Mechanisms of Singlet Oxygen Oxidation and

1

Mechanisms of Singlet Oxygen Oxidation and
Nutritional and Flavor Qualities of Food
David B. Min Department of Food Science and
Technology The Ohio State University Columbus,
Ohio USA
2
Challenging Research Areas in Food Chemistry

• Soybean Oil Reversion Beany Flavor Formation
• Sunlight Flavor Formation in Milk
• Mechanisms of Riboflavin Degradation under Light

• Nitrogen Packaging minimizes these reactions
  during storage

3
1772 Priestley Discovered Oxygen 1811
Avogadro Oxygen is a Diatomic Molecule 1848
Faraday Oxygen is a Paramagnetic Molecule 1934
Herzberg Observation of Singlet Oxygen 1964
Foote Wexler Rediscovered Singlet Oxygen  
               
History of Oxygen Molecule
4
Food Oxidation

• Triplet oxygen oxidation
• Singlet oxygen oxidation

5
Molecular Orbital of Triplet Oxygen
Molecular
?
Atomic
Atomic
?
?
?
?
2Px 2Py 2Pz
2Pz 2Py 2Px
?
Energy
?
2S
2S
?
?
1S
1S
?
6
Mechanism of Antioxidant
14 13 12 11 10 9
C
H
C
H
C
H
C
H
C
H
C
H
C
H
R
(
C
H
)
C
H
2
2
2
2
3
3
Initiation
Reactive oxygen species
- H
?
Preventive Antioxidant
13 12 11 10 9
(
C
H
)
C
H
C
H
R
C
H
C
H
C
H
C
H
C
H
2
4
3
2
?
E0 600mV
3O2
K109/sec
7
(
C
H
)
C
H
C
H
C
H
C
H
C
H
C
H
R
C
H
2
3
4
2
O
O
K 1 M-1sec-1
E01000mV
?
3O
R. ROO
H from RH (Lipid)
2
Propagation
?
E01000mV
.
H
O
O
C
(
C
H
)
C
(
)
C
H
Radical Scavenging Antioxidant
.
3
3
3
3
H from
O
C
H
O
C
H
3
3
E0 400mV
K 107 M-1sec-1
C
H
C
H
C
H
C
H
C
H
C
H
R
(
C
H
)
C
H
2
2
4
3
O
O
H
8
C
H
C
H
C
H
C
H
C
H
C
H
R
(
C
H
)
C
H
2
2
4
3
O
O
OH
E02300 mV
?
H
(
C
H
)
C
H
C
H
C
H
C
H
C
H
C
H
R
C
H
3
2
2
4
O
E01600 mV
?
Termination
C
H
(
C
H
)
C
HO
3
2
4
(
C
H
)
C
H
C
H
C
H
C
H
C
H
C
H
R
C
H
3
2
2
4
O
O
(
C
H
)
C
H
C
H
C
H
C
H
C
H
C
H
R
C
H
3
2
2
4
9
Molecular Orbital of Singlet Oxygen
Molecular
?
Atomic
Atomic
?
?
?
?
2Px 2Py 2Pz
2Pz 2Py 2Px
?
Energy
?
2S
2S
?
?
1S
1S
?
10
Reaction of 3O2 and 1O2 with Linoleic Acid
OOH
.
(?)
3
O
3
O
2
2
R
R
R
R
R
R
OOH
1
O
O
2
R
R
R
R
H
O
OOH
gtgtgt
3
1
R
R
O
O
2
2
11
Photosensitizers in Foods

• Chlorophyll
• Riboflavin

12
Chlorophyll
H
C
H
H
C
R
2
C
H
C
H
H
C
2
3
3
N
N
Mg
H
H
N
N
C
H
3
H
C
3
O
C
O
C
H
C
H
2
3
2
C
O
C
H

3
O
C
H
C
H
C
H
C
H
3
3
3
3
13
Riboflavin
C
O
H
H
2
C
H
O
H
C
H
O
H
C
H
O
H
C
H
2
C
H
O
N
3
N
H
N
N
C
H
3
O
14
Singlet Oxygen Formation by Photosensitizer
Excited State
1RF
K1-20?108/sec
ISC
Fluorescence K 2?108/sec
3RF
hv
K1-3?109/sec
Phosphorescence
3O2
K10-104/sec
1RF
Ground State
Singlet Oxygen
15
Singlet Oxygen Determination by Trapping
O
O
H3C
H3C
CH3
CH3
1O2
N
N
H3C
CH3
H3C
CH3
O
H

Tetramethyl-4-Piperidone-N-Oxyl
Tetramethyl-4-Piperidone
(TAN)
(TMPD)
16
Electron Spin Resonance Spectrum of
Tetramethyl-4-Piperidone-N-Oxyl
3370 G 3390 G
3410 G
17
Reversion Beany Flavor of Soybean Oil
18
Soybean Oil Reversion Beany Flavor Compounds
C
H
C
H
C
H
C
H
C
H
3
2
2
O
2-(1-pentenyl)-furan
C
H
C
H
C
H
C
H
C
H
2
2
3
O
2-(2-pentenyl)-furan
19
Soybean Oil Purification by Chromatography
Soybean oil
Silicic acid
Sugar
Charcoal-Celite
Silicic acid
Vacuum
Purified soybean oil
20
Identified Compounds in Soybean Oil

• Phospholipids
• Tocopherols
• Monoglycerides
• Diglycerides
• Oxidized triglycerides
• Free fatty acids
• Chlorophylls

21
Purified Soybean Oils
Colorless Tasteless Odorless
22
Light Box for Soybean Oil Flavor Study
23
Effect of 0 and 5 ppm Chlorophyll on
2-Pentenylfurans in Soybean Oil
0 ppm
No beany flavor ESR No singlet oxygen No singlet
oxygen, no
2-pentenylfuran
5 ppm
trans-2-heptenal
2-pentenylfuran

Strong beany flavor ESR Singlet oxygen
Singlet oxygen, 2-pentenylfuran
24
Mass Spectrum of 2-Pentenylfuran
Relative Abundance
m/z
25
2-Pentenylfuran from Linolenic Acid by 1O2
C
H
C
H
C
H
C
H
C
H
C
H
C
H
C
H
C
H
C
H
C
H
(
C
H
)
C
O
O
H
2
2
2
6
3
2
2
1O2
C
H
C
H
C
H
C
H
C
H
C
H
C
H
C
H
C
H
C
O
O
H
(
C
H
)
C
H
C
H
3
2
2
2
2
6
2
O
O
C
H
C
H
C
H
C
H
C
H
C
H
C
H
C
H
C
H
C
O
O
H
(
C
H
)
C
H
C
H
2
2
6
2
3
2
2
O
O


C
H
C
H
C
H
C
H
C
H
C
H
C
H
C
H
C
H
2
3
2
2
O
1O2
C
H
C
H
C
H
C
H
C
H
C
H
C
H
C
H
C
H
2
3
2
2
O
O
O
26
C
H
C
H
C
H
C
H
C
H
C
H
C
H
C
H
C
H
2
3
2
2

O
O
O
H
- OH

C
H
C
H
C
H
C
H
C
H
C
H
C
H
C
H
C
H
2
3
2
2

O
O

C
H
C
H
C
H
H
C
H
C
C
H
C
H
C
H
C
3
2
2
2
2
O
O
C
H
C
H
C
H
C
H
C
H
C
C
H
C
H
C
H
3
2
2
O
H
O
H
- H2O
2-(2-pentenyl)-furan
C
H
C
H
C
H
C
H
C
H
3
2
2
O
27
Chlorophyll Effect on the Volatile Compounds of
Soybean Oil under Light at 10oC
Headspace Volatile Content
Storage in Hours
ESR
28
Carotenoid
C
H
O
b -
Apo - 8'- carotenal
Carotene
b -
O
Canthaxanthin
O
29
Effects of ?-Apo-8-Carotenal, ?-Carotene or
Canthaxanthin on Soybean Oil Oxidation
Peroxide Value (meq/kg oil)
Storage Time (hr)
30
Effects of ?-Apo-8-Carotenal, ?-Carotene or
Canthaxanthin on Soybean Oil Oxidation
Peroxide Value (meq/kg oil)
Storage Time (hr)
ESR
31
Diagram for Chlorophyll Photosensitized Singlet
Oxygen Soybean Oil Oxidation
3O2
A
hv
ISC
Sensitizer
3Sen
AO2
1Sen
1O2
ko
kr
Q
Q
Q
kQ
Q
kd
kQ
kox-Q
Sen
Sen
3O2
3O2
QO2
dAO2
ko3O2
k rA
K

ko3O2kQQ
k rA (kox-Q kQ)Q kd
dt

Plot of AO2 -1 vs. A -1 at various Q
32
Singlet Oxygen Quenching Plot
-1
K
Slope
Q4
?
1/AO2
?
?
Q3
Intercept K-1
?
?
Q2
?
?
?
1 K
?
Q1
?
?
?
?
1/A
33
Triplet Sensitizer Quenching
Q3
?
Q2

?

1/ AO2
?
Q1
?

?
?
?

Slope K-1kd (ko 3O2 kQQ) /
ko3O2kr Intercept K-1(ko 3O2 kQQ) /
ko3O2
?
1/A
34
Quenching Mechanism of b-Carotene
1/ Peroxide Value ( 1/ M )
35
Singlet Oxygen Quenching Rate (kqkox-Q) of
Carotenoids
Carotenoids Number of Conjugated
Quenching Rate Double Bonds
(M-1 sec-1) ?- apo - 8- Carotenal
10 2.86 ? 109 ?-
Carotene 11
4.60 ? 109 Canthaxanthin
13
1.12 ? 1010

36
Singlet Oxygen Quenching Mechanism of Carotenoid
1O2 1?-Carotene 3O2 3?-Carotene
3 ?-Carotene
1?-Carotene
Energy Transfer
Radiationless Transfer
37
Sunlight Flavor of Milk
38
Sunlight Flavor of Milk
C
H
S
S
C
H
3
3
39
Electron Spin Resonance Spectrum of Singlet
Oxygen in Milk during Light Exposure
0 Minutes
No sunlight flavor
30 Minutes
Sunlight flavor
3370G
3390G
3410G
40
Singlet Oxygen Formation by Riboflavin
Excited State
1RF
K1-20?108/sec ISC
Fluorescence K 2?108/sec
3RF
hv
K1-3?109/sec
Phosphorescence
3O2
K10-104/sec
1RF
Ground State
Singlet Oxygen
41
Riboflavin Free Milk by Chromatography
Fluorosil
Vacuum
Riboflavin Free Milk
42
Electron Spin Resonance Spectrum of Singlet
Oxygen in Riboflavin Free Milk under Light
0 Minutes
No singlet Oxygen No sunlight flavor
30 Minutes
No singlet oxygen No sunlight flavor
No riboflavin
43
Storage Effect on Volatiles of Milk under Light
D
D
D
44
Mass spectra of Gas Chromatographic Peak D
M/Z
No singlet oxygen No dimethyl disulfide
45
Effect of Ascorbic acid on Dimethyl Disulfide
Formation in Milk
0 ppm Ascorbic Acid
Dimethyl disulfide
200 ppm Ascorbic Acid
1000 ppm Ascorbic Acid
Singlet oxygen quencher
46
Mechanism of Dimethyl Disulfide Formation from
Methionine by Singlet Oxygen
C
O
H
O

O
O
C
O
O
H

1
O
2
N
H
C
H
C
H
C
H
S
C
H
N
H
C
H
C
H
C
H
S
C
H
2
2
3
2
2
2
3
2
O
C
O
O
H
O
H
O
C
O
O
H

N
H
C
H
C
H
C
H
S
C
H
O
H
N
H
C
H
C
H
C
H
S
C
H
2
2
3
2
2
3
C
O
O
H
O
C
O
O
H
N
H
C
H
C
H
C
H
O

S
C
H
N
H
C
H
C
H
C
H
S
C
H
2
2
3
2
2
3
C
H
S
S
C
H
S
C
H
2
3
3
3
from riboflavin under light

1
O
2
47
Mechanisms and Kinetics of Riboflavin
Degradation under Light
48
Riboflavin in Body

• Riboflavin is an important part of coenzyme FAD
• Riboflavin is essential in
• Cell respiration
• Red blood cell formation
• Metabolism of protein, fats, carbohydrates
• Resistance to stress

49
Effects of Light on the Riboflavin in Milk ()
50
HPLC Chromatograms of Riboflavin in Waterat pH
6.5 Under Light
Riboflavin
Riboflavin
51
Mass Spectrum of Riboflavin Photodegradation
Product
Lumichrome

m/z
52
HPLC Chromatogram of Riboflavin in Waterat pH
8.5 Under Light
2 hours
Lumichrome
New peak at pH 8.5
Riboflavin
Lumiflavin
No effect by oxygen concentration
53
Diagram for Riboflavin Degradation by
Photosensitization Reaction
hv
KISC
3Riboflavin
1Riboflavin
1Riboflavin
54
Lumichrome Lumiflavin Formation from Riboflavin
Lumichrome
H
N
N
O
H
C
3
N
H
H
C
N
R
3
R
pH lt 8.5
H
O
H
O
O
H
H
O
H
H
C
H
C
2
2
C
C
H
R

2
N
N
O
O
N
N
H
C
H
C
3
h
v
3
N
H
N
H
I
S
C
N
H
C
H
C
N
Lumiflavin
3
3
O
O
pH gt 8.5
H
C
H
H
1
Riboflavin
3
Riboflavin
N
N
O
H
C
3
N
H
N
H
C
3
O
H
O
C
R

55
Volatile Compound Formation from Riboflavin
under Light
56
Light Effect on Gas Chromatographic Volatile
Compounds from Riboflavin
0 hr
6 hr
12 hr
Gas Chromatographic Retention Time
57
Mass Spectrum of Gas Chromatographic Peak from
Riboflavin Stored under Light
CH3
C
O
C
O
CH3
m/z
58
Diagram for Riboflavin Degradation by
Photosensitization Reaction
CH3
KISC
3O2
RF
hv
C
O
1O2
3RF
RF
1RF
kr
C
ko
O
CH3
kq
Q
Q
kd
kQ
Kox-Q
Q
RF
3O2
QO2
3O2
Singlet oxygen quencher Sodium azide
59
2, 3-Butanedione from Riboflavin by Singlet
Oxygen
R
R
H
H
C
H3C
C
H3C
N
N
N
N
O
O
1O2
10
1
9
O
2
8
O
7
3
N
H
N
H
H3C
5
6
H3C
4
C
C
N
N
H
H
O
O
Riboflavin
Riboflavin Endoperoxide
R
R
H
H
H3C
C
H3C
C
N
N
O
N
N
O
O
O
1O2
O
O
O
O
O
O
N
H
N
H
C
H3C
H3C
C
N
N
H
H
O
O
Riboflavin Endoperoxide Dioxetane
R
O
CH3
C
H
N
N
O
C
Scission
O

C
O
N
H
C
H
CH3
N
O
O
2, 3-Butanedione
60
Reaction Rate between Singlet Oxygen and
Riboflavin
kr (M-1s-1)
S/I (mM)
Intercept
Slope (S) (1/mM)
5.7 x 109
0.29
4.00
1.16
RF
Steady State Kinetic Equation
61
(No Transcript)
62
(No Transcript)
63
Diagram for Chlorophyll Photosensitized Singlet
Oxygen Soybean Oil Oxidation
Q Carotenoids
hv
3O2
A
(Soybean Oil)
ISC
AO2
1O2
Sen
3Sen
1Sen
ko
kr
(Chlorophyll)
Q
Q
Q
kQ
Q
kd
kQ
kox-Q
Sen
Sen
3O2
3O2
QO2
dAO2
ko3O2
k rA
K

k rA (kox-Q kQ)Q kd
ko3O2kQQ
dt

64
Singlet Oxygen Quenching Mechanism
-1
dAO2
k rA (kox-Q kq)Q kd
-1
ko3O2kQQ
K
ko3O2
kr A
dt
If there is only singlet oxygen quenching, the
equation is

-1
dAO2
-1
1
K
dt

Plot of AO2 -1 vs. A -1 at various Q
65
Triplet Sensitizer Quenching Mechanism
If carotenoids quenched triplet chlorophyll, the
steady state equation is
dAO2
ko3O2
k rA
K

ko3O2kQQ
k rA (kox-Q kQ)Q kd
dt

(kox-Q kQ)Q 0
dAO2
ko3O2
k rA
K

ko3O2kQQ
k rA kd
dt
Plot of AO2 -1 vs. A -1 at various Q