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

Authors: Kagan, Isabelle; Harlow, Brittany; Flythe, Michael

Submitted to: Review Article
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/26/2022
Publication Date: 4/26/2022
Citation: Kagan, I., Harlow, B.E., Flythe, M.D. 2022. A chromatographic method to monitor fructan catabolism in two cool-season grasses fermented by mixed bovine ruminal microbiota. Journal of the Science of Food and Agriculture Report. 2:264-271. https://doi.org/10.1002/jsf2.50.
DOI: https://doi.org/10.1002/jsf2.50

Interpretive Summary: Cool-season grasses contain chains of fructose (a simple carbohydrate) of varying length, called fructans. In the rumen of cattle, bacteria can break down fructans to fructose, thus providing a source of energy for the animal. Little is known about the process of fructan breakdown, such as whether longer or shorter chains of fructose are broken down first in the process of breaking fructans down into fructose. A method was developed to track changes in fructan length during a laboratory simulation of rumen bacterial utilization of fructan. Milled tissue from timothy and orchardgrass was suspended in a weak nutrient solution so that the main source of energy would be from the grasses. Bacteria taken from the rumen of cattle were added and allowed to grow in the solution for different lengths of time so that changes in carbohydrate utilization could be monitored. After zero, two, and five hours, a portion of the solution was separated to determine how much fructan, as well as small sugars like sucrose, had been utilized. Timothy and orchardgrass contained different initial amounts of glucose and sucrose, and the extent of disappearance of these sugars differed between orchardgrass and timothy solutions, although the same bacteria fermented both. Timothy and orchardgrass had different amounts of short-chain fructan initially, and those amounts changed over time. Over the first two hours that bacteria were allowed to grow in the presence of timothy or orchardgrass, long orchardgrass fructans became less abundant while short ones became more abundant. Long timothy fructans became shorter, and short timothy fructans became more abundant. These results suggest that bacteria in the rumen of cattle convert long fructans into shorter ones before breaking them down completely to fructose.

Technical Abstract: Cool-season grasses contain fructans (fructose polymers), whose monomers are linked by beta-2,6 or beta-1,2 bonds. Fructans in the rumen are degraded to fructose by bacterial enzymes, but little is known about the kinetics of fructan catabolism in the rumen. A chromatographic method, using high-performance liquid chromatography (HPLC) with pulsed amperometric detection, was developed to monitor changes in fructan profiles during the in vitro fermentation of timothy (Phleum pratense L.) and orchardgrass (Dactylis glomerata L.) by mixed rumen microbes. Sterile suspensions of freeze-dried, 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 hours post-inoculation. Timothy and orchardgrass differed in initial concentrations of glucose and sucrose, and in the extent of disappearance of these sugars (P < 0.01). The grasses also differed in initial concentrations of short-chain fructan, and in the relative increases in short-chain fructan during the first two hours of incubation (P = 0.01). Timothy and orchardgrass also differed in extent of catabolism of long-chain fructans (P = 0.03), and in the change in maximum degree of polymerization (P < 0.01). Over the first two hours, long-chain fructans in orchardgrass decreased while short-chain fructans increased. In timothy, short-chain fructans doubled in concentration, and the degree of polymerization decreased 15%. The results support a model of degradation of the longest fructan chains and conversion into shorter ones.