Outline Toksikologi Pakan: Tannins A. Introduction B. Structure C. Interaction D. Analytical methods E. Biological effects F. Treatments/processing methods Anuraga Jayanegara A. Introduction Plant secondary compounds - Do not function in primary metabolism such as biosynthesis, biodegradation and other energy conversions of intermediary metabolism - Do have diverse biological activities ranging from toxicity to hormonal mimicry - May play a role in protecting plants from herbivory and disease - Examples: alkaloids, terpenes, phenolics Classification Condensed tannin (CT, proanthocyanidins): oligomers of two or more flavan-3-ols such as catechin, epicatechin, or gallocatechin. Hydrolysable tannin (HT): consisting of a central core of carbohydrate to which phenolic carboxylic acids are bound by ester linkage. Tannins, definition Water-soluble phenolic compounds that have ability to precipitate proteins
Why plants produce phenolics? A strategy adopted by plants to deter attack by microorganisms, insects and higher animals. Factors affecting tannin levels: - Nutrient stress (N, P, K, S deficiencies) - High light intensity - High temperature - Severe drought - Tissue damage Increase tannin levels B. Structure Diversity of tannin structures Hydrolysable tannin (HT) Condensed tannin (CT) Source: Mueller-Harvey (2006) J. Sci. Food Agric. 86, 2010-2037.
C. Interaction Illustration of protein precipitation by tannins Multiple hydroxyl groups: enable phenolics to form complexes with proteins, polysachharides and minerals. Tannin-protein complex: 1. Hydrogen bonds: free phenolic hydroxyl groups 2. Hydrophobic bonds: aromatic ring structures 3. Covalent bonds: polymerization reactions due to heating, exposure to UV radiation and the action of polyphenol oxidase Marangon et al. (2010) Analytica Chimica Acta 660, 110-118 D. Analytical methods - Categorized into: (1) chemical methods, (2) protein precipitation methods, (3) gravimetric assays, (4) tannin bioassay, (5) Others. - Chemical assays: - Total phenols and total tannins: Folin-Ciocalteu (oxidation-reduction), ferric chloride (metal-complexing property of phenolics) - Condensed tannins: vanilin assay, butanol-hcl assay - Hydrolysable tannins: rhodanine method, HPLC - Protein precipitation assays: ninhydrin assay, BSA method, radial diffusion assay - Gravimetric methods: ytterbium acetate, PVPP - Tannin bioassay: in vitro gas production + PEG (e.g. Jayanegara & Sofyan, 2008) E. Biological effects Negative effects: - Decreasing nutrient utilization, in particular protein utilization - Decreasing feed intake - Toxicity and death of animals Positive effects: - Prevention of bloat - Protection of protein from rumen degradation - Mitigating methane emission - Modulation of PUFA biohydrogenation in the rumen - Controling of gastro-intestinal nematode parasites More detailed methods...
Decreasing nutrient utilization Toxicity problems Prevention of bloat Protection of protein from rumen degradation
Mitigating methane emission Global warming Loss of energy Evidences: - Jayanegara et al. (2009), Animal Feed Science and Technology 150, 230-237 - Jayanegara et al. (2009), Media Peternakan 32, 120-129 - Jayanegara et al. (2010), Sustainable Improvement of Animal Production and Health (Odongo, N.E., Garcia, M., Viljoen, G.J. (Eds.)), FAO, Rome, pp. 151-157 - Jayanegara et al. (2011), Animal Feed Science and Technology 163, 231-243 - Jayanegara et al. (2012), Journal of Animal Physiology and Animal Nutrition 96, 365-375 - Jayanegara et al. (online first), British Journal of Nutrition Source: Morgavi et al. (2010) Animal 4, 1024-1036 Ruminal methanogens attached to protozoal species interspecies H transfer Protozoa-associated methanogens contribute up to 37% of total rumen methane emissions Removal of protozoa from the rumen (defaunation) may CH 4 emission Source: Bhatta et al. (2009) J. Dairy Sci. 92, 5512-5522 Phenolics and microbial population Protozoa colonized by methanogens
Modulation of PUFA biohydrogenation Reduce the risk of cardiovascular disease Lowering plasma cholesterol level Linoleic acid α-linolenic acid Rumenic acid Vaccenic acid Prevent cancer proliferation Decrease atherosclerosis Improve immune system Stearic acid Source: Chilliard et al. (2007) Eur. J. Lipid Sci. Technol. 109, 828-855 Condensed tannins (CT) Source: Khiaosa-ard et al. (2009) J. Dairy Sci. 92, 177-188 CH: grass-clover hay (control) TF: dried sainfoin (7.9% CT) TH: CH + A. mearnsii extract (7.9% CT) SH: CH + Y. schidigera extract (1.1% saponins) Influence of tannins on biohydrogenating bacteria Inhibition of phenolics on lipase activity Containing 6.4% tannins from quebracho powder Source: Vasta et al. (2010) Appl. Environ. Microbiol. 76, 2549-2555 Source: Van Ranst et al. (2011) Animal 5, 512-521
F. Treatment methods Other evidences: - Jayanegara et al. (2011), Animal Production Science 51, 1127-1136 - Jayanegara et al. (2012), Livestock Science 147, 104-112 - Palupi et al. (online first), Journal of the Science of Food and Agriculture - Wood ash: a good source of alkali; 10% solution of oak wood ash decreased the content of TP, CT and protein precipitation capacity by 66, 80 and 75% in oak leaves, respectively. - Urea-ammoniation: 4% urea in fresh leaves. - Drying: but not effective. - Chemicals: extraction with organic solvents (30% acetone, 50% methanol, 40% ethanol) removed ca 70% tannins from oak leaves. - Solid-state fermentation: biodegradation of tannins using white-rot fungi. - Addition of tannin-binding polymers: PEG (polyethylene glycol) incorporation, PVPP.
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