Project Number: 3655-31000-023-02
Project Type: Specific Cooperative Agreement

Start Date: Sep 01, 2010
End Date: Aug 31, 2015

Objective:
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.

Approach:
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.