Master of Science in Wine and Beer Science of T.E.I. of Athens (Athens University of Applied Science) 14 May 2018

2. www.fmach.it

3. FEM metabolomic platform: instruments
Targeted analysis
Untargeted metabolomics
Isolation
FEM metabolomic platform: methods
www.fmach.it

4. Η Αλληγορία του Σπηλαίου

5. Metabolomics: definitions
What can the cell potentially do?
What is currently being turned on?
What enzymes are currently active?
What is being produced/consumed?
System biology

6. Metabolomics: definitions
metabolomics is the "systematic study of the unique chemical fingerprints that
specific cellular processes leave behind", the study of their small-molecule
metabolite profiles (Daviss 2005)
metabolomics is a newly emerging field of "omics" research concerned with the
comprehensive characterization of the small molecule metabolites in biological
systems. (Metabolomics Society)
metabolomics is the comprehensive and holistic study of the metabolome
the complete set of small-molecule
metabolites to be found within a biological
sample
metabonome: the complete set of metabologically
regulated elements in cells

7. From targeted to untargeted

8. Targeted versus Untargeted

9. Targeted versus Untargeted

10. Targeted versus Untargeted

11. Metabolomics: metabolite
What is a metabolite?
Product and intermediate of the metabolism – small organic molecules

12. Wine Chemistry – Wine Composition
How big is the wine metabolome?

13. Metabolomic: what is and what is not
How big is the metabolome?
Sigma-Aldrich has ~55K commercial available chemicals
Kegg contains 18K metabolites
HMDB is based in ~42K metabolites
Plant metabolome is estimated to cover 200K metabolites
PubChem ID contains more than 10M entries
CAS contains over 90M unique organic
and inorganic chemicals
ChemSpider contains over 57M
compounds from 530 data sources

14. Plant metabolome is estimated to cover
200 000 metabolites
#ofmetabolites5-21% ethanol
g/L
mg/L
µg/L
ng/L
pg/L
fg/L
How big is the metabolome?
Wine Chemistry – Wine Composition

15. #ofmetabolites5-21% ethanol
g/L
mg/L
µg/L
ng/L
pg/L
fg/L
Plant metabolome is estimated to cover
200 000 metabolites
How big is the metabolome?
Wine Chemistry – Wine Composition

16. Wine Chemistry – Wine Composition
#ofmetabolites
g/L
mg/L
µg/L
ng/L
pg/L
fg/L
95-79% water
5-21% ethanol
0-100 g/L sugars (glucose/fructose)
1-6 g/L tartaric acid
0-4 g/L malic acid
0-4 g/L lactic acid
0.5-1.5 g/L succinic acid
0.2-1.5 g/L acetic acid
4-15 g/L glycerol
0.1-10 g/L CO2
0.02-1.3 g/L argine
glutamine, alanine, glutamic acid, proline, glycine, histidine
arabinose, xylose, rhamnose
Fe, Cu, B, Mn
methanol, acetaldehyde,
SO2
proteins
thiamine, riboflavin, niacin, biotin, pantothenate and vitamin C
phenolics
polysaccharide

17. Wine Chemistry – Wine Composition
Waterhouse, Understanding wine chemistry, 2016

18. Lisec et al. Anal Chem 2016
Currently, the best dynamic range of modern MS is 106
approximately and is significantly lower than the estimated
concentration range of cellular metabolites as 1012 or more (Lei
et al JBC 2011)
Metabolomics: what is and what is not

19. Grape/Wine Chemistry

20. Wine Chemistry – Grape
Koutroumanidis et al

21. Grape Sensorial analysis: grape
What is the odor of a grape berry?

22. Grape Sensorial analysis: grape
Aromatic grape
Moscato, Gewϋrztraminer
Wine smell as the grape
Non-aromatic grape
a) silenced and b) not-silenced
Sauvignon blanc
Must non-aromatic, wine aromatic

23. Grape Sensorial analysis: grape
aromatic part aromatic part
sugar
O
OOH
OH
OH
OH
OH
enzymatic
or chemical
hydrolysis

24. Grape aromatic compounds: terpenoids
Isoprene
Hydrocarbon
5 x Carbons
2 x Isoprene
Monoterpenes
10 x Carbons
3 x Isoprene
Sesquiterpene
15 x Carbons
4 x Isoprene
Diterpenes
20 x Carbons
5 x Isoprene
Sesteterpenes
25 x Carbons
6 x Isoprene
Triterpenes
25 x Carbons
Terpene + Oxygen or Nitrogen = terpenoids

25. OH
α-terpineol
linalool
OH
geraniol
OH
OH
citronellol
myrcene
limonene
O
eucaliptolo
α-pinene
Grape aromatic compounds: terpenoids

26. Grape aromatic compounds: volatiles thiols
O
SH
OH
SH
O
SHO
4-mercapto-4-methylpentan-2-one
4MMP
3-mercaptohexan-1-ol
3MH
3-mercaptohexyl-acetate
3MHA

27. Grape aromatic compounds: volatiles thiols

28. OH
S
O
NH2
OH
Grape aromatic compounds: volatiles thiols
aromatic part
cysteine

29. β-carotene
O
O
O
O
O
Grape aromatic compounds: norisoprenoids

30. O
Grape aromatic compounds: norisoprenoids
megastigmane non-megastigmane
TDNβ-damascenone vitispirane
O
β-ionone

31. Grape aromatic compounds: norisoprenoids
Time
Quantity
carotenoids
more sun
Veraison Maturity Over maturity

32. Grape aromatic compounds: methoxypyrazine
N
N
R

33. Grape Sensorial analysis: grape

34. Grape Sensorial analysis: grape
Cultivar Free
terpenoids
(µg/L)
Bound
terpenoids
(µg/L)
Muscat Ottonel 1679 2873
Muscat Alessandria 1513 4040
Muscat Hamburg 594 1047
Gewürztraminer 282 4323
Riesling 73 262
Chardonnay 41 12
Syrah 13 65
Sauvignon 5 108
Cabernet Sauvignon - 13
Rapp et al 1984

35. Grape Sensorial analysis: grape
Lanaridis et al 2002
Muscat Blanc à Petits Grains Muscat d’Alexandrie

36. Grape Sensorial analysis: must
Aromatic grape
Moscato, Gewϋrztraminer
continuity

37. Grape Sensorial analysis: must
O
1-hexanol
OH OH OH
OO
Non-aromatic grape
(but aromatic in taste)
1-hexanal
3-trans-hexan-1-ol
3-trans-hexan-1-al
3-cis-hexan-1-ol
3-cis-hexan-1-al

38. Grape Sensorial analysis: must
O
1-hexanol
OH OH OH
OO
1-hexanal
3-trans-hexan-1-ol
3-trans-hexan-1-al
3-cis-hexan-1-ol
3-cis-hexan-1-al
O
OH
O
OH
linoleic acid
linolenic acid

39. Grape Sensorial analysis: must
Cabernet Sauvignon, Sauvignon blanc,
Cabernet Franc, Merlot
N
N
R
• Amplification of herd odor
• Not mature grapes

40. Grape Sensorial analysis: must
White grapes: Honey, dry fruits, chamomile
Red grapes: comfiture of red berries

41. Grape Sensorial analysis: alcoholic fermentation
OH
O
O
O OH
OH
OH
OH
OH
O
O
OH
O
glucose
pyruvic acid acetaldehyde ethanol

42. O
NH2
OH
O
NH2
R
OH
O
O
R
OH
O
R
OH
R
O
NH2
OH
O
NH2
NH
OH
O
NH2
OH
O
NH2
OH
Must sensorial analysis: alcoholic fermentation / Ehrlich reaction
tryptophanphenylalanine leucine isoleucinealanine

43. O
NH2
OH
O
NH2
R
OH
OH
R
O
NH2
OH
O
NH2
NH
OH
O
NH2
OH
O
NH2
OH
Must sensorial analysis: alcoholic fermentation / Ehrlich reaction
tryptophanphenylalanine leucine isoleucinealanine

44. OH
O
NH2
OH
O
NH2
OH
O
NH2
NH
OH
OH
NH
O
NH2
OH
O
NH2
OH
OH
OH
Must sensorial analysis: alcoholic fermentation / Ehrlich reaction
tryptophanphenylalanine leucine isoleucinealanine
OH
phenyl-ethanol ethanol 3-methyl-butanol tryptophol2-methyl-butanol
4-197 mg/L 6-490 mg/L 2-150 mg/L 2-30 mg/L

45. OH
O
NH2
OH
O
NH2
OH
O
NH2
NH
OH
OH
NH
O
NH2
OH
O
NH2
OH
OH
OH
Must sensorial analysis: alcoholic fermentation / Ehrlich reaction
tryptophanphenylalanine leucine isoleucinealanine
OH
phenyl-ethanol ethanol 3-methyl-butanol tryptophol2-methyl-butanol
amino acid in must ft
Cultivar
Maturity
Viticulture practices
Turbidity

46. OH
OH
OH
Must sensorial analysis: alcoholic fermentation / Ehrlich reaction
phenyl-ethanol 3-methyl-butanol 2-methyl-butanol
volatiles
threshold
300 mg/L
Adolf and Versini, 1999

47. Must sensorial analysis: alcoholic fermentation
OH
O
O
O OH
OH
OH
OH
OH
O
O
OH
O
glucose
pyruvic acid acetaldehyde ethanol
O
OH
acetic acid

48. Must sensorial analysis: alcoholic fermentation
O
OH
acetic acid
OH
O
OH
O
OH
O
OH
O
OH
O
2-methylpropanoic acid
3-methylbutanoic
octanoic acid acid
(caprilic acid)
Hexanoic acid
(capronic acid)
butanoic acid
(butyric acid)
OH
Odecanoic acid acid
(caprinic acid)

49. Must sensorial analysis: alcoholic fermentation / half time
OH
O
OH
O
OH
O
OH
O
3-methylbutanoic
octanoic acid acid
(caprilic acid)
Hexanoic acid
(capronic acid)
butanoic acid
(butyric acid)
O
O
O
O
O
O
O
O
isoamyl acetate
ethyl butanoate
ethyl hexanoate
ethyl octanoate

50. Wine sensorial analysis: the first battle
O
O
O
O
O
O
ethyl butanoate
ethyl hexanoate
ethyl octanoate
O
O
isoamyl acetate
OH
OH
OH
phenyl-ethanol
3-methyl-butanol
2-methyl-butanol
superior alcohol versus esters

51. Wine sensorial analysis: the first battle
O
O
O
O
O
O
ethyl butanoate
ethyl hexanoate
ethyl octanoate
O
O
isoamyl acetate
OH
OH
OH
phenyl-ethanol
3-methyl-butanol
2-methyl-butanol
superior alcohol versus esters

52. Wine sensorial analysis: the first battle
O
O
O
O
O
O
ethyl butanoate
ethyl hexanoate
ethyl octanoate
O
O
isoamyl acetate
OH
OH
OH
phenyl-ethanol
3-methyl-butanol
2-methyl-butanol
superior alcohol versus esters

53. O
OHOH
OH
OO
Wine sensorial analysis: the layers
O
OH
OH
O
OH
O
OH
O
OH
O OH
O
OH
OH
OH
O
O
O
O
O
O
O
O
Moio, il respiro del vino, 2016

54. Wine sensorial analysis: the layers
O O
OH
OH
OH
O
SH
OH
SH
O
SHO
N
N
R
Moio, il respiro del vino, 2016

55. Wine sensorial analysis: aging
Time

56. Wine sensorial analysis: aging
5 oC
10 oC
20 oC
30 oC
Time

57. Wine sensorial analysis: aging
isoamyl acetate
Time
ethyl esters
O
O
ethyl butanoate
O
O
isoamyl acetate

58. Wine sensorial analysis: aging
Time
ethyl esters
Alcohols superior
Phase I Phase II Phase III

59. Wine sensorial analysis: aging
Time
esters
Alcohols superior
Phase II

60. Wine sensorial analysis: aging
Time
esters
Alcohols superior
Phase II

61. Wine sensorial analysis: barrel aging
Time
Fermentation
barrique
aromatic or non-aromatic silenced cultivar

62. Wine sensorial analysis: barrel aging
Time
Fermentation
barrique
non-aromatic cultivar

63. Wine aroma perception

64. Grape Sensorial analysis: human perception
Choi and Han, book: How flavor Works (2012)

65. Grape Sensorial analysis: human perception
Primary gustatory cortex
Working memory and
behavioral strategies
Primary gustatory cortex and emotion Primary gustatory/olfactory cortex and memory

66. Grape Sensorial analysis: human perception

67. Wine Chemistry – Phenolics

68. Wine Chemistry – Phenolics – Classification according the skeleton
C6 C6-C3 C6-C3-C6
O
OH
O
OH
C6-C2-C6
phenolic acids cinnamics acids stilbenoids flavanoids
O
OH
OH
O
OH
OH
OH
O
OH
OH
OH
OH
OH
OH
OH
O
OH
OH
OH
O
OH
OH
O
OH
OH
OH
OH
OH
OH
OH
OH
O
OH
OH
OH
O
OH
OH
O
OH
O

69. Wine Chemistry – Phenolics – Phenolic acids
shikimic acid
O
OH
OH
O
OH
OH
OH
O
OH
OH
OH
OH
gallic acid
UV-Vis spectrum
nm250 300 350 400 450 500 550
270 nm
nm250 300 350 400 450 500 550
280 nm
• Wooden barrel
• Non vinifera
• Hydrolysable tannins
gallic acid
O
HO
HO
OH
O
HO
O
OH
OH
OH
glucogallin

70. Wine Chemistry – Phenolics - Cinnamates
shikimic acid phenylalanine cinnamic acid
coumaric acid
coutaric acidfertaric acid caftaric acid
caffeic acidferulic acid

71. Wine Chemistry – Phenolics - Cinnamates
coutaric acid
Wine 15 mg/L

72. fertaric acid
Wine Chemistry – Phenolics - Cinnamates
Wine 4 mg/L

73. Wine Chemistry – Phenolics - Cinnamates
nm250 300 350 400 450 500 550
caffeic acid
UV-Vis spectrum
324 nm

74. fertaric acid
Wine Chemistry – Phenolics - Cinnamates
coutaric acid 4-vinyl phenol
4-vinyl guaicol 4-ethyl guaicol
4-ethyl phenol
OH
O
O
OH
O
OH
OH
O
OH
O
O
OH
O
OH
OH
O
O CH3
OH
CH2
O CH3
OH
CH3
O CH3
OH
CH3OH
CH2

75. Wine Chemistry – Phenolics - Flavonols
shikimic acid phenylalanine cinnamic acid
naringenin
myricetinsyringetin
quercetin
keampferol
isorhamnetin
naringenin chalcone

76. Wine Chemistry – Phenolics - Flavonols
quercetin
quercetin 3-rabinoside
quercetin 3-glucuroside
quercetin 3-galactose quercetin 3-rhamnosidequercetin 3-glucose

77. Wine Chemistry – Phenolics - Flavonols
Mattivi et al JAFC 2006
myricetin
syringetin
quercetin
keampferol
isorhamnetin

78. Wine Chemistry – Phenolics - Flavonols
Mattivi et al JAFC 2006
myricetin
syringetin
quercetin
keampferol
isorhamnetin

79. Wine Chemistry – Phenolics - Flavonols
UV-Vis spectrum

80. Wine Chemistry – Phenolics - Anthocyanins
shikimic acid
phenylalanine cinnamic acid
naringenin
naringenin chalcone
cyanidin
delphinidin
pelargonidin
malvidin
peonidin

81. Wine Chemistry – Phenolics - Anthocyanins
O
+
OH
OH
O
OH
O
OO
OH
OH
OH
OH
O O
OH
OH
OH
OH
O
+
OH
O
OH
O
OO
OH
OH
OH
OH
OH
O
+
OH
O
OH
O
OO
OH
OH
OH
O
O
OH
OH
O
+
OH
O
OH
O
OO
OH
OH
OH
O
O
OH
OH
OH
O
+
OH
O
OH
O
OO
OH
OH
OH
O
O
OH
O
+
OH
O
OH
O
OH

82. Wine Chemistry – Phenolics - Anthocyanins
OH
OOH
OH
OH
OH
OH
O
+
OH
O
OH
O
OO
OH
OH
OH
OH
O
+
O
OH
OH
O
O OH
OH
OH
OH
OH
O
OH
O
OH
OH
OH
OH
O
+
O
OH
OH
O
O
OH
OH
OH
OH
O
OH
OH
O
OH
OH
OH
OH
O
+
OH
OH
O
OH
O
OO
OH
OH
OH
OH

83. Wine Chemistry – Phenolics - Anthocyanins
O
+
OH
OH
O
OH
O
OO
OH
OH
OH
OH
O
+
O
OH
OH
O
O OH
OH
OH
OH
O
O
O
+
O
OH
OH
O
O OH
OH
OH
OH
O
O OH
O
O
+
OH
O
OH
O
OO
OH
OH
OH
OH
OH
p-coumaric acid

84. Wine Chemistry – Phenolics - Anthocyanins
UV-Vis spectrum
nm250 300 350 400 450 500 550
O
OH
HO
OH
OH
OH
cyanidin

85. Wine Chemistry – Phenolics - Anthocyanins

86. Wine Chemistry – Phenolics - Anthocyanins
Bobeica et al Frontiers 2016

87. Mattivi et al JAFC 2006
cyanidin
delphinidin
pelargonidin
malvidin
petunidin
Wine Chemistry – Phenolics - Anthocyanins

88. Wine Chemistry – Phenolics - Anthocyanins
O
+
O
OH
OH
OH
O
O OH
OH
OH
OH
O O
+
OH
OH
OH
OH
O
O OH
OH
OH
OH
OH
O
+
O
OH
OH
OH
O
O OH
OH
OH
OH
O
+
OH
OH
OH
OH
O
O OH
OH
OH
OH
O
+
O
OH
OH
OH
O
O OH
OH
OH
OH
OH
+
+ +

89. Wine Chemistry – Phenolics - Anthocyanins
Scientific research

90. Wine Chemistry – Phenolics - Anthocyanins

91. Wine Chemistry – Phenolics - Anthocyanins

92. Wine Chemistry – Phenolics - Anthocyanins

93. DAD trace at
520 nm
mlv 3-glu vitisin B
mlv p-coum 3-glu
pinotins
Wine Chemistry – Phenolics - Anthocyanins

94. DO WHITE GRAPES REALLY EXIST?
6.00 6.50 7.00 7.50
%100
Cabernet
Sauvignon
Arapitsas et al Food Res Inter 2014

95. DO WHITE GRAPES REALLY EXIST?
6.00 6.50 7.00 7.50
%100
%
100
6.00 6.50 7.00
Chardonnay
Riesling
Sauvignon B.
Gewürztraminer
Muscateller
Cabernet
Sauvignon
Arapitsas et al Food Res Inter 2014

96. DO WHITE GRAPES REALLY EXIST?
Arapitsas et al Food Res Inter 2014

97. Wine Chemistry – Phenolics - Anthocyanins
Table 2. Anthocyanins concentration in red berries expressed in mg/Kg of grape
Sangiovese Cabernet Sauvignon Merlot
2011 2012 2011 2012 2011 2012
mg/Kg (%CV) mg/Kg (%CV) mg/Kg (%CV)
Dp 3-glu 142.325 (16) 84.507 (19) 323.664 (10) 118.683 (10) 135.812 (15) 116.543 (20)
Cy 3 glc 85.017 (15) 43.041 (17) 23.463 (19) 5.071 (12) 26.263 (17) 8.584 (23)
Pt 3-glu 77.391 (11) 48.817 (13) 134.140 (9) 33.842 (6) 58.784 (16) 35.580 (13)
Pn 3-glc 66.499 (11) 54.577 (17) 129.499 (19) 34.979 (6) 87.036 (16) 27.852 (13)
Mv 3-glc 163.243 (10) 126.62 (13) 815.296 (14) 193.910 (8) 285.486 (18) 137.371 (14)
Pl 3-glc 0.403 (6) 0.349 (23) 1.190 (14) 0.281 (12) 0.813 (13) 0.114 (30)
B-type vitisin 0.035 (16) 0.003 (28) 0.053 (6) nd 0.042 (18) nd
A-type vitisin 4.286 (20) 0.509 (17) 29.607 (7) 0.451 (11) 6.421 (18) nd
total acylated 55.220 (12) 16.465 (14) 1607.174 (7) 299.997 (10) 882.531 (14) 267.493 (9)
Total 594.419 (11) 374.888 (16) 3064.086 (12) 687.214 (8) 1483.188 (11) 593.537 (17)
Dp: delphinidin; Cy: cyanidin; Pt: petunidin; Pn: peonidin; Mv: malvidin; Pl: pelargonidin; glu: glucoside; nd:
not detected
Arapitsas et al Food Res Inter 2014

98. O
+
R
OH
OH
OH
O
O OH
OH
OH
OH
R
O
+
R
OH
OH
O
O OH
OH
OH
OH
O
R
O
+
R
OH
OH
O
O OH
OH
OH
OH
O
O OH
R
O
+
R
OH
OH
O
O OH
OH
OH
OH
O
OH
O
R
O
+
O
OH
OH
O
O OH
OH
OH
OH
OH
O
OH
O
OH
OH
OH
OH
O
+
O
OH
OH
O
O
OH
OH
OH
OH
O
OH
OH
O
OH
OH
OH
OH
Pigments & Wine ageing
Arapitsas et al J Agric Food Chem 2012

99. O
+
O
OH
OH
OH
O
O OH
OH
OH
OH
O O
+
OH
OH
OH
OH
O
O OH
OH
OH
OH
OH
O
+
O
OH
OH
OH
O
O OH
OH
OH
OH
O
+
OH
OH
OH
OH
O
O OH
OH
OH
OH
O
+
O
OH
OH
OH
O
O OH
OH
OH
OH
OH
+
+ +
Pigments & Wine ageing
Arapitsas et al J Agric Food Chem 2012

100. O
+
R
OH
OH
OH
O
O OH
OH
OH
OH
R
O
+
R
OH
OH
O
O OH
OH
OH
OH
O
R
delphinidin
cyanidin
peonidin
petunidin
malvidin
Pigments & Wine ageing
Arapitsas et al J Agric Food Chem 2012

101. Wine storage
Arapitsas et al Metabolomics 2015

102. 1 2 3 4 5
6 7 8 9 10
11 12 13 14 15
16 17 18 19 20
Wine storage
Arapitsas et al. Metabolomics 2015

103. 1
2010
Tempo 0
~4 oC Cellar DomesticWine storage
Arapitsas et al. Metabolomics 2015

104. 2010
~4 oC Cellar Domestic
6 mesi
Wine storage
Arapitsas et al. Metabolomics 2015

105. 2010
~4 oC Cellar Domestic
12 mesi
2011
Wine storage
Arapitsas et al. Metabolomics 2015

106. ~4 oC Cellar Domestic
2011
18 mesi
Wine storage
Arapitsas et al. Metabolomics 2015

107. ~4 oC Cellar Domestic
2012
24 mesi
2011
Wine storage
Arapitsas et al. Metabolomics 2015

108. Wine storage
O
+
O
OH
OH
O
OH
O
OH
O
OH
OH
OH
OH
R
O
+
OH
OH
OH
O R
O
O
O
+
O
OH
OH
O
O
OH
OH
O
R
cellar
domestic
cellar
domestic
Arapitsas et al. Metabolomics 2015

109. Wine storage
cellardomestic
time zero
cellar
domestic
*SO2 bleaching, Glories index
cellar
domestic
cellar
domestic
Arapitsas et al. Metabolomics 2015

110. O
OH
OH
OH
OH
OH
O
+
OH
O
OH
OH
O
O
C6H5
O
OH
OH
OH
OH
OH
O
+
OH
O
OH
OH
O
O
C6H5
O
+
OH
O
OH
O
O
O
C6H5
O OH
O
+
OH
O
OH
O
O
O
C6H5
OH
OH
O
+
OH
O
OH
OH
O
O
C6H5
Wine storage

111. Wine storage
cellardomestic
time zero
cellar
domestic
O
+
OH
OH
OH
O R
O
O
O
+
O
OH
OH
O
O
OH
O
O
R

112. Wine storage
O
+
O
OH
OH
O
OH
O
OH
O
OH
OH
OH
OH
R
O
+
OH
OH
OH
O R
O
O
O
+
O
OH
OH
O
O
OH
OH
O
R
cellar
domestic
Domestic storage  loss of red color
Pinotins  possible markers of bad storage
O
+
O
OH
OH
O
OH
O
R
OH
O
OH
OH
OH
OH
CH3
O
+
O
OH
OH
O
O
R
O CH3
Arapitsas et al. Metabolomics 2015

113. Wine Chemistry – Phenolics
Back to theory

114. Wine Chemistry – Phenolics - Flavanols
cyanidin
delphinidin
pelargonidin
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OH
OOH
OH
OH
OH
OOH
OH
OH
OH
+
+
+
epicatechin catechin
epigallocatechin gallocatechin
epiafzelechin afzelechin
OOH
OH
O
OH
O
OH
OH
OH
OH
OOH
OH
O
OH
O
OH
OH
OH
OH
OH
(epi)catechin-gallate
(epi)gallocatechin-gallate

115. Wine Chemistry – Phenolics – Flavanols (dimers)
(epi)catechin
epigallocatechin
OOH
OH
O
OH
O
OH
OH
OH
OH
OOH
OH
O
OH
O
OH
OH
OH
OH
OH
(epi)catechin-gallate
(epi)gallocatechin-gallate
OOH
OH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
O
OH
OH
OH
OH
O
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
O
OH
OH
O
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
O
OH
OH
OH
O
OH
OH
OH

116. Wine Chemistry – Phenolics – Flavanols (oligomers)
(epi)catechin
epigallocatechin
OOH
OH
O
OH
O
OH
OH
OH
OH
OOH
OH
O
OH
O
OH
OH
OH
OH
OH
(epi)catechin-gallate
(epi)gallocatechin-gallate
OOH
OH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH

117. Wine Chemistry – Phenolics – Flavanols (oligomers)
(epi)catechin
epigallocatechin
OOH
OH
O
OH
O
OH
OH
OH
OH
OOH
OH
O
OH
O
OH
OH
OH
OH
OH
(epi)catechin-gallate
(epi)gallocatechin-gallate
OOH
OH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH

118. Wine Chemistry – Phenolics – Flavanols (polyomers)
(epi)catechin
epigallocatechin
OOH
OH
O
OH
O
OH
OH
OH
OH
OOH
OH
O
OH
O
OH
OH
OH
OH
OH
(epi)catechin-gallate
(epi)gallocatechin-gallate
OOH
OH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
O
OH
OH
OH
OH
O
OH
OH
OH

119. Wine Chemistry – Phenolics – Flavanols (degree of polymerization)
terminal unit
4 upper units
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
O
OH
OH
OH
O
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
terminal unit
4 upper units

120. Wine Chemistry – Phenolics – Flavanols (astringent and bitter taste)
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OH
epicatechin catechin
gallocatechin
OOH
OH
O
OH
O
OH
OH
OH
OH
(epi)catechin-gallate
epicatechin is more bitter than catechin
OOH
OH
OH
OH
OH
catechin
The galloyl-ation increase the astringency
Cheynier et al. ACS Symposium 1997

121. Wine Chemistry – Phenolics – Flavanols (astringent and bitter taste)
Polymerization decrease bitterness (dimers, trimers and tetramers are more bitter than astringent)
polymerization slightly increase the astringency
(high degree or polymerization may decrease astringency)
(the maximum is reached ~ 8 units)
Cheynier et al. ACS Symposium 1997
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH

122. Wine Chemistry – Phenolics – Flavanols (astringent and bitter taste)
Skin versus seeds
Skin
Higher mDP
Galloyl
Epicatechin the major extension subunit
OOH
OH
OH
OH
OH
OH
OOH
OH
O
OH
O
OH
OH
OH
OH
Seeds
Lower mDP
galloyl esters

123. O
OH
OH
OH
OH
OH
O
OH
O
+
OH
OH
OH
H
H
Wine Chemistry – Phenolics – Flavanols (degree of polymerization)
terminal unit
OH
OHOH
H+, Cl-
50 oC
OOH
OH
OH
OH
OH
OH
OH OH
O
OH
OH
OH
OH
OH
O
OH
OH
OH
OH
OH
O
OH
OH
OH
OH
OH
O
OH
OH
OH
OH
OH
+

124. Wine Chemistry – Phenolics – Flavanols (degree of polymerization)
terminal unit
4 upper units
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OH
OHOH
H+, Cl-
50 oC
OOH
OH
OH
OH
OH
OH
OH OH
Degree of polymerization =
4 upper + 1 terminal unit
1 terminal unit
= 5
OOH
OH
OH
OH
OH
OH
OH OH
OOH
OH
OH
OH
OH
OH
OH OH
OOH
OH
OH
OH
OH
OH
OH OH
O
OH
OH
OH
OH
OH

125. Wine Chemistry – Phenolics – Flavanols (degree of polymerization)
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
O
OH
OH
OH
OH
O
OH
OH
OH
O
OH
OH
OH
OH
OH
O
OH
OH
OH
OH
OH
O
OH
OH
OH
OH
OH
O
OH
OH
OH
OH
OH
Mean degree of polymerization = mDP
O
OH
OH
OH
OH
OH

126. Wine Chemistry – Phenolics
skinpulp
seed
anthocyanins
tannins
flavonols
stilbenes
flavanols
tannins
gallic acids
cinnamates
flavonols
stilbenes

127. Scientific research
Wine Chemistry – Phenolics – Flavanols

128. Degree of tannin polymerization Phloroglucinolysis – before

129. Degree of tannin polymerization Phloroglucinolysis – before

130. Degree of tannin polymerization Phloroglucinolysis – before

131. Degree of tannin polymerization Phloroglucinolysis – before

132. Degree of tannin polymerization Phloroglucinolysis – before

133. Degree of tannin polymerization Phloroglucinolysis – before

134. catechin
epicatechin
gallocatechin
epigallocatechin
catechin gallate
%
Degree of tannin polymerization Phloroglucinolysis – before

135. Degree of tannin polymerization Phloroglucinolysis – before

136. catechin
epicatechin
gallocatechin
epigallocatechin
catechin gallate
Degree of tannin polymerization Phloroglucinolysis – after % terminal

137. Floroglucinolo post % upper+extension

139. Wine storage
Arapitsas et al Metabolomics 2015

140. Wine storage
O
OH
OH
OH
OH
OH
O
OH
OH
OH
OH
OH
cellar
domestic
cellar
domestic
Arapitsas et al. Metabolomics 2015

141. Wine storage
O
OH
OH
OH
OH
OH
O
OH
OH
OH
OH
OH
S
O
O
-
O
O
OH
OH
OH
OH
OH
O
OH
OH
OH
OH
OH
S
O
O
O
-
O
OH
OH
OH
OH
OH
O
OH
OH
OH
OH
OH
cellar
domestic
cellar
domestic
Arapitsas et al. Metabolomics 2015

142. Wine storage
O
OH
OH
OH
OH
OH
O
OH
OH
OH
OH
OH
S
O
O
O
-
O
OH
OH
OH
OH
OH
O
OH
OH
OH
OH
OH
Free SO2
Total SO2
cellar
domestic
cellar
domestic
Arapitsas et al. Metabolomics 2015

143. Arapitsas et al. Scientific Reports 2018
Wine Chemistry – Phenolics – Flavanols
O
OH
OH
OH
OH
OH
O
OH
OH
OH
OH
OH
O
OH
OH
OH
OH
OH
epicatechin
procyanidin B2
ECAT-SO3H
epicatechin-SO3H
ECAT-SO3H
procyanidin B2-SO3H
O
OH
OH
OH
OH
OH
S
O
O
OH
O
OH
OH
OH
OH
OH
O
OH
OH
OH
OH
OH
S
O
O
OH

144. Arapitsas et al. Scientific Reports 2018
Wine Chemistry – Phenolics – Flavanols
red
sparkling
white
sparkling
white
red
ECAT-SO3H
epicatechin-SO3H
ECAT-SO3H
procyanidin B2-SO3H
O
OH
OH
OH
OH
OH
S
O
O
OH
O
OH
OH
OH
OH
OH
O
OH
OH
OH
OH
OH
S
O
O
OH

145. Arapitsas et al. Scientific Reports 2018
Wine Chemistry – Phenolics – Flavanols
Amarone Tannat Sagrantino
6 8 10 14 16 19 24 26 31 5 6 7 14139
Age
10
Age
O
OH
OH
OH
OH
OH
O
OH
OH
OH
OH
OH
O
OH
OH
OH
OH
OH
epicatechin
procyanidin B2
136 7 11 12
Age

146. Arapitsas et al. Scientific Reports 2018
Wine Chemistry – Phenolics – Flavanols
ECAT-SO3H
epicatechin-SO3H
ECAT-SO3H
procyanidin B2-SO3H
O
OH
OH
OH
OH
OH
S
O
O
OH
O
OH
OH
OH
OH
OH
O
OH
OH
OH
OH
OH
S
O
O
OH
6 8 10 14 16 19 24 26 31 5 6 7 14139
Age
10
Age
136 7 11 12
Age

147. Arapitsas et al. Scientific Reports 2018
Wine Chemistry – Phenolics – Flavanols
Amarone Tannat Sagrantino

148. Arapitsas et al. Scientific Reports 2018
Wine Chemistry – Phenolics – Flavanols
O
OH
OH
OH
OH
OH
O
OH
OH
OH
OH
OH
O
OH
OH
OH
OH
OH
epicatechin
procyanidin B2
ECAT-SO3H
epicatechin-SO3H
ECAT-SO3H
procyanidin B2-SO3H
O
OH
OH
OH
OH
OH
S
O
O
OH
O
OH
OH
OH
OH
OH
O
OH
OH
OH
OH
OH
S
O
O
OH

149. Wine Chemistry – Phenolics
Back to theory

150. Wine Chemistry – Tryptophan metabolites
OH
NH2
N
H
O
O
NH2
OH
O
OH
N
H
OH
O
OH
N
H
OH
N
H
TOL
N
OH
O
OH
N
H
O
NH O
N
H
OH
NH2
shikimic
OH
OH
OH
OOH
OOH
NH2
O
NH2
O
O
N
H
OH
O
OH
OH
OH
OH
OH
O
OH
OH
OH
OH
OH
O
OH
OH
OH
OH
OH
O
NH
N
H
O
O
O
NH2
N
H
O
epicatechin
procyanidin B2
phenylalanine
tryptophan melatoninserotonin
tryptophan ethyl ester
N-acetyl-tryptophan ethyl ester
kynurenic
anthranillic
kynurenine
indole-3-pyruvic acid
indole-3-lactic acid
indole-3-acetic acid
tryptophol
2-aminoacetophenone
OOH
NH2
anthranillic

151. Scientific research
Wine Chemistry – Phenolics – Flavanols

152. Wine Metabolomics – Head space O2
-1200
-1100
-1000
-900
-800
-700
-600
-500
-400
-300
-200
-100
0
100
200
300
400
500
600
700
800
900
1000
1100
1200
-600 -500 -400 -300 -200 -100 0 100 200 300 400 500 600
t[1]
t[4]
Scores Comp[4] vs. Comp[1] colored by Variety
C
G
In
M
P
Q
T
Hotelling’s T2 Ellipse (95%) = (559.1; 1139)
R2X[4] = 0.06998
R2X[1] = 0.2906
EZinf o 2 - nomacorc2_1QC (M3: PCA-X) - 2015-01-23 12:22:22 (UTC+1)
3 x Grillo
5 x Pinot gris
1 x Inzolia
1 x Muller Thurgau
1 x Chardonnay
1 x Traminer
6 x varieties
12 x wines
N2 O2
2 x bottling conditions
2 months storage
(4+4)x12 = 96 bottles (+)
~8000 features
QC
Arapitsas et al. J Chromatogr A 2016

153. B
amino acids
cinnamics
phenolic acids
flavonoids
amines
organic acids
carbohydrates
fatty acids and their esters
tannins
pigments
Wine LC-MS metabolomics
Arapitsas et al. PlosOne 2012

154. Wine Metabolomics – Head space O2
LO: Low O2
HO: High O2
Arapitsas et al. J Chromatogr A 2016

155. LO: Low O2
HO: High O2
Arapitsas et al. J Chromatogr A 2016
Wine Metabolomics – Head space O2

156. O
OH OH
O
OH
OH
O
O O
O
OH
OH
O2
LO: Low O2
HO: High O2
Arapitsas et al. J Chromatogr A 2016
Wine Metabolomics – Head space O2

157. O
NHNH
O
OH
OH S
S
O
O
OH
O
NHNH
O
OH
OH SH
N
H
OH
S
O
O
OH
N
H
OH
O2, HSO3
- O2, HSO3
-
O
NH2
S OH
S
O
O
OH
O
NH2
SH OH
O2, HSO3
-
Glutathione reactivity – Reactions in model wine solution
Arapitsas et al. J Chromatogr A 2016

158. N
H
O
O
OH
O
OH
OH
OH
OH
N
H
O
O
OH
O
OH
OH
OH
OH
S O
OH
O
O2, HSO3
-
Glutathione reactivity – Reactions in model wine solution
Arapitsas et al. J Chromatogr A 2016

159. N
H
O
O
OH
O
OH
OH
OH
OH

160. Wine Metabolomics – public repositories
N
H
O
O
OH
O
OH
OH
OH
OH
http://www.ebi.ac.uk/metabolights/MTBLS137Franceschi et al. Frontiers 2014
indole-3-lactic glucoside
0
100
200
300
400
500
600
700
800
Germany Italy
area
Phoenix
Regent

161. N
H
O
O
OH
O
OH
OH
OH
OH

162. Wine Metabolomics – public repositories
N
H
O
O
OH
O
OH
OH
OH
OH
Top 30 red grape cultivars Top 30 white grape cultivars
271 entries 181 entries

163. Wine Chemistry – Tryptophan metabolites
OH
NH2
N
H
O
O
NH2
OH
O
OH
N
H
OH
O
OH
N
H
OH
N
H
TOL
N
OH
O
OH
N
H
O
NH O
N
H
OH
NH2
shikimic
OH
OH
OH
OOH
OOH
NH2
O
NH2
O
O
N
H
OH
O
OH
OH
OH
OH
OH
O
OH
OH
OH
OH
OH
O
OH
OH
OH
OH
OH
O
NH
N
H
O
O
O
NH2
N
H
O
epicatechin
procyanidin B2
phenylalanine
tryptophan melatoninserotonin
tryptophan ethyl ester
N-acetyl-tryptophan ethyl ester
kynurenic
anthranillic
kynurenine
indole-3-pyruvic acid
indole-3-lactic acid
indole-3-acetic acid
tryptophol
2-aminoacetophenone
OOH
NH2
anthranillic

164. Wine Chemistry – Tryptophan metabolites
O
OH
N
H S O
O OH
OH
N
H
TOL
tryptophol
red
sparkling
white
O
OH
N
H
OH
indole-3-lactic acid
O
OH
N
H
OH
S
O
O
OH
red
sparkling
white
O
OH
N
H
indole-3-acetic acid
O
OH
N
H S O
O OH
red
sparkling
white

165. Wine Chemistry – Tryptophan metabolites

166. Wine Metabolomics – Head space O2
-1200
-1100
-1000
-900
-800
-700
-600
-500
-400
-300
-200
-100
0
100
200
300
400
500
600
700
800
900
1000
1100
1200
-600 -500 -400 -300 -200 -100 0 100 200 300 400 500 600
t[1]
t[4]
Scores Comp[4] vs. Comp[1] colored by Variety
C
G
In
M
P
Q
T
Hotelling’s T2 Ellipse (95%) = (559.1; 1139)
R2X[4] = 0.06998
R2X[1] = 0.2906
EZinf o 2 - nomacorc2_1QC (M3: PCA-X) - 2015-01-23 12:22:22 (UTC+1)
3 x Grillo
5 x Pinot gris
1 x Inzolia
1 x Muller Thurgau
1 x Chardonnay
1 x Traminer
6 x varieties
12 x wines
N2 O2
2 x bottling conditions
2 months storage
(4+4)x12 = 96 bottles (+)
~8000 features
QC
Arapitsas et al. J Chromatogr A 2016

167. O2
OH NH
NH
O
NH2
O
S
O
O
OH
OH NH
NH
O
NH2
O
S
O
O
OH
OH NH
NH
O
NH2
O
SH
O
O
OH
Arapitsas et al. J Chromatogr A 2016
Wine Metabolomics – Head space O2

168. Wine Metabolomics – Head space O2
Nikolantinaki et al Food Chem 2014

169. O2
OH NH
NH
O
NH2
O
S
O
O
OH
OH NH
NH
O
NH2
O
S
O
O
OH
OH NH
NH
O
NH2
O
SH
O
O
OH
O2, HSO3
-
OH NH
NH
O
NH2
O
S
O
O
OH
S
O OH
O
Arapitsas et al. J Chromatogr A 2016
Wine Metabolomics – Head space O2

170. Glutathione reactivity – Reactions in model wine solution
Arapitsas et al. J Chromatogr A 2016

171. OH NH
NH
O
NH2
O
S
O
O
OH
OH NH
NH
O
NH2
O
S
O
O
OH
GSSG
OH NH
NH
O
NH2
O
S
O
O
OH
S
O OH
OGSSO3H
GSSG GSSG/SO2
10/1
GSSG/SO2
1/1
GSSG/SO2
1/10
GSSG GSSG/SO2
10/1
GSSG/SO2
1/1
GSSG/SO2
1/10
Glutathione reactivity – Reactions in model wine solution
Arapitsas et al. J Chromatogr A 2016

172. OH NH
NH
O
NH2
O
S
O
O
OH
OH NH
NH
O
NH2
O
S
O
O
OH
GSSG
OH NH
NH
O
NH2
O
S
O
O
OH
S
O OH
OGSSO3H
GSSG GSSG/SO2
10/1
GSSG/SO2
1/1
GSSG/SO2
1/10
GSSG GSSG/SO2
10/1
GSSG/SO2
1/1
GSSG/SO2
1/10
Glutathione reactivity – Reactions in model wine solution
Arapitsas et al. J Chromatogr A 2016

173. OH NH
NH
O
NH2
O
SH
O
O
OH
OH NH
NH
O
NH2
O
S
O
O
OH
SO OH
O
SO2
+ H2O
HSO3
-
OH NH
NH
O
NH2
O
S
O
O
OH
OH NH
NH
O
NH2
O
S
O
O
OH
O2
Glutathione reactivity – Reactions in model wine solution
Arapitsas et al. J Chromatogr A 2016