The in vitro precipitation of ribulose-1,5-bisphosphate carboxylase (Rubisco) by condensed tannin (CT) extracted from Lotus corniculatus and Lotus pedunculatus and the effect of these CT on the in vitro rumen degradation of Rubisco was used to compare the reactivity of these CT. The chemical structure of CT from Lotus corniculatus was homogenous with epicatechin stereochemistry and mostly procyanidin units. The CT from Lotus pedunculatus was heterogenous with mostly prodelphinidin units. The amount of CT required to precipitate all the Rubisco when total soluble leaf protein was incubated with CT from Lotus corniculatus and Lotus pedunculatus was similar. Although CT from both species were able to reduce the in vitro degradation of Rubisco, CT from Lotus corniculatus was less effective than CT from Lotus pedunculatus at reducing this degradation

Aerts, R J

Brand, A

Foo, Y L

McNabb, W C

Peters, J S

Waghorn, G C

English

University of Kentucky

University of Kentucky UKnowledge IGC Proceedings (1997-2023) XVIII IGC (1997) Manitoba & Saskatchewan Effect of Condensed Tannin in Lotus Corniculatus and Lotus Pedunculatus on Digestion of Rubisco in the Rumen W C. McNabb Grassland Research Centre, NZ R J. Aerts Massey University A Brand Wageningen Agricultural University J S. Peters Grassland Research Centre, NZ Y L. Foo New Zealand Institute for Industrial Research Development See next page for additional authors Follow this and additional works at: https://uknowledge.uky.edu/igc  Part of the Agricultural Science Commons, Agronomy and Crop Sciences Commons, Plant Biology Commons, Plant Pathology Commons, Soil Science Commons, and the Weed Science Commons This document is available at https://uknowledge.uky.edu/igc/1997/session8/12 <>Grasslands 2000</> This Event is brought to you for free and open access by the Plant and Soil Sciences at UKnowledge. It has been accepted for inclusion in IGC Proceedings (1997-2023) by an authorized administrator of UKnowledge. For more information, please contact UKnowledge@lsv.uky.edu. Presenter Information W C. McNabb, R J. Aerts, A Brand, J S. Peters, Y L. Foo, and G C. Waghorn This event is available at UKnowledge: https://uknowledge.uky.edu/igc/1997/session8/12 Session 8  - Tannins: Plant Breeding and Animal Effects 8-9ID NO. 1410EFFECT OF CONDENSED TANNIN IN LOTUS CORNICULATUS AND LOTUSPEDUNCULATUS ON DIGESTION OF RUBISCO IN THE RUMENW. C. McNabb1, R. J. Aerts2, A. Brand3, J. S. Peters1, Y. L. Foo4 and G. C. Waghorn11AgResearch, Grasslands Research Centre, Private Bag 11008, Palmerston North, New Zealand2Department of Animal Science, Massey University, Palmerston North, New Zealand3Department of Animal Nutrition, Wageningen Agricultural University, Wageningen, The Netherlands4New Zealand Institute for Industrial Research and Development, P.O. Box 31-310, Lower HuttABSTRACTThe in vitro precipitation of ribulose-1,5-bisphosphate carboxylase(Rubisco) by condensed tannin (CT) extracted from Lotuscorniculatus and Lotus pedunculatus and the effect of these CT onthe in vitro rumen degradation of Rubisco was used to compare thereactivity of these CT.  The chemical structure of CT from Lotuscorniculatus was homogenous with epicatechin stereochemistry andmostly procyanidin units.  The CT from Lotus pedunculatus washeterogenous with mostly prodelphinidin units.  The amount of CTrequired to precipitate all the Rubisco when total soluble leaf proteinwas incubated with CT from Lotus corniculatus and Lotuspedunculatus was similar.  Although CT from both species were ableto reduce the in vitro degradation of Rubisco, CT from Lotuscorniculatus was less effective than CT from Lotus pedunculatus atreducing this degradation.KEYWORDSCondensed tannin, protein degradation, reactivity, Rubisco, rumenINTRODUCTIONThe CT in Lotus corniculatus (22 g CT kg-1 dry matter (DM);Waghorn et al. 1987) fed to sheep, decreased nitrogen (N) digestibilityby 8 percentage units but increased apparent absorption of essentialamino acids (EAA) from the small intestine by 63%.  The CT inLotus pedunculatus (55 g CT kg-1 DM; Waghorn et al. 1994) alsoreduced N digestibility but the apparent absorption of EAA from thesmall intestine was unaffected by CT.  In sheep fed ryegrass (Loliumperenne) and Lotus pedunculatus with a final dietary CTconcentration of 18 g CT kg-1 DM, N digestibility was reduced by13 percentage units (Waghorn and Shelton 1995) and the effects ofCT were similar to Lotus pedunculatus (55 g CT kg-1 DM) fed tosheep.  Nutritional responses to CT are affected by concentrationand the chemical structure and reactivity of CT.  The objective ofthis study was to measure the reactivity of CT extracted from Lotuscorniculatus and Lotus pedunculatus.  The in vitro precipitation ofRubisco by CT and the effect of CT on the in vitro degradation ofRubisco when total soluble plant protein was incubated with rumenfluid was used to compare the reactivity of CT.  Rubisco is theprincipal leaf protein, representing 30-50% of the total protein present(Mangan, 1982).METHODSCondensed tannins were extracted from Lotus corniculatus and Lotuspedunculatus and total soluble leaf protein was extracted from whiteclover (Trifolium repens) using the methods described by Jackson etal. (1996).  The chemical structure of the CT were analysed by 13C-Nuclear Magnetic Resonance (NMR) and by thin layerchromatography (TLC) according to the methods described by Fooet al. (1996).The precipitation of Rubisco by CT was determined by adding either0, 0.1, 0.5, 1, 10, 25 or 50 µg of the CT extracted from Lotuscorniculatus and Lotus pedunculatus to 10 mg of total soluble leafprotein.  Barry and Forss (1983) reported that 1.8-2 mg ofpolyethylene glycol (PEG) g-1 CT was sufficient to release all proteinfrom CT-protein complexes.  Therefore, identical reactions were alsoprepared except 100 µg of PEG (molecular weight (MW) 3500) wasadded to remove all the effects of the CT.  All reactions were dilutedto 100 ml with McIlvaineís Buffer, pH 7.0 (Elving et al. 1956) andincubated at 39°C for 90 min.  After incubation reactions werecentrifuged at 12,000 g for 10 min and the resulting pellets werewashed three times in McIlvaineís Buffer, pH 7.0 to remove allsoluble leaf protein that was not precipitated by CT.The effect of CT on the degradation of Rubisco was measured usingin vitro rumen incubations.  A sheep with a large rumen cannula andfed fresh perennial ryegrass/white clover pasture ad. lib. was usedto supply rumen fluid for the incubations which were carried outusing a modification of the method described by McNabb et al.(1996).  Either 0, 0.5, 1, 5 and 10 mg of CT from Lotus corniculatusand Lotus pedunculatus were incubated with 5.6 mg of total solubleleaf protein and 0.75 ml of rumen fluid.  The incubations were dilutedto 3.75 ml with artificial saliva (McDougall, 1948) and incubated at39°C for 8 h.  Identical incubations were undertaken but with 20 µgof PEG added.Both in vitro experiments were undertaken in duplicate.  Pellets andin vitro rumen samples were resuspended in loading buffer (McNabbet al. 1996) and stored at -20oC for sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE).  Protein wasfractionated by SDS-PAGE and the Rubisco quantified by imagingdensitometry (BioRad, Model GS-670 Imaging Densitometer withMolecular AnalystTM/CP imaging analysis software) using methodsdescribed by McNabb et al. (1996).RESULTS AND DISCUSSIONThe distinguishing features of the chemical structure of the CT fromLotus corniculatus were its relatively homogenous monomer unitswith epicatechin type stereochemistry and the predominance ofprocyanidin units.  In contrast, CT from Lotus pedunculatus wasmore heterogenous with the monomer units being a mixture ofepicatechin and catechin type stereochemistry and a clear dominanceof prodelphinidin to procyanidin units.  The reactivity of CT increaseswith increasing delphinidin: cyanidin (PD:PC) ratio (Jones et al.1976).  Therefore, the CT from Lotus pedunculatus should be ableto precipitate more protein per unit weight than the CT from Lotuscorniculatus.The in vitro precipitation of Rubisco by CT from Lotus corniculatusand Lotus pedunculatus is shown in Fig. 1, whilst the effect of theseCT on the in vitro degradation of Rubisco when total plant proteinwas incubated with rumen fluid is shown in Fig. 2.  The amount ofCT required to precipitate all the Rubisco when 10 µg of total solubleleaf protein was incubated with CT from Lotus corniculatus and Lotuspedunculatus was similar, with between 25 and 50 µg required.Reactivity, which can be defined as the ability of CT to precipitateprotein per unit weight, was similar for the CT from Lotuscorniculatus and Lotus pedunculatus.However, there were considerable differences in the effect of CTfrom Lotus corniculatus and Lotus pedunculatus on the in vitrodegradation of Rubisco by microorganisms in rumen fluid.  Theinclusion of 5 or 10 mg of Lotus corniculatus CT reduced thedegradation of Rubisco to 54% and 61% of the protein added to theincubation, respectively, whilst Lotus pedunculatus CT (5 mg and10 mg) reduced the degradation of Rubisco to 28% of the proteinadded to the incubation.  When 5 mg and 10 mg of CT was added tothe in vitro rumen degradation assay, the ratio of CT to protein wasabout 1:1 and 2:1, respectively.  At these ratios, the precipitation ofRubisco by CT from Lotus corniculatus and Lotus pedunculatus wassimilar, and about 0.9.  However, CT from Lotus corniculatus wasless effective than CT from Lotus pedunculatus at reducing theSessison 8 - Tannins: Plant Breeding and Animal Effects 8-10degradation of Rubisco by rumen microorganisms.  This suggeststhat factors other than just protein precipitation may be important indetermining the final effect of CT in the rumen of sheep.The precipitation of Rubisco by CT from Lotus corniculatus wascompletely reversible by PEG.  However, about 0.2 of the Rubiscowas still precipitated when 50 µg of CT from Lotus pedunculatuswas incubated with PEG.  Jackson et al. (1996) demonstrated thatCT prepared from a range of forages by affinity chromatographyusing Sephadex LH-20 as a matrix, contained variable amounts ofnon-CT phenolic compounds together with the CT.  Similarcompounds may have been present in the CT which was extractedand purified from Lotus pedunculatus and may have been responsiblefor precipitating Rubisco in the presence of PEG.REFERENCESBarry, T.N. and D.A. Forss. 1983. The condensed tannin contentof vegetative Lotus pedunculatus, its regulation by fertiliserapplication, and effect upon protein solubility. J. Sci. Food Agric.34: 1047-1056.Elving, P.J., J.M. Markowitz and I. Rosenthal. 1956. Preparationof buffer systems of constant ionic strength. Analyt. Chem. 28: 1179-1180.Foo, L.Y., R. Newman, G.C. Waghorn, W.C. McNabb and M.J.Ulyatt. 1996. Proanthocyanidins from Lotus corniculatus.Phytochem. 41: 617-624.Jackson, F.S., W.C. McNabb, T.N. Barry, Y.L. Foo and J.S. Peters.1996. The condensed tannin content of a range of subtropical andtemperate forages and the reactivity of condensed tannin withribulose-1,5-bisphosphate carboxylase (Rubisco) protein. J. Sci. FoodAgric. (In press).Jones, W.T., R.B. Broadhurst and J.W. Lyttleton. 1976. Thecondensed tannins of pasture legume species. Phytochem. 15: 1407-1409.McDougall, E.I. 1948. Studies on ruminant saliva. 1. Thecomposition of output of sheepís saliva. Biochem. J. 43: 99-109.McNabb, W.C., G.C. Waghorn, J.S. Peters and T.N. Barry. 1996.The effect of condensed tannins in Lotus pedunculatus upon thesolubilization and degradation of ribulose-1,5-bisphosphatecarboxylase (Rubisco) protein in the rumen and the sites of Rubiscodigestion. Br. J. Nutr. (In press).Mangan, J.L. 1982. The nitrogenous constituents of fresh forages.in D.J. Thomson, D.E. Beever and R.G. Gunn, eds, Forage proteinin ruminant animal production. Occasional publication no. 6. BritishSociety of Animal Production, United Kingdom.Waghorn, G.C. and I.D. Shelton. 1995. Effect of condensed tanninsin Lotus pedunculatus on the nutritive value of ryegrass (Loliumperenne) fed to sheep. J. Agric. Sci. (Cam.) 125: 291-297.Waghorn, G.C., I.D. Shelton, W.C. McNabb and S.N.McCutcheon. 1994. Effects of condensed tannins in Lotuspedunculatus on its nutritive value for sheep. 2. Nitrogenous aspects.J. Agric. Sci. (Cam.) 123: 109-119.Waghorn, G.C., M.J. Ulyatt, A. John and M.T. Fisher. 1987. Theeffect of condensed tannins on the site of digestion of amino acidsand other nutrients in sheep fed on Lotus corniculatus L. Br. J. Nutr.57: 115-126.Figure 2The proportion of ribulose-1,5-bisphosphate carboxylase(Rubisco) protein degraded by rumen microorganismswhen condensed tannins (CT) extracted from Lotuscorniculatus (LC) and Lotus pedunculatus (LP) wereincubated with total soluble leaf protein extracted fromwhite clover (Trifolium repens) and rumen fluid from asheep fed fresh perennial ryegrass (Lolium perenne)/whiteclover pasture.  The incubations were done with   (-   -   -) and without (-  -  -) the addition of polyethylene glycol(PEG; molecular weight (MW) 3500).  All incubationswere done in duplicate.Figure 1The proportion of ribulose-1,5-bisphosphate carboxylase(Rubisco) protein precipitated when condensed tannins (CT)extracted from Lotus corniculatus (LC) and Lotuspedunculatus (LP) were incubated with total soluble leafprotein extracted from white clover.  The incubations weredone with (-  -  -) and without (-  -  -) the addition ofpolyethylene glycol (PEG; molecular weight (MW) 3500).All incubations were done in duplicate.

Effect of Condensed Tannin in Lotus Corniculatus and Lotus Pedunculatus on Digestion of Rubisco in the Rumen

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Effect of Condensed Tannin in Lotus Corniculatus and Lotus Pedunculatus on Digestion of Rubisco in the Rumen

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