Two molecules newly identified by Mass Spectrometry in fermented cocoa beans have a strong impact on chocolate sensory quality.

Two molecules newly identified by Mass Spectrometry

in fermented cocoa beans have a strong impact on

chocolate sensory quality.

Noémie Fayeulle a, Anna Vallverdú-Queralt a, Jean-Claude Boulet a, Jean-Michel Roger b, Clotilde Hue c, Renaud Boulanger d, Véronique Cheynier a, Nicolas Sommerer a

a Plateforme Polyphénols, UMR SPO, INRA, Montpellier Supagro, Université de Montpellier, 34060 Montpellier, France b UMR ITAP, IRSTEA, 361 rue J.F. Breton, BP 5095, 34196 Montpellier, France

c Valrhona SA, 8 Quai du Général de Gaulle, 26600 Tain l’Hermitage, France

Polyphenols & Fermentation process

Polyphenols in plants

• Secondary metabolites produced by plants

• Wide family of molecules

• Defence, protection, colour

• Polyphenols in cocoa

• Mostly flavan-3-ols

• Highly reactive molecules

• Bitterness and astringency in raw cocoa beans

O OH OH OH O H OH O OH OH OH O H OH O OH OH OH O H OH pH and T°C variations + metabolites Epicatechin Procyanidin dimer Wide range of potentially new molecules

Impact on chocolate taste

Standardized

process

Applied to the corresponding cocoas 12 fermented cocoas 4 sensory groups 1 2 3 4 Mass spectrometry

• widely used for polyphenols detection • Fast and accurate

• Detection, identification and quantification after extraction

Focus on polyphenol content : Polyphenols extraction from the beans

Use of the polyphenols relative quantity in fermented cocoas beans to evaluate the sensory poles

(molecules separation)

Low resolution mass spectrometry

(mass signal (m/z) and relative quantity)

Problematic

link between cocoa molecules and chocolate taste

• Mainly flavan-3-ols

• Among them two signals with significant impact

Purpose of this work :

identify these two mass signals

Statistical treatment of the polyphenols relative quantity :

Which molecules allow

the grouping of the

sensory poles ?

Pole 1

Pole 2

Pole 3

Pole 4

List of 32 mass signals

(=type of molecules)

Reported in cocoa not identified

Hypothesis: ethyl bridged flavanols

• Ethyl Bridged flavanols ?

O OH OH OH O H OH O OH OH OH O H OH C H₃ Ethyl Bridge Molecular Weight : 606.5734 g.mol O OH OH OH O H OH O OH OH OH O H O H C H₃ O OH OH OH O H OH Molecular Weight : 894.8256 g.mol O OH OH OH O H OH O OH OH OH O H OH O OH OH OH O H OH O OH OH OH O H OH O OH OH OH O H OH Epicatechin Epicatechin Epicatechin Procyanidin dimer

Ionized Molecular Weight : 605.1659 g.mol

Ionized Molecular Weight : 893.2292 g.mol

Low pH Low pH

Acetaldehyde + H+ Acetaldehyde + H+ O

How to confirm this hypothesis ?

Structural information Compare with a _reference

Determine the number of molecules in the

samples

High resolution mass

spectrometry

Low resolution mass spectrometry Model solution Chromatographic separation Chromatographic separation Polyphenol extract from cocoa beans

Polyphenol extract from cocoa beans

epicatechine + dimers + acetaldehyde at low pH

Structural information

Exact mass signal & chemical formula

Theoretical Ionized mass 605.1659 893.2292

Experimental Ionized mass 605.1641 893.2282

Mass defect (mDA) 1,8 1

O OH OH OH O H OH O OH OH OH O H OH C H₃ O OH OH OH O H OH O OH OH OH O H O H C H3 O OH OH OH O H OH

Theoretical chemical formula C₃₂H₂₉O₁₂ C₄₇H₄₁O₁₈ Experimental chemical formula C₃₂H₂₉O₁₂ C₄₇H₄₁O₁₈

High resolution mass signals

Structural information :

fragmentation scheme

The fragmentation pattern is specific from ethyl bridged flavanols

The structure is confirmed for both signals at m/z = 605 and m/z = 893

O OH OH OH O H OH O OH OH OH O H OH C H₃ O OH OH OH O H OH O OH OH OH O H OH C H₂ O CH₂ O H OH O OH OH OH O H OH C H₃

1120-F-SC204-3_10066.d: EIC 605.00 -All MS

1121-NFA179-solmodepiB2-T48h_10067.d: EIC 605.00 -All MS 0.0 0.2 0.4 0.6 0.8 1.0 6 x10 Intens. 0.00 0.25 0.50 0.75 1.00 1.25 8 x10 Intens. 16 18 20 22 24 26 Time [min] 1120-F-SC204-3_10066.d: EIC 893.00 -All MS

1121-NFA179-solmodepiB2-T48h_10067.d: EIC 893.00 -All MS 0.0 0.5 1.0 1.5 2.0 2.5 5 x10 Intens. 0 2 4 6 7 x10 Intens. 10 12 14 16 18 20 22 Time [min]

4 isomers

Cocoa extract Cocoa

extract

Model solution _Model

solution m/z = 605 _{m/z = 893} O OH OH OH O H OH O OH OH OH O H OH O OH OH OH O H OH C H3

5 isomers

Time (min) Time (min)

Intensity (UA) Intensity (UA)

Low resolution mass spectrometry

Conclusion

After statistical analysis, two unknown mass signals have been highlighted

These molecules discriminate the chocolate sensory poles

Mass spectrometry allowed to identify these two mass signals as

ethyl bridged flavanols

O OH OH OH O H OH O OH OH OH O H OH C H₃ O OH OH OH O H OH O OH OH OH O H O H C H3 O OH OH OH O H OH

Thanks for your attention

Special thanks to