Leaf phenolics and seaweed tannins : analysis, enzymatic oxidation and non-covalent protein binding

Abstract

Upon extraction of proteins from sugar beet leaves (Beta vulgaris L.) and oarweed (Laminaria digitata) for animal food and feed purposes, endogenous phenolics and proteins can interact with each other, which might affect the protein’s applicability. Sugar beet leaf proteins might become covalently modified by phenolics through polyphenol oxidase (PPO) activity. Oligomeric phenolics from seaweed (so-called phlorotannins (PhT)) might bind non-covalently to protein. The first aim of this thesis was to study factors involved in protein modification by phenolics. The second aim was to investigate the effect of PhT supplementation to feed on in vitro ruminal fermentation. Besides PPO activity and the amount of low molecular weight phenolic substrates present, brown colour formation in sugar beet leaves was dependent on the amount of phenolics, which do not serve as a substrate of PPO. These non-substrate phenolics can engage in browning reactions by oxidative coupling and subsequent coupled oxidation of the products formed. Similar reactions might also be involved in covalent protein modification by phenolics, and therewith protein properties. High molecular weight PhT from L. digitata could potentially modify protein properties by non‑covalent interactions. L. digitata contained PhT with subunits mainly connected via C‑O-C linkages, as determined using NMR spectroscopy. Further mass spectrometric analysis revealed the presence of a wide range of oligomers with degrees of polymerisation between 3 and 27. The interaction between PhT and proteins (b-casein and bovine serum albumin) was studied using model systems with different pH values, representing the various environments throughout the ruminants digestive tract. Phlorotannins bound to protein independent of pH, and broadened the pH range of protein precipitation from 0.5 to ~1.5 pH unit around the protein’s pI. At the pH of the abomasum of 2-3, the proteins re-solubilised again, presumably by increase in their net charge. Due to their ability to form water insoluble complexes, PhT could improve ruminal fermentation in vitro in a dose dependent manner, resulting in lower methane production and ammonia (NH3) concentration. The decreased NH3 concentration reflected decreased dietary protein breakdown in the rumen, which is considered a nutritional and environmental benefit.