Date Published: March 30, 2017
Publisher: Public Library of Science
Author(s): Brittany E. Harlow, Laurie M. Lawrence, Patricia A. Harris, Glen E. Aiken, Michael D. Flythe, George-John Nychas.
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 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°C, 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.
With the use of modern horses for high level performance activities, there has been a concomitant increase in demand to feed horses to maximize their athletic performance. Typically, concentrate is increased and forage is decreased in the diet in order to meet caloric needs. Cereal grains, which are high in starch, are important calorie sources in concentrate feeds used for horses. Previous research in our laboratory demonstrated that grain type can influence microbial changes in equine feces both ex vivo  and in vivo . For example, at equal starch intakes, cracked corn produces more marked changes in the fecal microbial ecosystem than whole cleaned oats, most notably in total amylolytic bacteria (corn: 100,000-fold increase, oats: 10-fold increase). Furthermore, these studies also identified a strong negative correlation between the viable number of lactobacilli and Group D Gram-positive cocci (GPC; Enterococcus spp., Streptococcus bovis/equinus) and the viable number of lactobacilli and total amylolytic bacteria, indicating a potential competitive relationship between these bacteria in the hindgut. It is noteworthy that these effects were observed both in vivo and ex vivo, even though the grain substrates in the latter experiments were not subjected to foregut digestion or any simulation thereof.
When ground corn, oats, or wheat was fermented by equine fecal microflora, the suspension pH declined (Fig 1). The average initial pH values of the cell suspensions were 6.8, and the extent of pH decline over the 24 h incubation was dependent on grain type and starch concentration. The lowest starch concentration eliciting maximal pH effects for all starch sources after 24 h (3.5, 4.4, and 4.2 for corn, oat and wheat incubations, respectively) was 1.6% w/v starch. Based on these results, all subsequent experiments were performed with 1.6% w/v starch.
It is counter-intuitive to propose inhibiting starch fermentation by adding a starch-fermenting organism, like a lactobacillus. However, previous studies in our laboratory have identified a strong negative relationship between the number of lactobacilli and the total number of amylolytic bacteria with grain fermentation, indicating competitive relationships among these bacteria (Harlow et al. 2015; Harlow et al. 2016). Based on the aforementioned observations, the objective of the current study was to determine if exogenous lactobacilli additions could mitigate both pH and microbial changes associated with corn, oat and wheat fermentation ex vivo.
The results from the current ex vivo study indicate that exogenous lactobacilli, most notably L. reuteri, can impact the microbial community composition, fermentation end-products and pH of cereal grain fermentations by equine hindgut microorganisms. These effects were independent of viability; i.e. autoclaved lactobacilli had the same effects as live. Additionally, the initial rate of starch disappearance with grain fermentation was slower with both live and dead L. reuteri addition, but these effects were substrate dependent. A slower rate of starch disappearance could permit adaptation of the fecal microflora allowing for greater stability. Thus, fermentations containing added L. reuteri either had similar or higher total starch disappearance in comparison to substrate only controls. This study provides a potential targeted treatment strategy for grain-induced hindgut acidosis in horses. Future research is needed to evaluate the effect of targeted probiotic therapies in vivo.