A chromatographic method to monitor fructan catabolism in two cool‐season grasses fermented by mixed bovine ruminal microbiota

Abstract

BACKGROUND: Cool-season grasses contain fructans (fructose-based polymers), whose monomers are linked by β-2,6 or β-1,2 bonds. Fructans in the rumen are hydrolysed to fructose by bacterial enzymes, but little is known about the kinetics of fructan catabolism in the rumen. A chromatographic method, using high-performance anion-exchange chromatography (HPAEC) with pulsed amperometric detection, was developed to monitor changes in fructan profiles during the in vitro fermentation of timothy (Phleum pratense L.) and cocksfoot (Dactylis glomerata L.) by mixed rumen microbes. Sterile suspensions of freeze-dried and milled tissue in minimal media were inoculated with washed rumen microbial cells under anaerobic conditions, and aliquots of the incubations were profiled 0, 2, and 5 h post-inoculation.RESULTS: Timothy and cocksfoot differed in initial concentrations of glucose and sucrose, and in the amounts remaining 2 h post-inoculation (p < 0.01). The grasses also differed in initial concentrations of short-chain fructan, and in concentrations present 2 h post-inoculation (p = 0.01). Timothy and cocksfoot also differed in amounts of longchain fructans remaining 2 and 5 h post-inoculation (p = 0.03), and in the maximum degree of polymerisation after 2 h (p < 0.01).CONCLUSIONS: Over the first 2 h, long-chain fructans in cocksfoot decreased while short-chain fructans increased. In timothy, concentrations of short-chain fructans doubled, and the degree of polymerisation decreased by 15%. The results support a model of degradation of the longest fructan chains and conversion into shorter ones.

Seasonal and species variation in raffinose, short‐chain fructan, and long‐chain fructan accumulation in tall fescue (Festuca arundinacea Schreb.) and timothy (Phleum pratense L.) grown in Central Kentucky

“…These extracts were put through end‐capped C 18 solid‐phase extraction columns (Clean‐Up, United Chemical Technologies, Bristol, PA, USA) containing 100 mg sorbent. Columns were conditioned with methanol and water as described by Kagan et al (2022), after which 0.9 mL of sample was vacuum‐filtered through the column and collected. SPE‐cleaned filtrates were stored at −20°C until analysis.…”

“…Columns were conditioned with methanol and water as described by Kagan et al (2022), after which 0.9 mL of sample was vacuum-filtered through the column and collected. SPE-cleaned filtrates were stored at À20 C until analysis.…”

“…Those results suggest that the presence of fructans in cool‐season grasses, like the presence of WSC as a whole, may affect rumen fermentation and ruminant performance. In addition, fructan chain length may affect fermentation patterns, because in vitro studies with mixed bovine ruminal microbiota resulted in long‐chain fructans being metabolized prior to short‐chain fructans (Kagan et al, 2022). An understanding of seasonal and species variation in fructan concentration and DP may complement assessment of forage WSCs when managing ruminant performance.…”

Fructan Catabolism by Rumen Microbiota of Cattle and Sheep

“…Despite the relevance of fructans in ruminant nutrition, research evaluating ruminal degradation of fructans is relatively limited. Overall, there are indications that DP may influence fermentation [19][20][21][22]. While specific bacteria may have differential capacities for degrading higher-DP long-chain fructans or lower-DP short-chain fructans [19,21], mixed bacteria harvested from equine digesta have been shown to degrade long-chain fructans prior to short-chain fructans [20].…”

“…While specific bacteria may have differential capacities for degrading higher-DP long-chain fructans or lower-DP short-chain fructans [19,21], mixed bacteria harvested from equine digesta have been shown to degrade long-chain fructans prior to short-chain fructans [20]. More recently, Kagan et al [22] found bovine rumen bacteria degraded long-chain fructan within the first two hours of fermentation, which was coupled with an increase in short-chain fructans, suggesting hydrolysis of long-chain fructans to short-chain fructans prior to subsequent degradation.…”