Isabelle A. Kagan

Inhibition of fructan‐fermenting equine faecal bacteria and Streptococcus bovis by hops (Humulus lupulus L.) β‐acid

The goals of this study were to determine if β‐acid from hops (Humulus lupulus L.) could be used to control fructan fermentation by equine hindgut micro‐organisms, and to verify the antimicrobial mode of action on Streptococcus bovis, which has been implicated in fructan fermentation, hindgut acidosis and pasture‐associated laminitis (PAL) in the horse.

A Validated Method for Gas Chromatographic Analysis of γ-Aminobutyric Acid in Tall Fescue Herbage

Gamma-aminobutyric acid (GABA) is an inhibitory neurotransmitter in animals that is also found in plants and has been associated with plant responses to stress. A simple and relatively rapid method of GABA separation and quantification was developed from a commercially available kit for serum amino acids (Phenomenex EZ:faast) and validated for tall fescue (Festuca arundinacea). Extraction in ethanol/water (80:20, v/v) at ambient temperature yielded detectable amounts of GABA. Clean separation from other amino acids in 28 min was achieved by gas chromatography (GC) with flame ionization detection (FID), using a 30 m, 5% phenyl/95% dimethylpolysiloxane column. The identity of the putative GABA peak was confirmed by GC with mass spectrometric (MS) detection. The relatively small effects of the sample matrix on GABA measurement were verified by demonstrating slope parallelism of GABA curves prepared in the presence and absence of fescue extracts. Limits of quantification and detection were 2.00 and 1.00 nmol/100 microL, respectively. Method recoveries at two different spike levels were 96.4 and 94.2%, with coefficients of variation of 7.3 and 7.2%, respectively.

Thin-layer Chromatographic (TLC) Separations and Bioassays of Plant Extracts to Identify Antimicrobial Compounds

A common screen for plant antimicrobial compounds consists of separating plant extracts by paper or thin-layer chromatography (PC or TLC), exposing the chromatograms to microbial suspensions (e.g. fungi or bacteria in broth or agar), allowing time for the microbes to grow in a humid environment, and visualizing zones with no microbial growth. The effectiveness of this screening method, known as bioautography, depends on both the quality of the chromatographic separation and the care taken with microbial culture conditions. This paper describes standard protocols for TLC and contact bioautography with a novel application to amino acid-fermenting bacteria. The extract is separated on flexible (aluminum-backed) silica TLC plates, and bands are visualized under ultraviolet (UV) light. Zones are cut out and incubated face down onto agar inoculated with the test microorganism. Inhibitory bands are visualized by staining the agar plates with tetrazolium red. The method is applied to the separation of red clover (Trifolium pratense cv. Kenland) phenolic compounds and their screening for activity against Clostridium sticklandii, a hyper ammonia-producing bacterium (HAB) that is native to the bovine rumen. The TLC methods apply to many types of plant extracts and other bacterial species (aerobic or anaerobic), as well as fungi, can be used as test organisms if culture conditions are modified to fit the growth requirements of the species.

Hops ( Humulus lupulus L.) Bitter Acids: Modulation of Rumen Fermentation and Potential as an Alternative Growth Promoter

Antibiotics can improve ruminant growth and efficiency by altering rumen fermentation via selective inhibition of microorganisms. However, antibiotic use is increasingly restricted due to concerns about the spread of antibiotic-resistance. Plant-based antimicrobials are alternatives to antibiotics in animal production. The hops plant (Humulus lupulus L.) produces a range of bioactive secondary metabolites, including antimicrobial prenylated phloroglucinols, which are commonly called alpha- and beta-acids. These latter compounds can be considered phyto-ionophores, phytochemicals with a similar antimicrobial mechanism of action to ionophore antibiotics (e.g., monensin, lasalocid). Like ionophores, the hop beta-acids inhibit rumen bacteria possessing a classical Gram-positive cell envelope. This selective inhibition causes several effects on rumen fermentation that are beneficial to finishing cattle, such as decreased proteolysis, ammonia production, acetate: propionate ratio, and methane production. This article reviews the effects of hops and hop secondary metabolites on rumen fermentation, including the physiological mechanisms on specific rumen microorganisms, and consequences for the ruminant host and ruminant production. Further, we propose that hop beta-acids are useful model natural products for ruminants because of (1) the ionophore-like mechanism of action and spectrum of activity and (2) the literature available on the plant due to its use in brewing.

Effects of Sampling Time, Cultivar, and Methodology on Water- and Ethanol-Soluble Carbohydrate Profiles of Three Cool-Season Grasses in Central Kentucky

Abstract Cool‐season grasses (CSGs) accumulate variable amounts of water‐soluble carbohydrates (WSCs, monosaccharides and disaccharides, and fructans), depending on climate, time of day and year, and genotype. Fructan concentrations in CSG are sometimes estimated as the difference between concentrations of WSC and ethanol‐soluble carbohydrates (ESCs, monosaccharides and disaccharides, and variable amounts of fructan). Characterizing both WSC and ESC may improve understanding of soluble carbohydrate profiles in pastures and inform grazing management decisions, particularly for horses at risk for laminitis. Three CSG cultivars from Kentucky pastures were collected in the morning and afternoon on two springtime dates. Water‐soluble carbohydrates and ESC were extracted with water or 80% ethanol, respectively, and analyzed by high‐performance liquid chromatography (HPLC) and a colorimetric assay. Method of analysis (HPLC or colorimetry), and extraction solvent, affected the composition of WSC or ESC determined in CSG samples, demonstrating the need to consider methodology when interpreting results. Total soluble carbohydrate (mean of WSC and ESC) concentrations across cultivars and harvests were generally higher in the afternoon than in the morning, based on both HPLC (P = .0023) and colorimetric (P < .0001) analysis. The diurnal variation was mainly due to sucrose (P < .0001). Among cultivars, monosaccharides and disaccharides constituted the majority of the averaged WSC and ESC concentrations. The proportions of water‐ and ethanol‐soluble fructan (P = .0101), and fructan chain lengths detected in water and ethanol extracts (P < .0001), differed among CSG cultivars. In choosing CSG cultivars for pastures, both soluble carbohydrate composition and concentrations should be considered. HighlightsMonosaccharides and disaccharides constituted the majority of total water‐soluble carbohydrate (WSC) or ethanol‐soluble carbohydrate (ESC) in three cultivars of different cool‐season grasses collected in mid‐springtime from central Kentucky plots.Relative amounts of glucose, fructose, and sucrose detected depended on extraction parameters.Most diurnal variation in the averaged WSC and ESC concentrations was due to sucrose, not to fructan.Fructan concentration and chain length varied with cultivar.In one cultivar, short‐chain fructans were abundant in both WSC and ESC extracts. Therefore, using the difference between WSC and ESC concentrations to calculate fructan is not a correct practice.