Comparison of protein precipitation ability of structurally diverse procyanidin-rich condensed tannins in two buffer systems

Authors: Zeller, Wayne; Reinhardt, Laurie; ROBE, JAMISON - University Of Wisconsin; Sullivan, Michael; Panke-Buisse, Kevin

Submitted to: Journal of Agricultural and Food Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/27/2020
Publication Date: 2/19/2020
Citation: Zeller, W.E., Reinhardt, L.A., Robe, J.T., Sullivan, M.L., Panke-Buisse, K. 2020. Comparison of protein precipitation ability of structurally diverse procyanidin-rich condensed tannins in two buffer systems. Journal of Agricultural and Food Chemistry. 68(7):2016-2023. https://doi.org/10.1021/acs.jafc.9b06173.
DOI: https://doi.org/10.1021/acs.jafc.9b06173

Interpretive Summary: Condensed tannins (CTs) are a class of compounds present in a variety of forage plants including big trefoil, birdsfoot trefoil and sainfoin. When fed, forages and other plants containing CTs have been shown to have an impact on animal production systems including decreased loss of protein during harvest and storage, a higher level of escape protein into the hindgut of ruminants where it is more efficiently utilized by the animal, reduced methane (a greenhouse gas) production during rumination, and reduced nitrogen waste in manure. These effects are tied to the CTs’ ability to bind to and precipitate protein. Unfortunately, current in vitro protein binding and precipitation results, among other factors, typically are not good predictors of outcomes during livestock feeding trials. A major reason for these discrepancies could be the medium in which most of these in vitro studies are conducted. We selected a subset of CTs which had nearly identical subunit composition but varied in structure (number of subunits and how subunits are linked together, and the extent of derivatization with gallic acid). The CTs used in this study were purified from grapeseed (GS), Tilia flowers (TF), cranberry (C), and red clover flowers (RCF). These CTs were tested for their protein precipitation ability against bovine serum albumin (BSA), lysozyme (LYS), and extracted alfalfa leaf protein (ALF) in 2-[N-morpholino]ethanesulfonic acid (MES, a buffer commonly used in this type of CT study) and in a buffer which simulates rumen fluid, Goering-Van Soest (GVS) buffer. Our results show that the GVS buffer system may better reflect the impact tannin structure (mDP [mean degree of polymerization, a measure of tannin size], incorporation of gallic acid, and how CT subunits are linked) have on protein complexation and precipitation. Although additional studies are warranted, our findings suggest that in vitro protein precipitation studies using GVS buffer may offer better guidance when selecting CT-containing forages and amendments for ruminant feeding studies.

Technical Abstract: The protein precipitation (PP) of bovine serum albumin (BSA), lysozyme (LYS), and alfalfa leaf protein (ALF) by four procyanidin-rich condensed tannin (CT) samples in both 2-[N-morpholino]ethanesulfonic acid (MES) and a modified Goering-Van Soest (GVS) buffer is described. Purified CT samples examined include Vitis vinifera seed (mean degree of polymerization [mDP] 4.1, 16.5 % galloylated), Tilia sp. flowers (B-type linkages, mDP 5.9), Vaccinium macrocarpon berries (mDP 8.7, 31.7% A-type linkages) and Trifolium pratense flowers (B-type linkages, mDP 12.3) and were characterized by 2D NMR (>90% purity). In general, CTs precipitated ALF >LYS=BSA. PP in GVS buffer was 1 to 2.25 times greater than in MES buffer (25 °C). The GVS buffer system better reflects the results/conclusions from the literature on the impact mDP, galloylation and A-type linkages have on PP. Determinations of PP using the MES buffer at 37 °C indicated that some of these differences may be attributed to the temperature GVS buffer determinations are conducted. In vitro PP studies using the GVS buffer may offer better guidance when selecting CT-containing forages and amendments for ruminant feeding studies.