Comparison of Mixolab and Falling Number
Comparison of Mixolab and Falling Number
Ab s t r a c t . Falling number method and amylograph test are methods widely used to determine
the alpha-amylase activity of wheat flour. By comparison, mixolab might be used to measure dough
characteristics such as protein quality, starch gelatinization, enzymatic activity and starch retrogra-
dation in a single test. The objective of this study was to determine the variability of, and relation-
ship among falling number, amylograph properties and mixolab parameters which characterised
starch complex. Test was performed on 76 samples of wheat flour type 550 and type 750 produced
in Poland. Mixolab parameters which characterise starch gelatinisation (C3), amylolytic activity
(C4) and starch retrogradation (C5) allowed to better differentiate types of flour than falling number
method or amylograph test. Falling number, maximum viscosity and final temperature of gelatinisa-
tion were positively correlated with starch gelatinisation (C3), amylolytic activity (C4, difference of
the points C3C4) and starch retrogradation (C5, difference of the points C5C4).
K e y w o r d s : alpha-amylase activity, wheat flour, amylograph, falling number, mixolab
INTRODUCTION
Wheat flour, annual production of which in Poland is approx. 2.2 million tons
(GUS 2013), is classified into eight types based on ash content, according to the
Polish Standard PN-A-74022:2003. Two types of flour are most important in the
baking and pastry industries. Wheat flour type 550 is used for the production of
bread and pastry products. Wheat flour type 750 is used for the production of
mixed bread, wafers and sticks.
The technological properties of flour and the quality of bread depend on both
protein and starch characteristics. Starch quality is related to the amount of starch,
starch damage and the activity of amylolytic enzymes of flour (Đurić et al. 2008,
494 A. SZAFRAŃSKA
Koksel et al. 2009). Starch damage affects the ability of flour to absorb water
during dough formation, and the ability to produce carbon dioxide in the fermen-
tation process. Higher starch damage is required for bread production, lower for
pastry production (Dubat 2004). Amylolytic activity of flour influences the quali-
ty of bread. The falling number method and the amylograph test are methods
commonly used to estimate alpha-amylase activity of flour by millers and bakers
(Rothkaehl 2003, Papouškova et al. 2011). Bread baked from wheat flour with too
low alpha-amylase activity (falling number over 300 s, maximum viscosity over
800 AU) has diminished loaf volume, pale crust and dry crumb. Bread baked
from wheat flour with too high alpha-amylase activity (falling number below 150
s, maximum viscosity below 100 AU) is characterised by low volume, dark crust
and crumb, sticky crumb and large holes inside.
The rheological properties of a dough determined by mixolab are recorded on a
graph. Measuring the consistency of a dough over time with gradual increase in the
applied temperature enables the user to determine the quality of wheat flour and
provides, in a single test, information on the protein and starch characteristics
(dough development time, protein breakdown, starch gelatinisation, starch retrogra-
dation, enzymatic activity) and interaction between these features (Dubat 2010).
The use of results obtained by mixolab requires much research and compari-
son with the results of traditionally used quality parameters (Codina et al. 2010,
Banu et al. 2011, Szafrańska 2011). In recent years, a growing interest in the pos-
sibilities of using mixolab in research has been seen. Most of the research has
been done in scientific units outside of Poland. The growing interest in mixolab
by milling and baking industries means a need to adapt the interpretation of its
results for wheat flours produced in Poland. For this purpose, two types of wheat
flour produced in Poland were analysed using the starch damage, falling number
and amylograph properties. The data were compared to those obtained from mix-
olab in order to establish some predictive models for evaluating the falling num-
ber and amylograph parameters as a function of mixolab values.
Materials
Thirty eight samples of commercial wheat flour type 550 and thirty eight
samples of wheat flour type 750 (harvest 2009 and 2010) were collected from
nineteen milling companies located in Poland. The ash content varied between
0.45 and 0.63 for tested wheat flour type 550 and between 0.58 and 0.85 for
wheat flour type 750 (Tab. 1).
COMPARISON OF ALPHA-AMYLASE ACTIVITY OF WHEAT FLOUR 495
Methods
For each wheat flour sample, the following qualities were determined: ash
content (PN-EN ISO 2171), particle size distribution (PN-A-74015), falling num-
ber (PN-EN ISO 3093), starch damage (Górniak 2006). Wheat flour colour was
evaluated using Minolta Chroma Meter Cr-310. The flour colour results were
reported in terms of 3-dimensional colour values based on L*, a*, b*. The amylo-
lytic properties of flours (maximum viscosity, initial temperature of gelatinisation,
final temperature of gelatinisation) were determined by Brabender amylograph
(PN-ISO 7973). Rheological properties of a dough were studied using Chopin
mixolab (Dubat 2010). The protocol has the following settings: mixing speed 80
rpm, total analysis time 45 min, dough weight 75.0 g, tank temperature 30°C,
hydratation water temperature 30°C. Flour and water were added accordingly to
obtain 75 g of dough with a maximum consistency of 1.10 Nm (±0.05) during the
first test phase. Mixolab test was performed using standard protocol: 8 min at
30°C, heating at a rate 4°C min-1 for 15 min, holding at 90°C for 7 min, cooling to
50°C at a rate 4°C min-1 for 10 min and holding at 50°C for 5 min. A typical mix-
olab curve (Fig. 1) is divided into five different phases: phase 1, initial kneading;
phase 2, protein weakening; phase 3, starch gelatinisation; phase 4, cooking sta-
bility; phase 5, starch retrogradation (Rosell et al. 2007).
Fig. 1. Typical mixolab curves of wheat flour type 550 and type 750
496 A. SZAFRAŃSKA
The parameters that are obtained from the curve are water absorption (%),
dough development time (time T1, min), dough stability (min), protein weakening
(C2, N m), starch gelatinisation (C3 and the difference between points C3 and C2,
abbreviated C3C2, N m), amylolytic activity (C4 and the difference between
points C3 and C4, abbreviated C3C4, N m), starch retrogradation (C5 and the
difference between points C5 and C4, abbreviated C5C4, N m). Time and dough
temperature for C2 (time T2, min; D2, oC) and for C3 (time T3, min; D3, oC) as
well as curve slopes between C2 and C3 (slope β, Nm min-1) and between C3 and
C4 (slope γ, Nm min-1) were also recorded.
Statistical analysis
Y = b0 + b1 · x1 + b2 · x2 + … + bn·xn (1)
where b0 is a constant value and bi, i = 1,…,n, are the regression coefficients of
the predictive model (Tab. 7). The predictive models were well correlated with
the measured data and were significant at p = 0.05. Student’s t-test was used to
determine the significance of each coefficient in the regression model.
Flour samples were obtained from wheat from 2009 and 2010 crops in which
grain was characterised by low alpha-amylase activity. Therefore the data was not
characterised according to the year of production of flour. Tested flours were
characterised in terms of particle size distribution, flour colour and parameters
COMPARISON OF ALPHA-AMYLASE ACTIVITY OF WHEAT FLOUR 497
Table 1. Ash content, particle size distribution and flour colour of tested wheat flours
ab – Mean values marked by different letters differ significantly at p < 0.05 (*) and p < 0.01 (**),
respectively.
Flour colour often affects the colour of the final product and therefore is one
of many wheat flour quality parameters included in specifications required by
end-users. Flour colour is related to the type of wheat, bran layer content, bran
colour, moisture content and particle size distribution (Jankowski 1981). Tested
wheat flours were characterised by high brightness, L*, in the range of 88-92,
which is required for production of pastry and many assortments of bread. Wheat
flour type 550 had a significantly higher brightness than wheat flour type 750
(Tab. 1), due to lower content of bran particles. The a* values, with all measure-
ments below zero, confirm that the green tone was dominating over the red in
tested wheat flours. Significantly more expressed green tone was detected for
wheat flour type 750. The b* values, with all measurements above zero, confirm
that yellow tone was dominating over the blue in tested wheat flours.
498 A. SZAFRAŃSKA
The falling number test is a relatively simple and rapid method of estimation
of alpha-amylase activity in flours. Optimal alpha-amylase activity of wheat
flours for baking purposes should be on the average level (falling number in the
range of 220-280 s) (Jurga 2003). Tested wheat flours were characterised by low
alpha-amylase activity (falling number in the range of 288-476 s) (Tab. 2). These
results agree with the observations of Cichoń and Ptak (2005) and Achremowicz
et al. (2010) who indicated medium and low alpha-amylase activity of wheat
flours produced in Poland.
Amylograph test measures the effect of alpha-amylase on the peak viscosity
of a cooked flour slurry. It requires a sample size of approx. 80 g of flour and
each test has a running time of 45 min. There were no significant differences in
the values of maximum viscosity of tested wheat flours. Gelatinisation tempera-
ture is an important factor determining the technological quality of starch (Shuey
and Tipples, 1994). Wheat flour type 550 was characterised by lower initial tem-
perature of gelatinisation and higher final temperature of gelatinisation than wheat
flour type 750 (Tab. 2).
Table 2. Falling number, amylograph properties and starch damage of tested wheat flours
Initial temperature of
58.5a** 57.5 59.5 58.8b** 57.0 60.0
gelatinisation (oC)
Final temperature
86.1b* 78.5 91.5 84.2a* 78.0 91.0
of gelatinisation (oC)
ab – Mean values marked by different letters differ significantly at p < 0.05 (*) and p < 0.01 (**),
respectively.
The level of starch damage directly affects the water absorption and the dough
mixing properties of flour and has a strong influence on dough preparation and
baking process. The optimum damage starch value varies with the use of the flour
and is greatly dependent upon the flour protein content, the alpha-amylase activity
and the type of bread to be made from the flour (Dubat 2004, Górniak 2006).
COMPARISON OF ALPHA-AMYLASE ACTIVITY OF WHEAT FLOUR 499
Starch damage in tested wheat flours was in the range of 4.9-16.0 and was signifi-
cant dependent on the type of flour (Tab. 2).
Rheological properties of dough was measured using mixolab. For this study
only mixolab parameters related to the characteristics of starch were shown.
Wheat flour type 550 was characterised by significant higher torque in points C3
(starch gelatinisation), C4 (amylolytic activity) and C5 (starch retrogradation)
than wheat flour type 750 (Tab. 3). These results agree with the observations of
Codina et al. (2010) who indicated a higher torque in point C3 in flours with low-
er ash content. Ash content determined in wheat flour type 550 and type 750 was
negatively correlated with the torque in point C3 (r = –0.431 and r = –0.504, re-
spectively) (Tab. 5 and Tab. 6).
Table 3. Mixolab characteristics (torque in chosen points and slopes) of tested wheat flours
ab – Mean values marked by different letters differ significantly at p < 0.05 (*) and p < 0.01 (**),
respectively.
cosity showed a stronger connection with the torque in points C3, C4 and C5 than
falling number (Tab. 5 and Tab. 6). The reasons for the differences should be seen
in the methodology for determining both parameters. During amylograph and
mixolab test there is a gradual heating of the sample, similar to the conditions
inside bread dough during baking. The final temperature of gelatinisation of
wheat flour type 550 and type 750 determined by amylograph was positively cor-
related with mixolab parameters C3, C4 and C5, with correlation coefficients
ranging from 0.576 to 0.802 (p<0.01) and from 0.349 to 0.795 (p<0.01), respec-
tively (Tab. 5 and 6).
The results obtained by Koksel et al. (2009) and Banu et al. (2011) indicate
that the difference between the torque in points C3 and C2 (abbreviated C3C2),
C3 and C4 (C3C4), C5 and C4 (C5C4) better characterised changes in the proper-
ties of a dough than analysing these parameters separately. The difference be-
tween points C3C2, which is associated with starch gelatinisation, decreased with
increase in the ash content in wheat flour type 550 and type 750 (r = –0.458 and
r = –0.496, respectively). The difference C3C2 of wheat flour type 550 was posi-
tively correlated with falling number and maximum viscosity (Tab. 5). The differ-
ence between the points C3C4, which is defined as an enzyme activity, was nega-
tively correlated with falling number (r = –0.643 and r = –0.835, respectively),
maximum viscosity (r = –0.698 and r = –0.853, respectively) and final tempera-
ture of gelatinisation (r = –0.769 and r = –0.892, respectively) (Tab. 5 and Tab. 6).
The difference between points C5C4, which is related to staling of bread and may
be a determinant of the shelf life of bread (Papouškova et al. 2011), was positive-
ly correlated with falling number (r = 0.727 and r = 0.782, respectively), maxi-
mum viscosity (r = 0.861 and r = 0.823, respectively) and final temperature of
gelatinisation (r = 0.706 and r = 0.794, respectively).
Dough temperature, which is measured at each point of mixolab curve, allows
to get information about initial temperature of gelatinisation (D2) and final tem-
perature of gelatinisation (D3) (Szafrańska 2013). Initial and final temperature of
gelatinisation measured by mixolab was lower than that measured by amylograph,
by 4.5oC and 5.5oC, respectively, because of the differences in consistency of
tested material (Tab. 4). The consistency of a dough tested by mixolab is approx.
500 BU while the amylograph test measures a flour-and-water slurry.
Mixolab measures the time required to obtain a torque in characteristic points
of a curve. Time T2 is related to time in which starch begins to gelatinise, where-
as time T3 is related to the end time of gelatinisation. Difference between these
two parameters is connected to gelatinisation time (abbreviated Time T3T2) and
in tested wheat flours was in the range of 6.0 to 8.7 min (Tab. 4). The gelatinisa-
tion time (Time T3T2) was significantly shorter for wheat flour type 550, with
higher starch damage. Gelatinisation time determined by mixolab was shorter
COMPARISON OF ALPHA-AMYLASE ACTIVITY OF WHEAT FLOUR 501
Table 5. Correlation coefficients between ash content, falling number, amylograph parameters and
chosen mixolab characteristicsa of tested wheat flour type 550
Initial Final
Ash Falling Maximum
temperature temperature
content Number viscosity
of gelatinisation of gelatinisation
C3 –0.431** 0.377* 0.502** 0.576**
C4 0.582** 0.681** 0.763**
C5 0.704** 0.829** 0.802**
C3C2 –0.458** 0.338* 0.470** 0.490**
C3C4 –0.643** –0.698** –0.769**
C5C4 0.727** 0.861** 0.706**
γ slope
Temperature D2
Temperature D3
Time T2 0.384*
Time T3 0.380* 0.513** 0.529**
Time T3T2 0.409* 0.549** 0.595**
a
*, ** Correlation coefficients significant at p < 0.05 and p < 0.01, respectively.
502 A. SZAFRAŃSKA
Table 6. Correlation coefficients between ash content, falling number, amylograph parameters and
chosen mixolab characteristicsa of tested wheat flour type 750
In this study a multiple procedure indicated that there was more than one signifi-
cant independent variable. The relationships between the studied parameters are given
in Table 7. The regression equation models for falling number contain two mixolab
variables: starch gelatinisation (C3) and starch retrogradation (C5) for wheat flour
type 550, and four mixolab variables: C3, C5, slope β and time T3 for wheat flour
type 750. The coefficients of all variables are statistically significant. The regression
models obtained accounted for, respectively, 59.6 and 81.9% of the variance in the
dependent variable falling number (Tab. 7). The standard errors of estimation show
the standard deviation of the residuals to be 24 and 17 s, respectively.
The models for maximum viscosity explain 79.4 and 86.7%, respectively, of
the variation of maximum viscosity variable of wheat flour type 550 and type
750. These models contain such mixolab parameters as starch gelatinisation (C3)
and starch retrogradation (C5), slope β, slope γ and time T3 (Tab. 7). The stand-
ard errors of estimation show the standard deviation of the residuals to be 105 and
78 AU, respectively.
The regression models for final temperature of gelatinisation include three
mixolab variables: dough torque in points C3, C5 and slope β. The values of the
determination adjusted coefficient show that the models explain 74.7 and 85.6%,
COMPARISON OF ALPHA-AMYLASE ACTIVITY OF WHEAT FLOUR 503
Table 7. Equations for the empirical relations between falling number, maximum viscosity, final
temperature of gelatinisation and parameters of the mixolab curve
Type
Dependent
of wheat Regression equation R2adjusted
variable
flour
CONCLUSIONS
1. Wheat flour types tested in this study were characterised by low alpha-
amylase activity. According to the results of falling number and maximum viscos-
ity there was no significant difference between the alpha-amylase activity of test-
ed wheat flour types.
2. Rheological properties of dough measured by mixolab: starch gelatiniza-
tion (C3), amylolytic activity (C4) and starch retrogradation (C5) were different
in tested wheat flour types.
3. Wheat flour type 550 was characterised by significant lower initial tem-
perature of gelatinisation than wheat flour type 750, determined both by amylo-
graph and mixolab. Gelatinisation time (Time T3T2) was different between the
flour types and was positively correlated with falling number, maximum viscosity
and final temperature of gelatinisation.
4. Statistical analysis on wheat flour type 550 and type 750 using multiple
regression method showed that mixolab values can be used to elaborate predictive
models for estimating values of the falling number (R 2adjusted = 0.596 and 0.819,
respectively), maximum viscosity (R2adjusted = 0.794 and 0.867, respectively) and
final temperature of gelatinisation (R2adjusted = 0.747 and 0.859, respectively).
504 A. SZAFRAŃSKA
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