Formation of selected compounds in model systems and food Tomas Davidek, Ondrej Novotny, IMRE BLANK

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1 9th Wartburg Symposium on Flavor hemistry & Biology April 13 16, 2010, Eisenach, Germany Formation of selected compounds in model systems and food Tomas Davidek, ndrej Novotny, IRE BLANK

2 Formation of diketones & cyclic enolones through the aillard reaction cascade bjective: Elucidate the formation mechanisms of diketones and cyclic enolones in aillard reaction systems based on glucose and amino acids as affected by ph (5 / 7), moisture (dry / wet), and amino acid (Gly / Pro) Examine the relative importance of different formation pathways by using equimolar mixtures of unlabelled and fully 13 -labelled glucose (ALA approach) Extend the model study to food systems using extrusion conditions (135, 20 min) Poster no. 45 (Davidek et al.) 2

3 arbohydrate module labelling (ALA): Elucidation of formation mechanisms Examine the relative importance of different formation pathways by using defined mixtures of unlabelled and fully 13 -labelled sugar + [ 12 6 ]-Glucose [ 13 6 ]-Glucose Recombination... x. x + 3 x : 2 : 1 x x : 1 Fragment labelling technique (Hofmann and Schieberle, 2001) arbon module labelling (Frank and Hofmann, 2002) arbohydrate module labelling (Schieberle et al., 2003) 3

4 ALA to elucidate the formation of furaneol from glucose/proline -heating (180, 10 min) H Aqueous solution (145, 20 min) 1 1 1: Furaneol formed via acetylformoine exclusively from intact carbon skeleton of the hexose sugar Furaneol formed via acetylformoine from - intact hexose sugar (40 %) and - from 3 -sugar fragments (60 %) (Hofmann and Schieberle, 2001) 4

5 ALA study on formation of selected compounds from glucose 2,3-Butanedione 2,3-Pentanedione yclotene H H H H H H 3 4-Hydroxy-2,5-dimethyl- 3(2H)-furanone (Furaneol ) Dihydromaltol 2,3-Dihydro-3,5-dihydroxy- 6-methyl-4H-pyran-4-one echanism of formation: - from intact sugar skeleton - recombination of sugar fragments - recombination of sugar fragments and formaldehyde (generated from glycine) Impact of: Water content, ph, amino acid co-reactant 5

6 Experimental setup 0.15 mmol [ 12 6 ]-glucose monohydrate 0.15 mmol [ 13 6 ]-glucose 0.1 mmol glycine or proline Aqueous buffer 1 ml 0.5 phosphate buffer ph 5 or 7 heating conditions mmol NaH 2 P 4 or Na 3 P 4 ixing in 20ml headspace vial Heating 135 /20min ooling in ice bath Dissolution of residue in 1 ml water (low moisture system only) Addition of 2g anhydr. Na 2 S 4 Analysis by SPE-GxG/TFS 6

7 Appearance of mixtures Glc/Gly with Na 3 P 4 Buffer ph 5 Buffer ph 7 Before heating After heating Aqueous buffer System ph Before heating Glc/Gly, buffer ph Glc/Gly, buffer ph Glc/Pro, buffer ph Glc/Pro, buffer ph After heating heating conditions ph System Before heating After heating Glc/Gly, NaH 2 P Glc/Gly, Na 3 P Glc/Pro, NaH 2 P Glc/Pro, Na 3 P

8 Glucose + Proline 2,3-Butanedione Acidic Basic WET 31% 2,3-butanedione % % Intact skeleton % +1 3% +2 21% +3 26% +4 27% 2,3-butanedione No Intact skeleton 19% % ,3-butanedione 2,3-butanedione 44% % Intact skeleton 44% Intact skeleton 39% 1 5% 12% 34% 39% 9% 6% 11% 35%

9 Formation of 2,3-Butanedione From fragments ( ): H 2 H H H,H H H Glucose H H H H β-dicarbonyl cleavage H H Hydroxy- 2-propanone Formaldehyde H H - H 2 2,3-Butanedione H 2 N Glycine Strecker degradation (Schieberle et al., 2003) From intact skeleton: H H H H H H H - H 2 H - H H 2,3-Butanedione (Weenen, 1998) 9

10 Glucose + Glycine 2,3-Butanedione Acidic 2,3-butanedione % 3Glc + 1 (7 Gly / 16% Glc) Basic 2,3-butanedione 82% 3Glc + 1 (52% Glc / 3 Gly) WET 47% 4% 3% 39% 7% <5% Intact skeleton <5% Intact skeleton 36% 13% 5% 28% 18% ,3-butanedione 46% 3Glc + 1 (26% Gly / Glc) 2,3-butanedione 62% 3Glc + 1 (48% Glc / 14% Gly) 51% 33% Intact skeleton 5% 3% 18% 23% 41% <1 Intact skeleton 12% 8% 19% Glc/Pro (2,3-B)

11 Extrusion experiment: Glucose + Gly 2,3-Butanedione aliper - sample "2D Glc/Glc*/Gly 135/20 split1: V:1" % 2,3-Butanedione easured distribution 3% 3% % +3 12% +4 45% 3 15% 45% 3 15% 1.5% 3% 1.5% % 3% 3% 3% Recombination Glc- 2 + Glc % Recombination Glc- 3 + Glc- 1 12% % 3 15% 45% 3 15% 7.5% 7.5% 3 3 Intact Glc skeleton Glc- 4 15% Recombination Glc- 3 + Gly ontribution of different formation pathways to 2,3-butanedione: Recombination of 3 (sugar) and 1 (mainly from Gly) fragments (~7) lose to mainly Wet/Acidic and /Acidic model systems 11

12 Formation of Furaneol H 2 H H H,H H H H H H - H 2 H H H Acetylformoin H N H H N, H 2, 2 roasting: solution: H HDF H H H H H - H 2 H H H H N H H N, H 2, 2 roasting: solution: H HDF (Schieberle et al., 2003; Schieberle, 2005) 12

13 Glucose + Proline Furaneol Acidic HDF Basic HDF WET 48% 47% HDF 48% 48% Intact skeleton Intact skeleton % 49% 2% 4% ~70 % Fragmentation (48% 3Glc+ 3Glc) % HDF % 4% % Intact skeleton % +6 49% +6 13

14 Glucose + Glycine Furaneol Acidic Basic HDF HDF WET 47% 46% Intact skeleton % 1% 2% ~65% Intact skeleton % % 5% % +6 HDF HDF 47% 47% Intact skeleton % 1% 3% 6% 2% 2% ~70 % Intact skeleton % +6 14

15 Extrusion experiment: Glucose + Gly Furaneol aliper - sample "2D Glc/Glc*/Gly 135/20 split1: V:1" H relative Intensity (% ) 57% HDF Intact skeleton % +6 H H H - H H 2 H 1-Deoxy-2,3-hexodiulose H - H 2 H 2,3-Dihydro-3,5-dihydroxy- 6-methyl-4H-pyran-4-one H H H [H] H H H [H] H H - H 2 Acetylformoin H H - H 2 H H Furaneol (HDF) H [H] H H H - H 2 H 3 yclotene yclic enolones mainly formed from intact glucose skeleton 15

16 Formation of Furaneol H 2 H H H,H H H H H H - H 2 H H H Acetylformoin H N H H N, H 2, 2 roasting: solution: 37% H HDF H H H H H - H 2 H H H H N H H N, H 2, 2 roasting: solution: 63% H HDF (Schieberle et al., 2003; Schieberle, 2005) odel systems: Furaneol is mainly formed from intact glucose skeleton (incl. Wet/Acidic) Except in Wet/Basic (Pro) where fragmentation is dominating (~7) Food system (extrusion): Furaneol is almost exclusively formed from the intact glucose skeleton 16

17 onclusions Flavour formation mechanisms via aillard-type reactions depend very much on the reaction conditions Aqueous systems versus dry-heating conditions (molecular mobility) ph (degree of sugar fragmentation / recombination) Results from model experiments cannot be extrapolated to food The ALA approach opens new avenues to examine the role of different formation pathways Relative effectiveness of reaction pathways under different reaction conditions (e.g. dry-heating versus aqueous solutions) Possibility to extend approach to other precursors and intermediates Specific labelling experiments in case of small signals (<5%) Role of amino acids In the presence of Glycine, the 1 moiety is preferably delivered by Gly In the presence of Pro, more sugar fragmentation observed Need to study more amino acids (source of fragments vs catalysis) 17

18 Back-up slides 18

19 Semiquantitative data 2.8E+07 HDF Glucose / Glycine Glucose / Proline H Peak area 2.1E E E+06 H 3 Higher turnover with Pro 0.0E+00 Wet ph 5 Wet ph 7 acidic basic Wet ph 5 Wet ph 7 acidic basic 2,3-butanedione 8.0E+07 Glucose / Glycine Glucose / Proline 6.0E+07 Peak area 4.0E E+07 Glc/Gly: Source of 1 is Gly Glc/Pro: Source of 1 is Glc 0.0E+00 Wet ph 5 Wet ph 7 acidic basic Wet ph 5 Wet ph 7 acidic basic 19