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

8

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

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• 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

12

LIPOXYGENASE

Polyunsaturated

fatty acids + O2

Intermediary

free radicalsHydroperoxides

Acting on lipid

13

• Bleaches dough

LIPOXYGENASE

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

15

• Bleaches dough

• Modifies crumb aroma

LIPOXYGENASE

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

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• 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

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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

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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• 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

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Glucose

O2

δ-GL

H2O2+ 2 Fer 2 Fer*

2 H2O

Dimers

GOX

RSSR 2 RSH

POD

SH oxidation by GOX

Acting on oses

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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

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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

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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

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Thank you for your attention