oxidoreductasesand related enzymes in breadmaking
TRANSCRIPT
1
Oxidoreductases and related
enzymes in breadmaking
Jacques NICOLAS
UMR SCALE 1211 (ENSIA-CNAM-INRA), Conservatoire
National des Arts et Métiers, Chaire de Biochimie Industrielle et
Agro-Alimentaire Case 306, 292 rue Saint Martin - 75141 Paris
Cedex 03, France
2
Oxidoreduction reactions are of importance
in breadmaking
Most of these reactions take place during
dough mixing
Are catalyzed by enzymes
Are dependent on oxygen
3
• Endogenous oxidoreductases in wheat flour
• Oxygen consumption during mixing
• Relations with endogenous and exogenous
oxidoreductases (and some hydrolases) –
Reactions catalyzed and technological effects
4
Mean absolute levels of
oxidoreductase activities in wheat flour
Lipoxygenase 40 µkat.kg-1 (linoleic acid)
Catalase 5000 µkat.kg-1 (hydrogen peroxide)
Peroxidase 7000 µkat.kg-1 (ferulic acid)
17000 µkat.kg-1 (gaiacol)
Polyphenoloxidase 0.08 µkat.kg-1 (DOPA)
Acid ascorbic acid oxidase 0.7 µkat.kg-1 (ascorbic acid)
GSH-DHA oxidoreductase 400 µkat.kg-1 (glutathione and
dehydroascorbic acid)
Germ
Germ
Germ
Shorts
Shorts
Bran
5
• Endogenous oxidoreductases in wheat flour
• Oxygen consumption during mixing
• Relations with endogenous and exogenous
oxidoreductases (and some hydrolases) –
Reactions catalyzed and technological effects
6
THE MIXER BIOREACTOR
Gas circulation
Interface
DC1
Gas analyzer
COSMA
Motor
IKA
FilterCold point
Mixer arm
Thermoregulation circuit
Thermostat
Liquid seal
Rubber seal
O2
CO2
Torque
7
Side cover
airtight
Thermostated (water jacket) mixer bowl
volume ca 10 L ���� 5 kg of dough
Measurement of temperatures
(dough, water jacket in and out)
Motor with torque
measurement
Upside cover with a
glass window airtight
Static
arm
Gaz Analyzer (O2and CO
2)
atmosphere of the mixer bowl
Storage of data
(PC with labview®)
THE SITOXYGRAPH
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Oxygen uptake of wheat flour
during mixing
0
1
2
3
4
5
6
7
0 10 20 30 40 50 60
Mixing time (min)
O2 uptake (µmol/g dm)
Sitoxygraph
Bioreactor
9
This apparatus allows to discriminate flours by their O2 uptake curve
Total oxygen uptake
Instant rates in the initial and intermediary periods of mixing
For flour without additives, O2 uptake is mainly related to
free polyunsaturated fatty acids (PUFA) and lipoxygenase activity
50 to 75 % of O2 uptake during mixing is explained by the PUFA oxidation
THE SITOXYGRAPH
10
• Distribution of endogenous oxidoreductasesin the wheat grain and in the millingfractions
• Oxygen consumption during mixing
• Relations with endogenous and exogenousoxidoreductases (and some hydrolases) –Reactions catalyzed and technologicaleffects
11
LipoxygenaseOxygen uptake
(% vs final value of control)
0
20
40
60
80
100
120
140
0 10 20 30 40 50 60
Time of mixing (min)
Oxygen uptake (%)
Control
LOX
Lipoxygenase : 40 µkat/kg
Acting on lipid
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LIPOXYGENASE
Polyunsaturated
fatty acids + O2
Intermediary
free radicalsHydroperoxides
Acting on lipid
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LIPOXYGENASE
Polyunsaturated
fatty acids + O2
Intermediary
free radicalsHydroperoxides
Dough bleaching effect
Carotenoid pigments Oxidised products
Acting on lipid
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LIPOXYGENASE
Polyunsaturated
fatty acids + O2
Intermediary
free radicalsHydroperoxides
Carotenoid pigments Oxidised products
Baking
Volatiles in
bread crumb
(hexanal,…)
Effect on bread aromaDough bleaching effect
Acting on lipid
17
• Bleaches dough
• Modifies crumb aroma
• Increases mixing tolerance
• Enhances bread volume
• Improves dough handling properties
LIPOXYGENASE
Acting on lipid
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LIPOXYGENASE
Polyunsaturated
fatty acids + O2
Intermediary
free radicalsHydroperoxides
2 RSH RSSR
Carotenoid pigments Oxidised products
Baking
Volatiles in
bread crumb
Effect on dough rheology
Dough bleaching effect Effect on bread aroma
Acting on lipid
19
Lipase and phospholipase
Oxygen uptake
(% vs final value of control)
0
20
40
60
80
100
120
140
160
180
0 10 20 30 40 50 60
Time of mixing (min)
Oxygen uptake (%
)
Control
L 0.25
PL 0.17
Lipopan
L 0.25 + PL 0.06
Lipase (L) 0.25 mkat/kg
Phospholipase (PL) 0.17 mkat/kg
Lipopan (0.25 L + 0.06 PL) mkat/kg
L 0.25 mkat + PL 0.06 mkat/kg
Acting on lipid
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LIPASE
Triglyceride + H2O Diglyceride Fatty acid+
+ +Diglyceride Monoglyceride Fatty acidH2O
• Modifies the balance non-polar to polar lipids
• Increases the amount of polyunsaturated free fatty acids
(lipoxygenase substrate)
Acting on lipid
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LIPASE, PHOSPHOLIPASE, LIPOPAN FBG™
(L) (PL) (LPP)
Triglyceride + H2O Diglyceride Fatty acid+
+ +Diglyceride Monoglyceride Fatty acidH2O
• Modifies the balance non-polar to polar lipids
• Increases the amount of polyunsaturated free fatty acids
(lipoxygenase substrate)
+ +Phospholipid Lysophospholipid Fatty acidH2O
+ +Glycolipid Lysoglycolipid Fatty acidH2O
L, LPP
L, LPP
PL, LPP
LPP
Acting on lipid
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• Bleaches dough
• Increases the gluten strength (G’ modulus)
• Improves fermentation tolerance
• Modifies crumb structure (more uniform)
• Enhances bread volume (depends on flour)
LIPASE, PHOSPHOLIPASE, LIPOPAN™
Acting on lipid
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LaccaseOxygen uptake
(% vs final value of control)
0
20
40
60
80
100
120
0 10 20 30 40 50 60
Time of mixing (min)
Oxygen uptake (%
)
ControlLAC 2LAC 3.4
Laccase 2 µkat/kg
Laccase 3.4 µkat/kg
Acting on phenol
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PEROXIDASE, POLYPHENOLOXIDASE, LACCASE
(POD) (PPO) (LAC)
• Main phenolic compounds in wheat flour are ferulic acid in pentosans (0.5 mmol/kg) andtyrosine in proteins (20 mmol /kg)
• PPOs act more rapidly on o-diphenols
POD
LAC
PPO
Polyphenols + H2O2 Semiquinones Polymers and / or quinones
Polyphenols + O2 Semiquinones Polymers and / or quinones
Polyphenols + O2Quinones Polymers
Enz Non enz
Acting on phenol
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Dimers from tyrosine residues
(POD, PPO or LAC on proteins)
CH2
CH2
OH
OH
P1
P2 CH2
CH2
O
OH
P1
P2
DityrosineIsodityrosine
Acting on phenol
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Dimers from ferulic acid
(POD or LAC on pentosans)
OH
OCH3
OH
OCH3
OHO
O
HO
55'
5-5' diferulate
O
OHO
HO
O
HO
OCH3
OCH3
5'
8
8-5' benzofuran diferulate
OH
CH3O
OO
OH
O
OH
OCH3
88'
8-8'- γγγγ - lactone diferulate
O
OCH3
OH
CH3O
O
OHO
8
4’
8-O-4' diferulate
OH
Acting on phenol
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LACCASE and PEROXIDASE
Both enzymes strengthen dough.
Addition of laccase or peroxidase (with H2O2) results mainly in themodification of the pentosan fraction (decrease of the content in ferulic acid monomer and increase of the diferulate content)
No clear effect of peroxidase alone (in the absence of GOX) or laccase has been observed on tyrosine.
Laccase causes an increase of Rmax and a decrease of Emax. These effectsdecrease with the dough resting time (Selinheimo et al., 2006)
Attempts of crosslink pentosans with cysteine or tyrosine by theseenzymes failed (Figueroa-Espinoza et al., 1999)
Heterodimers (polymers) can be obtained with POD (HRP) if properconditions are selected (ratio [tyr] / [fer] close to 40) due to a large difference in efficiency of HRP towards these two phenols
(Oudgenoeg et al., 2001)
Acting on phenol
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POLYPHENOLOXIDASE
(Mushroom tyrosinase)
Oxidative effect on wheat dough
Extensigraph measurement :
increase of resistance Rmax and decrease of extensibility Emax
=> ratio Rmax / Emax X 3 after addition of 25 µkat of PPO / kg of flour
(Kuninori et al., 1978)
Alveograph measurement :
increase of tenacity (P) and decrease of elasticity (L)
(Aja et al., 2003)
Acting on phenol
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5-S-cysteinyl-3,4-DOPA(obtained with PPO = mushroom tyrosinase)
OH
CH2
OH
P1
PPO
+ O
PPO
+ O
CH2
P1
O
O
+ R-CH2-SH
CH2
OH
P1
Tyrosine
DOPA
3,4-dihydroxyphenylalanine
Dopaquinone
5-S-cysteinyl-DOPA
OH
CH2
OH
P1
R-CH2-S
Acting on phenol
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POLYPHENOLOXIDASE
(Mushroom tyrosinase)
Addition of tyrosinase (40 mg / kg) results in a 15 fold increasein the 5-S-cysteinyl-DOPA content in dough
(Takasaki and Kawakishi, 1997)
Tyrosinase is able to polymerize gliadin with the formation of5-S-cysteinyl-DOPA (intra and intermolecular bonds)
(Takasaki et al., 2001)
Acting on phenol
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POLYPHENOLOXIDASE and LACCASE
(Trichoderma reesei) (Trametes hirsuta)
Addition of tyrosinase (5 µkat / kg) results in a 10 % increaseof bread volume
(Selinheimo et al., 2007)
Addition of laccase (5 µkat / kg) results in a 13 % increase ofbread volume
(Selinheimo et al., 2007)
Acting on phenol
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Glucose oxidase and Hexose oxidase
Oxygen uptake
(% vs final value of control)
0
20
40
60
80
100
120
140
160
180
0 10 20 30 40 50 60
Time of mixing (min)
Oxygen uptake (%)
Control
GOX 1,5
HOX 1,5
GOX 7
Glucose oxidase 1.5 µkat/kg
Glucose oxidase 7 µkat/kg
Hexose oxidase 1.5 µkat/kg
Acting on oses
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GLUCOSE OXIDASE - HEXOSE OXIDASE
Glucose + O2δ-D-gluconolactone + H2O2
GOX
+ +Hexose D-hexolactoneO2H2O2
HOX
Both enzymes
produce hydrogen peroxide
activate the peroxidase system
Acting on oses
34
GLUCOSE OXIDASE - HEXOSE OXIDASE
• For the same activity the higher O2 uptake during mixing can be explained by
a better affinity of HOX for glucose and O2
Acting on oses
35
Glucose oxidase and Hexose oxidase
Oxygen uptake
(% vs final value of control)
0
20
40
60
80
100
120
140
160
180
0 10 20 30 40 50 60
Time of mixing (min)
Oxygen uptake (%)
Control
GOX 1,5
HOX 1,5
GOX 7
Glucose oxidase 1.5 µkat/kg
Glucose oxidase 7 µkat/kg
Hexose oxidase 1.5 µkat/kg
Acting on oses
36
GLUCOSE OXIDASE - HEXOSE OXIDASE• For the same activity the higher O2 uptake during mixing can be explained by
a better affinity of HOX for glucose and O2
• Both enzymes cause a decrease in the SH content of dough probably via the
phenol oxidation by peroxidase (Poulsen and Bak Hostrup, 1998)
Acting on oses
37
Glucose
O2
δ-GL
H2O2+ 2 Fer 2 Fer*
2 H2O
Dimers
GOX
RSSR 2 RSH
POD
SH oxidation by GOX
Acting on oses
38
GLUCOSE OXIDASE - HEXOSE OXIDASE
• For the same activity the higher O2 uptake during mixing can be explained by
a better affinity of HOX for glucose and O2
• Both enzymes cause a decrease in the SH content of dough probably via the
phenol oxidation by peroxidase (Poulsen and Bak Hostrup, 1998)
• These enzymes increase the loaf volume : Addition of 1.5 µkat / kg increases
the volume by 15 % (GOX) or 25 % (HOX) (Poulsen and Bak Hostrup, 1998)
• Addition of GOX increases both G’ (elastic modulus) and G’’ (viscous
modulus) with an higher effect on G’ than on G’’ (Vemulapalli et al., 1998)
• GOX caused the oxidative gelation of the water soluble fraction extracted
from flour, increasing its viscosity until 1 µkat/kg (Vemulapalli and Hoseney, 1998)
• Higher GOX dosages decrease the viscosity of the water soluble fraction
system (Vemulapalli and Hoseney, 1998 ; Schooneveld-Bergmans et al. (1999)
• Addition of HOX (2 µkat/kg) and glucose (1 g/kg) increases 5 times the
content of dityrosine in dough (Hanft and Koehler, 2005)
Acting on oses
39
Conclusions
• Most of the oxidoreducing enzymes presented
here increase the oxygen uptake during mixing
and have a strengthening effect on dough
• Possibly by forming new covalent bonds in gluten
and / or in pentosan
• Many reductants are present : polyunsaturated
fatty acids, phenolics, thiols, reducing sugars…
• But only one oxidant : oxygen….
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2 PSH PSSP
2 GSH
GSSG
GSHPSSG
OH
R
OH
R
Quinone +
AA
OH
Polyunsaturated
fatty acids
Unsaturated
fatty acids
Lipoxygenase
Hematin
compounds
Intermediary free radicals
Pigments Bleached
products
Ascorbic acid (AA)
AA
oxidase
DHA
DHA
Reductase
Catalase
Peroxidase
Sulfhydryl oxidase
Glucose oxidase
GSSG +
Gluconolactone +Glucose
2 GSH
Polyphenoloxidase
+ 2 AH
+ AH2
H2O2
Ferulic acid
(pentosans)
Tyrosine
(proteins)
DiFer
Fer-Cys
Fer-Tyr
DOPA-Cys
DiTyr
Enzymatic
browning
products
Hydroperoxides
Oxidative
rancid
products
Polyphenoloxidase
Laccase
PeroxidaseH2O2
H2O2
2 H2O2
H2O2
H2O2
2 H2O + O2
H2O
½ O2
O2
2 H2O + AA
2 H2O + A
O2
½ O2
½ O2½ O2
41
Conclusions
A good comprehension of the oxidoreducingenzymes effects needs to understand :
• How these enzymes (including yeast) compete for the different substrates (oxygen and reductants) in the dough
• The behaviour of the primary products formed(free radicals from lipids, quinones and semi-quinones from phenolics,…) in the dough