Directory UMM :Data Elmu:jurnal:A:Animal Feed Science and Technology:Vol87.Issue1-2.Sept2000:
Animal Feed Science and Technology
87 (2000) 41±56
Characterisation of tannins and in vitro protein
digestibility of several Lotus corniculatus varieties
Helena Hedqvista, Irene Mueller-Harveyb, Jess D. Reedc,
Christian G. Kruegerc, Michael Murphya,*
a
Department of Animal Nutrition and Management, Swedish University of Agricultural Sciences,
KungsaÈngen Research Centre, S-753 23 Uppsala, Sweden
b
Department of Agriculture, The University of Reading, P.O. Box 236, Reading RG6 6AT, UK
c
Department of Animal Sciences, University of Wisconsin-Madison, Animal Sciences Building, 1675
Observatory Drive, Madison, WI 53706-1284, USA
Received 7 March 2000; received in revised form 28 June 2000; accepted 11 July 2000
Abstract
Seven birdsfoot trefoil (BFT) varieties (Lotus corniculatus) grown in Sweden, were harvested at
the 50% ¯owering stage and analysed for tannins by the radial diffusion and HCl±butanol methods.
The ¯avan-3-ol composition of different BFT tannins was determined by HPLC. Tannins were
isolated and examined for their molecular weight distributions by HPLC gel permeation
chromatography (GPC) and MALDI-TOF mass spectrometry. Ruminal protein degradability was
determined in vitro and related to tannin chemistry.
Tannin concentrations of the BFT varieties were generally low and ranged between 0.3±1.0%
(radial diffusion assay) and 0.2±1.7% (HCl±butanol assay) on a DM basis. The delphinidin:cyanidin
ratios showed considerable variation ranging from 16:84 to 33:67 amongst the seven varieties. GPC
analysis revealed small differences between the varieties with most of the variation occurring in the
relative proportions of the higher molecular weight tannins. MALDI-TOF mass spectrometry of
tannins from two varieties gave well-resolved spectra of tetramers, pentamers and hexamers.
Oligomers up to the decamers were also detectable. Each of these oligomers had a subset of
structures incorporating catechin/epicatechin (CE) and gallocatechin/epigallocatechin (GE) units.
Some homopolymers containing CE units only (i.e. procyanidins), but none with GE units only (i.e.
prodelphinidins), were detected. Most mixed CE/GE oligomers of all sizes contained one or two GE
units.
There were signi®cant differences (P 0:05) in vitro N-degradability between four varieties. The
data suggest that degradability of the soluble proteins in birdsfoot trefoil were negatively correlated
to tannin concentrations (R2 0:93) despite the fact that their overall concentrations were very low.
# 2000 Elsevier Science B.V. All rights reserved.
*
Corresponding author. Tel.: 46-18-67-16-31; fax: 46-18-67-29-46
E-mail address: michael.murphy@huv.slu.se (M. Murphy).
0377-8401/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 3 7 7 - 8 4 0 1 ( 0 0 ) 0 0 1 7 8 - 4
42
H. Hedqvist et al. / Animal Feed Science and Technology 87 (2000) 41±56
Abbreviations: CE, catechin or epicatechin; GE, gallocatechin or epigallocatechin; GPC, gel permeation
chromatography; H/B, HCl±butanol; HPLC, high performance liquid chromatography; MALDI-TOF MS,
matrix assisted laser desorption ionisation-time of flight mass spectrometry; RD, radial diffusion; TLC, thin
layer chromatography
Keywords: Birdsfoot trefoil; Protein degradation; Proanthocyanidins; Tannins; HPLC gel permeation
chromatography; MALDI-TOF mass spectrometry
1. Introduction
Birdsfoot trefoil (Lotus corniculatus L.) is a drought resistant legume which tolerates
low soil fertility and can be used for grazing in many countries (Beuselinck and Grant,
1995; Foo et al., 1996). Birdsfoot trefoil (BFT) is of interest because of its condensed
tannins and their effects on animal production. Condensed tannins can form stable
complexes with dietary protein in the rumen (pH range 3.5±7.0), thereby increasing the
proportion of undegraded rumen protein. The tannin-protein complex is thought to
dissociate at the lower pH in the abomasum, resulting in a higher absorption of dietary
amino acids from the duodenum (Douglas et al., 1995). Some reports have also suggested
that the tannins may act directly as anthelmintics against parasitic nematodes or indirectly
by improving nitrogen supply (Niezen et al., 1995; Robertson et al., 1995; Butter et al.,
1998, 1999). Recent experiments in New Zealand also demonstrated a large increase in
milk production, which was attributed to the protein-binding properties of the tannins in
the rumen (Woodward et al., 1999).
Growing conditions in Sweden are not optimal for BFT production because of high
rainfall and low winter temperatures together with poorly drained soils. BFT tannin
concentrations in WI, USA, were much lower (
87 (2000) 41±56
Characterisation of tannins and in vitro protein
digestibility of several Lotus corniculatus varieties
Helena Hedqvista, Irene Mueller-Harveyb, Jess D. Reedc,
Christian G. Kruegerc, Michael Murphya,*
a
Department of Animal Nutrition and Management, Swedish University of Agricultural Sciences,
KungsaÈngen Research Centre, S-753 23 Uppsala, Sweden
b
Department of Agriculture, The University of Reading, P.O. Box 236, Reading RG6 6AT, UK
c
Department of Animal Sciences, University of Wisconsin-Madison, Animal Sciences Building, 1675
Observatory Drive, Madison, WI 53706-1284, USA
Received 7 March 2000; received in revised form 28 June 2000; accepted 11 July 2000
Abstract
Seven birdsfoot trefoil (BFT) varieties (Lotus corniculatus) grown in Sweden, were harvested at
the 50% ¯owering stage and analysed for tannins by the radial diffusion and HCl±butanol methods.
The ¯avan-3-ol composition of different BFT tannins was determined by HPLC. Tannins were
isolated and examined for their molecular weight distributions by HPLC gel permeation
chromatography (GPC) and MALDI-TOF mass spectrometry. Ruminal protein degradability was
determined in vitro and related to tannin chemistry.
Tannin concentrations of the BFT varieties were generally low and ranged between 0.3±1.0%
(radial diffusion assay) and 0.2±1.7% (HCl±butanol assay) on a DM basis. The delphinidin:cyanidin
ratios showed considerable variation ranging from 16:84 to 33:67 amongst the seven varieties. GPC
analysis revealed small differences between the varieties with most of the variation occurring in the
relative proportions of the higher molecular weight tannins. MALDI-TOF mass spectrometry of
tannins from two varieties gave well-resolved spectra of tetramers, pentamers and hexamers.
Oligomers up to the decamers were also detectable. Each of these oligomers had a subset of
structures incorporating catechin/epicatechin (CE) and gallocatechin/epigallocatechin (GE) units.
Some homopolymers containing CE units only (i.e. procyanidins), but none with GE units only (i.e.
prodelphinidins), were detected. Most mixed CE/GE oligomers of all sizes contained one or two GE
units.
There were signi®cant differences (P 0:05) in vitro N-degradability between four varieties. The
data suggest that degradability of the soluble proteins in birdsfoot trefoil were negatively correlated
to tannin concentrations (R2 0:93) despite the fact that their overall concentrations were very low.
# 2000 Elsevier Science B.V. All rights reserved.
*
Corresponding author. Tel.: 46-18-67-16-31; fax: 46-18-67-29-46
E-mail address: michael.murphy@huv.slu.se (M. Murphy).
0377-8401/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 3 7 7 - 8 4 0 1 ( 0 0 ) 0 0 1 7 8 - 4
42
H. Hedqvist et al. / Animal Feed Science and Technology 87 (2000) 41±56
Abbreviations: CE, catechin or epicatechin; GE, gallocatechin or epigallocatechin; GPC, gel permeation
chromatography; H/B, HCl±butanol; HPLC, high performance liquid chromatography; MALDI-TOF MS,
matrix assisted laser desorption ionisation-time of flight mass spectrometry; RD, radial diffusion; TLC, thin
layer chromatography
Keywords: Birdsfoot trefoil; Protein degradation; Proanthocyanidins; Tannins; HPLC gel permeation
chromatography; MALDI-TOF mass spectrometry
1. Introduction
Birdsfoot trefoil (Lotus corniculatus L.) is a drought resistant legume which tolerates
low soil fertility and can be used for grazing in many countries (Beuselinck and Grant,
1995; Foo et al., 1996). Birdsfoot trefoil (BFT) is of interest because of its condensed
tannins and their effects on animal production. Condensed tannins can form stable
complexes with dietary protein in the rumen (pH range 3.5±7.0), thereby increasing the
proportion of undegraded rumen protein. The tannin-protein complex is thought to
dissociate at the lower pH in the abomasum, resulting in a higher absorption of dietary
amino acids from the duodenum (Douglas et al., 1995). Some reports have also suggested
that the tannins may act directly as anthelmintics against parasitic nematodes or indirectly
by improving nitrogen supply (Niezen et al., 1995; Robertson et al., 1995; Butter et al.,
1998, 1999). Recent experiments in New Zealand also demonstrated a large increase in
milk production, which was attributed to the protein-binding properties of the tannins in
the rumen (Woodward et al., 1999).
Growing conditions in Sweden are not optimal for BFT production because of high
rainfall and low winter temperatures together with poorly drained soils. BFT tannin
concentrations in WI, USA, were much lower (