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

Anne M. Vissers

Research output: Thesisinternal PhD, WU

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. 

Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • Wageningen University
Supervisors/Advisors
  • Gruppen, H., Promotor
  • Hendriks, Wouter, Promotor
  • Vincken, Jean-Paul, Co-promotor
Award date23 Jun 2017
Place of PublicationWageningen
Publisher
Print ISBNs9789463432023
DOIs
Publication statusPublished - 2017

Fingerprint

protein binding
tannins
macroalgae
oxidation
leaves
proteins
catechol oxidase
sugar beet
ammonia
rumen fermentation
molecular weight
Laminaria
leaf protein
abomasum
Beta vulgaris
food animals
bovine serum albumin
methane production
digestive tract
polymerization

Keywords

  • phenols
  • leaves
  • seaweeds
  • tannins
  • beta vulgaris
  • laminaria
  • proteins
  • catechol oxidase
  • nuclear magnetic resonance spectroscopy
  • in vitro
  • mass spectrometry
  • browning
  • fermentation
  • animal feeding

Cite this

Vissers, Anne M.. / Leaf phenolics and seaweed tannins : analysis, enzymatic oxidation and non-covalent protein binding. Wageningen : Wageningen University, 2017. 154 p.
@phdthesis{5680c1ab45804ebb838c61f249f229fe,
title = "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. ",
keywords = "phenols, leaves, seaweeds, tannins, beta vulgaris, laminaria, proteins, catechol oxidase, nuclear magnetic resonance spectroscopy, in vitro, mass spectrometry, browning, fermentation, animal feeding, fenolen, bladeren, zeewieren, tanninen, beta vulgaris, laminaria, eiwitten, catechol oxidase, kernmagnetische resonantiespectroscopie, in vitro, massaspectrometrie, bruinkleuring, fermentatie, diervoedering",
author = "Vissers, {Anne M.}",
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doi = "10.18174/414167",
language = "English",
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publisher = "Wageningen University",
school = "Wageningen University",

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Leaf phenolics and seaweed tannins : analysis, enzymatic oxidation and non-covalent protein binding. / Vissers, Anne M.

Wageningen : Wageningen University, 2017. 154 p.

Research output: Thesisinternal PhD, WU

TY - THES

T1 - Leaf phenolics and seaweed tannins : analysis, enzymatic oxidation and non-covalent protein binding

AU - Vissers, Anne M.

N1 - WU thesis 6700 Includes bibliographical references. - With summaries in English and Dutch

PY - 2017

Y1 - 2017

N2 - 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. 

AB - 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. 

KW - phenols

KW - leaves

KW - seaweeds

KW - tannins

KW - beta vulgaris

KW - laminaria

KW - proteins

KW - catechol oxidase

KW - nuclear magnetic resonance spectroscopy

KW - in vitro

KW - mass spectrometry

KW - browning

KW - fermentation

KW - animal feeding

KW - fenolen

KW - bladeren

KW - zeewieren

KW - tanninen

KW - beta vulgaris

KW - laminaria

KW - eiwitten

KW - catechol oxidase

KW - kernmagnetische resonantiespectroscopie

KW - in vitro

KW - massaspectrometrie

KW - bruinkleuring

KW - fermentatie

KW - diervoedering

U2 - 10.18174/414167

DO - 10.18174/414167

M3 - internal PhD, WU

SN - 9789463432023

PB - Wageningen University

CY - Wageningen

ER -