2013 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):
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.
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.
This research supports Objective 2 of the parent project: Characterize polyphenol-containing plant extracts and determine how they can be used to alter dairy cattle nitrogen efficiency, reduce in-barn emissions of ammonia and greenhouse gases and modify manure nitrogen availability in the soil. During the last year, the university collaborator focused on screening a large number of plants (ca 50) in order to source condensed tannins with a wider range of chemical properties for protein binding and degradability studies and for other experiments. Samples ranged from foods and medicinal plants to trees and comprised flowers, leaves, nuts and barks. Contrasting tannins were extracted, purified and fractionated. Six samples were obtained covering small-sized fractions (ca 670 to 1570 Daltons) that had procyanidin/prodelphinidin (PC/PD) ratios from 7.6/92.4 to 100/0. The seven medium-sized fractions (ca 870 to 3570 Daltons) had PC/PD ratios from 18.3/81.7 to 100/0. One large-sized fraction (ca 7250 Daltons) had a PC/PD ratio of 5.6/94.4. The purity of these fractions ranged from 18.5 to 66.7 (small fraction), 63.8 to 100 (medium fraction) and 69.7 g tannins/100 g material (large fraction). Two of these plant sources will be selected for preparing gram quantities of a PC-rich and a PD-rich tannin. In order to properly assess the role of tannins in modulating protein digestion, work continues on an improved acetone-butanol-hydrochloric acid assay for accurately quantifying extractable and insoluble forms of condensed tannins in forages and other plant materials. A paper was published describing how this improved method could be used to estimate the total quantity of condensed tannins in forages.