Protein Precipitation Behavior of Condensed Tannins from Lotus pedunculatus and Trifolium repens with Different Mean Degrees of Polymerization

Abstract

The precipitation of bovine serum albumin (BSA), lysozyme (LYS), and alfalfa leaf protein (ALF) by two large- and two medium-sized condensed tannin (CT) fractions of similar flavan-3-ol subunit composition is described. CT fractions isolated from white clover flowers and big trefoil leaves exhibited high-purity profiles by 1D/2D NMR and purities >90% (determined by thiolysis). At pH 6.5, large CTs with a mean degree of polymerization (mDP) of ∼18 exhibited similar protein precipitation behaviors and were significantly more effective than medium CTs (mDP ∼9). Medium CTs exhibited similar capacities to precipitate ALF or BSA, but showed small but significant differences in their capacity to precipitate LYS. All CTs precipitated ALF more effectively than BSA or LYS. Aggregation of CT-protein complexes likely aided precipitation of ALF and BSA, but not LYS. This study, one of the first to use CTs of confirmed high purity, demonstrates that the mDP of CTs influences protein precipitation efficacy.

The Occurrence, Biosynthesis, and Molecular Structure of Proanthocyanidins and Their Effects on Legume Forage Protein Precipitation, Digestion and Absorption in the Ruminant Digestive Tract

“…Some forage legumes like sainfoin and birdsfoot trefoil contain proanthocyanidins in all parts of the plant [14], while in alfalfa, perennial ryegrass and tall fescue ( Festuca arundinacea ) they accumulate mainly in the seed coat [15,16] and in white clover and red clover ( Trefolium pratense L.) mainly in the flowers [17,18]. However, trace concentrations of proanthocyanidin were detectable in areal parts of most temperate forages [19,20].…”

“…Ropiak et al [58] found that mDP of proanthocyanidin was the most important factor determining PCC, while monomer composition of proanthocyanidin was more important in explaining the interaction of proanthocyandin with protein in terms of hydrophobic binding and changing protein secondary structure. Several studies compared the PCC of proanthocyanidin fractions of different MW and all found that PCC increased with increasing mDP [17,59,60,61]. Habertson et al [62], however, suggested that mDP above eight would not further improve PCC effectiveness.…”

“…The PCC of proanthocyanidin depends not only on the structure but also on characteristics of protein they bind to [17,70]. Protein precipitating capacity was found to be weaker for BSA than for alfalfa leaf protein (Rubisco) [17,54,70], rapeseed protein [70], and proline-rich protein (gelatine) [58], but similar to the PCC with the enzyme lysozyme [17].…”

“…Protein precipitating capacity was found to be weaker for BSA than for alfalfa leaf protein (Rubisco) [17,54,70], rapeseed protein [70], and proline-rich protein (gelatine) [58], but similar to the PCC with the enzyme lysozyme [17]. Results from Lorenz et al [70] suggested that high over low MW proteins were preferentially precipitated, however, this was more apparent with rapeseed than Rubisco protein.…”

“…This suggests that proanthocyanidins from different forage legumes differ in their biological activity in ruminal Rubisco degradation. The quaternary structure of Rubisco is relatively unstable compared to the quaternary structure of BSA, which might explain why Rubisco is more readily precipitated by proanthocyanidin than BSA [17]. Proanthocyanidin might also more readily bind with a mix of proteins, as is the case of Rubisco and rape seed protein, than with individual proteins as for BSA [17,70].…”