4. Phenolic compounds

Example of phenolic as glycoside in plants

• Naringenin has no sugars attached

• Prunin has one sugar attached

• Naringin has two sugars attached

Phenolic compounds in food

• They are minor constituents of food

• They have no noteworthy nutritional value

• Present in plant products

• Most important sources; fruits, beverages and vegetables

• Has an influence on food properties.

Definition of phenolic compounds

A molecule that contains at least one aromatic ring with one or more hydroxyl groups

Examples of phenolic rich foods

• Coffee

• Tea

• Chocolate

• Berries and grapes

  • And products that are made of these: wine and juices

• Herbs and spices (but are consumed in small amounts)

Can contain 10-30% phenolics on dry matter basis

Influence of phenolics on color & appearance

• Gives berries blue, purple & red color

• Responsible for browning upon phenolic oxidation

• Can cause turbidity in beer

Influence of phenolics on flavor

• Aroma formation reactions (e.g. during roasting of coffee beans)

• Volatile phenolics as odors (e.g. vanilla)

• Gives bitter taste in grapefruit

• Astringency (dry feel in mouth when drinking wine)

Influence of phenolics on stability & shelf-life

• Antioxidant activity

• Antimicrobial properties

Structural variations in phenolics

• Different groups create different properties

• Three important parameters:

  • Polarity

  • Reactivity

  • Size of the conjugated system

Polarity, reactivity and size of conjugated system in phenolics

• Polarity: Phenolics are typically medium polar → limited water solubility

• Reactivity: Reactive phenolics often also possess high antioxidant activity

• Size of conjugated system: Conjugated system < 8 bonds = no color

Effect of hydroxylation on phenolics

• Increases water solubility & increases reactivity

• O-diphenol moiety is formed

  • Highly relevant for oxidation reactions and antioxidant activity

Effect of methylation on phenolics

• Decreases water solubility & decreases reactivity

• Binds to one of aromatic OH groups

• Therefore water solubility is reduced as phenolic is less soluble

Effect of glycosylation on phenolics

• Increases water solubility & decreases reactivity

• The attachment of a glycosidic group to an aromatic OH group

Effect of carboxylation on phenolics

• Increases water solubility & decreases pKa

• Attaches to aromatic ring

• May lower food pH

Effect of extending conjugated system on phenolics

• Increases reactivity & increases light absorbance

• By attaching (for example) alkynyl groups to the aromatic ring

• Longer = possibly color

Effect of formation of fused ring system on phenolics

• Possible extension of conjugated system

Structure of o-diphenol & relevance

• Highly relevant for oxidation reactions and antioxidant activity

Core structures of monomeric phenolics

Simple phenolics

Hydroxybenzoic acids

Hydroxycinnamic acids

Hydroxycinnamyl alcohols

Relevance of hydroxycinnamic acids

• Hydroxycinnamic acids & derivatives are present in grains and coffee beans. 

Two core structures of monomeric phenolics

Stillbenoids are found in grapes and wines.

Four important classes of di, oligo and polymeric phenolics

• Lignans

• Lignins

• Condensed tannins

• Hydrolysable tannins

Lignans & lignins

• Monomeric building blocks: hydroxycinnamic acids and/or hydroxycinnamyl alcohols

• Many possible linkage types

• Lignan: Has diverse di- or oligomeric structures. + Is metabolized by intestinal bacteria

• Lignin: Part of plant cell walls, difficult to degrade

What are the two types of tannins?

• Oligo- and polymeric phenolic compounds that strongly interact with proteins

• Hydrolysable: hydrolysis releases gallic acid

• Condensed: can be formed by oxidative coupling

flavonoids

Isoflavonoids

What reaction is desirable for many classes of phenolics?

• Glycosylation

• Makes phenolics more water-soluble and more stable

• Glycoslyated phenolics are known as glycosides

• The corresponding structure without glycosylation is known as the aglycon

Cis/Trans in hydroxycinnamic acids

• In nature, the most abundant configuration is trans

• the configuration can change during storage and processing of food or raw material

• The trans and the cis hydroxy cinnamic acid have different properties

Phenolics as antioxidants

• Radicals and metals can initiate undesirable reactions

• Phenolic antioxidants can protect against oxidation

• Flavonoids are often reported to be good antioxidants

Two ways of antioxidant activity in phenolics

1. Reducing oxidized compounds → e.g. by radical scavenging

2. Chelating metals = binding metal ions

Radical scavengers

• Radical scavengers react with “free” radicals to form more stable, less reactive radicals

• More resonance = better stabilized

Desirable flavonoid structural features for radical scavenging

• In general, additional OH groups

• o-diphenol moiety on B-ring

• C3 OH group on C ring

Antioxidant activity by metal chelation

• O-diphenol moieties of phenolics bind metals → bound metals are less reactive

• Certain other moieties can also bind metals

Desirable flavonoid structural features for metal chelation

• o-diphenol moiety on B ring

• C2-C3 double bond

• C3 OH group on C ring

• C4 carbonyl group on C ring

• C5 OH group on A ring

Enzymatic oxidation by polyphenoloxidase

Polyphenoloxidase (PPO) is an oxidative enzyme:

• Phenolics as substrates

• Oxygen as electron acceptor

• Two copper ions in active site

Why does oxidation by PPO not happen when the plant is not damaged?

• Because oxygen is needed for the oxidation reaction, which is not present in the plant cell

• Therefore, when the plant becomes damaged oxidation by PPO can happen as there is access to oxygen.

• PPO activity occurs upon damaging plant tissue during harvesting or processing

• PPO converts phenolics to reactive electron deficient o-quinones (electron deficient structures)

Two ways that quinones are formed by use of PPO

• PPO preferentially converts o-diphenols to o-quinones (= catecholase activity)

• Some PPOs can also convert monophenols to o-diphenols (=cresolase activity)

• The second reaction can lead back to the first reaction.

  • Monophenol → O-diphenol → o-quinone

Factors influencing formation of o-quinones

1. Overall PPO activity varies between sources

2. Characteristics and properties of the specific PPO

3. Structure of the phenolic compound

4. Conditions in the food process, ingredient, or product

Effect of o-quinone reactions on food properties

• O-quinones are very reactive because they are electron deficient

• O-quinones can therefore react with:

  • Other food molecules → leads to changes in flavor, color and appearance

  • Phenolic dimers → phenolic oligomers → phenolic polymers → insoluble brown pigments

    • The phenolic dimers and oligomers are soluble orange brown pigments and have interactions with proteins

Initiation step of browning from o-diphenol

• Nucleophilic addition of a non-oxidized phenolic to the electron-deficient o-quinone

• Forms a covalent carbon-carbon bond → connects conjugated systems

• This forms a dimer

Three reactions to form brown pigment from dimer which was made from o-quinone

• Route A: PPO acts on the dimer to form a dimer ortho-quinone

• Route C: Coupled oxidation, meaning that an oxidized compound is reduced at cost of oxidizing another compound. This too forms dimer ortho-quinone

  • This can be coupled to route A, where a dimer is oxidized to a dimer ortho-quinone

• The dimer o-quinone (no matter how it is formed) is turned into a trimer

• Route B: o-quinone reacts directly with a dimer to form a trimer

The larger = more color and reduced solubility

How to control enzymatic browning

• Eliminate oxygen

• Lower pH (away from optimum for PPO)

• Cool (lowers PPO activity)

• Add chelating agents (bind to copper ions that PPO needs)

The above methods are not permanent (if temp increases, PPO will become active again). To completely inactivate PPO:

• Heat-induced denaturation of PPO

• Add ascrobic acid or other antioxidants

• Add sulphite

• Remove phenolics (using PVPPP followed by precipitation & filtration)

Anthocyanins

• Flavonoids that have an extended conjugaed system → A and B ring connected via the C ring

• pH dependent red-purple-blue color

pH dependent color of anthocyanins

Addition of water to anthocyanins

The two types of protein-phenolic interactions

• Protein-phenolic conjugates

  • Irreversible & covalent

• Protein-phenolic complexes

  • Reversible & non-covalent

Protein-phenolic conjugation

1. Formation of o-quinones from o-diphenols by oxidation

2. Nucleophilic side chains in proteins attack the electron deficient o-quinones → protein bound o-diphenol

3. If this structure becomes oxidized again (→ protein bound o-quinone) it can be attacked by another protein or a protein bound o-diphenol creating a cross link (irreversible)

Protein-phenolic complexation

• Hydrogen bonds - can be formed between OH group and a group in the amino acid side chain/the backbone

• Hydrophobic interactions - take place between aromatic ring and hydrophobic groups/amino acid side chains

  • The main groups that play a role in these interactions are the ring structures of proline residues. And the aromatic side chains of tyrosine and phenylalanine residues.

• Ionic bonds - a type of electrostatic interaction. Occurs mainly between deprotonated OH group and positively charged group in amino acid side chain.

Effect of phenolic/protein ratio

• Low molar ratio: cross linking will likely happen (covalent or non-covalent or both). Causes aggregation and precipitation.

• High molar ratio: proteins coated with hydrohpobic layer of covalently or non-covalently attached phenolics. Will also cause aggregation → precipitation

Preventing protein phenolic interactions

• Removal of phenolic compounds

  • This can be done by reacting the phenolic to a proline rich protein or a protein analog (e.g. PVPP, strongly interacts with phenolics).

  • This will cause precipitation and makes the phenolic easy to remove.

• Hydrolysis of (proline-rich) proteins → enzymatic hydrolysis with proteases. (prevents interactions with phenolics)

• Controlling oxidation of phenolics → prevent formation of reactive o-quinones

Example phenolic protein interaction

• Haze formation in beverages

• Often considered to be undesirable

• Caused by protein-phenolic complexes and conjugates with reduced protein solubility

Effect of addition of EDTA

Reversibly inactivates PPO by binding Cu2+

Effect of addition of ascorbic acid

Dual action: reduces o-quinone back to o-diphenol

Effect of addition of sulphite

Dual action: can bind to o-quinones and to the active site of PPO

Effect of blanching

Irreversibly inactivates PPO by denaturation

Effect of replacing air with nitrogen

Lack of oxygen prevents oxidation

Effect of addition of acetic acid

Reduces activity of PPO by moving away from optimum pH

What affects the color of anthocyanins

A reaction with water

When do phenolics affect the taste of citrus fruit?

• If they are present as glycosides

• More specifically the taste-active forms are the (1→2) rhamnosyl-glucosides

When are condensed tannins most astringent?

At a DP of 5 to 7

Different flavanones and their bitterness

What makes phenolics astrigent?

• Oligomeric phenolic compounds can strongly interact with proteins because they have multiple binding sites

• They can even undergo cross linking

• This will lead to aggregation and precipitation leading to an astrigent taste

Strecker + what is its function

• Strecker degradation yields an amino acid derived aldehyde (aroma) and aminophenol

• Amino acid + o-quinone → imine intermediate → aldehyde + aminophenol

• Desirable: The reaction produces highly odorous Strecker aldehydes (e.g., phenylacetaldehyde, methional) which contribute significantly to the desirable sensory properties and flavor profiles of many foods and beverages, such as chocolate and wine.

• Undesirable: The same reaction can lead to the formation of off-flavors, contribute to the loss of beneficial antioxidants, and form potentially unstable intermediate compounds in certain contexts (like wine oxidation)

Phenolics in tea

• Tea leaves are rich in phenolics (20-30% DM) → mainly catechins

• Most importantly epicatechin gallate and epigallocatechin gallate

• A type of flavanol

The tea production process (green & black tea)

• Black tea is crushed because it exposes the leaves to air → causing oxidation and allowing PPO to work

• This oxidation step is written in the slide as “fermentation”

Follow up reaction of theaflavins

Process of theaflavin & bisthea flaving production

All reactions

Why should you not use “polyphenols”?

The prefix “poly” in the name polyphenol refers to the multiple hydroxyl groups that often occur in the structures of phenolic compounds. However, many common phenolics, such as p-coumaric acid and p-hydroxybenzoic acid, only possess one hydroxyl group on an aromatic ring.

What do flavonoids collectively refer to?

• 2-phenylbenzopyrans

• 3-phenylbenzopyrans

Detection of phenolic compounds by visual observation

• By increasing the pH of e.g. a juice, you can tell if there are anthocyanins.

• If the juice turns from red to purple to blue and eventually yellow it is highly likely that anthocyanins are present.

Detection of phenolics by UV-Vis spectrophotometry

• The absorbance of UV and visible light by compounds in solution can be measured.

• This works very well for phenolics because they always have at least one aromatic ring, which absorbs light in the UV range.

• Larger phenolics = absorb light in the visible light range

• Additionally, there are many colorimetric assays that rely on a reaction that results in a change in the absorbance of light of a specific wavelength.

How to quantify total amount of phenolics

• Folin-Ciocalteu assay

• Which is a colorless mixture of two metals that can oxidize phenolic compounds and in the process the metals become reduced.

• Their reduced form has a bright blue color, that can be visually observed an dmeasured by UV-Vis spectrophotometry.

• Total Phenolic content (TPC), is often expressed as gallic acid equivalents.

How to measure antioxidant activity?

• There are different ways

• Most of which are colorimetric assays that measure radial scavenging activity

• Not very reliable and may be interfered by other compounds present in the sample.

Advanced methods for analysis of phenolics

• Most commonly used: combination of liquid chromatography with detection by UV-Vis spectrophotometry and/or mass spectrometry.

• More accurate and less interfered by other compounds + gives info on structure of phenolic.

• Downside: expensive, takes time, more complicated.