Testing of an enzymatic conditioner with proteasic activity, used for the

improvement of technological qualities of several wheat flours dough

RADIANA TAMBA-BEREHOIU, N. C. POPA, STELA POPESCU, VASILICA STAN,

DANA BALAN, A. IANCU*

Faculty of Biotechnology, Bd. Marasti No. 59, 71331, Bucharest, Romania

* S.C. Boromir S.A.

Abstract
The using of proteasic conditioners is practiced for strong flours, with highly resistent and elastic
glutenic nets, but small extensibility. The proteases addition reduce the kneading times and the doughs’
weakening. The experiment consisted in the comparative evaluation of technological qualities of several
control flours and the same flours improved by the addition of an enzymatic conditioner with proteasic
activity (26 samples). We explored some physical-chemical parameters as: moisture, protein, wet gluten,
the gluten deformation index and the hydration capacity. Secondly, we explored the rheological
characteristics, respectively the proper farinograms and alveograms.
Observing the statistical evaluations of laboratory results, we may ascertain that the most
sensitive parameters for the proteasic action, were: gluten deformation index, stability, energy W,
resistance P and P/L index. Least sensitive parameter at the protease treatment was hydration capacity.
Gluten deformation index in improved flours, can be strictly controlled per the protease addition,
by the regression equation; dough’s deformation energy (W), resistance P and P/L ratio, in improved
flours, can also be anticipated starting from the initial value of these parameters in control flours, relied on
the regression equations, which depends on enzymatic conditioner quantity. The farinographic stability
parameter in improved flours, was not significantly influenced by the starting value of the stability in the
control flours, but correlated negatively distinctly significant with the enzymatic conditioner quantity.

Keywords: wheat flours, improvement, proteases, quality parameters, farinogram,

alveogram etc.

Introduction

The using of proteasic conditioners is practiced for the strong flours, which form

highly resistent and highly elastic glutenic nets, with small extensibility, respectively for

the products obtained in short technological processes, in which the fermentation times

are reduced or do not exist (sticks, rolls, biscuits, waffers). The proteases addition takes

into consideration the reducing of kneading times and the doughs’ weakening, as a result

of the peptidic bonds hydrolysis in gluten. As a result of the enzymatic reaction,

1
extensibility and others several dough’s rheological properties, appears to be

increased (2).

The most frequent proteases’ sources are Aspergillus oryzae, Bacillus subtillis,

and a range of vegetal products (like papain and bromelin). All the same, malt is an

important proteases’ source, but its strong amilolytic effect makes it less desirable (1).

The proteases’ hydrolitic effect is strongly dependent on the preparation source, the best

results upon the dough’s rheology being obtained for fungic and bacterian proteases. The

vegetal proteases produce a profound protheoliysis of the gluten, reason for which they

are used extremely seldom.

The correct dosage of the proteasic conditioner added to the dough, is very

important for obtaining good quality products. The proteases’ excess gives sticky dough

with difficult processing, a reduced capacity of gases’ retention and a strong coloured

crust. An underdosage gives small volume products, with a weak symmetry and

breakable core.

The most illustrative analysis methods, for the effects of protease addition upon

the flour proteins’ behaviour, are represented by the farinographic method and

respectively the alveographic method. The farinographic method investigates the flour

quality, according to the main farinogram characteristics, which are: development time,

stability, softening, tolerance value. The farinogram represents the dough’s evolution film

in specific kneading conditions, after obtaining a standard consistency of 500 U.B.

(Brabender units). The alveographic method analyses the stretching resistance of a dough

sheet, under the air’s pressure. It swells like a bubble until it breaks. We register: the

2
maximum stretching resistance of the dough (P), the extensibility (L), the total energy

(W) and the P/L ratio (synthetic indicator for quality).

The present paper’s aim allude to the rheological behaviour analysis of some

wheat flours dough, treated with proteases, in comparison with dough obtained from

flours which have not been treated with enzymatic conditioners. We followed in the same

time the identification of the statistic legislation to express the interdependency between

the flours’ quality parameters and the protease quantity used for the improvement, in

order to identify a mathematical method to simplify the dosage decision and to reduce the

chances of mistake induction at this level, in the technological process.

Materials and Methods

The experiment consisted in the comparative evaluation of technological qualities

of several control flours and the same flours improved by the addition of an enzymatic

conditioner with proteasic activity. The quality parameters of the dough, resulted from

the control flour and the improved flour samples, have been tested firstly, by exploring

some physical-chemical properties as: moisture, protein, wet gluten, the gluten

deformation index, the hydration capacity, and secondly, by exploring the rheological

characteristics, respectively the proper farinograms and alveograms.

The utilized analysis methods are those ones described by the national and

international in force standards, as: STAS 90-88 for the determination of the flour’s

physical-chemical parameters (moisture, protein, wet gluten quantity, gluten deformation

index), respectively ICC-Standard No. 121 and ICC-Standard No. 115/1 for the execution

of farinograms and alveogramms. The apparatus includes a Brabender farinograph and a

Chopin alveograph (3, 4, 5).

3
 The studied material was represented by 26 samples of unimproved 480 type flour

(control), derived from different production lots, produced at S.C Boromir S.A. These

have been differently improved with an enzymatic conditioner having proteasic activity,

named Veron W, furnished by the AB Enzymes Company. Its active substance is

represented by a metalproteinase resulted from specific Bacillus subtilis cultures (IUB-

No.: 3.4.24.28).

The enzymatic conditioner quantity which has been added, was within 10 and 600

ppm. The manner for improvement is shown in table 1.

Table 1. The improvement variants for flours, with Veron W

No. Enzymatic conditioner No. Enzymatic conditioner

flour lot quantity (ppm) flour lot quantity (ppm)

1 140 14 100
2 170 15 170
3 150 16 190
4 140 17 210
5 130 18 200
6 200 19 180
7 160 20 170
8 140 21 150
9 500 22 110
10 10 23 100
11 50 24 100
12 70 25 100
13 80 26 600

The differences between the control flours and the enzymatic treated flours were

analyzed by statistical analysis methods.

Results and discussion

Table 2 presents the average values of the control flours’ quality parameters, and

the main variability statistical indices.

4
 Table 2. The variability estimates for the control flours’ quality parameters

Parameter X ± sx s CV %
Moisture (%) 14.052 ± 0.294 0.086 2.094
Proteins (%) 9.690 ± 0.344 0.119 3.966
Wet gluten (%) 26.457 ± 1.254 1.573 4.739
Gluten deformation (mm) 5.722 ± 2.562 6.565 44.777
Hydration capacity (%) 56.738 ± 0.723 0.522 1.274
Stability (min) 8.000 ± 1.680 2.062 21.001
Deformation energy W (10-4 J) 220.000 ± 66.762 4459.583 30.351
Extensibility L (mm) 92.162 ± 24.218 586.587 26.879
Resistance P (mm) 70.980 ± 13.346 178.126 18.990
P/L 0.848 ± 0.321 0.103 37.950

The average of the quality parameters’ values for the improved flours, is shown in

table 3.

Table 3. The variability estimates for the improved flours’ quality parameters

Parameter X ± sx s CV %
Moisture (%) 13.753 ± 0.409 0.167 2.970
Proteins (%) 9.487 ± 0.172 0.003 1.820
Wet gluten (%) 25.675 ± 0.467 0.218 1.819
Gluten deformation (mm) 10.970 ± 5.447 29.659 49.660
Hydration capacity (%) 56.925 ± 0.798 0.636 1.401
Stability (min) 6.15 ± 1.593 2.735 25.585
Deformation energy W (10-4 J) 162.709 ± 45.580 2077.606 28.014
Extensibility L (mm) 105.100 ± 14.138 199.884 13.451
Resistance P (mm) 54.05 ± 10.816 116.997 20.012
P/L 0.537 ± 0.138 0.019 25.710

We can notice important modifications of the variability coefficients for some

enzymatic treated flours, compared with the control flours, which have not been treated.

This is the result for applying different treatments with enzyme. The parameters which

registered significant modifications of the variability coefficients (Fisher test) were:

protein (F = 4. 714**), gluten deformation (F = 0.005***), the alveogram extensibility L (F

= 2.934*), and the P / L (F = 6.894***) ratio.

5
 The average values for the quality parameters emphasized statistical differences

(t test) between the control flours and the improved flours, according to table 4.

Table 4. The significance of the differences between the control flours’ and the

improved flours’ quality parameters

Parameter Control Improved t Significance

Flour Flour level
Moisture (%) 14.052 13.753 2.560 *
Proteins (%) 9.690 9.487 2.318 *
Wet gluten (%) 26.457 25.675 2.365 *
Gluten deformation (mm) 5.722 10.970 3.499 **
Hydration capacity (%) 56.358 56.625 0.689 ns
Stability (min) 8.000 6.150 3.180 **
Deformation energy W (10-4 J) 220.000 162.708 3.272 **
Extensibility L (mm) 92.160 105.100 1.237 *
Resistance P (mm) 70.980 54.005 4.590 ***
P/L 0.848 0.537 4.405 ***
(* significant; ** distinctly significant; *** very significant)

We can notice that the gluten deformation index has increased distinctly

significant for the improved flours (t=3.499**); stability (t=3.180**) and deformation

energy W (t=3.272**) decreased distinctly significant for the improved flour, resistance P

and the P/L report decreased very significantly for the improved flours (t=4.590*** and

respectively t=4.405***). Moisture, protein, wet gluten and extensibility L, showed only

significant differences between the flours, according to table 4.

We appreciate that the least sensitive quality parameter in the proteasic activity

was the hydration capacity, while the enzyme’s specific action determined a distinctly

significant decrease of the breaking resistance. The very significant decrease of the total

6
quantity of energy absorbed by the dough in stretching (W), was realized on the

decreased of the breaking resistance (P).

Analysing the correlation between the quality parameters of the enzymatic treated

flours and the used quantity of enzymatic conditioner, we observed interesting aspects.

Although, there were significant differences between some quality parameters of the

control and improved flours, these did not always correlate with the used enzymatic

conditioner quantity. This proves that some differences shown in table 4 are due to the

interaction of the improving agent with the characteristics of each flour. The effect of the

added enzyme can be estimated taking into consideration the flours’ lots individuality,

with the help of the correlation coefficients and the regression curves, in order to interpret

the improving effect by a general statistic law.

Table 5 presents the significant correlation between the used enzymatic

conditioner quantity and some physical-chemical and rheological parameters of the

flours, and also the adequate regression equations. We mention that we used different

regression models, in order to obtain the best determination coefficients (R2). Thus, we

used the linear regression equation for gluten’s deformation index, extensibility (L) and

hydration capacity, depending on the enzymatic conditioner quantity. For stability-

enzymatic conditioner quantity relationship, we used the exponential regression equation

and for energy (W) – conditioner quantity dependence, we used the logarithmic

regression equation.

Table 5. Correlation and regressions between flours’ quality parameters and enzymatic

conditioner quantity, used for improvement

Parameter - Correla- Determi-

Enzymatic conditioner tion coe- Regression nation Signifi
quantity (x) fficient r equation coeffi- cance

7
 cient R2
Gluten deformation-x 0.862 y = 4.400 + (0.032 x) 0.744 ***
Stability-x -0.843 y = 8.287e-0.0016 x 0.711 **
Energy W-x -0.508 y = -36.254 ln(x)+335.82 0.258 *
Extensibility L-x -0.544 y = 123.460 + (-0.140 x) 0.296 *

We also show the significant correlation coefficients and the respective regression

equations, for the differences ∆ , registered between the quality parameters for improved

flours, and the quality parameters for control flours, depending on the enzymatic

conditioner quantity (table 6). We mention that we used the following types of regression

equations: logarithmic equations for the difference ∆ of gluten deformation, resistance P

and energy W values, between improved flour and control flour, in relationship with

enzymatic conditioner quantity; polynomial equation for the difference ∆ , between the

P/L ratio, concerning improved and control flours, in relationship with Veron W quantity.

Table 6. Correlation and regressions of ∆ (difference between improved flours and

control flours quality parameters), related to the quantity of enzymatic conditioner

Difference parameters Correla- Determi Sig-

(∆ ) - tion Regression -nation nifi-
Veron W quantity (x) coeffi- equation coeffi- cance
cient r cient R2
∆ Gluten deformation- 0.815 y = 8.717 ln(x)-38.781 0.664 **
x
∆ Energy W-x 0.696 y = -36.357ln(x)+72.876 0.0.484 *
∆ Resistance P-x 0.870 y = -8.626 ln(x)+21.44 0.758 ***
∆ P/L-x 0.774 y = -2E-0.5 x2+0.002 x- 0.599 **
0.0073

8
 We notice a very significant positive correlation (r=0.862***) of gluten

deformation index with the quantity of enzymatic conditioner (figure 1). The

determination coefficient R2 shows that the proteolytic activity, very significantly

different in the enzymatic treated flours in comparison with the control flours, is due

75 % to added protease and only in a small extent, to the activity of some internal factors

of the flour.
Gluten deformation

y=0.0322x+4.3994
25
R 2 =0.7436
20
(mm)

15
10
5
0
0 200 400 600 800
VeronW(ppm)

Figure 1. Gluten deformation regression, depending on Veron W quantity

The transgression probability of the regression: deformation index-enzymatic

conditioner quantity, is smaller than 0.01 %, which means that the value of this parameter

can be very strictly controlled by protease addition.

The regression equation for the difference of gluten deformation index ∆ G

between enzymatic treated and control flours, related to the quantity of enzymatic

conditioner (equation presented in figure 2), strengthened the significant influence of the

9
enzyme upon this parameter (r=0,815**). At the same time, the value of gluten

deformation index in control flours, as indicator of their initial proteasic activity, must be

taken into consideration at the time of dosing the enzymatic conditioner, because this

value influences decisively the difference between the improved and the control flours.

y = 8.717 ln(x) - 38.781

Gluten deformation

20
difference (mm)

R2 = 0.6645
15

10

0
0 200 400 600 800
Veron W (ppm)

Figure 2. Regression of gluten deformation index difference ∆ G, between

improved and control flour, depending on Veron W quantity

The increasing of enzymatic conditioner’s quantity determined a distinctly

significant decreasing of the stability farinogram parameter (r = -0.843**), in proportion

of over 71 % (R2 = 0.71), as seen in figure 3.

Stability (min)

10
y = 8.287e -0.0016x
8
R2 = 0.7113
6
4
2
0
0 200 400 600 800
Veron W (ppm)

10
 Figure 3. Regression of dough’s stability, depending on Veron W quantity

The quantity of enzymatic conditioner did not influence significantly the

difference between the dough’s stability, for improved flours in comparison with control

flours (r = 0.285).This fact shows, that the effect of the added enzyme upon the dough’s

stability, does not depend decisively on the control flours’ initial stability. The correlation

of the stability, between the control flour and the improved flour, was also insignificant

(r = 0.369).

The energy W absorbed by the dough can be decreased by protease addition

(r=-0.508*), the enzyme influencing in proportion of 26 % the energy W parameter value,

in the improved flours (figure 4).

y = -36.254 ln(x) + 335.82

300
R2 = 0.2579
250
200
W (E-4 J)

150
100
50
0
0 50 100 150 200 250
Veron W (ppm )

Figure 4. Regression of dough’s energy W, depending on Veron W quantity

Decreasing of the energy quantity is due to the enzyme’s attack upon the peptidic

bonds in dough, concomitantly with a decreasing of its extensibility and breaking

resistance.

The significant correlation is also kept for dependency of the energy differences

∆ W, between improved and control flours, with the used quantity of enzymatic

11
conditioner (figure 5). The added enzyme contributed with approximate 48 % to the

modification of this difference (r = 0.696*).

y = -36.357ln(x) + 72.876
50
R2 = 0.4845
W differance (E-4 J)

0
0 50 100 150 200 250
-50

-100

-150

-200
Veron W (ppm)

Figure 5. Regression of energy W difference ∆ W, between improved and

control flour, depending on Veron W quantity

The enzyme’s action upon the extensibility of the dough has been determined in

proportion of 29.6 % and consisted in reducing its value, while the enzyme’s quantity

increased (figure 6).

150 y = -0.1401x + 123.46

R 2 = 0.2959
L (mm)

100

50

0
0 50 100 150 200 250
12Veron W (ppm)
 Figure 6. Regression of extensibility L, depending on Veron W quantity

We did not register significant correlation neither between the extensibility

parameter L for the control flours, respectively for the improved flours, nor between the

extensibility’s differences ∆ L and the used enzyme’s quantity.

Although, in improved flours, the average value of the extensibility L increased

significantly in comparison with the control flours (see table 3), this fact did not correlate

significantly with the added enzymatic conditioner quantity. We even noticed that, as the

added enzymatic conditioner quantity increased over a limit, there was a reducing of the

extensibility. An explanation in this sense could be due to the indirect effects of the

enzyme. So, the enzymatic proteolysis of the dough, activated several oxide-reduction

processes at the -SH groups’ level (by releasing sulfur aminoacids, through

conformational modifications able to set free the -SH groups of the proteins). The

peptides bonds’ splitting up, could be balanced or contrabalanced by the forming of

disulphydic bridges between protein remains. The intensity of the redox processes in

dough depends on the nature of the control flour, the added enzyme having the tendency

to unblock this potential. An argument in this sense is the negative correlation of the

stability parameter in the control flours, with the extensibility parameter L, for improved

flours (r = -0.540*), as we know that the more stable flours present preponderantly

oxidize activities.

We did not register significant correlation between the breaking resistance (P) of

the improved flours and the used enzymatic conditioner quantity, although this parameter

presented distinct significant differences in comparison with the control flours. The

13
protease addition contributed with 75.8 % to the decrease of the breaking resistance (P) in

improved flours, in comparison with the control flours, as seen in figure 7.

P difference (mm)

y= -8.6258 ln(x) + 21.44

10
R 2 = 0.7577
0
-10 0 50 100 150 200 250
-20
-30
-40
VeronW(ppm)

Figure 7. Regression of breaking resistance P difference ∆ P, between improved and

control flour, depending on Veron W quantity

As regards the P/L ratio, we noticed a significant decrease, while the added

protease quantity increased. The ∆ P/L difference between the enzymatical treated flour

and the control flour increased significantly, while the Veron W quantity increased, as

seen in figure 8. This difference increasing was realized especially as the breaking

resistance (P) decreased.

0
P\L difference

-0.2 0 50 100 150 200 250

-0.4
-0.6
-0.8 2
y = -2E-05x + 0.0021x - 0.0734
-1 2
R = 0.599
Veron
14 W (ppm)
 Figure 8. Regression of the P/L ratios difference ∆ P/L , between improved and

control flour, depending on Veron W quantity

In order to illustrate more suggestive, the protease addition effect upon the main

farinographic and alveographic quality parameters previously discussed, we presented the

results for the improvement of a control flour lot with 170, 190 and respectively 210 ppm

enzymatic conditioner. So, figures 9 – 14 mark out the whole range of laboratory tests,

and confirm the statistical estimations previously displayed.

Figure 9. Farinogram of improved flour with 170 ppm Veron W

Figure 10. Farinogram of improved flour with 190 ppm Veron W

Figure 11. Farinogram of improved flour with 210 ppm Veron W

Figure 12. Alveogram of improved flour with 170 ppm Veron W

Figure 13. Alveogram of improved flour with 190 ppm Veron W

Figure 14. Alveogram of improved flour with 210 ppm Veron W

In figure 15, we notice that certain farinographic parameters (stability, dough’s

development) decreased substantially, while the enzymatic conditioner quantity used for

improving the flour, increased. The gluten deformation index had a different behaviour,

as it increased with the increasing of the enzymatic conditioner quantity. All these

observations confirm the statistical estimations realized at total level, for the 26 tests (26

flour samples).

Gluten deformation (mm) 210 ppm

190 ppm
Stability (min)
170 ppm
Development (min)

0 5 10 15 20

15
 Figure 15. Evolution of main farinographic parameters,

depending on Veron W quantity

Regarding the alveographic parameters, the behaviour at the protease additon was

less uniform (figure 16).

200

150

100

50

0
P (m m) L (mm) W (10E-4j)

170 ppm 190 ppm 210 ppm

Figure 16. Evolution of main alveographic parameters,

depending on Veron W quantity

So, the parameter energy W showed a light decrease, almost non significant, at

the improvement with 190 ppm, towards 170 ppm, followed by a sudden large decrease

at the improvement with 210 ppm. The extensibility parameter L registered a relative

uniform decrease, as the enzymatic conditioner quantity increased. For the resistance

parameter P, the increase on the 170 – 190 ppm interval was followed by a significant

decrease, when treated with 210 ppm enzymatic conditioner. The decrease of the

alveogram’s P parameter was notable, in this last case the parameter’s value decreased

under the initial one.

Conclusions

16
 1. The most sensitive parameters for the proteasic action, tested with “t” test,

were: gluten deformation index, stability, energy W, resistance P and P/L index. Their

values registered distinct significant and very significant differences in improved flours,

in comparison with the control flours. Moisture, protein, wet gluten quantity and

extensibility of the dough made from improved flour, were only significantly different in

comparison with the control flours. Least sensitive parameter at the protease treatment

was hydration capacity, which did not differ significantly between the control and the

improved flours;

2. The value of the gluten deformation index parameter in improved flours, can be

controlled per the protease addition, by the regression equation, taking into consideration

the starting value of this parameter in the control flour;

3. The value of the farinographic stability parameter in improved flours,

correlated negatively distinctly significant with the enzymatic conditioner quantity. The

enzyme’s action upon this parameter was not significantly influenced by the starting

value of the stability parameter in the control flour;

4. The value of the alveographic energy parameter (W) in flours improved with

Veron W conditioner, can be anticipated starting from the initial value of this parameter

in the control flours, and also from the used enzymatic conditioner quantity, relied on the

regression equations;

5. The regression calculated equations, also allow the control of diminishing the

resistance parameter P, respectively the ratio P/L, for the improved flours in comparison

with control flours;

17
 6. The total statistical estimations, obtained at the level of 26 tested flours, were

confirmed by the rheological tests’ cycle, which consisted in different improvement, on 3

ppm levels, with Veron W, of the same control flour, as follows:

- farinogram’s stability and development parameters, registered significant

decreases, as the enzymatic conditioner’s quantity increased, while the gluten

deformation index increased significantly;.

- the extensibility L and the energy W parameters of the alveogram, decreased

significantly as the Veron dosage increased from 170 to 210 ppm, while the resistance P,

registered a light increase at 190 ppm enzymatic conditioner’s addition and a subsequent

decrease at the 210 ppm enzymatic conditioner’s addition.

References

1. Jurcoane, Şt., Săsărman, E., Lupescu, I., Roşu, A., Berehoiu – Tamba, R., Banu,
A., Rădoi, F., 2004, Tratat de Biotehnologie, vol I, Ed. Tehnică, Bucureşti.
2. Poutanen, K., 1997, Enzymes: An important tool in the improvement of the quality of
cereal foods, Trends in Food Science & Technology, 8.
3. STAS 90-88, Făină de grâu, Metode de analiză.
4. ICC-Standard No. 121, Method of using of the Chopin-Alveograph.
5. ICC-Standard No. 115/1, Farinograph.

18