Notes Advanced Molecular Gastronomy
1. Astringency
Astringency – dry, puckering sensation in the mouth, often experienced upon ingestion of tannin-rich foods
Two main types of tannins:
- Hydrolysable tannins – containing ester bonds which can be cleaved with an
alkaline solution
- Condensed tannins – very stable molecules
! C-C bonds extremely strong so won’t be released
Epicatechin and catechin are flavan-3-ol building blocks, able to polymerize:
Different classes of proanthocyanidins:
! Upper units are called extensional (E) units, bottom unit without 4-substitution is called terminal (T) unit
Different ways of connecting catechin building blocks:
- B-type proanthocyanidins – 1 linkage (single bond) between two catechin units
- A-type proanthocyanidins – 2 linkages (double bond) between two catechin units (also A-type when
both single and double bonds are present between different catechin units)
Substituents can be attached to proanthocyanidins:
Page 1 of 43
,Proanthocyanidins that are present in various foods (e.g. barley, beer, grapes, red wine, peanuts, cocoa
beans, cinnamon) differ in their degree of polymerization (DP), bond types (A/B), substitution with gallic
acid (yes/no), and quantity.
Normal-phase and reversed-phase chromatography:
In order to analyse a compound with MS you need to ionize the molecule in order to detect it:
- Negative mode – take off hydrogen (mass in spectrum is -1)
- Positive mode – add hydrogen (mass in spectrum is +1)
MS2 – molecule gets extra energy in mass spectrometer and is fragmented. Fragmentation pattern is
diagnostic for a particular molecule. Can e.g. be used to check whether a molecule is an A-type or B-type
proanthocyanidin
Column type affects the resolution of the mass spectra:
- Analytical column – smaller column particles, higher resolution, sharp peaks
- Preparative column – larger column particles, lower resolution, rounded off peaks
A higher degree of polymerization (DP) leads to a higher molecular diversity and a more complex mass
spectrum due to the variety of ways in which catechin molecules can bind to each other due to the
different bond types.
Relevance of analysing A- and B-type proanthocyanidins: more compact molecules are often less
astringent. A-type proanthocyanidins are more compact than B-type proanthocyanidins. Therefore, the
amount of A-type and B-type proanthocyanidins may give an indication of the astringency of a compound.
By converting B-type into A-type with e.g. the help of enzymes (tannase), astringency could be reduced.
! Note: important to remember that LC-MS can be used to really sequence the structure of
proanthocyanidins and get very detailed information on the location of double bonds or subunits.
Mechanisms of polyphenol-protein complexation:
1. Monodentate complexation:
- Monomeric polyphenol
- High concentration of polyphenols needed due to small size
- Hydrophobic surface caused by ‘polyphenol shell’ drives aggregation
2. Multidentate complexation:
- Multimeric polyphenol
- Low concentration of polyphenols needed due to large size
- Cross-linking drives aggregation
Salivary proline-rich proteins (PRPs) (random-coil: all residues readily accessible for polyphenols to make
interaction) > add multimeric phenols > cross-links created between proteins > aggregates formed >
precipitation of salivary proteins > impaired lubrication in mouth > mouth drying effect
Page 2 of 43
,Two possible ways of protein-polyphenol interaction:
1. Stacking/hydrophobic interaction between aromatic polyphenol rings and pyrrolidine (prolyl) ring of
proline (drives interaction)
2. Hydrogen bonding between B-ring hydroxyl groups and pyrrolidine ring (between OH groups of B-ring
from phenolics and oxygen of amide group of proline) (reinforces interaction)
Current view: bit of both?
Proline is a good binding site for polyphenols:
- Oxygens (carbonyl groups C=O) in tertiary amide of proline are good H-bond acceptors
- Pro-Pro repeats have huge impact on protein structure: exposure of subsequent prolyl rings
CH-π stacking interactions – electropositive hydrogen is craving for electrons and starts to ‘borrow’
electrons from the aromatic ring that is electron-rich (due to electronegative N-atom in ring)
! The driving force behind protein-polyphenol interactions is that proline residues are involved and that it’s
a mixture of CH-π stacking interactions (primary driver of the interactions) and the connection is
reinforced/secured by 2 hydrogen bonds.
Saliva facts:
- 3 major glands with distinct orifices and different composition of secretions.
- Composition of saliva is dynamic (different within time of day and between individuals) contrary to
plasma.
- Lower protein content (3) than plasma (17)
- Rich in random coil proteins (mucins, acidic PRPs, basic PRPs, basic PRG, histatins)
- Rich in proline
Functions of saliva:
1. Protective (tissue coating, lubrication, remineralization of teeth)
2. Host-defence (immunological activity, anti-viral, anti-microbial)
3. Digestion (digestive enzymes, bolus formation, taste)
Classes of salivary proteins with known affinity for tannins:
- Mucins (can be large peptides >600 Da, can be heavily glycosylated, MUC7 most important)
- Proline-rich proteins (PRPs) (between 6-66 Da, most abundant salivary proteins)
- Histatins (relatively small peptides ~1-4 Da, rich in histidine)
3 main classes of PRPs: acidic, basic, glycosylated (basic and glycosylated PRPs are related and have a
higher proline content than the acidic PRPs)
Page 3 of 43
, IB-5:
- C-terminal part of the pro-protein
- Contains 70 amino acids
- Flexible backbone with little secondary structure (random coil)
- Used as model protein for studying salivary protein-polyphenol interactions
Three strategies for the fining of wine:
1. Use proline rich proteins (e.g. gelatins) to remove polyphenols from the wine and therefore reduce
astringency
2. Use tannase to remove gallic acid units
3. Convert B-type to A-type proanthocyanidins (with e.g. laccase)
Tannins in wine:
1. Condensed tannins (proanthocyanidins)
2. Hydrolysable tannins (gallotannins) – bijv. pentagalloyl glucose (glucose + 5 gallic acids
bound via ester bonds)
3. Hydrolysable tannins (ellagitannins) – bijv. castalagin and grandinin (glucose + number of
gallic acid residues that are also bound to each other via C-C bonds)
Protein precipitation by tannins: add proteins and tannins to Eppendorf > incubate
> centrifuge (spin down aggregates) > protein analysis for the supernatant > plot x-
axis polyphenol concentration and y-axis amount of protein precipitated:
- Slope – measure for the affinity of protein to the polyphenol (high affinity
when curve shifts to the left/when the curve is steep)
- Plateau value – all protein added has precipitated and is thus bound to the
polyphenol
! Castalagin and grandinin are relatively rigid molecules, possibly explaining the lower affinity for proteins
in comparison with the more flexible procyanidin and pentagalloyl glucose
Summary: Criteria proteins for protein-polyphenol interactions:
- Presence of proline
- Preferably proline repeats
- Flexibility of protein – random coil (unstructured protein) promotes binding
Summary: Criteria polyphenols for protein-polyphenol interactions:
- Certain length of the tannin
- Presence of gallic acid residues – function as adaptable anchors (when not bound via C-C)
- Flexibility – rotational freedom (flexibility) of aromatic rings promotes binding to proteins
- Conformation – some molecules have a more compact or more extended conformation. A more
extended conformation leads to higher binding affinity due to better exposure of B-rings (A-type more
compact, B-type more extended)
Protein-polyphenol interactions are not the whole story:
- Most astringent compound with lowest taste threshold (grandinin) is not the most efficient protein-
precipitating agent
- Some compounds with high affinity for salivary proteins show relatively low astringency scores
- There are also molecules perceived as very astringent but do not show binding to proteins at all
- The glycosylation pattern to a very large extent determines astringency of the compounds
- Probably combination of protein-polyphenol interactions and astringency receptor-mediated
interactions
Page 4 of 43
1. Astringency
Astringency – dry, puckering sensation in the mouth, often experienced upon ingestion of tannin-rich foods
Two main types of tannins:
- Hydrolysable tannins – containing ester bonds which can be cleaved with an
alkaline solution
- Condensed tannins – very stable molecules
! C-C bonds extremely strong so won’t be released
Epicatechin and catechin are flavan-3-ol building blocks, able to polymerize:
Different classes of proanthocyanidins:
! Upper units are called extensional (E) units, bottom unit without 4-substitution is called terminal (T) unit
Different ways of connecting catechin building blocks:
- B-type proanthocyanidins – 1 linkage (single bond) between two catechin units
- A-type proanthocyanidins – 2 linkages (double bond) between two catechin units (also A-type when
both single and double bonds are present between different catechin units)
Substituents can be attached to proanthocyanidins:
Page 1 of 43
,Proanthocyanidins that are present in various foods (e.g. barley, beer, grapes, red wine, peanuts, cocoa
beans, cinnamon) differ in their degree of polymerization (DP), bond types (A/B), substitution with gallic
acid (yes/no), and quantity.
Normal-phase and reversed-phase chromatography:
In order to analyse a compound with MS you need to ionize the molecule in order to detect it:
- Negative mode – take off hydrogen (mass in spectrum is -1)
- Positive mode – add hydrogen (mass in spectrum is +1)
MS2 – molecule gets extra energy in mass spectrometer and is fragmented. Fragmentation pattern is
diagnostic for a particular molecule. Can e.g. be used to check whether a molecule is an A-type or B-type
proanthocyanidin
Column type affects the resolution of the mass spectra:
- Analytical column – smaller column particles, higher resolution, sharp peaks
- Preparative column – larger column particles, lower resolution, rounded off peaks
A higher degree of polymerization (DP) leads to a higher molecular diversity and a more complex mass
spectrum due to the variety of ways in which catechin molecules can bind to each other due to the
different bond types.
Relevance of analysing A- and B-type proanthocyanidins: more compact molecules are often less
astringent. A-type proanthocyanidins are more compact than B-type proanthocyanidins. Therefore, the
amount of A-type and B-type proanthocyanidins may give an indication of the astringency of a compound.
By converting B-type into A-type with e.g. the help of enzymes (tannase), astringency could be reduced.
! Note: important to remember that LC-MS can be used to really sequence the structure of
proanthocyanidins and get very detailed information on the location of double bonds or subunits.
Mechanisms of polyphenol-protein complexation:
1. Monodentate complexation:
- Monomeric polyphenol
- High concentration of polyphenols needed due to small size
- Hydrophobic surface caused by ‘polyphenol shell’ drives aggregation
2. Multidentate complexation:
- Multimeric polyphenol
- Low concentration of polyphenols needed due to large size
- Cross-linking drives aggregation
Salivary proline-rich proteins (PRPs) (random-coil: all residues readily accessible for polyphenols to make
interaction) > add multimeric phenols > cross-links created between proteins > aggregates formed >
precipitation of salivary proteins > impaired lubrication in mouth > mouth drying effect
Page 2 of 43
,Two possible ways of protein-polyphenol interaction:
1. Stacking/hydrophobic interaction between aromatic polyphenol rings and pyrrolidine (prolyl) ring of
proline (drives interaction)
2. Hydrogen bonding between B-ring hydroxyl groups and pyrrolidine ring (between OH groups of B-ring
from phenolics and oxygen of amide group of proline) (reinforces interaction)
Current view: bit of both?
Proline is a good binding site for polyphenols:
- Oxygens (carbonyl groups C=O) in tertiary amide of proline are good H-bond acceptors
- Pro-Pro repeats have huge impact on protein structure: exposure of subsequent prolyl rings
CH-π stacking interactions – electropositive hydrogen is craving for electrons and starts to ‘borrow’
electrons from the aromatic ring that is electron-rich (due to electronegative N-atom in ring)
! The driving force behind protein-polyphenol interactions is that proline residues are involved and that it’s
a mixture of CH-π stacking interactions (primary driver of the interactions) and the connection is
reinforced/secured by 2 hydrogen bonds.
Saliva facts:
- 3 major glands with distinct orifices and different composition of secretions.
- Composition of saliva is dynamic (different within time of day and between individuals) contrary to
plasma.
- Lower protein content (3) than plasma (17)
- Rich in random coil proteins (mucins, acidic PRPs, basic PRPs, basic PRG, histatins)
- Rich in proline
Functions of saliva:
1. Protective (tissue coating, lubrication, remineralization of teeth)
2. Host-defence (immunological activity, anti-viral, anti-microbial)
3. Digestion (digestive enzymes, bolus formation, taste)
Classes of salivary proteins with known affinity for tannins:
- Mucins (can be large peptides >600 Da, can be heavily glycosylated, MUC7 most important)
- Proline-rich proteins (PRPs) (between 6-66 Da, most abundant salivary proteins)
- Histatins (relatively small peptides ~1-4 Da, rich in histidine)
3 main classes of PRPs: acidic, basic, glycosylated (basic and glycosylated PRPs are related and have a
higher proline content than the acidic PRPs)
Page 3 of 43
, IB-5:
- C-terminal part of the pro-protein
- Contains 70 amino acids
- Flexible backbone with little secondary structure (random coil)
- Used as model protein for studying salivary protein-polyphenol interactions
Three strategies for the fining of wine:
1. Use proline rich proteins (e.g. gelatins) to remove polyphenols from the wine and therefore reduce
astringency
2. Use tannase to remove gallic acid units
3. Convert B-type to A-type proanthocyanidins (with e.g. laccase)
Tannins in wine:
1. Condensed tannins (proanthocyanidins)
2. Hydrolysable tannins (gallotannins) – bijv. pentagalloyl glucose (glucose + 5 gallic acids
bound via ester bonds)
3. Hydrolysable tannins (ellagitannins) – bijv. castalagin and grandinin (glucose + number of
gallic acid residues that are also bound to each other via C-C bonds)
Protein precipitation by tannins: add proteins and tannins to Eppendorf > incubate
> centrifuge (spin down aggregates) > protein analysis for the supernatant > plot x-
axis polyphenol concentration and y-axis amount of protein precipitated:
- Slope – measure for the affinity of protein to the polyphenol (high affinity
when curve shifts to the left/when the curve is steep)
- Plateau value – all protein added has precipitated and is thus bound to the
polyphenol
! Castalagin and grandinin are relatively rigid molecules, possibly explaining the lower affinity for proteins
in comparison with the more flexible procyanidin and pentagalloyl glucose
Summary: Criteria proteins for protein-polyphenol interactions:
- Presence of proline
- Preferably proline repeats
- Flexibility of protein – random coil (unstructured protein) promotes binding
Summary: Criteria polyphenols for protein-polyphenol interactions:
- Certain length of the tannin
- Presence of gallic acid residues – function as adaptable anchors (when not bound via C-C)
- Flexibility – rotational freedom (flexibility) of aromatic rings promotes binding to proteins
- Conformation – some molecules have a more compact or more extended conformation. A more
extended conformation leads to higher binding affinity due to better exposure of B-rings (A-type more
compact, B-type more extended)
Protein-polyphenol interactions are not the whole story:
- Most astringent compound with lowest taste threshold (grandinin) is not the most efficient protein-
precipitating agent
- Some compounds with high affinity for salivary proteins show relatively low astringency scores
- There are also molecules perceived as very astringent but do not show binding to proteins at all
- The glycosylation pattern to a very large extent determines astringency of the compounds
- Probably combination of protein-polyphenol interactions and astringency receptor-mediated
interactions
Page 4 of 43
