Laminaria digitata phlorotannins decrease protein degradation and methanogenesis during in vitro ruminal fermentation.

BACKGROUND: Phlorotannins (PhTs) are marine tannins consisting of phloroglucinol subunits connected via carbon-carbon and ether linkages. These have non-covalent protein binding properties and are, therefore, expected to be beneficial in protecting protein from hydrolysis during ruminal fermentation. In this study, the effectiveness of a methanolic PhTs extract from Laminaria digitata (10, 20, 40, 50, 75 and 100 g kg-1 tannin-free grass silage, with or without addition of polyethylene glycol (PEG), was investigated in vitro on protection of dietary protein and reduction of methane (CH4 ) in ruminal fluid. RESULTS: Addition of PhTs had linear (P < 0.0001) and quadratic (P = 0.0003) effects on gas and CH4 production, respectively. Optimal dosage of PhTs was 40 g kg-1 as at this point CH4 decreased (P < 0.0001) from 24.5 to 15.2 mL g-1 organic matter (OM), without affecting gas production (P = 0.3115) and total volatile fatty acids (P = 1.000). Ammonia trended (P = 0.0903) to decrease from 0.49 to 0.39 mmol g-1 OM, indicating protection of protein. Addition of PEG inhibited the effect of tannins at all dosage levels, and none of the fermentation parameters differed from the control. CONCLUSION: PhTs effectively protected protein from fermentation and reduced ruminal methanogenesis. © 2017 Society of Chemical Industry.

Resolubilization of Protein from Water-Insoluble Phlorotannin-Protein Complexes upon Acidification.

Marine phlorotannins (PhT) from Laminaria digitata might protect feed proteins from ruminal digestion by formation of insoluble non-covalent tannin-protein complexes at rumen pH (6-7). Formation and disintegration of PhT-protein complexes was studied with ß-casein (random coil) and bovine serum albumin (BSA, globular) at various pH. PhT had similar binding affinity for ß-casein and BSA as pentagalloyl glucose, as studied by fluorescence quenching. The affinity of PhT for both proteins was independent of pH (3.0, 6.0, and 8.0). In the presence of PhT, the pH range for precipitation of tannin-protein complexes widened to 0.5-1.5 pH units around the isoelectric point (pI) of the protein. Complete protein resolubilization from insoluble PhT-protein complexes was achieved at pH 7 and 2 for ß-casein and BSA, respectively. It was demonstrated that PhT modulate the solubility of proteins at neutral pH and that resolubilization of PhT-protein complexes at pH deviating from pI is mainly governed by the charge state of the protein.

Phlorotannin Composition of Laminaria digitata.

INTRODUCTION: Phlorotannins are complex mixtures of phloroglucinol oligomers connected via C-C (fucols) or C-O-C (phlorethols) linkages. Their uniformity in subunits and large molecular weight hamper their structural analysis. Despite its commercial relevance for alginate extraction, phlorotannins in Laminaria digitata have not been studied. OBJECTIVE: To obtain quantitative and structural information on phlorotannins in a methanolic extract from L. digitata. METHODOLOGY: The combined use of 13 C and 1 H NMR spectroscopy allowed characterisation of linkage types and extract purity. The purity determined was used to calibrate the responses obtained with the colorimetric 2,4-dimethoxybenzaldehyde (DMBA) and Folin-Ciocalteu (FC) assays. Using NP-flash chromatography, phlorotannin fractions separated on oligomer size were obtained and enabled structural and molecular weight characterisation using ESI-MS and MALDI-TOF-MS. RESULTS: The fucol-to-phlorethol linkage ratio was 1:26 and the extract was 60.1% pure, determined by NMR spectroscopy. For DMBA, the response of the extract was 12 times lower than that of phloroglucinol, whereas there was no difference for FC. By accounting for differences in response, the colorimetric assays were applicable for quantification using phloroglucinol as a standard. The phlorotannin content was around 4.5% DM. Fucol- and phlorethol-linkage types were annotated based on characteristic MSn fragmentations. Structural isomers of phlorotannins up to a degree of polymerisation of 18 (DP18) were annotated and identification of several isomers hinted at branched phloroglucinol oligomers. With MALDI-TOF-MS phlorotannins up to DP27 were annotated. CONCLUSION: By combining several analytical techniques, phlorotannins in L. digitata were quantified and characterised with respect to fucol-to-phlorethol linkage ratio, molecular weight (distribution), and occurrence of structural isomers. Copyright © 2017 John Wiley & Sons, Ltd.