The Flavor Principle of Fenugreek - Dr. Imre Blank's Homepage

The Flavor Principle of Fenugreek
I. Blank, Nestlé Research Center
211 th ACS Symposium, New Orleans, March 24-28, 1996
• SPICE : Fenugreek (Trigonella foenum-graecum L.)
• FLAVOR : Strong, spicy, seasonig-like, sweet
• CHEMISTRY : Formation of sotolon
OH
NH 2
OH
NH 2
O
OH
O
O
O
O
4-Hydroxy-L-isoleucine
(HIL)
HIL-Lactone
Sotolon
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Structure of the presentation
CHEMISTRY
FLAVOR
FENUGREEK
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Fenugreek is used as a food and flavoring
• BOTANY : Trigonella foenum-graecum (Fabaceae)
Annual herb with pods containing 10-20 seeds
• OCCURRENCE : Cultivated in North Africa, France, India
• USE : Flavoring (curries, condiments, pickles, chutneys)
Food (mixed with flour for bread, yellow dye)
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4-Hydroxy-L-isoleucine (HIL) is the major
free amino acid in fenugreek
• CONCENTRATION :
5-7 g HIL/kg seeds
about 80% of total free amino acids
(Sauvaire et al., 4th Int. Workshop on seeds, Angers, France, 1993)
• MATURATION :
Maximum at 30-40 days after flowering
10-12 g HIL/kg seed
(Sauvaire et al., Phytochem.: 23, 479, 1984)
OH NH 2
OH
O
(2S,3R,4S)-HIL
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‘Sensory Directed Chemical Analysis’
to elucidate key flavor compounds
• Sample preparation : Direct extraction with diethyl ether,
Purification by vacuum sublimation (Sen et al., JAFC 39: 757, 1991)
• Screening of potent odorants : GC-Olfactometry (GC-O), Aroma
Extract Dilution Analysis (Grosch, Trends Food Sci. Technol. 4: 68, 1993)
• Identification : GC-MS
• Quantification : Isotope Dilution Assay (IDA) using labeled
sotolon as internal standard (Blank et al., JAFC, submitted)
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Sotolon is the character impact flavor compound
of fenugreek
Results of GC-Olfactometry :
O
OH
O
FD-factor : 10000
O
O
O
O
O
OH
OH
OH
100
10
10
10
10
OH
OH
O
FD-factors
O
OH
N
N
O
N
N
O
10
10
10
10
10
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‘Isotope Dilution Assay’ is an accurate
quantification method of labile compounds
Using labeled sotolon as internal standard
Abundance
150000
100000
50000
Ion m/z 128
sotolon
12.56
O
OH
O
9.00 10.00 11.00 12.00 13.00 14.00
Abundance
80000
60000
40000
20000
Ion m/z 130
labeled sotolon
12.56
*
*
O
OH
O
9.00 10.00 11.00 12.00 13.00 14.00
Retention time (min)
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Concentration of sotolon depends on the
geographical origin and variety
Sample Sotolon
(mg/kg)
Fenugreek (Egypt) 25
Fenugreek (Australia) 4
Fenugreek (India) 7
Fenugreek (France) 2-6
Curry powder (containing fenugreek) 40
Liquid seasonings 0.1-90
Odor threshold in water (detection/nasal) : 0.3 µg/L
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Sotolon is most likely formed from
4-hydroxy-L-isoleucine (HIL)
H +
OH
OH
NH 2
O
(2S,3R,4S)-HIL
O
NH 2
O
R
O
O
R
H +
R
O
OH
O
R
O
O
(5S)-Sotolon
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The lactone of 4-hydroxy-L-isoleucine is a
better precursor of sotolon than the amino acid
Carbonyl compound
Methylglyoxal
2,3-Butanedione
Sotolon yield (mol%)
(from HIL-Lactone)
35.9
0.3
Sotolon yield (mol%)
(from HIL)
7.4

Lactonization of 4-hydroxy-L-isoleucine (HIL)
is favored under acidic conditions
Molar ratio HIL-Lactone / HIL
0.6
0.5
0.4
OH NH 2
OH
O
0.3
H +
0.2
0.1
NH 2
0
pH 7 pH 6 pH 5 pH 4 pH 3
O O
HIL
Reaction conditions : 100°C, 1 h, phosphate buffer
Analytical technique : FAB-MS
HIL-Lactone
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Sotolon is formed from 4-hydroxy-L-isoleucine
by thermally induced oxidative deamination
OH
NH 2
O
MG
OH
O
NH 2
H + H N CH C
H
O
O O O
H 2 O
O O
H 2 O
CH 3
HO
N
CH
C
CH 3
N
OH
H
O
O
O
O
O
CO 2
H O 2
N H 2
O
HO
N
HO
H 2 O HO
N H 2
O
O
Strecker aldehyde
O
Sotolon
OH
O
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Formation of sotolon from HIL and its lactone :
Influence of pH
60.0
50.0
Methylglyoxal + HIL-Lactone
Methylglyoxal + HIL
Yield (mol %)
40.0
30.0
20.0
10.0
0.0
2.5 3.5 4.5 5.5 6.5 7.5
pH
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Influence of the reaction time :
Continuous increase of sotolon within 10 h
30.0
25.0
Yield (mol %)
20.0
15.0
10.0
5.0
Methylglyoxal + HIL
0.0
0 5 10 15 20 25
Time (h)
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Effect of the temperature :
Sotolon formation is favored at T > 80°C
8.0
Yield (mol %)
7.0
6.0
5.0
4.0
3.0
2.0
1.0
0.0
Methylglyoxal + HIL
20 40 60 80 100
Temperature (°C)
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The key flavor compound of fenugreek and
its formation from precursors is elucidated
Limiting factors :
Lactonization of 4-hydroxy-L-isoleucine (HIL)
HIL-Lactone is a better precursor than HIL
Reactivity of carbonyl compounds
Methylglyoxal is an efficient reactant
Optimal conditions :
pH : pH optimum 5-6
Time : continuous increase (10 fold within 10 h)
Temperature : significant amounts at > 80°C
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Several skillful people contributed to this
work
Mrs. Jianming LIN : Diploma work
Mrs. Stéphanie DEVAUD : Preparation of flavor extracts
Dr. Laurent FAY : MS measurements
Mr. René FUMEAUX : Synthesis of HIL
Dr. Dieter WELTI : NMR measurements
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