by other equine bacteria in the laboratory, which is consistent with the previous observations in horses that total starch-using bacteria were fewest when lactobacilli were most numerous. Because the lactobacilli did not have to be alive, we believe this is an example of "allelopathy", or chemical competition between ecological competitors. Currently, this work is more informative for scientists than for horse owners and veterinarians, but understanding how probiotics work will allow them to be selected and used rationally.

Technical Abstract: Cereal grains are often included in equine diets. When starch intake exceeds foregut digestion starch will reach the hindgut, impacting microbial ecology. Probiotics (e.g., lactobacilli) are reported to mitigate GI dysbioses in other species. This study was conducted to determine the effect of starch source and exogenous lactobacilli on pH and the growth of amylolytic and lactate-utilizing bacteria. Feces were collected from 3 mature geldings fed grass hay with access to pasture. Fecal microbes were harvested by differential centrifugation, washed, and re-suspended in anaerobic media containing ground corn, wheat, or oats at 1.6% (w/v) starch and one of five treatments: Control (substrate only), L. acidophilus, L. buchneri, L. reuteri, or an equal mixture of all three (107 cells/mL, final concentration). After 24 h of incubation (37 ºC, 160 rpm), samples were collected for pH and enumerations of total amylolytics, Group D Gram-positive cocci (GPC; Enterococci, Streptococci), lactobacilli, and lactate-utilizing bacteria. Enumeration data were log transformed prior to ANOVA (SAS, v. 9.3). Lactobacilli inhibited pH decline in corn and wheat fermentations (P < 0.0001). Specifically, addition of either L. reuteri or L. acidophilus was most effective at mitigating pH decline with both corn and wheat fermentation, in which the greatest acidification occurred (P < 0.05). Exogenous lactobacilli decreased amylolytics, while increasing lactate-utilizers in corn and wheat fermentations (P < 0.0001). In oat fermentations, L. acidophilus and L. reuteri inhibited pH decline and increased lactate-utilizers while decreasing amylolytics (P < 0.0001). For all substrates, L. reuteri additions (regardless of viability) had the lowest number of GPC and the highest number of lactobacilli and lactate-utilizers (P < 0.05). There were no additive effects when lactobacilli were mixed. Exogenous lactobacilli decreased the initial (first 8 h) rate of starch catalysis when wheat was the substrate, but did not decrease total (24 h) starch utilization in any case. These results indicate that exogenous lactobacilli can impact the microbial community and pH of cereal grain fermentations by equine fecal microflora ex vivo. Additionally, dead (autoclaved) exogenous lactobacilli had similar effects as live lactobacilli on fermentation. This latter result indicates that the mechanism by which lactobacilli impact other amylolytic bacteria is not simple resource competition.