2011 Annual Report 1a.Objectives (from AD-416) 1. Investigate the in vitro solubility and ruminal-gastrointestinal digestibility of alfalfa proteins treated with various condensed and hydrolysable tannin preparations of differing composition. 2. Identify subfractions within tannin preparations that most effectively precipitate and protect alfalfa proteins from pregastric proteolysis while permitting efficient gastrointestinal digestion. 1b.Approach (from AD-416) Objective 1. Experiment 1. ARS SY will prepare five condensed tannins with approximate prodelphinidin to procyanidin ratios of 100:0 from white clover seed, 70:30 from big trefoil leaves, 70:30 from sainfoin leaves, 30:70 from birdsfoot trefoil leaves, and 0:100 from alfalfa seed using Sephadex LH-20 column chromatography. University SY will synthesize pentagalloyl glucose and isolate galloylated condensed tannins from grapeseed with Sephadex LH-20, and isolate gallotannins from Sumac and ellagitannin from chestnut by countercurrent chromatography. University of Reading will analyze tannins for composition/structure and molecular weight distribution by thiolysis, MALDI-TOF MS and HPLC-gel permeation chromatography. ARS will apply each tannin to macerated alfalfa at 0, 10, 20, 30, and 40 mg/g of dry matter for protein fractionation and degradability studies. Alfalfa proteins will be fractionated according to the Cornell Net Carbohydrate Protein System. Ruminal protein degradability of alfalfa will be assessed by the inhibitor in-vitro procedure. Total tract degradability of protein will be determined by sequential in vitro incubation of alfalfa with rumen microflora followed by enzymatic hydrolysis with acid-pepsin and neutral-pancreatin. Objective 2. Experiment 2. University of Reading will prepare low, moderate, and high molecular weight condensed tannin fractions from birdsfoot trefoil and big trefoil by Toyopearl column chromatography and analyze the fractions for composition/structure by thiolytic degradation and molecular weight distribution by HPLC-gel permeation chromatography. ARS will add the tannin fractions at ~30 mg/g of dry matter to macerated alfalfa for protein fractionation and degradability studies according to methods described for Experiment 1. 3.Progress Report Contrasting types of tannins are being synthesized or isolated from different plant materials in 8 gram quantities. Pentagalloyl glucose was synthesized from tannic acid as the starting material and its purity was confirmed by nuclear magnetic resonance (NMR). We then isolated galloylated condensed tannins from grapeseed extracts, gallotannins from a commercially available ‘Sumac’ tannin preparation, and ellagitannins from a commercially available ‘chestnut wood’ tannin mixture. Condensed tannins differing in procyanidin/prodelphinidin composition will be isolated from birdsfoot trefoil, big trefoil, and several populations of willow or apple. (We determined that willow and apple were more suitable sources for obtaining condensed tannins with contrasting composition than sainfoin herbage, alfalfa seed, or white clover seed.) These tannin preparations will be analyzed for their composition, structural characteristics, and molecular weight distribution by thiolysis, MALDI-TOF mass spectrometry, and HPLC-gel permeation chromatography. Tannin preparations will then be added to mechanically macerated alfalfa to assess how tannin composition affects the degradability of protein by rumen bacteria and gastrointestinal enzymes. Research is also progressing on optimizing a Sephadex LH-20 column fractionation method for isolating 2.5 g quantities of low, moderate and high molecular weight condensed tannin fractions from birdsfoot trefoil and big trefoil. These plant materials were chosen because their condensed tannins differ in procyanidin/prodelphinidin composition. These tannin fractions will be analyzed as above and applied to mechanically macerated alfalfa to assess how the molecular weight and composition of condensed tannins affects the degradability of protein by rumen bacteria and gastrointestinal enzymes. In addition, small quantities of high purity condensed tannins from birdsfoot trefoil and big trefoil are being used to develop an improved HCl/butanol assay for condensed tannin analysis. ADODR monitoring was accomplished through conference and individual phone calls, and e-mails with the cooperator.