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United States Department of Agriculture

Agricultural Research Service


Location: Dairy Forage Research

2012 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:
This project is related to the following objective of the parent project: Objective 3. Determine the biochemical/chemical/genetic basis for biological systems needed to inhibit degradation of forage proteins during harvest, storage and utilization to minimize nitrogen waste from dairy production systems. The large-scale isolation of 2.5 g quantities of contrasting tannins has been completed. This yielded a series of low, medium, and high molecular weight fractions covering 1,200 to 12,000 Daltons. The lowest size fractions (1,200 to 2,100 Dalton) had procyanidin/prodelphinidin (PC/PD) ratios from 28/72 to 79/21. The medium size fractions (3,000 to 4,800 Dalton) had PC/PD ratios of 18/82 to 68/32 and the highest size fraction (12,000 Dalton) had a PC/PD ratio of 35/65. The purity of these fractions ranged from 35 to 94%. NMR (Nuclear Magnetic Resonance) analysis revealed that carbohydrates and phospholipids were the main contaminants of the tannin fractions. Research continues on developing methods for further purifying these tannin fractions. Once purified, the effect of these tannin fractions on the ruminal and gastrointestinal degradability of alfalfa protein will be assessed by in vitro techniques. In order to improve condensed tannin (CT) quantitation, we tested various co-solvents for the widely used butanol-HCl assay and found that acetone increased anthocyanidin yields from birdsfoot trefoil (Lotus corniculatus L.) and big trefoil (Lotus uliginosus Schkuhr) herbage. An optimized acetone-butanol-HCl assay with iron gave consistent linear responses with CT standards and increased estimates of total CT in herbage and leaves of both Lotus species by at least 2-fold over the standard method run without acetone. Overall, we found that the acetone-butanol-HCl assay, when used with CT standards of defined purity, dramatically improved quantitation of total CT in forage legumes.

4. Accomplishments

Last Modified: 2/23/2016
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