Condensed Tannins in Forages Diverse, Poorly Understood

Abstract

Wayne Zeller, of the U.S. Dairy Forage Research Center, recently reviewed the state of CT research in Crop Science (http://doi.org/10.2135/cropsci2018.05.0323). He stresses that CTs are not monolithic, but rather vary widely in their size and structure, and describes the current methods for purifying and studying CTs, along with each method's limitations. Just as the amino acids that comprise proteins can be combined in nearly infinite combinations and configurations, the flavan-3-ols that comprise CTs can be combined in various ways. The resulting CT molecules are characterized by the way the flavan-3-ol subunits are linked as well as by stereochemistry, hydroxylation patterns, and the addition of other functional groups. Most of the CTs in common forage species are comprised of four main flavan-3-ols that can be bonded to each other in a combination of linear or branched formations (see illustration above). Even CTs with the same components can have them linked in different order, and each plant contains “an extremely diverse set of structures,” according to Zeller. These are not currently separable by available methods, and thus are studied as a mixture. The formation of CT–protein complexes is impacted by the structures of both the CT and the protein in question as well as the pH, the isoelectric point (pI) of the protein, and the temperature. Formation of CT–protein complexes is known to rely on both hydrogen-bonding and hydrophobic interactions although it is not clear which factor is paramount or occurs first. Condensed tannins with more hydroxyl groups or other functional groups available for hydrogen bonding bond to proteins more effectively, and Zeller points out that they tend to be more effective anti-parasitic agents. Protein precipitation capacity and enzyme inhibition have been demonstrated to increase along with the size of the CT. Other CT structural features that affect protein binding and precipitation include type of linkages between the flavan-3-ol subunits and whether hydroxyl groups on these subunits are derivatized. Pure CTs are highly desired for meaningful experimental results, but they are difficult to obtain. High-performance liquid chromatography (HPLC) can yield only small amounts of pure CTs, and it is expensive and labor intensive. Some researchers have used Sephadex LH-20 as an adsorbent resin to purify CTs, but these CT samples are generally contaminated with carbohydrates and lipids. Moreover, these techniques may oxidize CTs, and thus provide artefactual components when samples are analyzed. If a sample can be obtained, nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry, and other methods can be used to start to elucidate the structure of the CT under investigation. Farmers want to use CTs and/ or CT-containing forages to improve the health of their ruminants and the productivity and sustainability of their farms. However, given that CTs are structurally so diverse, determining how they interact with proteins is difficult to define. Studies must be done not only on how CT structure affects protein precipitation in vitro, but also on how that translates to protein release during the pH changes between an animal's rumen, abomasum, and hindgut. Zeller suggests studies to determine how the other components in forage, like carbohydrates and lipids, affect CT–protein interactions as well as studies examining the interactions between CTs and an animal's microbiome population and any impacts they might have on the animal's health and productivity. Even when a plant's CT content is accurately measured, structural information is still required to assess any biologically relevant activity. The CT content and structure of a forage can be affected not only by the plant's cultivar, but by its harvest time, preservation method, and even storage time. Condensed tannin content must therefore be measured anew each time stored feed is to be used. Only in this way can we determine the best way to utilize CTs to promote healthy animals and healthy farms, according to Zeller. “We need the right level of CTs to get the most benefit,” he says. “Too much CT binds up all of the forage protein, so none will be available to feed the animal's microbiome. There are many papers reporting on CTs, but people don't know the content or the structure, and those molecular details matter.” Read the full Crop Science article, “Activity, Purification, and Analysis of Condensed Tannins: Current State of Affairs and Future Endeavors” at http://doi.org/10.2135/cropsci2018.05.0323.