Profiling of cool-season forage arabinoxylans via a validated HPAEC-PAD method

Authors: JOYCE, GLENNA - University Of Kentucky; Kagan, Isabelle; Flythe, Michael; Harlow, Brittany; SCHENDEL, RACHEL - University Of Kentucky

Submitted to: Frontiers in Plant Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/27/2023
Publication Date: 3/13/2023
Citation: Joyce, G.E., Kagan, I., Flythe, M.D., Davis, B.E., Schendel, R.R. 2023. Profiling of cool-season forage arabinoxylans via a validated HPAEC-PAD method. Frontiers in Plant Science. 14. Article 1116995. https://doi.org/10.3389/fpls.2023.1116995.
DOI: https://doi.org/10.3389/fpls.2023.1116995

Interpretive Summary: The fiber in cool-season grasses consists partly of arabinoxylans, a class of sugars that help to determine the structure of plant cell walls. Arabinoxylans (AX) exist in different lengths (different numbers of sugars linked together), and different AX have different amounts of phenolic compounds (natural products that can be antioxidants) bound to them. These structural differences among AX are likely to affect how grasses are digested by grazing animals, but too little is known about the structures of AX in cool-season grasses to consider AX when choosing feed for forage animals. The purpose of this study was to develop a method to profile AX structures in different cool-season forage grasses (timothy, perennial ryegrass, tall fescue, and Kentucky bluegrass. Individual sugars in the cell walls of the grasses were also measured, as were some of the phenolic compounds bound to AX. The four grasses differed in the amounts of simple sugars recovered from the cell walls, suggesting differences in cell wall structure. More small chains of sugars were recovered from perennial ryegrass cell walls than from the cell walls of timothy, Kentucky bluegrass, or tall fescue. The method that was developed is ideally suited to monitor structural changes of AX in forages as a result of plant breeding, pasture management, and fermentation of plant material.

Technical Abstract: Cool-season pasture grasses contain arabinoxylans (AX) as their major cell wall hemicellulosic polysaccharide. AX structural differences correspond to differing enzymatic degradability, so structural profiling of forage AX may be useful for assessing forage quality and suitability for ruminant feed. The main objective of this study was to optimize and validate a high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) method for the simultaneous quantification of 10 endoxylanase-released xylooligosaccharides (XOS) and arabinoxylan oligosaccharides (AXOS) in cool-season forage cell wall material. The following analytical parameters were determined or optimized: chromatographic separation and retention time (tR HPAEC), internal standard suitability, working concentration range (CR), limit of detection (LOD), limit of quantification (LOQ), relative response factor (RRF), and quadratic calibration curves. The developed method was used to profile the AX structure of four cool-season grasses commonly grown in pastures (timothy, Phleum pretense L.; perennial ryegrass, Lolium perenne L.; tall fescue, Schedonorus arundinaceus (Schreb.) Dumort.; and Kentucky bluegrass, Poa pratensis L.). In addition, the cell wall monosaccharide and ester-linked hydroxycinnamic acid (HCA) contents were determined for each grass. The developed method revealed unique structural aspects of the AX structure of these forage grass samples that complemented the results of the cell wall monosaccharide analysis. For example, xylotriose, representing an unsubstituted portion of the AX polysaccharide backbone, was the most abundantly-released oligosaccharide in all the species. Perennial rye samples tended to have greater amounts of released oligosaccharides compared to the other species. This method is ideally suited to monitor structural changes of AX in forages as a result of plant breeding, pasture management, and fermentation of plant material.