Journal of the Science of Food and Agriculture J Sci Food Agric 79 :79–85 (1999)

Condensed tannins fromLotus corniculatus andLotus pedunculatus exert different effects onthe in vitro rumen degradation ofribulose-1,5-bisphosphatecarboxylase/oxygenase (Rubisco) proteinRobert J Aerts ,1,2 Warren C McNabb,2* Abdul Molan,2 Andre� Brand,3Tom N Barry1 and Jas on S Peters 21 Ins titute of Food, Nutrition and Human Health, Mas s ey Univers ity, Palmers ton North, New Zealand

2 Nutrition Group, AgRes earch, Gras s lands Res earch Centre, Private Bag 11008, Palmers ton North, New Zealand

3 Wageningen Agricultural Univers ity, Department of Animal Nutrition, Wageningen, The Netherlands

Abstract : Condensed tannins (CT) or proanthocyanidins (PA), which occur in a restricted range of

forages, have the ability to interact with proteins and enzymes and can inýuence the digestion of plant

protein in the rumen. We compared the eþ ects of CT extracts from Lotus corniculatus and peduncu-

latus on degradation of the principal leaf protein, ribulose-1,5-bisphosphate carboxylase/oxygenase

(Rubisco), by rumen microorganisms. Total soluble leaf protein extracted from white clover

(Trifolium repens) was incubated with fresh rumen ýuid from sheep and a range of concentrations of

each CT extract. The rate of degradation of the large (LSU) and small subunit (SSU) of Rubisco was

quantiüed by fractionating the proteins in samples taken from in vitro rumen incubations using

sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) and imaging densitometry.

To deduce the eþ ects of the CT extracts, experiments were performed in the presence (CT inactive)

and absence (CT active) of polyethylene glycol (PEG; MW 3350). The two CT extracts diþ ered mark-

edly in their eþ ects on the degradation of the LSU and SSU of Rubisco. At concentrations of 0.89 and

1.79 mg CT mg—1 total soluble leaf protein, the CT extract from L pedunculatus was more eþ ective at

preventing the degradation of the LSU and SSU by rumen microorganisms than the CT extract from

L corniculatus. At a concentration of 1.79 mg CT mg—1 total soluble leaf protein, the CT extracts from

L corniculatus and pedunculatus prevented about 0.75 and 0.83 of the LSU and about 0.69 and 0.86 of

the SSU, respectively, from being degraded. Addition of PEG removed the inhibition and almost

complete degradation of these proteins occurred, as was the case in incubations without CT extracts.

The results of this study suggest that the concentration of CT in the diet and the chemical structure

which aþ ects the activity of the CT needs to be considered when assessing the eþ ects of CT on protein

metabolism in ruminants.

1999 Society of Chemical Industry(

Keywords: condensed tannins ; Lotus corniculatus ; Lotus pedunculatus ; protein degradation; ribulose-1,5-bis-phosphate carboxylase/oxygenase

INTRODUCTION

Condensed tannins (CT) or proanthocyanidins (PA)are phenolic compounds that act as chemicaldefences in plants.1 These compounds have detri-mental eþects on herbivores, although CT may playa wide variety of roles on plants.2 Condensed tanninscan also have positive eþects on vertebrates, forinstance by reducing the harmful eþects of otherplant secondary metabolites in the diet.3,4 A veryimportant eþect exerted by CT when present in the

diet of ruminants, is to increase the efficiency ofprotein digestion.5 This is due to reduced ruminalprotein degradation and an enhanced nitrogen (N)ýow to the small intestine. As a result, CT improvedlive weight gain, wool growth and milk yield in sheepconsuming the legume Lotus corniculatus.6,7

Condensed tannins in L corniculatus (22g kg~1 drymatter (DM)) reduced rumen ammonia concentra-tion by 0.27, increased non-ammonia nitrogen(NAN) outýow from the rumen, and increased the

* Corres pondence to : Warren C McNabb, Nutrition Group,

AgRes earch, Gras s lands Res earch Centre, Private Bag 11008,

Palmers ton North, New Zealand

Contract/grant s pons or : Wools of New Zealand

Contract/grant s pons or : New Zealand Foundation for Res earch,Science and Technology

(Received 6 June 1997; revis ed vers ion received 11 May 1998;

accepted 1 June 1998)

( 1999 Society of Chemical Industry. J Sci Food Agric 0022–5142/99/$17.50 79

RJ Aerts et al

apparent absorption of essential amino acids (EAA)from the small intestine of sheep by 0.63.8 In asimilar experiment, CT from L pedunculatus(55g kg~1 DM) also reduced rumen ammonia andmarkedly increased NAN outýow from the rumenbut did not improve the apparent absorption of EAAfrom the small intestine.9 The CT in a mixed dietconsisting of ryegrass (Lolium perenne) and Lotuspedunculatus with a ünal concentration in the mixeddiet of 18g CT kg~1 DM, had a similar eþect on Ndigestion as the CT in L pedunculatus (55g kg~1DM) when that forage was fed as a sole diet tosheep.10 These results suggest that, although theimpact of CT on the digestion of dietary protein isaþected by concentration, the type and source of theCT also has a role to play.

The CT in L pedunculatus reduced the degradationof ribulose-1,5-bisphosphate carboxylase/oxygenase(Rubisco) protein by rumen microorganisms andincreased the proportion of this protein that wasdigested in the small intestine of sheep.11 In thatstudy, Rubisco represented 0.39 of the protein inthe diet and was the principal dietary protein. Theobjective of the present study was to measure theeþects of CT extracted from L corniculatus and Lpedunculatus on the in vitro degradation of Rubiscoby rumen microorganisms. The CT in Lcorniculatus12 and L pedunculatus13 have similarmolecular weights (MW) but their ýavan-3-ol com-position is vastly diþerent ; the CT in L corniculatusconsists predominantly of procyanidin-type subunitswhilst prodelphinidin-type subunits dominate theCT in L pedunculatus. Therefore, comparing the CTextracted from these forages provides an idealopportunity to determine if the diþerences in ýavan-3-ol composition can result in diþerent eþects on thedegradation of dietary protein in the rumen.

MATERIALS AND METHODS

Experimental design

An in vitro experiment was undertaken to determinethe extent to which CT extracted from L corniculatusand L pedunculatus altered the in vitro degradation ofRubisco when total soluble leaf protein from whiteclover (Trifolium repens) was incubated with rumenýuid. Condensed tannins are generally preparedusing chromatography with Sephadex LH-20(Pharmacia, Uppsala, Sweden) as a matrix. Jacksonet al14 reported that CT prepared from L cor-niculatus in this way, although containing predomi-nantly CT, also contained other phenoliccompounds. In this study, the term ‘CT extract’ waspreferred to reýect this composition rather thanmore speciüc terms like ‘CT’ or ‘PA’. The eþect ofCT extracts was determined with and without theaddition of polyethylene glycol (PEG). The additionof PEG prevents CT from binding toprotein15enabling the eþect of CT to be deduced bycomparing incubations with added PEG (CT

inactive) to incubations without added PEG (CTactive). Rubisco was quantiüed using imaging densi-tometry after separation of proteins in samples takenfrom in vitro rumen incubations by sodium dodecylsulphate–polyacrylamide gel electrophoresis (SDS-PAGE). Measurement of the rate of in vitro degrada-tion of Rubisco when total soluble leaf protein wasincubated with rumen ýuid provided a means tocompare the CT extracts from L corniculatus and Lpedunculatus.

Preparation of condensed tannin extracts

The CT extracts were prepared from the leaves of Lcorniculatus L (cv Goldie) and L pedunculatus Cav(cv Maku) using a column containing SephadexLH-20 (Pharmacia, Sweden) according to themethod described in Ref 14. Lotus corniculatus andpedunculatus leaves were harvested on 12 separateoccasions over a month period and Sephadex LH-20extracts prepared. The Sephadex LH-20 extractswere stored at [ 20¡C and pooled for use in thisstudy.

In vitro rumen incubations

Total soluble protein was extracted from 45g offresh white clover (which contain only trace amountof CT16) by mincing the leaves in a food processor(Electronic 1500, Italy) with 135ml of freshly pre-pared artiücial saliva,17 pH 6.8 (mM : 117;NaHCO3 ,

26; NaCl, 8; KCl, 8; 0.2;Na2HPO4 , CaCl2 ,0.3) that was saturated with gas. TheMgCl2 , CO2

resulting slurry was strained through cheese clothand the extract ültered through very üne cloth(Miracloth, Calbiochem, USA). The concentrationof total soluble leaf protein in the extract was deter-mined using the method described in Ref 18.

A castrated male sheep ütted with a rumencannula (55mm id) was maintained on pasture con-sisting predominantly of perennial ryegrass (Loliumperenne ; containing only trace amounts of CT16) andwhite clover for 30 days prior to the experiment.After fasting the sheep for 2h, rumen ýuid was col-lected and stained through cheese cloth into a pre-warmed Dewar ýask that had been ýushed with CO2gas. The pH of the rumen ýuid ranged from 6.30 to6.90. After collection, the rumen ýuid was againýushed with and used immediately for the inCO2 ,vitro rumen incubations.

In 10ml test tubes ütted with one-way valves, thefollowing was added; 2ml of the white clover proteinextract (containing about 5.6mg total soluble leafprotein), 1ml of artiücial saliva (bothCO2-saturatedprewarmed to 39¡C), 0.75ml of rumen ýuid, and ali-quots containing either 0,0.5, 1, 5 or 10mg of CTextract from either L corniculatus or L pedunculatus.The concentrations of CT extract in the incubationswere therefore approximately 0, 0.1, 0.2, 0.9 and1.8mg CT mg~1 total soluble leaf protein. Poly-ethylene glycol (MW 3350; Union Carbide, USA)was included in half the in vitro rumen incubations.

80 J Sci Food Agric 79 :79–85 (1999)

Condensed tannin reactivity and protein degradation in the rumen

Figure 1. The degradation of the LSU of ribulos e-1,5-

bis phos phate carboxylas e/oxygenas e (Rubis co) during in vitroincubation of total s oluble leaf protein extracted from white clover

(Trifolium repens ) and rumen fluid from s heep. Incubations were

performed (A; without and with the addition of (B) 0.5, (C)>, È)1.0, (D) 5.0 and (E) 10.0mg of CT extract from Lotus(…,L)corniculatus or Lotus pedunculatus . Incubations were(=,K)als o undertaken (CT active ; without and (CT inactive ;>,=,…)

with the addition of PEG (20mg; MW 3350). SamplesÈ,K,L)were removed prior to and after 1, 2, 3, 4, 5, 6.5 and 8 h of

incubation. Total protein in the s amples was fractionated by

SDS-PAGE and the LSU was quantified by imaging dens itometry.

The amount of LSU protein prior to incubation was s et to 100%,and the percentage of LSU protein remaining during incubation

was determined. Means of quadruplicate incubations are s hown.

Barry and Forss19 reported that 2mg PEG mg~1 CTwas required to completely inactivate CT so it wasassumed that 20mg of PEG should be added to thein vitro rumen incubations requiring PEG.

The tubes were ýushed with and incubatedCO2 ,at 39¡C in a waterbath. Prior to and after 1, 2, 3, 4, 5,6.5 and 8h of incubation, 25ll aliquots from the invitro rumen incubations were removed, added to afreshly prepared mixture containing 5ll 10% SDS(w/v), 5ll 10% Triton (w/v), 2ll 25% PEG (w/v),2.5ll b-mercaptoethanol, and 10ll protein loadingbuþer (0.3M Tris (pH 6.8), 60% glycerol (w/v), 10%

Figure 2. The degradation of the SSU of ribulos e-1,5-

bis phos phate carboxylas e/oxygenas e (Rubis co) during in vitroincubation of total s oluble leaf protein extracted from white clover

(Trifolium repens ) and rumen fluid from s heep. Incubations were

performed (A; without and, with the addition of (B) 0.5, (C)>, È)1.0, (D) 5.0 and (E) 10.0mg of CT extract from Lotus(…,L)corniculatus or Lotus pedunculatus . Incubations were(=,K)als o undertaken (CT active ; without and (CT inactive ;>,=,…)

with the addition of PEG (20mg; MW 3350). SamplesÈ,K,L)were removed prior to and after 1, 2, 3, 4, 5, 6.5 and 8 h of

incubation. Total protein in the s amples was fractionated by

SDS-PAGE, and the SSU was quantified by imaging dens itometry.

The amount of SSU protein prior to incubation was s et to 100%,and the percentage of SSU protein remaining during incubation

was determined. Means of quadruplicate incubations are s hown.

SDS (w/v), 0.25mg ml~1 bromophenol blue), andstored at [ 20¡C for SDS-PAGE. After each sam-pling, the in vitro rumen incubation were ýushedwith gas. All in vitro rumen incubations wereCO2repeated in quadruplicate.

SDS-PAGE and analysis of Rubisco

Prior to SDS-PAGE, samples were heated at 97¡Cfor 4min to denature all proteins and dissociate CT–protein complexes. After centrifugation at 12000] gfor 5min, total soluble protein in the supernatantwas fractionated by SDS-PAGE using the method

J Sci Food Agric 79 :79–85 (1999) 81

RJ Aerts et al

Figure 3. The percentage of (A) the LSU and (B) the SSU of

ribulos e-1,5-bis phos phate carboxylas e/oxygenas e (Rubis co)protein which remained undegraded after 6.5 and 8 h when total

s oluble leaf protein from white clover (Trifolium repens ) was

incubated with rumen fluid from s heep. The in vitro rumen

incubations were undertaken without and with the addition of CT

extracts from either Lotus corniculatus or Lotus(L) (=)pedunculatus . The percentage of undegraded protein at the end

of the incubation period was calculated as the average for the 6.5

and 8 h time points . Means of quadruplicate incubations are

s hown.

described in Ref 11. After SDS-PAGE the protein inthe gels was üxed by washing the gels in methanol/acetic acid/water (40 : 10 : 50, v/v) for 15min.Protein in the gels was visualised by staining withCoomassie Brilliant Blue R250 (0.1% CoomassieBrilliant Blue R-250 (w/v) in methanol/acetic acid/water (40 : 10 : 50, v/v)) for 20min. Gels weredestained in methanol/acetic acid/water(10 : 7.5 : 82.5, v/v) for 24h. Developed gels werequantiüed by imaging densitometry (Bio-Rad,Model GS-670 Imaging Densitometer with Molecu-lar AnalystTM@CP imaging analysis software, USA)Mature Rubisco consists of 8 LSU (MW 54000) and8 SSU20 (MW 16000) and accounts for 30–50% ofthe total plant protein.21 Therefore, the LSU andSSU represented the predominant proteins presentin the total soluble leaf protein extract and werereadily detectable on stained gels.

Calculation of data

The intensity of the LSU and SSU of Rubisco onSDS-PAGE gels prior to incubation (0h) was set at

100%, and the percentage of each protein remainingafter 1, 2, 3, 4, 5, 6.5 and 8h incubation was calcu-lated (for the data in Figs 1 and 2). For each concen-tration of added CT, the percentage of undegradedLSU and SSU protein remaining after the in vitrorumen incubations were complete was calculated.The percentage of LSU and SSU protein degradedafter 6.5 and 8h of incubation was similar so thesetime points were averaged (for the data in Fig 3).

RESULTS

The degradation of the LSU and SSU of Rubisco by

rumen microorganisms

In the absence of rumen ýuid, the LSU and SSU ofthe Rubisco in the total soluble leaf protein extractedfrom white clover is stable for the duration of the invitro rumen incubations (data not shown). In theincubations consisting of total soluble leaf proteinextract and rumen ýuid only, the LSU of Rubiscowas rapidly degraded by the rumen microorganisms(Fig 1A). About 50% of the protein was degraded inthe ürst hour of incubation, followed by furthergradual degradation of the protein over the remain-ing 7h of incubation. Degradation of the SSU ofRubisco also occurred, although the kinetics wheresomewhat slower than those observed for the LSU(Fig 2A). The addition of PEG had no substantialeþect on the rate of degradation of either the LSU orSSU (Fig 1A and 2A).

The effect of CT extracts on LSU and SSU protein

degradation

When 1, 5 and 10mg of CT extract from L peduncu-latus was added to in vitro rumen incubations, italways tended to be more eþective at reducing thedegradation of the LSU than the CT extract from Lcorniculatus (Fig 1C–E). At the lowest level of CTextract that was added to in vitro rumen incubations(0.5mg), the CT extracts from L corniculatus andpedunculatus tended to be ineþective at reducingprotein degradation (Fig 1B). In the absence of PEG,the addition of 1, 5 and 10mg of CT extract from Lcorniculatus (Fig 2D and E) reduced the degradationof the SSU compared to incubations without this CTextract (Fig 2A). However, addition of 5 and 10mgof CT extract from L pedunculatus to in vitro rumenincubations tended to be more eþective at reducingthe degradation of the SSU (Fig 2D and E). Also, atthe lower concentration of 0.5mg of CT extract, theextracts from L corniculatus and pedunculatus wereineþective at reducing the degradation of the SSU(Fig 2B). At most concentrations of both CTextracts, addition of PEG to the in vitro rumen incu-bations tended to increase the degradation of theLSU and SSU proteins, with the eþect generallybeing similar for both CT extracts.

Concentrations of CT extract from L corniculatusand pedunculatus of 0.2mg mg~1 total soluble leaf

82 J Sci Food Agric 79 :79–85 (1999)

Condensed tannin reactivity and protein degradation in the rumen

protein or less had similar (P [ 0.05) eþects andtended to protect about 0.35 of the LSU and 0.46 ofthe SSU, respectively, from degradation after 6 and8h incubation (Fig 3A and 3B). However, at concen-trations of 0.89 and 1.79mg CT mg~1 total solubleleaf protein respectively, the CT extract from Lpedunculatus was more eþective at reducing the deg-radation of the LSU (P \ 0.013 and P \ 0.054) andSSU (P \ 0.039 and P \ 0.034) by rumen micro-organisms than the CT extract from L corniculatus(Fig 3A and 3B).

DISCUSSION

The microbial population in the rumen is able toeþectively utilise structural components of plant cellssuch as cellulose and hemicellulose and as such,rumen microorganisms are essential for the contin-ued survival of all ruminants. However, rumenmicroorganisms also utilise a large proportion of theprotein in the diet ; as much as 0.7 of the dietaryprotein can be degraded to ammonia in the rumen ofsheep fed fresh forages.22 Condensed tanninsincrease duodenal NAN ýow per unit total N eaten,and the presence of CT in the diet reduces ammoniaconcentration in the rumen.8,9 These eþects are pre-sumably due to the interaction between CT andprotein which occurs in the rumen. There is con-siderable variation in the ýavan-3-ol composition ofCT23 and CT can interact with enzymes and pro-teins in several ways.2,24 Indeed, the ultimate eþectof CT on protein metabolism in ruminants appearsto be eþected by the type of CT in the diet.8,9 In thepresent study, we compared the eþects of CTextracts from L corniculatus and L pedunculatus onthe degradation of the major leaf protein, Rubisco,by rumen microorganisms. The results of this studyclearly demonstrate that these CT extracts diþerenteþects on the kinetics of Rubisco degradation duringin vitro rumen incubations containing total solubleleaf protein. The CT extract from L pedunculatuswas more eþective at reducing the degradation of theLSU and SSU of Rubisco than the CT extract fromL corniculatus.

Tanner et al25 reported that CT from L cor-niculatus and pedunculatus had similar eþects on thedegradation of the LSU of Rubisco when the con-centration of CT added to in vitro rumen incubationswas 0.2mg mg~1 protein. At this concentration, thediþerence between CT extracts from these twoforages was also small in the present study. However,in the present study at concentrations of 0.89 and1.79mg CT mg~1 soluble leaf protein, there weresigniücant diþerences between the two CT extracts.Condensed tannins may exert their eþects onRubisco degradation in the rumen by interactingdirectly with Rubisco and/or with proteolyticenzymes of microbial origin ; in the ürst case steri-cally interfering with binding of proteases to the

plant protein and, in the second case, by directlyinhibiting the proteases themselves. The CT extractsof L corniculatus and L pedunculatus had similareþects on the in vitro precipitation of the LSU andSSU of Rubisco.26 This suggests that these extractsinteracted in a similar way with this plant protein.Condensed tannins have inhibited virtually everyenzyme tested24 and were found to inhibit rumenmicrobial proteolytic, ureolytic, and cellulolyticenzymatic activities, as well as general fermentativeactivities, and cell multiplication.27 Moreover, bac-terial proteases are present outside the cells in a cell-bound form, and binding of CT on the surface ofbacterial cells has been observed.25,28 Condensedtannins from L corniculatus and L pedunculatus diþerconsiderably in their chemical structures.12,13 Theaverage MW of the CT from L pedunculatus is 2200and this is slightly higher than the MW of CT fromL corniculatus (1900). Furthermore, the CT fromL pedunculatus contains a predominance ofprodelphinidin-type subunits with epigallocatechin(0.64) representing the prevalent subunit.13 Con-versely, the CT from L corniculatus has predomi-nantly procyanidin-type subunits with epicatechin(0.67) dominating.12 Generally, CT with high MWinteract more strongly with enzymes and other pro-teins than CT with low MW,29h31 and the reactivityof CT increases with increasing prodelphinidincontent.32 Although the diþerences between CTfrom L corniculatus and L pedunculatus were insuffi-cient to cause any appreciable diþerence in the invitro precipitation of Rubisco when these CTextracts were reacted with total soluble leaf proteinfrom white clover,26 the CT extract from L peduncu-latus was more eþective at reducing Rubisco degra-dation than the CT extract from L corniculatus in thepresent study. Taken together, the results in Ref 26and those presented in this study suggest that proteinprecipitation by CT may be more responsive to themolecular weight of the CT, and to a lesser extent,eþected by the prodelphinidin content, whilst theeþect of CT on the degradation of protein by rumenmicroorganisms may be more responsive to diþer-ences in the ýavan-3-ol composition of the CT. Thiscertainly warrants further investigation.

Generally, concentration of CT in the diet only isconsidered when assessing the eþect of CT-containing forages on animal performance. Typi-cally, concentrations of 20–40g CT kg~1 DM isconsidered optimal for improving animal per-formance.33,34 When the CT from L pedunculatusexceeded 55g kg~1 DM, voluntary intake and Ndigestibility were depressed by 12% and 12 percent-age units, respectively.9 In that study, the liveweightgain of the sheep fed L pedunculatus was uneþectedby the CT in the diet. However, when the concentra-tion of CT exceeded 90g kg~1 DM, N digestibilitywas depressed by 21 percentage units, and voluntaryintake by 12%.35h37 In those studies, animal pro-ductivity was also aþected. Waghorn and Shelton10

J Sci Food Agric 79 :79–85 (1999) 83

RJ Aerts et al

demonstrated that, in sheep fed perennial ryegrass(Lolium perenne) and L pedunculatus with a CT con-centration in the mixed diet of 18g kg~1 DM, Ndigestibility of that diet was depressed by 13 per-centage units compared to a similar diet fed to sheepbut where the eþect of CT had been removed byinclusion of PEG with the diet. Taken together withthe results of the present study, factors other thanconcentration also need to be considered when evalu-ating the eþect of CT. These factors should includethe chemistry and reactivity of the CT being con-sidered.

ACKNOWLEDGEMENTS

The authors are indebted to Wools of New Zealandand the New Zealand Foundation for Research,Science and Technology for their ünancial assist-ance.

REFERENCES1 Swain T, 1978 Plant–animal coevolution: Asynoptic view of

the paleozoic and mesozoic, in Biochemical Aspects of Plant

and Animal Coevolution (Ann Proc Phytochem Soc Eur), Edby Harborne JB. Academic Press, London, UK. pp 3–19(1978).

2 Bernays EA, Copper Driver G and Bilgener M, Herbivoresand plant tannins, in Advances in Ecological Research, Ed byBegon M, Fitter AH, Ford ED and MacFadyen A. Aca-demic Press, London. pp 263–302 (1989).

3 Goldstein W and Spencer KC, Inhibition of cyanogenesis bytannins. J Chem Ecol 11:847–858 (1985).

4 Freeland WJ and Janzen DH Strategies in herbivory bymammals : the role of plant secondary compounds. Amer

Natur 108:268–289 (1974).5 Barry TN and Manley TR, Interrelationships between the

concentrations of total condensed tannin, free condensedtannin and lignin in Lotus sp. and their possible conse-quences in ruminant nutrition. J Sci Food Agric 37 :248–254(1986).

6 Wang Y, Douglas GB, Waghorn GC, Barry TN, Foote AGand Purchas RW, Eþect of condensed tannins upon the per-formance of lambs grazing Lotus corniculatus and lucerne(Medicago sativa). J Agric Sci (Camb) 126:87–98 (1996).

7 Wang Y, Douglas GB, Waghorn GC, Barry TN and FooteAG, Eþect of condensed tannins in Lotus corniculatus uponlactation performance in ewes. J Agric Sci (Camb) 126:353–362 (1996).

8 Waghorn GC, Ulyatt MJ, John A and Fisher MT, The eþectof condensed tannins on the site of digestion of amino acidsand other nutrients in sheep fed on Lotus corniculatus. Br J

Nutr 57:115–126 (1987).9 Waghorn GC, Shelton ID, McNabb WC and McCutcheon

SN, Eþects of condensed tannins in Lotus pedunculatus onits nutritive value for sheep. 2. Nitrogenous aspects. J Agric

Sci (Camb) 123:109–119 (1994).10 Waghorn GC and Shelton ID, Eþect of condensed tannins in

Lotus pedunculatus on the nutritive value of ryegrass (Lolium

perenne) fed to sheep. J Agric Sci (Camb) 125:291–297(1995).

11 McNabb WC, Waghorn GC, Peters JS and Barry TN, Theeþect of condensed tannins in Lotus pedunculatus upon thesolubilization and degradation of ribulose-1,5-bisphosphatecarboxylase (Rubisco) protein in the rumen and the sites ofRubisco digestion. Br J Nutr 76:535–549 (1996).

12 Foo LY, Newman R, Waghorn G, McNabb WC and UlyattMJ Proanthocyanidins from Lotus corniculatus. Phyto-

chemistry 41:617–624 (1996).13 Foo LY, Li Y, McNabb WC, Waghorn GC and Ulyatt MJ,

The proanthocyanidins of Lotus pedunculatus. Phyto-

chemistry 45:1689–1695 (1997).14 Jackson FS, McNabb WC, Barry TN, Foo YL and Peters JS,

The condensed tannin content of a range of subtropical andtemperate forages and the reactivity of condensed tanninwith ribulose-1,5-bisphosphate carboxylase (Rubisco)protein. J Sci Food Agric 72:483–492 (1996).

15 Jones W and Mangan J, Complexes of the condensed tanninsof sainfoin (Onobrychis viciifolia Scop.) with fraction 1 leafprotein and with submaxillary mucoprotein, and theirreversal by polyethylene glycol and pH. J Sci Food Agric

28:126–136 (1977).16 Terrill TH, Rowan AM, Douglas GB and Barry TN, Deter-

mination of extractable and bound condensed tannin con-centrations in forage plants, protein concentrate meals andcereal grains. J Sci Food Agric 58:321–329 (1992).

17 McDougall E, 1948 Studies on ruminant saliva. 1. The com-position of output of sheep’s saliva. Biochem J 43:99–109(1948).

18 Bradford MM, A rapid and sensitive method for the quantitat-ion of microgram quantities of protein utilizing the principleof protein-dye binding. Anal Biochem 72:248–254 (1976).

19 Barry TN and Forss DA, The condensed tannin content ofvegetative Lotus pedunculatus, its regulation by fertiliserapplication, and eþect upon protein solubility. J Sci Food

Agric 34 :1047–1056 (1983).20 Kawashima N and Wildman SG, Fraction 1 protein. Ann Rev

Plant Physiol 21:325–358 (1970).21 Mangan JL and West J , Ruminal digestion of chloroplasts and

the protection of protein by gluteraldehyde treatment. J

Agric Sci (Camb) 89:3–15 (1977).22 Ulyatt MJ, MacRae JC, Clarke RT and Pearce PD, Quantitat-

ive digestion of fresh herbage by sheep. IV. Protein synthe-sis in the stomach. J Agric Sci (Camb) 84:453–458 (1975).

23 Foo LY, Jones WT, Porter LJ and Williams VM, Proanthocy-anidin polymers of fodder legumes. Phytochemistry 21:933–935 (1982).

24 Haslam E, Natural polyphenols (vegetable tannins) as drugs :possible modes of action. J Nat Prod 59:205–215 (1996).

25 Tanner GJ, Moore AE and Larkin PJ, Proanthocyanidinsinhibit hydrolysis of leaf proteins by rumen microýora in

vitro. Br J Nutr 71:947–958 (1994).26 McNabb WC, Peters JS, Foo LY, Waghorn GC and Jackson

FS, Eþect of condensed tannin prepared from severalforages on the in vitro precipitation of ribulose-1,5-bisp-hosphate carboxylase (Rubisco) protein and its digestion bytrypsin (EC 2.4.21.4) and chymotrypsin (EC 2.4.21.1). J Sci

Food Agric (in press) 1998.27 Mueller-Harvey I and McAllan AB, Tannins, their biochem-

istry and nutritional properties, in Advances in Plant Cell

Biochemistry and Biotechnology, JAI Press, pp 151–217(1992).

28 Jones GA, Jakober KD, Bae HD, McAllister TA, Cheng KJand Muir AD, Some interactions between condensedtannins of forage legumes, bovine serum albumin, and üvestrains of proteolytic rumen bacteria. Proceedings of the

World Conference on Animal Production, Edmonton, Canada

pp 68–69 (1993).29 Beart JE, Lilley TH and Haslam E, Plant polyphenols – sec-

ondary metabolism and chemical defence: some obser-vations. Phytochemistry 24 33–38 (1985).

30 Horigome T, Kumar R and R Okamoto K, Eþects of con-densed tannins prepared from leaves of fodder plants ondigestive enzymes in vitro and in the intestine of rats. Br J

Nutr 60:275–285 (1988).31 Kawamoto H, Nakatsubo F and Murakami K, Stoichiometric

studies of tannin-protein co-precipitation. Phytochemistry

41:1427–1431 (1996).

84 J Sci Food Agric 79 :79–85 (1999)

Condensed tannin reactivity and protein degradation in the rumen

32 Jones W, Broadhurst R and Lyttleton J, The condensedtannins of pasture legume species. Phytochemistry 15:1407–1409 (1976).

33 Barry TN, Condensed tannins : their role in ruminant proteinand carbohydrate digestion and possible eþects upon therumen ecosystem, in The Roles of Protozoa and Fungi in

Ruminant Digestion, Ed by Nolan JV, Leng RA andDemeyer DI, Penambul Books, Armidale, Australia. pp153–169 (1989).

34 Waghorn GC, Jones WT, Shelton ID and McNabb WC, Con-densed tannins and the nutritive value of herbage. Proc NZ

Grassl Assoc 51:171–176 (1990).

35 Barry TN and Duncan SJ, The role of condensed tannins inthe nutritional value of Lotus pedunculatus for sheep. 1. Vol-untary intake. Br J Nutr 51 :484–491 (1984).

36 Barry TN and Manley TR, The role of condensed tannins inthe nutritional value of Lotus pedunculatus for sheep. 2.Quantitative digestion of carbohydrates and proteins. Br J

Nutr 51 :493–504 (1984).37 Barry TN, Manley TR and Duncan SJ, The role of condensed

tannins in the nutritional value of Lotus pedunculatus forsheep. 4. Sites of carbohydrate and protein digestion asinýuenced by dietary reactive tannin concentration. Br J

Nutr 55 :123–137 (1986).

J Sci Food Agric 79 :79–85 (1999) 85