J Sci Food Agric 1997, 74, 513È523

Comparison of the Precipitation of Alfalfa LeafProtein and Bovine Serum Albumin by Tannins inthe Radial Diffusion MethodB I Giner-Chavez,1 P J Van Soest,1 J B Robertson,1 C Lascano2 and A N Pell1,*1 Department of Animal Science, Cornell University, New York 14853, USA2 Centro Internacional de Agricultura Tropical-CIAT. A.A. 6713, Cali, Colombia

(Received 17 June 1996 ; revised version received 9 December 1996 ; accepted 19 February 1997)

Abstract : The precipitation of protein by condensed and hydrolysable tanninswas evaluated with the radial di†usion method of Hagerman (1987) using bovineserum albumin (BSA) and isolated leaf protein from fresh alfalfa (Medicagosativa). Alfalfa leaf protein (AALP) was included at two concentrations, 25 and156 mg N litre~1, at pH 6É8 and 39¡C to simulate rumen conditions. The con-densed tannins were puriÐed from lyophilised samples of Arachis pintoi, Desmo-dium ovalifolium, Gliricidia sepium, Manihot esculenta and quebracho (Schinopsisbalansae). Hydrolysable tannins from tannic acid (TA) were used as well. Therewas a signiÐcant interaction (P\ 0É001) between tannin and protein source, andprotein level on protein precipitation. Most puriÐed condensed tannins (CTs)precipitated more AALP than BSA when protein was included at the same level.PuriÐed CT from quebracho and hydrolysable tannin from TA failed to precipi-tate AALP at both protein levels. In a second experiment, tannins from crudeplant extracts were studied in the radial di†usion method using BSA and twolevels of AALP. The crude plant extracts were obtained from lyophilised plantsamples of A pintoi, Centrosema macrocarpum, Clitoria ternatea, D ovalifolium,Erythrina berteroana, E poepigiana, G sepium, M esculenta, Pueraria montana andP phaseoloides. The protein precipitated by soluble tannins in the plant sampleswas correlated to the total phenolic content and to the soluble CT estimated bythe acid butanol assay or by the radial di†usion method. Tannins from di†erentplant species precipitated di†erent amounts of BSA and AALP. Therefore, themeasures of the biological activity of tannins based on BSA precipitation maynot reÑect the ability of tannins to precipitate proteins of plant origin such asthose commonly found in the diets of herbivores. The present study o†ers thepossibility of using the radial di†usion method with plant proteins at precipi-tation conditions similar to those in the rumen.

J Sci Food Agric 74, 513È523 (1997)No. of Figures : 2. No. of Tables : 4. No. of References : 35

Key words : condensed tannins, alfalfa leaf protein, bovine serum albumin,protein precipitation

INTRODUCTION

Legumes and other plant foliages are important sourcesof feed for ruminants in the tropics, where they play asigniÐcant role in supplementing grass-based diets(Norton 1994). Forage legumes can contain high levelsof tannins, particularly condensed tannins, that can

* To whom correspondence should be addressed.

have either negative or beneÐcial e†ects on protein andcarbohydrate digestibility (Mueller-Harvey andMcAllan 1992 ; Reed 1995).

Tannins have been deÐned as “any phenolic com-pound of sufficiently high molecular weight containingsufficient hydroxyl and other suitable groups (i.e.carboxyls) to form e†ectively strong complexes withprotein and other macromolecules under the particularenvironmental conditions being studiedÏ (Horvath

5131997 SCI. J Sci Food Agric 0022-5142/97/$17.50. Printed in Great Britain(

514 B I Giner-Chavez et al

1981). Di†erent species of plants synthesise tannins thatvary greatly in structure. The two major structuralclasses of tannins are hydrolysable and condensedtannins (proanthocyanidins). Although they di†er bio-synthetically and chemically, they both are phenolicsand can precipitate protein. Because the affinity oftannins for di†erent substrates varies with the physi-cochemical conditions, it is important to study theseinteractions using conditions that are similar to those ofthe environment under studyÈin this case, the rumen.

There are two types of protein precipitation assays todetermine the biological activities of tannins. One groupestimates the amount of tannins precipitated by a stan-dard protein such as bovine serum albumin (BSA)(Hagerman and Butler 1978) and the second groupmeasures the amount of protein in the tanninÈproteincomplex (Hagerman and Butler 1980 ; Martin andMartin 1983 ; Asquith and Butler 1985). The disadvan-tages of these methods are that they require carefulsample preparation and special equipment. In addition,solvents such as acetone interfere with protein precipi-tation (Hagerman 1987).

The radial di†usion method developed by Hagerman(1987) does not su†er from these disadvantages. In thisassay, the amount of tannin present in a crude plantextract is indirectly measured by the ability of thetannin to precipitate BSA. The tannin di†uses througha protein-containing gel, resulting in the formation of atanninÈprotein precipitate ring. The area of the precipi-tate is believed to be proportional to the amount oftannin in the extract. An advantage of this procedure isthat many samples can be handled with limited labor-atory facilities.

Since di†erent proteins are precipitated to di†erentextents by tannins (Martin and Martin 1983), it isappropriate to use a protein source that is similar tothose in animal feeds if the goal is to evaluate whethertanninÈprotein binding makes dietary protein unavail-able. The Ðrst objective of this study was to comparethe amount of protein precipitated by tannins in thestandard BSA assay and in a modiÐed version usingalfalfa leaf protein. In addition, the pH of the alfalfa leafprotein gels and the incubation temperature were modi-Ðed to simulate the ruminal environment. A secondobjective was to compare the precipitation of BSA andalfalfa protein by both puriÐed condensed tannins andunpuriÐed soluble condensed tannins from variousplant species.

MATERIAL AND METHODS

Tannin sources

Commercial tannin sourcesTannic acid (TA, purity 97%, Lot 40-57B) was pur-chased from Chem Service (PO Box 3108, Westchester,

PA 19381, USA). Crude quebracho was kindly donatedby the Trask Chemical Corporation, 3200 W. SomersetCourt, Marrietta, GA 30067, USA). The TA and queb-racho were puriÐed using Sephadex LH-20 (Hagerman1991).

Plant samplesPlant samples were frozen and lyophilised shortly afterharvest. Plant samples of Arachis pintoi, Centrosemamacrocarpum, Clitoria ternatea, Desmodium ovalifolium,Erythrina berteroana, E poepigiana, Gliricidia sepium,Manihot esculenta, Pueraria montana and P phaseoloideswere donated by Dr Carlos Lascano and Dr CarlosVicente Duran from CIAT (Centro Internacional deAgricultura Tropical, Cali, Columbia). A second set ofplant samples of D ovalifolium and G sepium wasdonated by the Escuela Agricola Panamericana (ElZamorano, Honduras). A third set of plant samples of Gsepium was donated by Dr Edwin Perez (San Jose� ,Costa Rica).

Extraction of crude plant extracts for condensed tanninpuriÐcationBecause condensed tannins (CTs) were not present in allplant species obtained, only puriÐed CTs from A pintoi,D ovalifolium, G sepium and M esculenta were studied.Crude plant extracts were obtained by three sequentialextractions of 2 g of the lyophilised plant material using70% aqueous acetone. The CTs were puriÐed fromthese crude extracts using the ytterbium methoddescribed by Giner-Chavez et al (1997).

Condensed tannin puriÐcationThe same procedure used for the puriÐcation of queb-racho CT (Hagerman 1991) was used for the plantsamples, with one modiÐcation. Since acetone preventedthe absorption of tannins to Sephadex LH-20, acetonewas removed from the crude plant extract undervacuum at less than 30¡C. The evaporated volume ofacetone was replaced by a smaller volume of ethanol todissolve some of the chlorophyll that might have pre-cipitated during evaporation. PuriÐcation of CT fromeach source was performed in triplicate. CTs from eachpuriÐcation were combined.

Extraction of crude plant extract for direct use in theradial di†usion assayThe extraction of tannin from plant samples was per-formed as previously described, with one modiÐcation.Extraction of 200 mg of lyophilised plant material wasperformed in a 15-ml snap-top plastic tube three timeswith 8 ml of 70% aqueous acetone (v/v) for 20 min intriplicate.

Extraction of alfalfa leaf protein

Fresh forage was collected at the Teaching andResearch Center (Harford, New York) of the Animal

Protein source and amount a†ects protein precipitation by tannins 515

Science Department of Cornell University. Fresh alfalfa(Medicago sativa) forage (2 kg) was separated intoleaves and stems, and the leaves were washed with dis-tilled water while the stems were discarded. The alfalfaleaf protein (AALP) was extracted by a modiÐcation ofthe procedure of Merodio and Sabater (1988). The pro-cedure was modiÐed in the following ways : (1) no heatwas used during the fractionation, (2) formic acidreplaced HCl to precipitate the coagulum and (3) pre-cipitated protein was washed with EtOH and dried withacetone instead of lyophilising the residue. After thecoagulum settled, the supernatant was decanted and theremaining slurry was rinsed 2È4 more times with EtOHuntil no yellow colour was evident in EtOH. Theresidual protein fraction was Ðltered through WhatmanÐlter paper (no. 54) and washed with acetone. The pre-cipitate was dried at room temperature. Drying washastened by pressing and crumbling the precipitate witha spatula into a thin Ðlm to minimise oxidation. Theprotein from Ðve extractions which were performedduring a 2-week period was combined and stored at[25¡C in a desiccator in the dark until further use.

Radial di†usion assay

The radial di†usion assay with agar containing BSAwas performed as described by Hagerman (1987). Tomimic rumen conditions, the standard assay was modi-Ðed by varying the source and level of protein, the gelpreparation procedure, pH and incubation temperature.

Selected protein levelsTwo levels of AALP were used. One was similar to theprotein content of the BSA-containing gel (0É1%) (highprotein level, AAH) and the second one was similar tothe soluble protein content of rumen Ñuid (low proteinlevel, AAL). The protein content was expressed on a Nbasis. The total protein content of BSA was determinedby the Kjeldahl digestion method using boric acidduring distillation (Pierce and Haenisch 1947). Themethod of Licitra et al (1996) was used to measure thesoluble protein content of clariÐed rumen Ñuid, exceptthat a 30-ml aliquot of clariÐed ruminal Ñuid wasplaced in a 125-ml Erhenmeyer Ñask and the volumemade up to 50 ml with cold distilled water.

Ruminal Ñuid (1 litre) was separately collected fromtwo dry Ðstulated Holstein cows fed a mixed, mostlygrass, hay diet. Samples were collected 3 h after feedingfor two consecutive days. Within 0É5 h after collection,rumen Ñuid was blended, Ðltered through two layers ofcheese cloth and centrifuged at 170] g for 20 min at5¡C. The supernatant then was centrifuged twice at18 000] g for 20 min at 5¡C.

Selected pH and incubation temperatureThe pH of the AALP-gels was 6É8 and the incubationtemperature was 39¡C to simulate rumen conditions.

The mean pH was obtained by sampling the pH of therumen liquid from four di†erent sections in the ruminalcompartment of two di†erent forage-fed cows.

Incorporation of alfalfa leaf protein in radial di†usionplatesTwo AALP aliquots of 0É1 g were separately suspendedin 5 ml of 0É5 M ethanolamine and allowed to dissolveovernight at 4¡C. The solutions were mixed and trans-ferred to a 50-ml plastic centrifuge tube. The combinedsolution was centrifuged at 5000 ] g for 20 min at 5¡C,and the supernatant was transferred to a 50-ml cylinderand the volume made up to 30 ml. The soluble proteincontent (Licitra et al 1996) of the solution was deter-mined on 2-ml aliquots in triplicate. The rest of thesolution was stored at 4¡C to be used in the radial di†u-sion assay.

After soluble protein content was determined, two ali-quots were taken, one equivalent to the amount ofprotein in 100 mg of BSA and another equivalent to theamount of soluble protein in 100 ml of clariÐed rumenÑuid. Both aliquots were separately placed in 50-mlbeakers and the volume made up to 25 ml with distilledwater. The pH of the solution was decreased to 6É8 with0É1 M acetic acid, and the volume made up to 50 mlwith distilled water. The solution was warmed to 45¡Cin a water-bath.

Agarose solution was prepared with 2 g of agaroseand 100 ml of 50 mM N-[-2-acetamido]-2-iminodiaceticacid bu†er (ADA, pH adjusted to 6É8 with 1É5 M

ethanolamine) by boiling while stirring in a water-bath.The agarose solution was divided into two equal por-tions (v/v) and placed in a water-bath at 45¡C. The solu-bilised AALP solutions (50 ml) were mixed with theagarose solution (50 ml) to a Ðnal agarose concentra-tion of 1%. The solution was dispensed in 9É5-ml ali-quots into Petri plates and the rest of the procedure wasas for the BSA-containing plates.

Condensed tannin content measurementThe soluble CT content of the crude plant extracts wasmeasured in four replicates using the radial di†usionassay with BSA-containing gels (Hagerman 1987) andby the radial di†usion modiÐcation with AAH- andAAL-containing gels. Each extract (8 kl) was dispensedinto individual 4-mm wells in the agarose gel. Toprevent formation of discrete concentric rings, the sub-sequent aliquot was dispensed before the initial aliquotwas absorbed completely. After incubation, the diam-eter of each ring was measured twice at right angles toone another with a Vernier caliper to the nearest0É1 mm. The height was measured with the caliper aswell. The measurements were averaged and the area ofthe ring was calculated. The standard used in this assaywas obtained by dispensing 16È48 kl of tannin-containing solution (2 mg ml~1) in aliquots of 8 kl into

516 B I Giner-Chavez et al

individual wells and measuring the diameter and heightof each ring.

Calculation of protein precipitatedTo estimate the biological activity of tannins in thepresent study, the amount of protein precipitated wasdetermined by multiplying the volume of the ringformed by the amount of protein per ml of the protein-containing gel. The following formula was used to cal-culate the protein precipitated (procedure developed byC Lascano, unpublished data) :

Protein precipitated (kg)\

[Volume (ml)]] [Protein concentration (kg) ml~1)]

where where is the ringVolume\ (r1[ r2)2 É h Én, r1radius, is the well radius and h is the well height (cm).r2

In the case of a sample plant extract, the totalamount of protein precipitated was divided by thesample dry matter weight at 100¡C.

Analytical assays

T otal phenolicsTotal phenolic (TP) content was measured in the crudeplant extracts in triplicate by HagermanÏs (1991) modiÐ-cation of the Prussian blue assay. All determinationswere performed using a Milton Roy Spectronic 21model DV spectrophotometer (820 Linden Avenue,Rochester, NY 14625, USA).

Condensed tannin content by the acid butanol assaySoluble CTs were measured in crude plant extracts intriplicate by the acid butanol assay (Porter et al 1986).A reagent blank, without sample, was prepared andused for the baseline absorbance reading. A standardcurve for each plant was generated using the puriÐedtannin to permit tannin quantiÐcation.

Analytical measurements of the alfalfa leaf proteinThe amino acid composition of AALP was determinedin duplicate by hydrolysing 0É075 g of AALP and75 kmol of norleucine internal standard in 25 ml of 6 M

HCl under nitrogen for 21 h at 110¡C. The sample wasÐltered through Whatman Ðlter paper (no. 1) into avolumetric Ñask and the volume was made up to 50 mlwith HPLC-grade distilled water. An aliquot of 1 mlwas evaporated under nitrogen and the volume madeup to 10 ml with lithium citrate bu†er (pH 2É8) foramino acid analysis. Samples were analysed by ion-exchange HPLC using Ñuorescence detection with o-phthalaldehyde (Model 334, Beckman Instruments,2350 Camino Ramon, PO Box 5101, San Ramon, CA94583-0701, USA).

SDS-PAGE was performed on the AALP asdescribed by Laemmli (1970). The composition of theseparating gel was 12% acrylamide, 0É3% bisacrylamide

and 0É1% SDS. The composition of the stacking gel was3É0% acrylamide, 0É08% bisacrylamide and 0É1% SDS.The AALP (10 mg) was dissolved in 0É5 ml of SDS-sample bu†er (1É52 g Tris base, 2 g SDS, 1 mg bromo-phenol blue and 20 ml glycerol in 100 ml of deionisedwater) at pH 6É8 and boiled 5 min at 100¡C to denaturethe proteins. The pH of the electrophoresis bu†er was8É3 (3É02 g Tris base, 14É4 g of glycine and 1É0 g of SDSin 1000 ml of deionised water). The gel was run at aconstant voltage of 60 V for 24 h at room temperature.

Total protein content of AALP was analysed in100-mg samples by the Kjeldahl digestion method usingboric acid during distillation (Pierce and Haenisch1947). Soluble carbohydrates were determined by theanthrone method (Bailey 1958) using fructose as thestandard. Dry matter content was determined by dryingat 100¡C for 24 h. Ash content was determined byashing at 520¡C for 8 h. Ether extract was determinedby a standard method (AOAC 1990).

Experiment 1. Precipitation of protein from AALP andBSA by di†erent sources of puriÐed condensed tannin

The protein precipitated by each source of puriÐed CT(A pintoi, D ovalifolium, G sepium, M esculenta, queb-racho and TA) was evaluated in AAL-, AAH- and BSA-containing gel plates. PuriÐed CT (2 mg ml~1) wasdissolved in distilled water and applied in successive ali-quots (2, 3, 4, 5 or 6 aliquots per well) of 8 kl in individ-ual wells. Two measurements of the diameter of eachring were taken and averaged to calculate the volume ofprecipitated tanninÈprotein. Four replicates of plateswere performed for each protein-level and CT source.

Experiment 2. Precipitation of protein from AALP andBSA by soluble condensed tannin present in variousplants

The proteins precipitated by tannins present in crudeplant extracts of various parts of A pintoi, C macro-carpum, C ternatea, D ovalifolium, E poepigiana, E ber-teroana, G sepium, M esculenta, P phaseoloides and Pmontana were evaluated in AAL-, AAH- and BSA-containing gel plates. Six successive aliquots of 8 klwere dispensed into individual wells. Two readings ofthe diameter of each ring were obtained and averagedto calculate the precipitated tanninÈprotein volume.Four replicates of plates were performed for eachprotein-level and plant extract.

Statistical analysis

Experiment 1A three-way analysis of variance was used to examinethe e†ects of protein level (standard vs rumen) andsource (BSA vs AALP), tannin source (A pintoi, D ovali-

Protein source and amount a†ects protein precipitation by tannins 517

folium, G sepium, M esculenta, quebracho and TA) andtannin level (16, 32, 48, 64, 80 and 96 kl ml~1) on theamount of protein precipitated. The equivalent factore†ects model used was

Yijkm

\ k É É É ] ai] b

j] c

k] (ab)

ij] (ac)

ik] (bc)

jk] (abc)

ijk] e

ijkmwhere a is the protein source and level, b is the tanninsource and c is the tannin level.

Orthogonal contrasts were constructed to investigatethe e†ects of level and source of protein : (1, BSA againstAALP; 2, AAL against AAH) and source of tannin (1,quebracho CT against TA; 2, quebracho CT againstplant CT; 3, legume CT against M esculenta CT; 4, treelegume CT against non-tree legume CT; 5, A pintoi CTagainst D ovalifolium CT). The gradient trend e†ect oftannin concentration on protein precipitation wasevaluated to verify which polynomial (linear, quadratic,cubic and quartic) trends were signiÐcant (Neter et al1990).

Experiment 2Correlation coefficients and their level of signiÐcancewere calculated to identify associations between TPcontent or soluble CT-acid butanol or soluble CT-radial di†usion content and protein precipitated bysoluble tannins present in the plant samples. Outlyingobservations were identiÐed by CookÏs distancemeasure analysis in the regression analysis (Neter et al1990). The GLM, correlation and regression procedures(SAS 1990) were used for the ANOVA and regressionanalyses.

RESULTS AND DISCUSSION

Analysis of alfalfa leaf protein

The yield of AALP from alfalfa foliage on a dry matter(DM) basis was 1É1 g kg~1. This yield represented 0É7and 1É1% of the total crude protein and soluble proteincontent, respectively, in alfalfa foliage. The yield ofAALP was low because no heat was applied duringfractionation to avoid proteolysis leading to decreasedcoagulation (Baraniak 1990). The chemical compositionof AALP (DM, 900É0 g kg~1 ; NPN, 62É0 g kg~1 ;soluble true protein, 677É0 g kg~1 ; soluble sugars,19É0 g kg~1 ; ether extract, 0É4 g kg~1 ; ash, 4É8 g kg~1 ;other unidentiÐed components, 236É8 g kg~1) and theamino acid proÐle (Table 1) were similar to other prep-arations (Betschart 1974 ; Bicko† et al 1975). Glutamicacid, aspartic acid, leucine, arginine and lysine were themost abundant amino acids. Glutamic acid, lysine andleucine are also the most abundant amino acids in BSA.

Two major bands with molecular weights (MWs) ofapproximately 50 000 and 14 200 were evident on SDS-PAGE. These bands probably correspond to the large

TABLE 1Amino acid composition (dry matter basis) of alfalfa leaf

protein

Amino acid Content (g kg~1)

Alanine 43É9Arginine 53É2Aspartic acid 71É2Cystine 8É6Glutamic acid 83É2Glycine 37É5Histidine 21É8Isoleucine 35É9Leucine 67É6Lysine 51É2Methionine 9É9Phenylalanine 45É6Serine 22É9Threonine 36É6Tyrosine 36É9Ornithine 0É6Valine 46É6

(MW 56 000) and small subunits (MW 16 000) ofribulose-1,5-biphosphate carboxylase, which is themajor soluble leaf protein (McNabb et al 1994). Theabsence of multiple bands of low MW indicates littleproteolysis occurred during AALP puriÐcation, a situ-ation observed when heat is used during AALP prep-aration (Free and Satterlee 1975).

BSA and ruminal Ñuid analysis

The N content of the BSA after tungstic acid precipi-tation was 15É6 mg N g~1. The soluble fraction of thetungstic acid precipitate of the clariÐed ruminal Ñuidcontained 25^ 1É4 kg N ml~1. The pH was 6É58 ^ 0É08and 6É97 ^ 0É08 from each cow, respectively. The AALand AAH gels contained 25 and 156 kg N ml~1 fromAALP, respectively. The pH of the gels was 6É8. The Nlevel in the BSA and AAH gels was six times higherthan the amount measured in clariÐed rumen Ñuid.However, soluble protein in clariÐed rumen liquid is notthe only source of protein in rumen digesta. Microbialand insoluble dietary protein also can complex withtannin and, therefore, the available protein pool mayvary signiÐcantly according to feed composition andDM intake.

Comparison between protein complex area and proteinprecipitated volume

Highly signiÐcant correlations were obtained from therelationship between tannin concentration and precipi-tated area (standard method) (r \ 0É97, P\ 0É001) orprotein precipitated volume (modiÐed method)

518 B I Giner-Chavez et al

(r \ 0É99, P\ 0É001). As expected, the relationshipbetween the two methods was also highly correlated(r \ 0É99, P\ 0É001) for each protein-level and eachpuriÐed tannin source.

Experiment 1. Precipitation of protein from BSA andAALP by di†erent puriÐed tannins

The relationship between the amount of protein precipi-tated and tannin concentration in the gel is shown inFig 1. Figure 2 presents the efficiency with whichtannins could precipitate protein (g g~1) when level andsource of protein in the gels was varied.

Fig 1. Protein precipitated by puriÐed condensed tannin from(A) Arachis pintoi, (D) Desmodium ovalifolium, (G) Gliricidasepium, (M) Manihot esculenta, (Q) quebracho and (TA) tannic

acid in (1) AAL-, (2) AAH- and (3) BSA-containing gels.

The three-way interaction among tannin source,protein source and level of protein precipitation wassigniÐcant (P\ 0É001). The relationship between CTconcentration and protein precipitation was linear(P\ 0É001). At the higher level of protein concentration(156 mg N litre~1), the efficiency with which CT from Apintoi, D ovalifolium, G sepium and M esculenta precipi-tated AALP was higher than for BSA. Less AALP wasprecipitated by tannins when AALP was incorporatedinto the agar at typical ruminal concentrations than atthe higher level. Neither puriÐed CT from quebrachonor hydrolysable tannins from TA precipitated AALPat either protein level. These results agreed with those ofPerez-Maldonado et al (1995), who found that moreplant leaf protein was precipitated by CT from Desmo-dium intortum and L otus pedunculatus than by TA whensamples were incubated in a ruminal environment. TAand puriÐed CT from quebracho precipitated more BSAthan puriÐed CT from forages. Martin and Martin(1983) also found that TA could precipitate BSA moreefficiently than quebracho.

PuriÐed CT precipitated more AALP than BSA, butthe opposite occurred with quebracho and TA. If thesources of tannin are ranked by their ability to precipi-tate protein, the order is the same (M esculenta[ Dovalifolium[ A pintoi) within protein level. The onlyexception was G sepium (Fig 1). A higher affinity of CTfor protein as the degree of polymerisation increases(Hagerman and Klucher 1986 ; Vaithiyanathan andKumar 1993) may explain this pattern. The di†erentbehaviour of the CT from G sepium may have occurredbecause the optimum protein level for maximum pre-cipitation by this tannin was not evaluated (Hagermanand Robbins 1987). The higher reactivity of the Gsepium CT may be related to the difficulty of extractingthem from plant tissue.

The efficiency with which tannins precipitated AALPdecreased with an increase in the amount of protein inthe gel. Although there was a six-fold di†erence inprotein between the AAL and AAH treatments, the dif-ference between AAL and AAH in the amount ofprotein precipitated was not proportional to theamount of protein added (1É6 times, M esculenta ; 2É2times, D ovalifolium ; 3É3 times, A pintoi). The amount oftannin precipitated by G sepium was proportional to theincrease in the amount of protein added.

Experiment 2. Precipitation of protein from BSA andAALP by soluble tannins present in various plant samples

The TP content of the plant samples is presented inTable 2. The soluble CT content from the acid butanolassay and the radial di†usion method using the threedi†erent types of protein-containing gels is presented inTable 3. The amount of protein precipitated in each ofthe three protein-containing gels is shown in Table 4.

Protein source and amount a†ects protein precipitation by tannins 519

Fig 2. Efficiency of protein precipitation (kg kg~1) by puriÐed tannins of AAL-, (0) AAH- and BSA-containing gels.(K) (=)

The TP and soluble CT-acid butanol contents werehighly correlated among samples (r \ 0É97, P\ 0É001)that contained CT. When samples with both TP andsoluble CT were ranked from high to low tannin levels,the order was D ovalifolium[ M esculenta, Gsepium[ A pintoi. The order of the samples that con-tained phenolics but no CT wasErythrina [ Centrosema[ Clitoria, Pueraria species.

The soluble CT-radial di†usion content had a lowcorrelation with TP (r \ 0É60, P\ 0É001) and solubleCT-acid butanol content (r \ 0É62, P\ 0É001). Thesoluble CT-radial di†usion content among species had anarrower range than the other two assays, which mayexplain low correlations with the TP and the acid-butanol methods. The rankings of the plants using theradial di†usion assay was D ovalifolium, M esculenta, Apintoi [ G sepium. The low correlations obtained arenot surprising when one considers the speciÐcity of theassays. The TP assay was designed to measure the totalnumber of phenolic hydroxyl groups present regardlessof the phenolic molecules on which they occur. In con-trast, the acid-butanol method, which involves depoly-merisation of CT resulting in the formation ofanthocyanins which are detected spectro-photometrically, measures the amount of proanthocya-nidins present. The radial di†usion assay yieldsinformation on the ability of a tannin to precipitateprotein and is related to the biological activity of thetannin (Hagerman and Butler 1989).

The amount of protein precipitated was more strong-ly correlated with the soluble CT-radial di†usioncontent than with the TP or the CT-acid butanolcontent. Other studies have also failed to obtain highcorrelations between protein precipitation and TP orCT content (Becker and Martin 1982). Extracts from Cmacrocarpum, C ternatea, E berteroana, E poepigiana, P

montana and P phaseoloides species did not precipitateprotein ; consequently, the TP in these samples are non-tannin phenolics that do not precipitate protein(Hagerman 1987). In descending order, the amount ofprotein precipitated by tannins in crude plant extractswas D ovalifolium[ M esculenta[ A pintoi [ G sepium.Among all samples, except G sepium, the immaturefoliages contained more tannins that precipitatedprotein than mature foliages. The tannins in the leafsamples were more efficient at precipitating protein andwere present in greater quantities than those in the leafand twig samples. The twig samples contained lesstannins than the others.

The tannins precipitated slightly more protein fromAALP than from BSA at the same concentration (Fig2). When as much as 96 kg of tannin was added to thewells of the agar, the relationship between added tanninand protein precipitated remain linear for both proteinlevels and for all of the tannins tested. Although sixtimes as much protein was added to the high proteintreatments compared to the low level, only in the caseof G sepium does the amount of protein precipitatedreÑect this ratio. With the other tannins, the ratio of theamount in the AAH treatment to the amount bound inthe AAL treatment varied from 1É75 : 1 (M esculenta) to4É5 : 1 (A pintoi). These ratios indicate that the bindingefficiency was higher (more protein bound per unit oftannin present) at the low level of protein inclusion.

The ranking of the samples by the amount of proteinprecipitated in AAL was not the same as for the higherprotein treatments. Nor was the ranking of the amountof protein precipitated in AAL the same as the rankingobtained from the TP and soluble CT-radial di†usioncontent. Therefore, it can be hypothesised that theseassays are of limited value and emphasise the impor-tance of using tannin measurements that relate to the

520 B I Giner-Chavez et al

TABLE 2Total phenolics content (g kg~1) in various plant samples

Sample species(plant part/maturity stage)

Precedence T otal phenolics

Meana SE

Desmodium ovalifolium1 Leaf immature CIAT 350 140É2 3É82 Leaf immature CIAT 350 171É6 3É83 Leaf immature Honduras 163É7 4É44 Leaf mature CIAT 13089 185É9 6É05 Leaf mature CIAT 350 198É9 3É16 Leaf mature CIAT 350 231É6 2É87 Leaf mature CIAT 350 278É5 1É69 Leaf and twig mature CIAT 350 96É0 4É88 Leaf and twig mature CIAT 350 176É1 4É210 Leaf and twig mature Costa Rica 118É0 0É911 Twig mature CIAT 350 56É3 1É512 Twig mature CIAT 13089 74É0 5É5

Gliricidia sepium13 Leaf immature CIAT 34É6 014 Leaf immature CIAT 59É1 0É515 Leaf immature Costa Rica 93É5 2É216 Leaf immature Honduras 22É3 0É217 Leaf immature Honduras 37É7 2É518 Leaf mature CIAT 41É7 0É919 Leaf mature CIAT 80É2 0É720 Leaf mature Costa Rica 80É2 2É421 Leaf mature Honduras 42É1 0É222 Leaf and twig immature Costa Rica 42É5 1É123 Leaf and twig mature Costa Rica 43É8 0É5

Manihot esculenta24 Leaf and stem immature CIAT MCOL12 43É5 0É425 Leaf and stem immature CIAT CM507 93É2 2É026 Leaf and stem mature CIAT MCOL13 36É6 0É627 Leaf and stem mature CIAT CM508 57É5 0É8

Arachis pintoi28 Leaf and twig immature CIAT 57É2 1É729 Leaf and twig mature CIAT 30É6 0É730 Leaf and twig mature Costa Rica 15É7 0É3

Erythrina31 E poepigiana leaf CIAT 19483 41É7 1É032 E poepigiana leaf CIAT 21730 52É4 1É7

Centrosema macrocarpum33 Leaf and twig immature CIAT 12É4 0É134 Leaf and twig immature CIAT 16É2 0É135 Leaf and twig mature CIAT 10É7 0É1

Pueraria36 P montana leaf and twig CIAT 17277 6É7 0É137 P phaseoloides leaf and twig CIAT 21052 10É1 0É3

Clitoria ternatea38 Leaf CIAT 8É2 039 Leaf CIAT 9É9 0É1

a Mean of six determinations.

Protein source and amount a†ects protein precipitation by tannins 521

TABLE 3Soluble condensed tannin content (g kg~1) in various plant samplesa

Sample species Acid butanol Soluble condensed tanninsb(plant part/maturity stage)

Meanc SE Radial di†usion

AAL AAH BSA

Meanc SE Meanc SE Meanc SE

Desmodium ovalifolium1 Leaf immature 194É8 3É0 188É7 2É1 159É4 0 193É8 3É12 Leaf immature 187É4 0 145É2 4É2 159É2 2É3 207É7 1É43 Leaf immature 169É1 3É5 165É1 3É6 159É4 0 170É9 04 Leaf mature 220É9 5É8 150É9 0 150É1 6É2 183É8 5É45 Leaf mature 198É8 5É18 266É6 9É9 231É9 6É7 273É4 10É26 Leaf mature 265É3 5É0 155É6 0 170É8 2É4 229É6 9É07 Leaf mature 261É8 6É81 134É9 0 151É2 0 181É3 8É18 Leaf and twig mature 107É4 4É8 142É0 4É5 154É1 5É0 186É0 6É09 Leaf and twig mature 207É3 4É7 144É7 4É1 151É2 0 182É5 8É110 Leaf and twig mature 143É8 9É45 156É8 0 183É8 8É2 178É1 2É911 Twig mature 57É0 0É88 111É3 6É0 127É0 0 140É1 3É512 Twig mature 72É5 2É39 107É8 0É7 143É0 0 149É7 0

Gliricidia sepium16 Leaf immature 25É4 1É2 0 0 017 Leaf immature 30É6 0É6 57É9 0 84É1 0 021 Leaf mature 39É0 1É1 86É3 0 81É4 5É9 186É5 3É8

Manihot esculenta24 Leaf and stem immature 32É5 0É7 91É2 0É4 99É1 1É9 129É2 4É825 Leaf and stem immature 124É2 0É4 148É0 5É3 156É6 0 168É6 2É626 Leaf and stem mature 26É3 0É5 103É5 0 78É5 4É9 119É4 2É227 Leaf and stem mature 71É0 3É7 103É5 0 95É2 3É5 119É4 2É2

Arachis pintoi28 Leaf and twig immature 98É7 0É1 185É9 6É6 148É8 8É0 166É9 7É529 Leaf and twig mature 40É4 0É1 135É0 8É0 126É2 6É3 030 Leaf and twig mature 48É3 0 108É3 4É9 136É7 5É1 132É1 0

a Soluble condensed tannin was not detected in samples 13, 14, 15, 18, 19, 20, 22, 23 (Gliricidia sepium) ; 31, 32(Erythrina) ; 33, 34, 35 (Centrosema) ; 36, 37 (Pueraria) ; 38, 39 (Clitoria).b Soluble condensed tannin content calculated by the acid butanol assay or by the radial di†usion methodusing the respective plant puriÐed condensed tannin as standard.c Mean of eight determinations.

biological activity and actual feeding conditions of theanimal. As stated by Martin and Martin (1982), level oftannins do not necessarily reÑect the nutritional qualityof the plants. Other factors must be considered, such asthe protein level of the plant, the presence of otherexogenous and endogenous sources of protein in thedigestive tract and the defensive mechanisms of theanimal to cope with dietary tannins.

When the correlations between the amount of proteinprecipitated and TP or soluble CT-acid butanol orsoluble CT-radial di†usion content were examined, thecorrelation coefficients increased when the data from Dovalifolium and A pintoi were deleted from the data set.Some of the D ovalifolium samples had high levels ofsoluble CT-acid butanol (198 g kg~1), which were not

reÑected in the amount of protein precipitated. Thesesamples were mature foliages and may have had ahigher degree of polymerisation than the less maturesamples (Makkar et al 1988). As the degree of polym-erisation increases, fewer active sites are available forinteraction with protein, and the molecule becomes lessopen and Ñexible (Hagerman and Butler 1991). Accord-ing to Jones et al (1976), the reactivity of tannins onprotein precipitation decreases as their molecularweight exceeds 20 000.

The A pintoi samples generally precipitated lessprotein in the radial di†usion assays than would beexpected based on the TP and acid-butanol assays. Apintoi may have contained a larger proportion of CTsthat were too small to precipitate protein (Hagerman

522 B I Giner-Chavez et al

TABLE 4Protein (g kg~1) from AALP and BSA precipitated by soluble condensed tannin in various

plant samplesa

Sample species Protein source and level(plant part/maturity stage)

AAL AAH BSA

Meanb SE Meanb SE Meanb SE

Desmodium ovalifolium1 Leaf immature 16É1 0É3 36É6 1É3 19É2 0É92 Leaf immature 11É6 0É6 26É3 1É0 31É9 0É53 Leaf immature 14É7 0É6 26É3 0 19É1 04 Leaf mature 12É5 0 22É6 1É3 23É4 1É85 Leaf mature 28É6 1É8 46É8 2É9 44É3 3É96 Leaf mature 13É4 0 31É9 1É1 41É1 3É67 Leaf mature 10É0 0 24É4 0 22É6 2É78 Leaf and twig mature 9É0 0É6 16É7 0É7 16É7 1É79 Leaf and twig mature 11É9 0É6 24É4 0 24É4 010 Leaf and twig mature 11É0 0 27É6 1É6 14É3 0É811 Twig mature 6É6 1É1 13É7 0 10É1 0É912 Twig mature 6É1 0É1 19É3 0 12É5 0

Gliricidia sepium17 Leaf immature 0É5 0 5É4 0É3 021 Leaf mature 1É7 0 6É8 0É4 1É9 0É1

Manihot esculenta24 Leaf and stem immature 3É2 0 4É4 0É2 5É9 0É125 Leaf and stem immature 12É3 0É6 30É6 0 21É1 1É126 Leaf and stem mature 0É6 0 1É7 0É1 3É4 0É127 Leaf and stem mature 5É6 0 5É8 0É3 6É2 0

Arachis pintoi28 Leaf and twig immature 5É8 0É2 7É7 0É2 7É4 1É429 Leaf and twig mature 2É1 0É1 2É9 0É1 030 Leaf and twig mature 1É0 0 5É2 0É1 1É9 0

a Protein nitrogen precipitation was not detected in samples 13, 14, 15, 16, 18, 19, 20, 22, 23(Gliricidia sepium) ; 31, 32 (Erythrina) ; 33, 34, 35 (Centrosema) ; 36, 37 (Pueraria) ; 38, 39(Clitoria).b Mean of eight determinations.

and Butler 1991). This explanation is supported by thelower efficiency of the A pintoi CT in precipitatingAALP and BSA. The CT content may have been over-estimated, because the low affinity of the tannin forprotein resulted in a calibration line with a low slope.

CONCLUSIONS

The modiÐed radial di†usion method was a valuabletool to study the interaction of di†erent sources of puri-Ðed tannins and protein under conditions representativeof those in the rumen. Tannin source, as well as theamount and type of protein included in the gel, a†ectedthe efficiency with which proteins were precipitated. The

likelihood of obtaining data that can accurately predicttannin e†ects on digestibility will be enhanced if theconditions in the in vitro system mimic those found invivo. Thus, in the radial di†usion assay, the types andamounts of proteins and tannins should be similar tothose found in animal diets. SigniÐcant nutritional infer-ences may be made only after these criteria have beensatisÐed.

To have a better understanding of the biologicalactivity of tannins, further development of proteinbinding assays is warranted. The available protein pre-cipitation assays (Hagerman 1987) do not lend them-selves to use with large numbers of samples. The radialdi†usion assay is convenient, but it su†ers from its ina-bility to detect soluble tanninÈprotein complexes, a dif-Ðculty common to all of the precipitation assays.Methods capable of measuring the formation of soluble

Protein source and amount a†ects protein precipitation by tannins 523

complexes would be an important advance in tanninmethodology. Currently, a combination of chemical andbiological assays with in vivo and in vitro digestibilitystudies is most likely to yield useful information topredict the impact of tannins on forage digestibility andanimal performance.

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