Characterisation of tannins and in vitro protein
digestibility of several
Lotus corniculatus
varieties
Helena Hedqvist
a, Irene Mueller-Harvey
b, Jess D. Reed
c,
Christian G. Krueger
c, Michael Murphy
a,*aDepartment of Animal Nutrition and Management, Swedish University of Agricultural Sciences, KungsaÈngen Research Centre, S-753 23 Uppsala, Sweden
bDepartment of Agriculture, The University of Reading, P.O. Box 236, Reading RG6 6AT, UK cDepartment 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 (P0:05) in vitroN-degradability between four varieties. The data suggest that degradability of the soluble proteins in birdsfoot trefoil were negatively correlated to tannin concentrations (R20:93) despite the fact that their overall concentrations were very low. #2000 Elsevier Science B.V. All rights reserved.
87 (2000) 41±56
*Corresponding author. Tel.:46-18-67-16-31; fax:46-18-67-29-46 E-mail address: michael.murphy@huv.slu.se (M. Murphy).
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 corniculatusL.) 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 (<1% DM) (Broderick and Albrecht, 1997) than in New Zealand (2.5±3.9% DM) (Lowther et al., 1987; Wang et al., 1994; Douglas et al., 1995). Some of these differences may be attributable to the use of different tannin assays (Reed, 1995) but they could also be due to the different environments (Barry and Forss, 1983). As the climate in Scandinavia is more similar to Wisconsin low tannin values can be expected too. It is not known if such low concentrations can also bene®t animal production.
The aim of the present investigation was to determine the extent of tannin variation in several BFT varieties when grown in Sweden and to study their effects on protein degradation in vitro using rumen ¯uid.
2. Material and methods
2.1. Crop production
fescue and Norcen was grown in a pasture together with timothy grass at RaÊdde in south-west Sweden (578370N, 900 mm annual rainfall).
2.2. Sampling of plant material
BFT varieties from pure stands and from stands co-cultivated with meadow fescue were harvested when 50% of the plants had open ¯owers on the main stems. The pasture at RaÊdde was sampled three times during the last week in June while cattle were grazing. The plants were cut 5 cm above ground and immediately placed into an insulated cooler with ice and within 30 min stored frozen at 208C. BFT samples from the co-cultivated stands were separated while frozen. All BFT samples were lyophilised (within 3 months from harvest) and then ground in a hammer mill to pass through a 1 mm screen.
2.3. Isolation of tannins
Four gram of the lyophilised and milled sample was mixed with 20 ml acetone/water (7:3, v/v) in polypropylene centrifuge tubes and extracted with a MSE ultrasonic probe (Tambro, MoÈlndal, Sweden) for 45 s. Extractions were repeated ®ve times (545 s) with the tubes placed in ice water to keep the temperature below 358C. Following sonication the extracts were centrifuged at 3250 g for 5 min and the supernatants transferred to 50 ml test tubes. Acetone was removed under a stream of nitrogen at 458C. The samples were centrifuged again at 13,000 g for 5 min to remove water insoluble material. The ®nal supernatant was applied to a Sephadex LH-20 column (8:01:5 cm)
and eluted with 125 ml ethanol/water (95:5, v/v) to remove low molecular weight phenolics (Strumeyer and Malin, 1975). Tannins were located in a brown band at the top of the Sephadex column, eluted with 30 ml acetone/water (7:3, v/v) and collected in 10 ml fractions. Acetone and ethanol were evaporated under a stream of nitrogen at 458C. The aqueous fractions were frozen and then freeze-dried. Tannins were stored under nitrogen at ÿ208C. Tannins isolated from Sephadex LH20 from four varieties (GA1, Norcen, Fergus, Viking) were mixed in equal amounts (on a weight basis) and used as the internal standard for the HCl±butanol and the radial diffusion assays (see Section 2.4).
2.4. Tannin analyses
2.4.1. Radial diffusion (RD) assay
Tannins were analysed according to Hagerman (1987). BFT samples (4 g) were extracted with 20 ml of acetone/water (7:3, v/v), after which 68ml of the supernatant was applied to wells in agar gel containing bovine serum albumin (Sigma Chemical Co., St. Louis, Missouri). After incubation at 308C for at least 96 h the diameter of the formed ring was measured.
2.4.2. HCl±butanol (H/B) assay
Proanthocyanidins were analysed using the HCl±butanol assay of Porter et al. (1986). BFT samples (4 g) were extracted with 20 ml of acetone/water (7:3, v/v). One millilitre of the extract was mixed with 6 ml of HCl±butanol (5:95, v/v) and 200ml iron reagent and then placed into a water bath at 1008C for 50 min. After cooling, absorbance was read at 550 nm. To correct for interferences, absorbance was read before and after heating. Each sample was analysed in triplicate.
2.4.3. Thin layer chromatography (TLC)
TLC was used to screen the anthocyanidins produced by the HCl±butanol reaction according to Mueller-Harvey et al. (1987) and to establish rapidly if there were qualitative varietal differences. The ground plant material (1 g) was extracted with 5 ml acetone/ water (7:3, v/v) in an ultrasonic bath for 30 min. The supernatant (1 ml) was mixed with 0.5 ml dichloromethane to remove chlorophyll. Then 100ml of the aqueous phase was mixed with 3 ml HCl±butanol (5:95, v/v) and heated at 958C for 50 min. The solution (15ml) was applied to 5 cm5 cm Cellulose MN 300 plates (Machery-Nagel, Dueren, Germany). Elution in the ®rst direction was with formic acid/HCl/water (10:1:3, v/v/v) and in the second direction with pentanol/glacial acetic acid/water (2:1:1, v/v/v).
2.4.4. Delphinidin/cyanidin (D:C) ratios by HPLC
The HCl±butanol reaction mixture (see Section 2.4.2) was examined for the relative proportions of delphinidin (derived from gallocatechin/epigallocatechin, GE, units) and cyanidin (from catechin/epicatechin, CE, units) by high performance liquid chromato-graphy (HPLC) according to Stewart et al. (2000). HCl and butanol were removed by evaporation at 508C under a stream of nitrogen. The residue was dissolved in 0.5 ml HCl± methanol (1:95, v/v). Aliquots of 20ml were injected onto an HPLCm-Bondapak Radial-Pak C18 column (8 mm i.d.) (Waters, Watford, UK), protected by a cartridge guard column containing 5mm Spherisorb ODS (Hichrom, Reading, UK). Acetic acid/water (95:5, v/v; solvent A) and methanol (solvent B) were used for gradient elution at 2 ml minÿ1using a Gilson HPLC-system with UNIpoint software (Gilson, Middleton, WI, USA). The gradient pro®le was: 5±40% solvent B (0±5 min); 40±50% B (5±10 min); 50±100% B (10±15 min); 100±5% (15±20 min). Absorbance was measured at 525 nm with a Kratos SF 769 variable wavelength detector (Kratos Analytical, Manchester, UK).
2.4.5. Molecular weight distributions by HPLC-GPC
30 columns (8mm; Polymer Laboratories Ltd, Church Stretton, UK). Tannin compounds were eluted in acetic acid/methanol (0.5:100; v/v) at 1.2 ml minÿ1. Absorbance was measured at 280 nm with the HPLC equipment described above.
2.4.6. MALDI-TOF mass spectrometry
MALDI-TOF mass spectra were collected on a Bruker Re¯ex II-TOF mass spectrometer (Billerica, MA) equipped with delayed extraction and a N2 laser set at
337 nm. For positive mode spectra in the re¯ectron mode, an accelerating voltage of 25.0 kV and a re¯ectron voltage of 26.5 kV were used. For positive mode spectra in the linear mode, an accelerating voltage of 25.0 kV was used. Spectra are the sum of 100± 500 shots. Spectra were calibrated with bradykinin (1060.6 MW) and glucagon (3483.8 MW) as internal standards.
Following previously developed methods (Krueger et al., 2000),trans-3-indoleacrylic acid (t-IAA) was chosen as the matrix. The dried samples were reconstituted in acetone/ water (8:2, v/v) to give sample concentrations of 1.8 mg/100ml. The ratio of 100ml of reconstituted sample to 5 mg of the matrix t-IAA was found to provide the most reproducible spectra with a high signal to noise ratio. The matrix solution (0.25ml) was deposited onto the target. Trans-3-indoleacrylic acid (Aldrich Chemical Company, Milwaukee, WI) bradykinin, and glucagon (Sigma Chemical Company, St. Louis, MO) were used as received.
2.5. In vitro protein degradation
The rate of protein degradation in four BFT varieties with differing tannin contents (Fergus, Viking (2 year old plants) and GA1 from Uppsala and Norcen from RaÊdde) was determined by the in vitro inhibitor method of Broderick (1987). Rumen ¯uid was taken from lactating cows. Samples of casein and timothy were used as reference materials and incubated at the same time as the BFT samples. Amino acids and ammonia were determined at 0, 0.5, 1, 3 and 4 h after the start of the incubation. Samples were analysed with a Technicon Auto Analyzer (Broderick and Kang, 1980). Results were corrected for soluble nitrogen, which was estimated from the proportion of N that disappeared after a 2 h incubation period in the rumen in sacco. Degradation results were ®tted to a non-linear curve with TableCurveTM software (Jandel Scienti®c, Erkrath, Germany). In replicate 1, cows were fed a grass silage diet supplemented with barley concentrate. In replicates 2 and 3, the rumen ¯uid was from cows fed an alfalfa silage diet with the barley supplement and in replicates 2 and 3, the method by Broderick (1987) was modi®ed by removing one tube at each sampling time instead of repeated sampling of the same tube.
2.6. Statistical analysis
The statistical analysis of the protein degradation data was performed using the General Linear Model described by SAS (1997). When testing for varietal differences, the following model was applied:
whereais the effect of variety,bthe effect of run andethe random error, independently and normally distributed. The signi®cance level was set atP<0:05.
3. Results
3.1. Tannin concentrations
The seven BFT varieties generally had low tannin concentrations with mean values of 0.7% DM by the RD assay and 0.9% DM by the H/B assay. There was considerable variation between varieties ranging from 0.3 to 1.0% (RD assay) and from 0.2 to 1.7 (H/B assay; Table 1). The H/B assay tended to give slightly higher values. Variety GA1 produced the highest tannin levels at Uppsala and also at RaÊdde. Some seasonal differences were noted: Viking from Uppsala had a higher content in 2 year old plants, 0.9% (RD) or 1.1% (H/B), than in 1 year old plants, 0.6% (RD or H/B).
3.2. Tannin composition
The delphinidin/cyanidin ratios varied from 16:84 to 33:67 with a mean value of 25:75 (Table 1). This means that there was a two-fold difference in the percentage of GE units, 16±33%, amongst the BFT varieties.
3.3. Gel permeation chromatography (HPLC-GPC)
The distribution of tannin molecular weights differed only slightly between the BFT varieties. In GPC analysis, the molecular weights of eluting tannins decreases over time. Therefore, peaks 1 and 2 have the highest molecular weights. It can be seen that most variation occurs in the peak height ratios of peaks 2 and 3 (Fig. 1). This means that Fergus had proportionally more large molecular weight tannins than the other varieties. Unfortunately, polystyrene standards, which are commonly used as molecular weight markers, did not elute from the HPLC GPC column under the conditions necessary for the
Table 1
Tannin concentrations and composition in seven varieties of birdsfoot trefoil (Lotus corniculatus) grown at Uppsala, Sweden and measured by the radial diffusion and HCl±butanol assays (expressed as % of DM)a
Variety Radial diffusion HCl±butanol Delphinidin/cyanidin ratio
GA1 1.0 (0.9) 1.7 (1.3) 28:72 (26:74)
Fergus 0.8 1.4 21:79
Leo 0.7 (0.5) 0.9 (0.5) 21:70
Norcen 0.7 (0.6) 0.9 (0.6) 21:79 (16:84)
Viking 0.6 0.6 24:76
Dawn 0.5 0.5 32:68
Empire 0.3 (n.d.)b 0.2 33:67
Fig. 1. HPLC gel permeation chromatography of condensed tannins isolated from threeLotus corniculatus
separation of these tannin molecules. Thus, it was not possible to determine the molecular weights of the tannins and MALDI-TOF mass spectrometry was used instead.
3.4. MALDI-TOF mass spectrometry
Two tannin varieties with contrasting GPC patterns were examined further by MALDI-TOF MS (peak height ratios of GLP peaks 2 and 3 were 1.2 for Fergus and 0.7 for Viking). The MALDI-TOF MS spectra revealed two series of tannin oligomers. The ®rst series consists of well resolved tannin tetramers to hexamers, but also includes oligomers up to the decamer (Fig. 2). A second series exists within each of these oligomers, i.e. there are several tetrameric, pentameric, etc. tannins (Fig. 3). Comparing the observed (Fig. 3) with the calculated molecular weights (Table 2)it can be seen that there are more heteropolymers than homopolymers. Tetrameric to hexameric homopolymers consisting of four to six catechin/epicatechin (CE) units, i.e. procyanidins, had molecular weights of 1177, 1465, 1754 Da, respectively. Several heteropolymers containing both CE- and gallocatechin/epigallocatechin (GE) units were more prominent in the MS spectra (Figs. 2a and 3). Most heteropolymers had one to two GE units (tetramers: 1193, 1209; pentamers: 1481, 1497; hexamers: 1770, 1786), but higher substitution patterns with three to four GE units were also detectable (tetramer: 1225; pentamer: 1513; hexamer: 1801, 1817). No GE homopolymers, i.e. prodelphinidins, were detected by MALDI-TOF MS.
3.5. Protein degradation
The extent of N-degradation differed signi®cantly (P<0:05) between Norcen and the
other three BFT varieties (Table 3). The proportion of soluble nitrogen that was degraded after 4 h was highest in Norcen and lowest in GA1. The ranking amongst the varieties
Fig. 2. MALDI-TOF mass spectroscopy (linear mode) of an oligomeric series of condensed tannins from two
was the same in all replicates but the absolute amounts differed. The correlation between the RD tannins and degraded protein fraction after 4 h was very high (R20:93). Curve
®tting revealed that the degradation rates did not follow a single exponential function. It would appear that degradation rates at times 0±1 h differed from those between 1 and 4 h.
Fig. 3. (Continued).
Table 2
Calculated molecular weights of oligomers (Dalton)
Flavanol composition
bIndicates a homopolymer containing only CE units. cGE: gallocatechin/epigallocatechin.
However, due to the small number of data points (n5), this could not be investigated further.
4. Discussion
4.1. Tannin concentrations in BFT varieties and their effects on protein degradation
In Sweden, the mean tannin concentration in BFT varieties was <1% of the DM and was, thus, comparable to the levels found in Wisconsin (Broderick and Albrecht, 1997). These were signi®cantly lower than the concentrations reported from New Zealand, i.e. 2.5±3.9% (Wang et al., 1994; Douglas et al., 1995; Lowther et al., 1987). However, not all reported tannin concentrations are necessarily comparable. The use of tannic acid (a hydrolysable tannin) as the standard in the R/D assay tends to yield underestimates (Hagerman, 1987), whereas the vanillin reaction can sometimes produce overestimates with catechin as the standard (Mole and Waterman, 1987). The present study attempts to report more reliable concentrations by using isolated BFT tannins as the standard. In addition to this precaution, two tannin assays were employed. Although the RD and H/B assays produced a similar ranking of the varieties (Table 1), the concentrations were not identical. One possible explanation could be that the two assays differ in their reactivities towards individual tannin molecules. We, therefore, suggest that the divergent data from the RD and H/B assays are due to differences in the relative composition of tannins (see below). Recent work on Calliandra calothyrsus tannins also demonstrated that intra-species tannin differences signi®cantly affected the assay results (Stewart et al., 2000). It would appear that tannin concentrations were in¯uenced most by varietal and less by geographic effects (Table 1) in Sweden.
One of the most surprising ®ndings from this study is the fact that even at concentrations below 1% of DM, tannins were negatively correlated (R20:93) to
protein degradation in vitro. In vivo feeding trials are needed to test their effects on protein digestion in the whole gut. However, for ruminants with little undegradable
Table 3
Tannin contents (as % of DM) and proportion of soluble nitrogen from four varieties of birdsfoot trefoil (Lotus corniculatus) degraded after 4 h in vitro using rumen ¯uid from lactating cows fed grass or alfalfa silage-based dietsa
Variety % Tannin Proportion of N degraded
RDb H/Bc Donor cow diet Mean
Grass silage Alfalfa silage Alfalfa silage
GA1 1.0 1.7 0.43 0.50 0.76 0.56 b
Viking 0.9 1.1 0.52 0.59 0.89 0.67 b
Fergus 0.8 1.4 0.53 0.57 0.93 0.68 b
Norcen 0.6 0.6 0.61 0.83 1.0 0.81 a
aMeans followed by different letters are signi®cantly different (P<0:05). bRD: diffusion assay.
protein in their diets, e.g. animals on pasture, this could mean an increase of between 18 and 25% of the ¯ow of feed protein to the duodenum. This in turn could improve the nutritional status of the animal and improve its ability to ®ght infections, such as helminths (Butter et al., 1999). Therefore, even though concentrations were low in BFT varieties, selection of varieties should also be based on tannin contents. It would be wrong to assume that all BFT varieties produce similar nutritional bene®ts. Instead, varieties with the higher tannin contents, i.e. GA1, Fergus and Viking, should be investigated further despite their somewhat inferior agronomic properties.
4.2. Tannin composition in BFT varieties
D:C ratios of the BFT tannins varied two-fold ranging from 16:84 to 33:67, which agreed with observations by Foo et al. (1982) who reported values from 27:73 to 50:50. In contrast, L. uliginosus (previously: L. pedunculatus) and other species appear to have more constant D:C ratios (Jones et al., 1976; Foo et al., 1982). However, BFT tannins from Sweden had much lower levels of GE-units (16±33%) than the New Zealand tannins (27±50%).
It is not known if there are nutritional implications of variations in D:C ratios. Broderick and Albrecht (1997) observed slower ruminal degradation of protein fromL. uliginosuscompared to BFT, which could be caused by their different tannin structures or concentrations. Foo et al. (1997) and Aerts et al. (1999) suggested that the nutritional differences between these two species could be related to differences in their D:C ratios, although this might not be the only reason as the D:C ratios in sainfoin, for example, are similar toL. uliginosusÐ unlike their nutritive values (Thomson et al., 1971; Jones et al., 1976; Foo et al., 1982; Koupai-Abyazani et al., 1993).
There are few reports of HPLC-GPC separations of condensed tannins (Karchesy et al., 1989). BFT tannins were successfully separated into six peaks by HPLC-GPC on PL-Aquagel OH columns (Fig. 1). Most variation between BFT varieties occurred amongst the higher molecular weight tannins, i.e. in the relative heights of peaks 2 and 3. The Fergus and Norcen varieties had the highest proportions of peak 2 tannins. The distribution of tannin molecular weights was similar between the GA1, Leo (chromatograms not shown) and Viking varieties. It remains to be seen if such differences will affect protein degradation in the rumen.
Our other MALDI-TOF MS data of tannins from sorghum,C. calothyrsusand grapes suggest that a multiplicity of structures can occur within a species and may be relatively widespread (Krueger et al., 2000). This is supported by previous reports of tannin heterogeneity in sorghum, barley andCroton lechleri(Brandon et al., 1982; Cai et al., 1991). Structural isomerism in condensed tannin chemistry, therefore, extends beyond the variation in inter¯avonoid linkages (4!8 or 4!6, a or b) and cis- or trans -stereochemistries (Stafford, 1990), but can also include distinctive patterns of B-ring hydroxylation within oligomers of the same degree of polymerisation. Unlike FAB MS or NMR specroscopy (Cai et al., 1991), MALDI-TOF MS can be used successfully to elucidate such mixtures of closely related tannin oligomers.
In conclusion, differences in tannin composition and protein degradation were observed between the BFT varieties. Further research is required to elucidate the relationship between CE/GE ratios, molecular weights and the nutritional effects of BFT tannins. Such information may provide guidance for plant breeders aiming to improve the agronomic performance and nutritional quality of BFT.
Acknowledgements
We would like to acknowledge Martha Vestling and Paul Treichel, UW-Madison Chemistry Instrumentation Center for assistance with the MALDI-TOF MS, Ron Brown for skilled assistance with HPLC analysis, HaÊkan Wallin for excellent technical assistance
with the AutoAnalyzer and Polymer Laboratories Ltd for advice in setting up the GPC analysis and loan of GPC-columns.
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