Notes Advanced Molecular Gastronomy (FPH31306). The notes are quite extensive but contain all information necessary to pass the course. They also contain parts of the lecture slides for better understanding of the course material.
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:
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,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
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,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
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
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