and related 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

1 Oxidoreduction reactions are of importance in breadmaking

Most of these reactions take place during dough mixing Are catalyzed by enzymes Are dependent on

2 • Endogenous oxidoreductases in wheat flour • Oxygen consumption during mixing • Relations with endogenous and exogenous oxidoreductases (and some ) – Reactions catalyzed and technological effects

3 Mean absolute levels of activities in wheat flour Shorts Peroxidase 7000 µkat.kg -1 (ferulic acid) 17000 µkat.kg -1 (gaiacol) Germ Catalase 5000 µkat.kg -1 (hydrogen peroxide)

Germ GSH-DHA oxidoreductase 400 µkat.kg -1 (glutathione and dehydroascorbic acid) Germ Lipoxygenase 40 µkat.kg -1 (linoleic acid)

Shorts Acid ascorbic acid oxidase 0.7 µkat.kg -1 (ascorbic acid)

Bran Polyphenoloxidase 0.08 µkat.kg -1 (DOPA)

4 • Endogenous oxidoreductases in wheat flour • Oxygen consumption during mixing • Relations with endogenous and exogenous oxidoreductases (and some hydrolases) – Reactions catalyzed and technological effects

5 THE MIXER BIOREACTOR

Filter Cold point Motor IKA

Gas circulation

Gas analyzer COSMA Interface Rubber seal DC1 Liquid seal

O2

Torque Mixer arm

CO 2

Thermoregulation circuit

Thermostat6 THE SITOXYGRAPH

Storage of data Upside cover with a (PC with labview ®) glass window airtight

Side cover airtight

Static arm

Motor with torque Gaz Analyzer (O 2 and CO 2) Thermostated (water jacket) mixer bowl measurement atmosphere of the mixer bowl volume ca 10 L  5 kg of dough Measurement of temperatures (dough, water jacket in and out)

7 Oxygen uptake of wheat flour during mixing

7

6

5

4

3

uptake (µmol/g dm) (µmol/g uptake 2 Sitoxygraph 2 O 1 Bioreactor

0 0 10 20 30 40 50 60 Mixing time (min)

8 THE SITOXYGRAPH

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, O 2 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

9 • Distribution of endogenous oxidoreductases in the wheat grain and in the milling fractions • Oxygen consumption during mixing • Relations with endogenous and exogenous oxidoreductases (and some hydrolases) – Reactions catalyzed and technological effects

10 Acting on lipid Lipoxygenase

Oxygen uptake Lipoxygenase : 40 µkat/kg (% vs final value of control)

140 Control LOX

120

100

80

60 Oxygen uptake (%) 40

20

0 0 10 20 30 40 50 60 Time of mixing (min) 11 Acting on lipid LIPOXYGENASE

Polyunsaturated Intermediary Hydroperoxides fatty acids + O2 free radicals

12 Acting on lipid LIPOXYGENASE

• Bleaches dough

13 Acting on lipid LIPOXYGENASE

Polyunsaturated Intermediary Hydroperoxides fatty acids + O2 free radicals

Carotenoid pigments Oxidised products

Dough bleaching effect

14 Acting on lipid LIPOXYGENASE

• Bleaches dough • Modifies crumb aroma

15 Acting on lipid LIPOXYGENASE Volatiles in bread crumb (hexanal,…)

Baking

Polyunsaturated Intermediary Hydroperoxides fatty acids + O2 free radicals

Carotenoid pigments Oxidised products

Dough bleaching effect Effect on bread aroma 16 Acting on lipid LIPOXYGENASE

• Bleaches dough • Modifies crumb aroma • Increases mixing tolerance • Enhances bread volume • Improves dough handling properties

17 Acting on lipid LIPOXYGENASE

Volatiles in bread crumb

Baking

Polyunsaturated Intermediary Hydroperoxides fatty acids + O2 free radicals

Carotenoid pigments Oxidised products

2 RSH RSSR Dough bleaching effect Effect on bread aroma Effect on dough rheology 18 Acting on lipid Lipase and phospholipase

Oxygen uptake Lipase (L) 0.25 mkat/kg (% vs final value of control) Phospholipase (PL) 0.17 mkat/kg Lipopan (0.25 L + 0.06 PL) mkat/kg L 0.25 mkat + PL 0.06 mkat/kg 180 Control 160 L 0.25 PL 0.17 140 Lipopan 120 L 0.25 + PL 0.06

100

80 Oxygen uptakeOxygen (%) 60

40

20

0 0 10 20 30 40 50 60 Time of mixing (min) 19 Acting on lipid LIPASE

Fatty acid Triglyceride + H2O Diglyceride +

Diglyceride+ H2O Monoglyceride+ Fatty acid

• Modifies the balance non-polar to polar lipids • Increases the amount of polyunsaturated free fatty acids (lipoxygenase )

20 Acting on lipid LIPASE, PHOSPHOLIPASE, LIPOPAN FBG™ (L) (PL) (LPP)

L, LPP Triglyceride + H2O Diglyceride+ Fatty acid

Diglyceride+ H2O Monoglyceride+ Fatty acid L, LPP

Phospholipid+ H2O Lysophospholipid+ Fatty acid PL, LPP

Glycolipid+ H2O Lysoglycolipid+ Fatty acid LPP

• Modifies the balance non-polar to polar lipids • Increases the amount of polyunsaturated free fatty acids (lipoxygenase substrate)

21 Acting on lipid

LIPASE, PHOSPHOLIPASE, LIPOPAN™

• Bleaches dough • Increases the gluten strength (G’ modulus) • Improves fermentation tolerance • Modifies crumb structure (more uniform) • Enhances bread volume (depends on flour)

22 Laccase Acting on phenol

Oxygen uptake (% vs final value of control) Laccase 2 µkat/kg Laccase 3.4 µkat/kg Control 120 LAC 2 LAC 3.4

100

80

60

Oxygen uptake (%) 40

20

0 0 10 20 30 40 50 60 Time of mixing (min)

23 Acting on phenol

PEROXIDASE, POLYPHENOLOXIDASE, LACCASE (POD) (PPO) (LAC)

Enz Non enz

Polyphenols + H2O2 Semiquinones Polymers and / or quinones POD

Polyphenols + O2 Semiquinones Polymers and / or quinones LAC

Quinones Polymers Polyphenols + O2 PPO

• Main phenolic compounds in wheat flour are ferulic acid in pentosans (0.5 mmol/kg) and tyrosine in proteins (20 mmol /kg) • PPOs act more rapidly on o-diphenols

24 Acting on phenol Dimers from tyrosine residues (POD, PPO or LAC on proteins)

P1 P1

CH 2 CH 2 OH

O OH OH

CH 2

P2 CH 2

P Dityrosine 2 Isodityrosine

25 Dimers from ferulic acid Acting on phenol (POD or LAC on pentosans)

HO O

HO O

O O 5'

OCH 3 HO HO 8 O 5 5' OCH 3

OH OH

OCH HO OCH 3 3

8-5' benzofuran diferulate 5-5' diferulate

O

OCH 3 O O OH OH HO O 8 8'

OH 8 O 4’ O OCH 3

CH 3O CH 3O OH OH γγγ 8-8'- - lactone diferulate 8-O-4' diferulate 26 Acting on phenol LACCASE and PEROXIDASE

Both enzymes strengthen dough.

Addition of laccase or peroxidase (with H2O2) results mainly in the modification 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 effects decrease with the dough resting time (Selinheimo et al., 2006) Attempts of crosslink pentosans with cysteine or tyrosine by these enzymes failed (Figueroa-Espinoza et al., 1999) Heterodimers (polymers) can be obtained with POD (HRP) if proper conditions 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)

27 Acting on phenol 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)

28 Acting on phenol 5-S-cysteinyl-3,4-DOPA (obtained with PPO = mushroom tyrosinase)

P1 P1 P1 P1

CH 2 CH 2 CH 2 CH 2

PPO PPO + R-CH 2-SH

+ O + O O OH R-CH 2-S OH OH OH O OH

Tyrosine Dopaquinone DOPA 5-S-cysteinyl-DOPA 3,4-dihydroxyphenylalanine

29 Acting on phenol POLYPHENOLOXIDASE (Mushroom tyrosinase)

Addition of tyrosinase (40 mg / kg) results in a 15 fold increase in the 5-S-cysteinyl-DOPA content in dough (Takasaki and Kawakishi, 1997)

Tyrosinase is able to polymerize gliadin with the formation of 5-S-cysteinyl-DOPA (intra and intermolecular bonds) (Takasaki et al., 2001)

30 Acting on phenol POLYPHENOLOXIDASE and LACCASE (Trichoderma reesei ) ( Trametes hirsuta )

Addition of tyrosinase (5 µkat / kg) results in a 10 % increase of bread volume (Selinheimo et al., 2007 )

Addition of laccase (5 µkat / kg) results in a 13 % increase of bread volume (Selinheimo et al., 2007 )

31 Acting on oses oxidase and Hexose oxidase

Oxygen uptake 1.5 µkat/kg (% vs final value of control) Glucose oxidase 7 µkat/kg Hexose oxidase 1.5 µkat/kg 180 Control 160 GOX 1,5

140 HOX 1,5 GOX 7 120

100

80

Oxygen(%) uptake 60

40

20

0 0 10 20 30 40 50 60 Time of mixing (min) 32 Acting on oses GLUCOSE OXIDASE - HEXOSE OXIDASE

GOX δ H2O2 Glucose + O2 -D-gluconolactone +

HOX H O Hexose+ O2 D-hexolactone + 2 2

Both enzymes produce hydrogen peroxide activate the peroxidase system

33 Acting on oses 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

34 Acting on oses Glucose oxidase and Hexose oxidase

Oxygen uptake Glucose oxidase 1.5 µkat/kg (% vs final value of control) Glucose oxidase 7 µkat/kg Hexose oxidase 1.5 µkat/kg 180 Control 160 GOX 1,5

140 HOX 1,5 GOX 7 120

100

80

Oxygen(%) uptake 60

40

20

0 0 10 20 30 40 50 60 Time of mixing (min) 35 Acting on oses 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)

36 Acting on oses SH oxidation by GOX

Glucose O2 Dimers GOX

δ-GL 2 H 2O POD H2O2 + 2 Fer 2 Fer*

RSSR 2 RSH

37 Acting on oses 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)

38 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….

39 Sulfhydryl oxidase H2O2 2 GSH GSSG + H2O2 Ferulic acid DiFer Peroxidase (pentosans) Fer-Cys O2 Fer-Tyr Tyrosine DOPA-Cys Polyphenoloxidase Glucose Gluconolactone + H O (proteins) DiTyr 2 2 ½ O Laccase Glucose oxidase 2 Polyphenoloxidase Catalase OH OH 2 H 2O2 2 H 2O + O2 OH Enzymatic 2 H O + AA browning H2O2 + 2 AH 2 Quinone + H2O products H O + AH 2 H O + A 2 2 2 2 ½ O 2 ½ O Peroxidase 2 R R

Ascorbic acid (AA) Lipoxygenase ½ O 2 Polyunsaturated AA fatty acids Intermediary free radicals Hydroperoxides oxidase O2

DHA 2 GSH Hematin Pigments Oxidative DHA Unsaturated compounds Bleached rancid fatty acids products GSSG Reductase products

AA 2 PSH PSSP

PSSG GSH 40 Conclusions

A good comprehension of the oxidoreducing enzymes 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

41 Thank you for your attention

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