Phenolic compounds in food

Lecture 3

Wouter de Bruijn Period 4

Laboratory of Food Chemistry 2024
Content of the lectures on phenolics

▪ Structure & biosynthesis

▪ Extraction, isolation, and analysis
→ Case 1: Extraction, characterization, and quantification of avenanthramides in
germinated oat
▪ Oxidation of phenolics
• Browning upon oxidation of tea phenolics
→ Case 2: Auto-oxidative browning of green tea catechins
• Case 3: Browning during processing of by-products from potato
▪ Inhibition of oxidation and browning of phenolics
• Insights in the reactivity of sulfur nucleophiles with phenolics
• Case 4: Unravelling the efficient inhibition of PPO by sulfite
▪ Wrap up phenolics in food: Future outlook, challenges, and conclusion
• Case 5: Oxidative coupling as a tool for bioactivation of phenolamides from barley by-
products
Inhibition of oxidation and browning
of phenolics
Established approaches to inhibit enzymatic browning

H2O O2
optimum T
sulfo-phenolics • Cooling (reversible)
active PPO
• Heating (irreversible)
• Remove O2
optimum pH 5-7
• Ascorbic acid
Brown • Lowering pH
pigments • Sulfite

Applicability of some approaches

dehydroascorbic acid ascorbic acid is very situational
Exploring new enzymatic browning inhibitors

• Demand for less processed food and less synthetic additives in food:
What natural compounds can be used as browning inhibitors?

• Thiol-containing compounds as alternatives for sulfite (first part of this lecture)

→ Insights in the reactivity of sulfur-containing compounds with o-quinones
→ Unravelling the efficient inhibition of PPO by sulfite (case 4)

• Phenolics and their derivatives as PPO inhibitors

• Some hydroxycinnamic and benzoic acids are (weak) PPO inhibitors 1
• Coumaric acid alkyl esters (R′ = alkyl) can potently inhibit PPO 2
• Candidate inhibitors are often substrate look-a-likes without o-diphenol moiety

1 Sukhonthara et al (2016) Food Chem. 190: 922-927

2 Varela et al. (2020) Bioorg. Chem. 103: 104108.
Insights in the reactivity of sulfur
nucleophiles with phenolics
Nucleophilic addition of thiols to o-quinones

• Thiols = R-SH → Natural thiols include cysteine (Cys) & glutathione (GSH)
• Thiols are nucleophiles → can attack o-quinones in a similar way as sulfite
• Which is the stronger nucleophile: thiol or sulfite?
Thiols are more nucleophilic than NaHSO3

• Thiols more nucleophilic than NaHSO3 100

Rel. amount of addition

→ electron withdrawing effect of 80
oxygens surrounding sulfur in HSO3−

product (%)
makes it less nucleophilic 60
• Cys more reactive than GSH, 40
probably due to more accessible 20
sulfur (less steric hindrance)
0
NaHSO₃ NaHSO₃ NaHSO₃ Cys +
+ Cys + + Cys + GSH GSH
GSH

Competition experiment: sulfite (NaHSO3),

cysteine (Cys), and glutathione (GSH),
incubated in different combinations with
catechin and tyrosinase (=PPO).
Position of sulfonation determined with NMR

• Influence of phenolics’ R-group on addition reactions with sulfite

→ position of sulfonation?
• Sulfo-phenolics: only two protons attached to the aromatic ring
• Magnitude of coupling between protons in 1H NMR used for identification
Raise your hand if you have some
experience with NMR

para protons meta protons ortho protons

Position of sulfonation of epicatechin by 2D COSY NMR
small
coupling small
no no coupling Coupling leads to peak
coupling coupling large
coupling
large
coupling
splitting in 1H spectrum
→ Small couplings not
always clearly visible

Along the diagonal

in COSY: proton
cross peaks with
identical protons
Position of sulfonation of epicatechin by 2D COSY NMR
small
coupling small
no no coupling
coupling coupling large
coupling
large
coupling
21%
ortho
protons

60%
meta
protons

19%
para
protons
Preference for sulfonation at specific positions
100

80 Sulfite addition to:

x
60 position x
%

position y
40 z
position z y
20

0
Preferred position of sulfonation
depends on R

As explained in lecture 2:
position x (= C2’) is preferred
for chlorogenic acid (= 5-CaQA)
Inductive and resonance effects: (partial) charge
localization in the o-quinone

Polar covalent bond

→ more electronegative
group withdraws electrons
δ+ δ−
C O

Vicinal (i.e. neighbouring) positive (+) or

partial positive (+) charges are unfavourable
Methoxycatechol: sulfite mainly on z-position

Methoxyl group is
electron-donating
via resonance
→ This effect is much
y stronger than its inductive
electron-withdrawing effect
x z

• Five possible resonance structures

• Positive charge on z-position preferred
• Therefore, higher occurrence of sulfite at z-position,
compared to the x- and y-positions
Chlorocatechol: sulfite mainly on y-position

Cl is inductively electron withdrawing

y
• Four possible resonance structures
x z
• Positive charge on x-position very
unfavourable
• Position y and z seem equivalent,
but y-position is the preferred site
of addition (experimental evidence!)
• This might be explained by the
stronger inductive electron-
withdrawing effect of chlorine
compared to oxygen
Effect of steric hindrance on sulfite positioning

y • Epicatechin: y-position is preferred, even

z though charge distribution is unfavourable
• Steric hindrance apparently overrules charge
x distribution
Recap: Reaction sulfur nucleophiles with o-quinones

⚫ Type of sulfur compound matters!

• Thiols are more nucleophilic and thus more reactive than sulfite
• Both nucleophilicity and accessibility of sulfur affect reactivity
⚫ Position of nucleophilic attack by sulfur compounds is mainly determined by
electron-donation and -withdrawal of the ring substituents:
• Resonance effects (conjugative electron delocalization) yielding positively
charged carbon atoms (i.e. carbocations) in the ring
• Inductive effects (mainly electron-withdrawing effect of electronegative
atoms) yielding partially positive carbon atoms (+) in the ring
• Substituents of the o-quinone (e.g. Cl, OMe) can modulate the electron
distribution in the o-quinone → some carbocations are more likely to occur
• Resonance and inductive effects can have opposite character
→ e.g. OCH3: inductive electron-withdrawal << resonance electron-donation
• Steric effects can also modulate site of addition of sulfur compound
Questions
Question: Can you draw the relevant resonance structures
underlying sulfite’s site of attachment on 5-CaQA o-quinone?

x
O2 y
z
PPO
5-CaQA o-quinone
Answer: 5-CaQA sulfite mainly on x-position

y
z

Two resonance structures that result in + on x-position

y
z

Narvaez et al. (2011) J. Agric. Food Chem. 59: 10247-10255.

Questions: Oxidation & reactivity of phenolics

1. Considering the groups in side chains of amino acid residues in a protein, what
would be the order of reactivity towards o-quinones: CH3, NH2, OH, or SH?
2. Besides inhibiting further oxidation by PPO/tyrosinase, sulfonation can also be
useful as a structural modification to change the properties of phenolics. What
could be a beneficial effect of sulfonation on the properties of a phenolic?
3. In the following structures, what type of proton coupling (no, small, or large)
would you observe in NMR?
a) b) c) d)
Answers: Oxidation & reactivity of phenolics

1. The most nucleophilic group would be the most reactive towards o-quinones,
so the order would be: SH > NH2 > OH > CH3
2. Sulfonation adds a polar sulfo-group to phenolics, thereby making them more
polar and better soluble in water.
3. a) b) c) d)

Only 1 proton, para meta protons, meta and ortho

no coupling protons, small coupling protons,
no coupling small and large
couplings
Inhibition of oxidation and browning
of phenolics
Sulfur compounds as enzymatic browning inhibitors

SH-compounds
or HSO3−
H2O O2
optimum T RS-phenolics or
active PPO sulfo-phenolics

optimum pH 5-7
Brown
pigments

• Thiol compounds like cysteine or

glutathione can function as
natural alternatives for sulfite
• Is this the full story of the anti-browning
mechanism of sulfite?

Kuijpers et al. (2012) J. Agric Food Chem. 60: 3507-3514.

 Case 4
Unravelling the efficient inhibition of PPO by sulfite
Refresher: PPO is a copper-dependent oxidase

Example PPO from grape (Vitis vinifera)

• PPO is also known as

tyrosinase Histidine
• Oxygen consumed as electron residues
acceptor during catalysis
→ PPO activity can be Copper ions
monitored by measuring
oxygen consumption
• Active site has two copper
ions, each coordinated by
three histidine residues

Virador, et al. (2010) doi:10.1021/jf902939q; RSCB PDB ID, 2P3X;

Image generated with Mol*, D. Sehnal, et al. (2018) doi:10.2312/molva.20181103
Sulfite slowly inactivates tyrosinase from mushroom

5-CaQA + NaHSO3 + PPO

(single addition)
5-CaQA + NaHSO3 + PPO
(2 additions)
5-CaQA + PPO

2nd PPO
addition
Investigate inactivation of tyrosinase by sulfite

PPO
+NaHSO3
PPO

NaHSO3
ultrafiltration

If PPO remains inactive, even after removal of NaHSO3 by ultrafiltration

→ strong indication of irreversible inactivation
NaHSO3 causes irreversible inactivation of tyrosinase

Experimental set-up: 100

Relative [O2] (%)

1. Pre-incubate tyrosinase 80
with sulfite or DTT
2. Ultrafiltration to remove 60
sulfite or DTT 40
NaHSO3
3. O2 consumption DTT
measurement using 20
tyrosine as substrate 0
control
0 500 1000
Incubation time (s)

Less oxygen consumption after tyrosinase has been pre-incubated with

sulfite or DTT → both sulfite and DTT inactivate tyrosinase

Kuijpers et al. (2013) FEBS J. 280: 6184-6195.

Inactivation by DTT is not irreversible

• Inactivation of PPO by DTT is

reversible by copper addition
→ CuCl2 restores activity
• DTT likely inhibits PPO by
chelation of Cu2+
• Inactivation by NaHSO3 is not
reversed by addition of CuCl2

Sulfite must irreversibly inactivate PPO by another mechanism

Inactivation of PPO by NaHSO3 is dose-dependent

100 10 mM NaHSO3
pre-treated
80
Relative [O2] (%)

60
1 mM NaHSO3
40 pre-treated

20
control
0
0 100 200 300 400
Incubation time (s)

How does NaHSO3 irreversibly inhibit PPO/tyrosinase?

→ Does it bind to the active site of the enzyme?

Kuijpers et al. (2013) FEBS J. 280: 6184-6195.

Hypothesis: one of the active site histidine residues of
mushroom tyrosinase is modified by sulfite
Sequence alignment of
active site amino acids
of 2 PPO isoforms
→ Thioether between
H (= histidine) and
C (= cysteine)
in active site

• Theoretically: If thiol from C can perform nucleophilic attack on H to form the

thioether, then sulfite might also be able to do this
→ Note similarity with formation of sulfo-phenolics!
• All histidine residues in copper-A and copper-B site are potential targets
• Hydrolysis of tyrosinase by protease followed by RP-LC-MS analysis of the
peptides to search for sulfite-modified histidine residues
Identified sulfite-modified peptides from copper-B site

Two proteases used for hydrolysis of PPO peptic peptides

prior to analysis of the resulting peptides chymotryptic peptides
sulfite-modified region

Sulfite modification detected in histidine-containing peptides

originating from the PPO copper-B site

Kuijpers et al. (2013) FEBS J. 280: 6184-6195.

Two candidate histidine residues: His263 or His259

MVHNTVHF
MS fragmentation of the
peptide MVHNTVHF resulted
in a highly abundant
fragment corresponding to
loss of sulfite
→ high abundance of this
fragment indicates that there
is a relatively weak bond
between sulfite and peptide

Kuijpers et al. (2013) FEBS J. 280: 6184-6195.

Possible reactions of His residues in active site

Thioether between
Cys and His formed
via attack of thiol on C

Sulfite could
attach to C or N

Virador et al. (2010) J. Agric. Food Chem. 58: 1189-1201.

Kuijpers et al. (2013) FEBS J. 280: 6184-6195.
Sulfite addition to N of His residues seems likely

• Based on ease of MS
fragmentation it was
proposed that HSO3 is
attached to N
→ C-S bond is stronger
than N-S
• Additionally, attachment
at N would directly
prevent coordination of
copper, thereby explaining
PPO inactivation

Virador et al. (2010) J. Agric. Food Chem. 58: 1189-1201.

Kuijpers et al. (2013) FEBS J. 280: 6184-6195.
The power of sulfite’s inhibitory effect

SH-compounds
or HSO3−
H2O O2 Colourless sulfo-phenolics
optimum T RS-phenolics or cannot react further to brown
active PPO sulfo-phenolics pigments & cannot be oxidised
to (sulfo-)o-quinones
optimum pH 5-7 AND
Brown Sulfite irreversibly inhibits PPO
pigments by modifying its active site

Important notes:
• Cysteine or glutathione do not modify the active site
of PPO (too large?)
• Inhibition of PPO by sulfite is quite slow

Kuijpers et al. (2012) J. Agric Food Chem. 60: 3507-3514.

Preserving phenolic structure in presence of PPO

• Addition of sulfite in presence of PPO prevents browning...

...but leads to formation of sulfo-phenolics
• It might be important to preserve the original phenolic structure
→ e.g. if the phenolic itself has desirable (bio-)functionality
• PPO inactivation by sulfite could be a solution but is quite slow
→ takes some time during which phenolics can be oxidised to o-quinones and
then converted to sulfo-phenolics

Challenge
Prevent PPO-catalysed browning by inactivation with sulfite
AND
preserve the original phenolic structure
Combining sulfite with other PPO inhibitors

To preserve phenolic structure: An additional approach is needed to delay

oxidation during the lag time for irreversible PPO inhibition by sulfite

What are the options for the additional approach?

Lower pH below pH optimum of PPO What happens to active species of sulfite?

SO2  H2SO3  HSO3-  SO32- *
decrease  pH → increase

Add ascorbic acid to reduce Competition between sulfite and ascorbic

o-quinones acid for reaction with o-quinones → Who wins?

Reduce temperature Is sulfite reactivity less affected than

enzyme activity?

*Green, 1976, Food Chem. 1: 103-123

Overview enzymatic oxidative browning inhibitors
Mode of action
Browning Inhibitor Directly on Quenching quinone’s Other remarks Conclusion
PPO reactivity
Sulfite   Cheap
Lag time for inactivation PPO

→ Changed phenolic structure
Allergic reactions?
More nucleophilic & reactive
Cysteine or other thiols   than sulfite
/
Too large? Changed phenolic structure

Vit C   Also lowers pH, cheap

Temporary (until used up)
/
Can react with phenolics

Cu2+ chelators  / Food grade?

Expensive?

DTT likely quenches reactivity due to its
two thiol groups. Other chelators
(e.g. EDTA) will not
Phenolics   Reversible
Concentration-dependent

Expensive?

pH   Reversible?
Compatible with purpose?
/
Heat   Irreversible
Compatible with purpose?

Questions
Questions: Action and inhibition of PPO

1. Sulfite has a dual action mechanism due to its reaction with o-quinones and
PPO. If you combine sulfite with packaging under O2-less conditions, then what
will be the effect on this dual action mechanism?
2. How many moles of O2 does PPO/tyrosinase use to oxidize tyrosine to
dopaquinone? Note: the amount of O2 was not specified in the lectures so far!
tyrosine ?? O2 dopaquinone

PPO
Answers: Action and inhibition of PPO

1. Sulfite reacts with o-quinones, which will not be formed in absence of O2.
Sulfite will most likely still react with PPO. So, under O2-less conditions, the
main anti-browning mechanism of sulfite will be via inactivation of PPO.
2. For these reactions in total 1 mole of O2 will be used by PPO, see below:
tyrosine DOPA dopaquinone
½ O2 ½ O2 H2O

PPO PPO
 Wrap up phenolics in food:
Future outlook, challenges, and conclusion
Future outlook: Exploiting desirable oxidative coupling

• Oxidative coupling leads to changes in properties of phenolics, such as:

• Colour
• Size & polarity
• Bioavailability & bioactivity
• Oxidation isn’t always a bad thing!

• Oxidative coupling can be a valuable tool for structural modification

→ Can be exploited to modulate phenolics’ properties

• Example application for future:

Bioactivation of phenolamides from brewing by-products (Case 5)
 Case 5
Oxidative coupling as a tool for bioactivation of
phenolamides from barley by-products
Malting & beer brewing by-products

• Barley malt is an essential ingredient in beer brewing and whiskey distillery

• The malting and beer brewing process produces several by-products

Barley
Wort
malt
Barley Malting
Mashing Brewing
(seeds) (germination)

Rootlets Spent grain Trub

Waste or animal feed

Phenolamides in barley

• Amides of a hydroxycinnamic acid and agmatine

R1 = R2 = H: coumaroylagmatine
R1 = OCH3, R2 = H: feruloylagmatine
R1 = R2 = OCH3: sinapoylagmatine

• Barley rootlets are rich in hydroxycinnamoylagmatines 1

• Oxidative coupling of these compounds occurs in the stressed plants

→ Oxidative coupling as a bioactivation mechanism?

• Our aim: Valorisation of barley rootlets as preservatives against microbial

spoilage via bioactivation of hydroxycinnamoylagmatines
1 Koistinen, et al. (2020), DOI: 10.1038/s41538-020-00081-0
Peroxidase as an oxidative coupling tool

• Hydroxycinnamic acids and their derivatives can by coupled with HRP + H2O2
• Coupling reactivity & products dependent on phenolic structure
→ Mainly influenced by substituents on aromatic ring (R1 and R2)

H2O2 + other
coupling
POD products

X = OH / N-R / O-R Intramolecular

R1 = R2 = H: p-coumaric rearrangements
R1 = OH, R2 = H: caffeic
R1 = OCH3, R2 = H: ferulic Dimers with diverse structures
R1 = R2 = OCH3: sinapic & various linkage types
Preservatives by bioactivation of barley side-streams

• Hydroxycinnamoylagmatines can be
extracted from beer brewing by-products
• Valorisation as natural food preservatives
after oxidative coupling

Barley Malting
(seeds) (germination)

Rootlets Natural food preservative

Extract Bioactivation
hydroxycinnamic (enzymatic
acid derivatives oxidative coupling)
Take-home messages lecture 3, including cases 4 & 5

• There is a demand for natural oxidative browning inhibitors

→ Candidates: thiols & hydroxycinnamic acid derivatives
• Sulfite & thiols can react with o-quinones
→ Reactivity affected by nucleophilicity & accessibility of the sulfur group(s)
• Sulfur addition prevents further reaction of o-quinones
→ Position of sulfur compound addition influenced by phenolics’ substituents
→ Resonance, inductive, and steric effects govern position of addition
• Determination of position of substituents on phenolics’ aromatic ring using
NMR (1H and COSY) spectra
• Sulfite can irreversibly inactive PPO by reacting with active site histidine
• Oxidative coupling: a tool to modulate structure and properties of phenolics
• Bioactivation of natural hydroxycinnamic acid derivatives via
radical-mediated oxidative coupling with peroxidase
Challenges & conclusion
Challenges: Unwanted reactions & interactions

• Oxidation: browning of phenolics and other food constituents

→ Loss of product quality
• Haze formation (e.g. in beer) due to interaction with proteins
• Difficult to remove from plant-derived food ingredients
→ e.g. plant proteins
• Unwanted off-taste (astringency & bitterness)
• Lignin can hinder valorisation of plant biomass
• Complexation with metals: Off-colour
formation and reduced bioavailability
→ A challenge in fortified food
Recap: Learning outcomes

At the end of these lectures, you should be able to...:

▪ Describe (draw) structure of simple phenolics and their derivatives
▪ Discuss the effect of phenolics’ structure on their properties and
reactivity
▪ Design a strategy for extraction and isolation of phenolics
▪ Select analytical methods for quantification and structural
characterization of phenolics
▪ Discuss mechanisms underlying oxidation, oxidative coupling, and
browning of phenolics
▪ Know applications of various browning inhibition strategies and
discuss the underlying mechanisms
Phenolic compounds in food

I hope you enjoyed learning more about phenolics in food! ☺

Do you have any questions?