The ingestion of large quantities of fructan or other rapidly fermentable carbohydrates has been associated with the development of laminitis. The fermentation of these compounds can lead to the proliferation of Gram-positive bacteria ( e.g.Streptococcus spp.) increasing lactic acid production and resulting in a decrease in hindgut pH. Antibiotics that inhibit Gram-positive bacteria ( e.g. Virginiamycin) can be employed to mitigate these microbial disturbances. However, natural products, such as plant-derived antimicrobials, also inhibit Gram-positive bacteria, and do not raise the same concern about antibiotic resistance. Hops are employed in breweries as a preservative due to the antimicrobial β-acids present in the flowers or ‘cones’ of the plant. These β-acids inhibit Gram-positive bacteria, like those involved in the pathogenesis of laminitis. This study was designed to determine if hops β-acid extract could mitigate changes associated with carbohydrate overload in vitro . Feces were collected from 3 horses (over multiple days) which were maintained on alfalfa hay with access to pasture. Upon arrival to the laboratory, each fecal sample was mixed with basal media and squeezed through a cheesecloth to remove large plant particles. Planktonic bacteria were then harvested by differential centrifugation. The bacteria pellet was re-suspended, sparged with N 2 and dispensed into serum bottles with either 0.1% or 0.6% inulin (model fructan). Hops β-acid extract was then added (45, 90, 180, 270, 360, and 450 ppm final concentrations) in duplicate. The bottles were incubated in a shaking water bath (37°C, 160rpm) for 24 hrs. Samples were obtained (0, 4, 6, 12 and 24 hrs of incubation) to determine pH. Furthermore, 0 and 24 hr samples were clarified by centrifugation and frozen (-20°C) for VFA analysis using HPLC (anion exchange column, refractive index detector). Data were analyzed using repeated measures ANOVA in the mixed procedure of SAS version 9.3. Incubations containing only inulin (0.1% or 0.6%) had decreased pH and increased lactic acid concentration at 24 hrs. However, β-acid treatments were able to mitigate pH disturbances in the 0.1% (excluding 45 ppm; P < 0.001) and 0.6% samples (excluding 45 and 90 ppm; P < 0.0001). At 24 hrs the 450 ppm treatment had a higher average pH of 6.24 and 5.52 in comparison to the 0.1% (pH 5.52) and 0.6% (pH 3.72) inulin-only controls, respectively ( P < 0.0001, in both instances). Total VFA concentrations (acetate+propionate+butyrate) increased with increasing β-acid concentration in 0.6% samples ( P< 0.0001); demonstrating that fermentation by non-lactic acid bacteria was not inhibited by β-acid treatment. Furthermore, lactate decreased with increasing β-acid concentration in 0.6% samples (excluding 45 ppm; P = 0.0011). This result indicates that hops β-acid extract could inhibit Gram-positive bacteria that produce lactate. In conclusion, hops β-acid extract could be a promising natural alternative to antibiotics in preventing the disturbances to the hindgut microflora associated with laminitis caused by carbohydrate overload.</P>
Journal of Equine Veterinary Science – Elsevier
Published: May 1, 2013
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