Tannin extracts and their BSA precipitation ability at different pH values in vitro M. Schweigmann, F. Taube, M. Gierus University of Kiel, Germany Institute of Crop Science and Plant Breeding – Grass and Forage Science/ Organic Agriculture

Content Issue and hypothesis Tannins Extracts Material & Methods Results & Discussion Summary

Issue and hypothesis Issue: dairy feeding: surplus of rumen degradable protein from grass = low nitrogen use efficiency (NUE) options: adding concentrates with low rumen degradability (soya) adding secondary plant compounds (PPO, tannins) Hypothesis: Tannin extracts are suitable as feed additives to increase NUE.

Condensed tannins secondary plant compounds flavan-3-ol units phenolic ring condensed to oligomers, polymers form complexes reversible, pH-dependent reaction? protect feed protein in the rumen (pH > 6), release again in the abomasum (pH < 3) = increasing feed protein use efficiency 5% CT to prevent bloat (Haslam, 1996; Aerts et al., 1999; Barry & McNabb, 1999)

Material & Methods tannin extracts from different sources: quebracho (heartwood): Schinopsis lorentzii mimosa (bark): Acacia mearnsii tara (pods): Caesalpinia spinosa gambier (leaves): Uncaria gambir Table 1. Tannin extracts and their content of condensed tannins (CT) and total phenols (TP) in g kg–1 for comparison# quebracho mimosa tara gambier birdsfoot trefoil CT* 138 253 5 49 19 TP** ca. 1000 967 772 48 * determined by butanol/HCl method (Terrill et al., 1992) ** determined by folin-ciocalteu method (Singleton and Rossi, 1965) # masterthesis, same equipment (Hamacher, 2012)

Material & Methods Precipitation: modulated method of Osborne & McNeill (2001) bovine serum albumin (BSA), dissolved in a sodium-acetate-solution; 60µg per tube pH values between 7/6 and 2 tannin extracts in amounts of 0 to 10mg per tube Bio-Rad Protein Assay, photometrical measurement of colour reaction log-logistic equation: f(x) = 0 + d / {1 + exp (b (log(x) – log(e)))}

Differences for extract plateau values Table 2. Plateau values in µg BSA with statistical differences. plateau quebracho mimosa tara gambier pH 7 69,850aA 74,376abA 65,648aA >100†aA pH 6 64,779baA 81,902aB 70,432aA >100†aAB pH 5 57,489cbA 79,158acB 72,329aB 74,042aAB pH 4 48,536cA 77,587abB 74,116aB 60,472aAB pH 3 35,669dA 69,964bB 78,732aC 46,238aA pH 2 25,125eA 71,079bcB 74,840aB a,b: different small letters indicate significant differences between pH values within an extract (P<0.05) A, B: different capital letters indicate significant differences between extracts at the same pH value (P<0.05) †: out of range Quebracho indicates potential for increased NUE of the dairy cow: high precipitation at rumen pH; low precipitation at abomasum pH

Conclusions Tannin source and pH value affected precipitation significantly. Quebracho formed complexes at simulated rumen pH, but barely at simulated abomasum pH. Precipitation profiles of mimosa, tara, and gambier did not support the given hypothesis. next step: validating results using in vivo / in situ techniques; testing reversibility of complexing in the abomasum  

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References Aerts, R.J., Barry, T.N., McNabb, W.C. (1999): Polyphenols and agriculture: beneficial effects of proanthocyanidins in forages. Agriculture, Ecosystems and Environment 75, 1- 12. Barry, T.N. und McNabb, W.C. (1999): The implications of condensed tannins on the nutritive value of temperate forages fed to ruminants. British Journal of Nutrition 81, 263- 272. Hamacher, M. (2012): Sekundäre Pflanzeninhaltsstoffe und Proteinbindungsvermögen von Wiesenkräutern und Futterleguminosen. masterthesis at university of Kiel. Haslam, E. (1996): Natural polyphenols (vegetable tannins) as drugs: Possible modes of action. Journal of Natural Production 59, 205-215. Osborne, N.J.T. und McNeill, D.M. (2001): Characterisation of Leucaena condensed tannins by size and protein precipitation capacity. Journal of the Science of Food and Agriculture 81, 1113-1119. Singleton, J. und Rossi, P. (1965): Colorimetry of total phenolics with phosphomolybdic phosphotungstic acid reagents. American Journal of Enology and Viticulture 16, 144- 158. Terrill, T.H., Rowan, A.M., Douglas, G.B., Barry, T.N. (1992): Determination of extractable and bound condensed tannin concentrations in forage plants, protein concentrate meals and cereal grains, Journal of the Science of Food and Agriculture 58, 321-329.

Precipitation at pH 7 and 2 o quebracho ∆ mimosa + tara x gambier Mimosa seemed to precipitate BSA most efficiently.

Differences for extract ED50 values quebracho mimosa tara gambier pH 7 4,028aA 2,587aB 7,927aC 42,781aABC pH 6 2,651bA 2,057bB 4,849bC 47,889aABC pH 5 1,960adA 1,463cA 1,687cA 6,784aB pH 4 10,623cA 0,596dB 0,733dB 3,289aC pH 3 1,009dA 1,630bcB 1,354cA 3,862aC pH 2 1,094abcdAB 3,256eA 4,029eB 41,390aAB a,b: different small letters indicate significant differences between pH values within an extract (P<0.05) A, B: different capital letters indicate significant differences between extracts at the same pH value (P<0.05)

Precipitation at pH 6 and 5 o quebracho ∆ mimosa + tara x gambier

Precipitation by quebracho o pH2 ∆ pH3 + pH4 x pH5 ◊ pH6 pH7 ∆

Precipitation at pH 7 for CT

Precipitation at pH 6 and 2 for CT