Loc Levavasseur1, Lalatiana Rakotozafy1, Alain Sommier2, Gabrielle Moulin1, Sarah Lanchas1, Jocelyn Rouill3, Jacques Georis4, Jacques Potus1 and Jacques Nicolas1.1Chaire de Biochimie IAA, UMR SCALE 1211 (ENSIA / CNAM / INRA), CNAM, 292 rue Saint-Martin, case 306, 75241 Paris Cedex 03, France.

2 UMR-GENIAL, 1 avenue des Olympiades, 91744 Massy Cedex, France.3J. Soufflet SA, Quai Sarrail, BP 12, 10400 Nogent sur Seine, France.4Beldem-Puratos Group, Rue Bourrie 12, B-5300 Andenne, Belgium.

Comparison of the oxygen consumption of wheat flour dough

recorded by two instrumented kneaders

using multivariate statistical analysis.

References1. Nicolas J and Drapron R. Lipoxygenase and some related enzymes in breadmaking, in Lipids in Cereal Technology, ed. By Barnes PJ. Academic Press, London, pp. 213236 (1983).

2. Eyoum A, Celhay F, Nron S, El Amrani F, Boussard A, Poiffait A, Potus J, Baret J-L and Nicolas J. Biochemical factors of importance in the oxygen consumption of unyeasted and yeasted wheat flours during dough mixing, in Recent Advances in Enzymes in Grain Processing, ed. by Courtin CM, Veraverbeke WS and Delcour JA. Katholieke Universiteit Leuven, Leuven, pp. 303309 (2002).

3. Levavasseur L, Rakotozafy L, Manceau E, Louarme L, Robert H, Baret JL, Potus J and Nicolas J. Discrimination of wheat varieties by simultaneous measurements of oxygen consumption and consistency of flour dough during mixing. J Sci Food Agric 86 (11) : 1688-1698 (2006)

4. Castello P, Jollet S, Potus J, Baret J-L and Nicolas J. Effect of exogenous lipase on dough lipids during mixing of wheat flours. Cereal Chem. 75: 595-601 (1998).

5. Delcros JF, Rakotozafy L, Boussard A, Davidou S, Porte C, Potus J, and Nicolas J. Effect of mixing conditions on the behaviour of lipoxygenase, peroxidase and catalase in wheat flour doughs. Cereal Chem 75: 85-93 (1998).

6. Morel MH, Dehlon P, Autran JC, Leygue JP, Bar-LHelgouach C. Effects of temperature, sonication time and power settings on size distribution and extractibility of total wheat flour proteins as determined by size-exclusion high performance liquid chromatography. Cereal Chem. 77: 685-691 (2000).

7. Statgraphics Plus 5.1 for Windows. Statpoint Inc. Herndon, Virginia, USA (2001). http://www.cnam.fr/biochimie

ConclusionIn our experimental conditions, we observed almost no difference concerning the oxidation of the lipid fraction (carotenoid pigments and free PUFA) of doughs obtained with the two kneaders. For the 13 flours tested, a similar discrimination

based on oxygen uptake during mixing was given by the two kneaders. Although the different hydrations of doughs changed the reactants concentrations and although the differences in mixers modified the intensity of the enzyme-substrates contacts,

the total O2 consumption and the kinetics of O2 uptake given by the two mixers were well correlated provided that the time scales were adjusted. With the pilot kneader (Sitoxygraph), the O2 uptake (estimated by the lipid oxidation reactions) is

approximately 2.5 times faster than with the experimental laboratory mixer (Bioreactor). Nevertheless, the more drastic mixing conditions with Sitoxygraph might lead to overmixing for some flours as illustrated by the decrease of the HMW / LMW

glutenins ratio in doughs compared to initial flours. Being close to industrial conditions, the O2 uptakes measured by Sitoxygraph gave a better estimation of the kinetics of O2 consumption during bread dough mixing. Using Sitoxygraph, further works

are in progress to follow simultaneously the oxygen uptake and the dough rheological properties as affected by the type of flours and the dough formulation.

NANTES 17-21 September 2006

Materials & Methods

* (Flour oxidable species) (Dough residual oxidable species) = Oxidized species

** (Flour extractable activity) (Dough residual extractable activity) = Loss in extractable enzyme activity

Figure 2. Schematic representation of the experiments performed in this study

The major part of the PUFA in the free fatty acids (FFA) fraction (Figure 4A) and carotenoid pigments (Figure 4B) are

oxidized during mixing. The oxidation levels for PUFA range from 85 to 98 % with both mixers and for carotenoid

pigments from 63 to 98 % with (B) and from 88 to 98 % with (S).

Biochemical data

Figure 4. Correlation between the levels of oxidized species for the bioreactor (B) and the sitoxygraph (S).

A. Levels of PUFA oxidized in the FFA fraction. B. Levels of carotenoid oxidation.

The ratios of O2 consumed to oxidize PUFA in the FFA

fraction are similar for both mixers (Figure 5). For the 10

flours obtained from pure wheat varieties and BPMF, only

20 to 50 % of the O2 consumed during mixing can be

explained by the PUFA oxidation in the FFA fraction. For

the two commercial flours (CN04 and CNS), 55 to 75 % of

the consumed O2 is explained by the PUFA oxidation.

Conversely to the 11 previous flours, these two flours were

not freshly milled and contained higher amounts of free

PUFA which were formed during the flour storage in the

mill. Similar ratios (50 to 70 %) were obtained by Eyoum et

al.2 using mature flours (not freshly milled) from pure wheat

varieties.

During mixing, the oxidation of polyunsaturated fatty acids (PUFA) in the monoacylglycerol and free forms by wheat lipoxygenase (LOX) in the presence of

oxygen (O2) leads to the co-oxidation of different endogenous dough reducing compounds such as thiols (SH), phenols (Ph), carotenoids, etc1 (Figure 1). We

defined2 and used3 the oxygen consumption and instant rate uptake of O2 (ViO2) of the flour dough during mixing to discriminate wheat flours related to

differences in their biochemical compositions. In the present study, we aimed to compare two different kinds of mixing (work input, mixer arm, dough hydration,

etc.). We studied the evolution of the redox enzymes present in the dough (LOX, peroxidase (POD) and catalase (CAT)), their effects on their direct and indirect

substrates (O2, PUFA and carotenoids) and the modification of the size repartition of the dough proteins. 13 different flours (3 commercial flours and 10 flours

obtained by industrial milling of pure wheat varieties) were mixed with the two kneaders (Figure 2) : the Bioreactor2 (B) and the Sitoxygraph (S).

Endogenous Redox enzymes

PSSP

2 PSH

ROOH

ROH

coupled oxidations

ROOH

RH

XSSX

2 XSH 2 Ph

Ph-Ph

2 H2O

H2O2

O2

Peroxidase

Lipoxygenase

CatalaseH2O + O2

Inhibition?

Carotenoid pigments

Bleached pigments

Endogenous Redox enzymes

PSSP

2 PSH

ROOH

ROH

coupled oxidations

ROOH

RH

XSSX

2 XSH 2 Ph

Ph-Ph

2 H2O

H2O2

O2

Peroxidase

Lipoxygenase

CatalaseH2O + O2

Inhibition?

Carotenoid pigments

Bleached pigments

Introduction

Figure 1. Exemples of redox systems involved in the modification of the dough rheology

In table 1, the comparison of the data given by both

apparatus Bioreactor (B) and Sitoxygraph (S) shows

a high Pearsons correlation coefficient (r = 0.78)

regarding total oxygen consumption. The best

correlation (r = 0.91) is obtained between V20O2B and

V6O2S. V40O2B is also positively correlated with

V14O2S (r = 0.58).

Similar distributions of the different flours studied are obtained using either (B) or (S) (Figure 3). A better

reproducibility is obtained with (S) as illustrated by the proximity of experimental points obtained from the same

flour (Figure 3B). For both apparatus :

# CN04 and ME gave the highest initial instant rate of oxygen uptake (V4O2B and V6O2S)

# CNS and QB showed the highest intermediary instant rates (V20O2B and V14O2S) and total O2 consumption.

# CA, AU, BPMF showed the highest final instant rate of oxygen uptake (V40O2B and V24O2S).

Figure 3. Principal component analysis7 (PCA) on oxygen data (Total O2 consumption at the end of mixing and

instant rate of oxygen uptake (ViO2)). A. Bioreactor. B. Sitoxygraph

Table 1. Pearsons correlation coefficients7 between O2 data obtained with

the Sitoxygraph and the Bioreactor (instant rates and total O2 uptake)

Oxygen data

There is a good correlation considering kinetic aspects of oxygen consumption between both mixers provided that the

time scale of (B) is 2.5 times higher than that of (S) to obtain the best correlations between instant rates of (B) and (S).

Concerning the proteins, no differences were

observed among flour and dough obtained from the

two kneaders in the mean total protein extractibility

(Figure 6A) as well as in the HMW/LMW glutenins

mean ratio (Figure 6B). For the total protein

extractibility, a loss of 20 % was observed for CH

doughs from the two mixers.

In addition, with (B) a significant loss was observed

with AP and OR whereas with (S) a significant loss

was observed with BPMF and AU, and a significant

increase with CN and OR.

For the HMW/LMW glutenins ratio, no significant

differences between flours and (B) doughs were observed

whereas a significant decrease was observed with (S)

doughs (CN, AU, NI and QB). These differences may be

due to an overmixing with (S) (confirmed by sensorial

estimation of the baker). Thus compared to (B), the more

drastic mixing with (S) may provoke a depolymerisation of

the gluten proteins. Therefore after 25 minutes of mixing

with (S), the doughs from CN, AU, NI and QB are

overmixed whereas the dough from CA (a strong flour) is

probably not overmixed (no modification of the high

HMW/LMW glutenins ratio).

Figure 6. Total proteins distribution (A. ) and HMW/LMW glutenins ratio (B.) of 13 different flours and their doughs obtained from Bioreactor

and Sitoxygraph

900

1000

1100

1200

1300

1400

1500

CN04

CNS

BPM

FAP AU CA CH M

E NI OR

QB S

1S2

Mea

n

Pro

teins

(UA/

g.m

s)

flour bioreactor dough sitoxygraph doughA.

0,7

0,8

0,9

1,0

CN04

CNS

BPM

FAP

AU

CA

CH

ME N

IOR

QB S

1S2

Mea

n

HMW

/ LM

W g

lutenin

ratio

flour bioreactor dough sitoxygraph doughB.

V2.5O2S V6O2S V12O2S V14O2S V24O2S O2total S

V4O2B 0.39 0.27 0.08 0.02 0.02 0.14V20O2B 0.69 0.91 0.84 0.72 0.74 0.85

V40O2B 0.13 0.36 0.53 0.58 0.55 0.52O2total B 0.65 0.82 0.76 0.67 0.65 0.78

VtO2S : instant rate of oxygen uptake (mol.g-1 dm.min-1) obtained with the Sitoxygraph (S) at the time t

VtO2B : instant rate of oxygen uptake (mol.g-1 dm.min-1) obtained with the Bioreactor (B) at the time t

O2total : total oxygen consumption (mol.g-1 dm) measured during mixing (S) and (B)

Bold type : Significant coefficient at 95% confidence level

Figure 5. Correlation between the oxidized PUFA/O2 consumed

ratios for the two mixers.

ox. PUFA : oxidized polyunsaturated fatty acids (mol.g-1 dm)O2 : total oxygen consumed (mol.g

-1 dm)

y = 0.93xR2 = 0.84

0.2

0.3

0.4

0.5

0.6

0.7

0.2 0.3 0.4 0.5 0.6 0.7

ox. PUFA/O2 ratio (S)

ox.

PU

FA

/O2

rati

o (

B)

CN04

CNS

BPMF

A. B.

-5

-4

-3

-2

-1

0

1

2

3

4

5

-6 -4 -2 0 2 4 6 8

PC 1 (66.69 %)

PC

2 (2

4.28

%)

CNSME

CH

OR

S1AP

CA

QB

AU

CN04

NI

BPMF

S2

B.

-0.5

-0.4

-0.3

-0.2

-0.1

0.0

0.1

0.2

0.3

0.4

0.5

0.0 0.1 0.2 0.3 0.4

PC 1 (66.69 %)

PC

2 (2

4.28

%)

V4O2

V10O2

V12O2

V16O2

V24O2

V20O2

V8O2V6O2

O2total

V2,5O2

V22O2

V18O2

V14O2

-5

-4

-3

-2

-1

0

1

2

3

4

5

-6 -4 -2 0 2 4 6 8

PC 1 (59.80 %)

PC

2 (2

3.82

%)

CNS

ME

CH

OR

S1

AP CA

QBAU

CN04

BPMF

S2

NI

A.

-0.5

-0.4

-0.3

-0.2

-0.1

0

0.1

0.2

0.3

0.4

0.5

0 0.1 0.2 0.3 0.4

PC 1 (59.80 %)

PC

2 (2

3.82

%)

V4O2 V8O2V12O2

V16O2

V24O2

V20O2

V28O2

V32O2

V44O2

V36O2

V40O2

V48O2V52O2

V56O2

V60O2

O2total

Lipids oxidation

Proteins

MIXING

Bioreactor :- 325 g flour;

- 227.5 g H 20 (70 %)

- 6.5 g NaCl (2 %)

- 60 min at 200 RPM

Sitoxygraph :- 2500 g flour;

- 1500 g H20 (60 %)

- 45 g NaCl (1,8 %)

- 2 min at 30 RPM followed by 23 min at 52.5 RPM

Flour Dough3 commercial Flours from 2004 and 2005 :

Corde Noire 2004 (CN04) ; Corde Noire sp

(CNS) ; BPMF 2005 (BPMF).

10 french pure varieties of flour harvested in 2005 :

Apache (AP), Aubusson (AU), Cap Horn (CA), Charger

(CH), Mendel (ME), Nirvana (NI), Orvantis (OR), Quebon

(QB), Sankara 1 (S1), Sankara 2 (S2).

Moisture content 13,54 to 15,42 %(hm) ; Protein content from 10,1 to 12,9 %( dm)

Oxidable species *

Extractable enzyme activity **

Protein structures

Residual oxidable species *

Structured proteins

Residual extractable enzyme activity **LOX, POD and CAT activities 5

Protein extraction and separation by SE -6

O2 depletion and instant rate of oxygen uptake 2

MIXING

Bioreactor :- 325 g flour;

- 227.5 g H 20 (70 %)

- 6.5 g NaCl (2 %)

- 60 min at 200 RPM

Sitoxygraph :- 2500 g flour;

- 1500 g H20 (60 %)

- 45 g NaCl (1,8 %)

- 2 min at 30 RPM followed by 23 min at 52.5 RPM

Flour DoughFlours from 2004 and 2005 :

Corde Noire 2004 (CN04) ; Corde Noire spciale 2005

(CNS) ; BPMF 2005 (BPMF).

10 french pure varieties of flour harvested in 2005 :

Apache (AP), Aubusson (AU), Cap Horn (CA), Charger

(CH), Mendel (ME), Nirvana (NI), Orvantis (OR), Quebon

(QB), Sankara 1 (S1), Sankara 2 (S2).

Moisture content 13,54 to 15,42 %(hm) ; Protein content from 10,1 to 12,9 %( dm)

Oxidable species *

Extractable enzyme activity **

Protein structures

Residual oxidable species *

Structured proteins

Residual extractable enzyme activity **

PUFA and Carotenoid pigments content 4

LOX, POD and CAT activities 5

Protein extraction and separation by SE-HPLC6

O2 depletion and instant rate of oxygen uptake 2

-

-

Loc Levavasseur

Ph-D student