Journal of Cereal Science 111 (2023) 103673

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Journal of Cereal Science

journal homepage: www.elsevier.com/locate/jcs

Improving microbial quality of wheat flour using ethanol-water mixture as

a tempering solution
Pervin Ari Akin a, b, Emine Kübra Tayyarcan b, Şefika Evran b, Ismail Hakki Boyaci b, *
a
Field Crops Central Research Institute, Republic of Turkey Ministry of Agriculture and Forestry, 06170, Ankara, Turkey
b
Department of Food Engineering, Hacettepe University, Beytepe, 06800, Ankara, Turkey

A R T I C L E I N F O A B S T R A C T

Keywords: Wheat flour may contain high microbial load due to the contamination of the wheat surface by different sources.
Wheat This situation leads to outbreaks of foodborne diseases caused by wheat and wheat-based products. In this
Ethanol research, a novel application has been developed to reduce microbial contamination at the tempering stage of
Tempering
wheat. The effect of water with different concentrations of ethanol (10–90% (v/v)) against the microbial load of
Microbial
Quality
wheat was evaluated. Although 70% ethanol is widely used as an antimicrobial agent, the significant reduction in
aerobic plate count (APC) and yeast-mold count (YMC) in wheat was 5.02 ± 0.20 log CFU/g and 5.96 ± 0.22
after tempering with 50% ethanol and above, respectively. The mechanism for the antimicrobial effect of 50%
ethanol is that wheat absorbs water faster than ethanol, causing a rise in ethanol concentration on the grain
surface over time. The chemical and technological properties of wheat flour were also investigated at 50%
ethanol concentration, which showed the highest microbial reduction. This study shows adding ethanol at the
tempering stage reduces the microbial load of wheat while maintaining some functional quality of the flour
without requiring any treatments or time.

1. Introduction (Berghofer et al., 2003).

Wheat (Triticum aestivum) plays an important role in feeding and
providing food security around the world. Wheat is generally processed
into flour, which is utilized in the production of bread, pasta, and other
bakery products. Since wheat products have a unique taste as well as a
light and leavened texture, they are universally popular as a food.
Due to the lower water content, wheat has been assumed as a
microbiologically safe food. Although wheat contains low moisture,
studies have shown that microorganisms can survive in a dormant state
for a long period of time and when they find a more suitable environ­
ment (e.g., batter or mixes), they can emerge from dormancy (Feng and
Archila-Godinez, 2021). Recent studies and reports show that flour
contamination may be more frequent and at higher levels than what was
previously known. Studies have shown that Salmonella and Escherichia
coli outbreaks can be traced back to cereal-based products (Forghani
et al., 2019). Since 2009, six foodborne outbreaks in Canada and the
United States have been connected to wheat flour and other flour-based
products, infecting almost 200 individuals with Salmonella and Escher­
ichia coli (Feng and Archila-Godinez, 2021). As expected, the microbial
quality of wheat flour is closely linked to the original wheat grain
Research has shown that milling was significantly effective in
reducing microbial contamination to a certain level since the bran
contains high concentration of microbial contaminants (Sabillón et al.,
2016a, 2016b). However, some microorganisms and molds can remain
in flour and have an important effect on the quality and safety of milling
products (Rose et al., 2012).
By tempering with water, the endosperm of wheat can be separated
from its bran. Tempering plays an important role in the quality of wheat
flour. Tempering wheat with lactic acid (5.0%) and NaCl (1.0% wheat
basis) caused almost 3.0–4.5 log CFU/g reductions for aerobic plate
count (APC) and Enterobacteriaceae (Eb). To the contrary, no significant
differences in the functionality of refined wheat flour and whole-wheat
flour were found by tempering wheat with NaCl and lactic acid (Sabillón
et al., 2016a, 2017). To temper the wheat grains, different levels of
active chlorine and slightly acidic electrolyzed water were used. The
most effective active chlorine concentration was 70 mg/L since it caused
0.93 log CFU/g in total plate count and 0.78 log CFU/g reduction in
yeast and mold count (Chen et al., 2020). The food industry uses chlo­
rine since it has an antimicrobial effect on food and sanitizes equipment.
However, chlorine produces trihalomethanes (THMs) and carcinogenic

* Corresponding author.
E-mail address: ihb@hacettepe.edu.tr (I.H. Boyaci).

https://doi.org/10.1016/j.jcs.2023.103673
Received 7 June 2022; Received in revised form 3 March 2023; Accepted 24 March 2023
Available online 26 March 2023
0733-5210/© 2023 Elsevier Ltd. All rights reserved.
P. Ari Akin et al.
compounds such as haloacetic acids and bromate ions (Chittrakorn,
2008). As an alternative to chlorine, the effectiveness of ozone was
studied. Wheat samples with ozonated water were milled to straight
flour and the total bacteria and yeast/mold in refined flour were
investigated. Tempering wheat with ozonated water at 11.5 ppm caused
1.0–2.0 log CFU/g reductions for total bacteria and yeast and mold
counts (YMC). The refined wheat flour, processed from wheat tempered
with ozonated flour up to 11.5 mg/L, had not significantly different
Farinograph and Extensograph characteristics compared to the control
(İbanoǧlu, 2001). Ozone can be used as a tempering agent in wheat to
decrease the microbial load. However, corrosion-resistant equipment is
a requirement to work with ozone, which increases the operational
costs. In addition, it is difficult to disseminate the use of ozone in the
milling industry, since the dose workers need to be exposed to during a
shift is 0.1 ppm (Magallanes López and Simsek, 2021).
Ethanol has been used as a disinfectant in the food industry; how­
ever, it shows the bactericidal effect with water. Especially optimum
cidal activity of ethanol is 60–90% solutions in water (v/v). The cidal
activity of ethanol drops sharply when the ethanol concentration is
below 50%, (v/v) (Centers for Disease Control and Prevention, 2016).
However, low ethanol concentrations in water can be increased on
biomaterial surfaces due to the difference in absorption rates of ethanol
and water in some biomaterials. For instance, ethanol adsorption rate in
wheat flour was found slower than water adsorption rate in wheat flour
(Bushuk and Winkler, 1957). When wheat grains interact with the
ethanol-water mixture, water is likely to be absorbed faster than ethanol
into the wheat kernel. Therefore, the concentration of ethanol in the
solution remaining on the surface of the wheat kernel increases. The
increase in ethanol concentration on the wheat surface is expected to
lower the microbial load on the kernel surface. In this method, in
addition to tempering, the number of microorganisms on the surface of
wheat is decreased or even sterilized during the tempering process.
While this technique improves the microbiological quality of wheat, it
should not have an adverse effect on the properties of tempered prod­
ucts. Consequently, it could be possible to produce flour and flour-based
products that pose no risk to food safety. However, no research has been
done on the use of ethanol to temper wheat prior to milling. The ob­
jectives of this study were to determine the effect of different ethanol
concentrations on microbial load on wheat, the diffusion coefficient of
ethanol and water in the wheat kernel, and the impact of ethanol
decontamination on the dough and bread quality. Thus, an innovative
approach has been developed to improve the microbial quality of wheat
flour without affecting the product quality.
2. Material and methods
2.1. Materials
Hard winter wheat samples with two different levels of microbial
loads (sample A-low microbial load and sample B-high microbial load)
were used to conduct this study. Sample A was obtained from the Central
Research Institute for Field Crops in Turkey and sample B was obtained
from a local source in Ankara, Turkey. Ethanol was obtained from Merck
KGaA (Darmstadt, Germany). Yeast (active dry) was purchased from Dr.
Oetker (Bilafat, Germany). Whey was provided by Gemici Gida Ticaret
Ltd. Şti. (Istanbul, Turkey). Ammonium phosphate and ascorbic acid
were purchased from Merck KGaA (Darmstadt, Germany). Shortening
was bought at B&G Foods (Roseland, NJ, USA). Salt, sucrose, and malt
syrup were obtained from local sources.
2.2. Preparation and application of tempering solution
To investigate the effectiveness of utilizing ethanol as an antimi­
crobial agent for wheat, various concentrations of ethanol were tested. A
100 ml of stock solution with ethanol and water was prepared at each
concentration in increments of 10% for the ratios between 10 and 90%.
2
Journal of Cereal Science 111 (2023) 103673
The amount of ethanol solution to temper wheat samples was deter­
mined based on optimum milling moisture and the initial moisture
content of Sample A and B which are 10.1 and 10.4%, respectively. As a
control, sterile distilled water was used. Then, each concentration of
ethanol solution was used to temper samples A and B overnight (18 h) to
15.5% moisture content, adhering to Method 26–95.01 (AACC Inter­
national, 2013). To achieve optimal tempering conditions, 63.9 mL of
water and 127.8 mL of 50% liquid ethanol solution were added to 1 kg of
wheat.
2.3. Microbiological analysis
2.3.1. Sample preparation procedures
Sterile, buffered phosphate diluent (225 ml) (BPW; Himedia, Italy)
was mixed with grain samples (25 g) in sterilized bags and homogenized
in paddle blenders (ECN 710–0873, Italy) for 90s (Sabillón et al.,
2016a). The suspensions were then diluted from 10− 1 to 10− 5 and
analyzed three times.
2.3.2. Analysis of microorganism counts
Aerobic plate count (APC): The spread plate method was used to
calculate plate count within the guidelines of AACC International
method 42–11.01 (AACC International, 2013) on plate count agar
(Oxoid Ltd., Basingstoke, Hampshire, England). The plates were incu­
bated at 35 ◦ C for 24–48 h. Duplicates were made as necessitated by
appropriate dilutions.
Yeasts and molds count (YMC): Yeast and molds were counted in
accordance with AACC International method 42–50.01 (AACC Interna­
tional, 2013) on Potato Dextrose Agar (Oxoid Ltd.) acidified with 10%
tartaric acid to pH 3.5 and Rose Bengal Chloramphenicol Agar Base
(Oxoid Ltd.) with chloramphenicol supplement (Oxoid Ltd.). The plates
were incubated at 22–25 ◦ C for five days. As necessitated, appropriate
dilutions were duplicated.
2.4. Soaking tests and calculation water and ethanol diffusion coefficients
of wheat kernels
The wheat kernels’ moisture levels were measured to ASAE standard
S352.2 (ASAE, 2000). Experiments were performed in distilled water
and anhydrous ethanol at 25 ◦ C. The containers and solution were kept
at 25 ◦ C for a few hours to reach the same temperature. Wheat samples
(10 g) were inserted into 50 ml tubes. Afterwards, 20 ml of ethanol or
water was added. At determined intervals, excess liquid from the tubes
containing wheat were transferred to another tube. Then, centrifugation
was carried out at 500 rpm 25 ◦ C to remove excess moisture on the
wheat samples. Absorbed ethanol or water was measured by finding the
difference between the weight of the dry samples and the weight of the
samples. The difference was then divided by the dry weight of the
kernel. The total analysis time was 400 min. To find the equilibrium
content of the wheat kernel, wheat samples with ethanol or water in
tubes were kept for two days. Then, the same procedure mentioned
above was followed to find equilibrium content of water and ethanol in
wheat kernel. Each trial was done in duplicate, and the average value
was presented.
The water and ethanol diffusion coefficients of wheat kernels were
calculated according to Kang and Delwiche (Kang et al., 2000).
Boundary conditions and assumptions for water and ethanol diffusion
through wheat kernels were defined as reported by Crank (1975) and
Kang et al. (2000).
Boundary conditions:
i. The initial concentration of water or ethanol in the kernel is
defined and uniform.
ii. Surface water or ethanol concentration is constant for the time t
≥ 0.
P. Ari Akin et al. Journal of Cereal Science 111 (2023) 103673

iii. The water or ethanol content of the kernel equalizes as soaking is 2.7. Dough properties
completed.
2.7.1. Farinograph parameters
Assumptions about the wheat kernel: Water was mixed with whole wheat flour samples in 50 g Farino­
graph bowls for 30 min in accordance with ICC Method 115/1 (Inter­
i. The diffusion coefficient is constant and not affected by water or national Association for Cereal Chemistry, 1992). At 12 min, the
ethanol levels. stability, dough development, softening, water absorption, and dough
ii. The composition is isotropic homogeneous. development were measured.
iii. While tempering occurs, the volume change is negligible.
iv. The temperature is constant throughout the process. 2.7.2. Mixolab parameters
v. The structure was considered spherical. According to AACCI 54–60.01, the amount of water and flour
required to make the total weight of the dough 75 g were mixed in a
One-dimensional diffusion equation for spherical geometry was Mixolab bowl for 45 min (AACC International, 2013). Mixolab param­
derived as equation (1) by Kang et al. (2000). eters are water absorption (the percentages of water needed to create 1.1
( 2 ) ± 0.05 Nm of torque (g/100 g), time needed for dough development
∂s ∂s ∂s (time for C1 (used in water absorption calculation (Nm)), starch, gela­
D +2 = (1)
∂r 2 r∂r ∂t tinization (C3, (Nm)), stability of the hot-formed gel (C4, Nm), and
starch retrogradation during the cooling phase. Mixolab also determines
where.
slope α (for the curve between C2 and the end of the period at 30 ◦ C),
slope β (of the curve bound by C2 and C3), and slope γ (of the curve
D = diffusion coefficient (m2/s)
bound by C3 and C4) (Dubat, 2016).
s = substance content (%, dry base)
r = radius (m)
2.8. Bread-making procedure
t = time (s)
The AACCI 100% Whole Wheat Pan Bread Method 10–13.02 was
Solving equation (1) with the defined boundary conditions and as­
used (AACC International, 2013). The flour, active dry yeast, salt, and
sumptions gives as follows (Kang et al., 2000).
sucrose were mixed. Then, the remaining ingredients were added and
( )∑ ( ) [ ]
st − sin 6 6 2 2 t
mixed to 10% past Farinograph peak. The dough was placed on a sheet
(2)
∞
SR = =1 − exp − Dn π
seq − sin π2 n=1 n2 r02 and divided by distances of 0.87, 0.47, and 0.32 cm, as well as molded,
panned, and proofed for 60 min. The fermentation time was 180 min at
where. 30 ◦ C. The dough was baked in a rotary oven (Despatch, Minneapolis,
MN, USA) at 204 ◦ C for 25 min. Loaves of bread were depanned after the
SR = substance ratio (dimensionless quantity ranging between 1 and baking procedure was completed. Then, the bread samples were placed
0) on wire racks for 2 h. After cooling, each bread sample was stored at
st = mean substrate content at a given time t (%, dry base) room temperature overnight in polyethylene bags.
seq = equilibrium substrate content at the surface of the kernel (%,
dry base) 2.9. Bread characteristics
sin = starting substrate content of the full kernel region
r0 = radius (m) 2.9.1. Volume and specific volume
The bread was measured using a loaf volumeter (National Mfg,
A nonlinear curve fitting method was applied to experimental data Lincoln, NE, USA) and the rapeseed displacement method. The specific
with equation (2) and water or ethanol diffusion coefficient in wheat volume was defined as the sample volume (cm3) divided by the sample
kernel were determined by iterative method using Orgine Software weight (g) in cubic centimeters per gram (cm3/g).
(OriginLab Corporation, Northampton, MA, USA).
2.9.2. Analysis of color
Crust, flour, and crumb were analyzed utilizing a HunterLab Min­
2.5. Experimental wheat milling and extraction rate iScan (Model EZ/4500 L, Hunter Associates Laboratory Inc., Reston, VI,
USA) to measure the color. On the Hunter scale,L* is expressed as a
The samples were milled to straight grade flour by a Buhler 202 number on a gradient bound by 0, representing black and 100, repre­
pneumatic mill (Uzwil, Switzerland) (AACC International, 2013) in senting white. A positive value for a* signals redness and negative a*
accordance with AACCI 26–50.01. Then the bran particles were reduced stipulates greenness. Positive b* and negative b* indicate yellowness and
using a 0.5 mm sieve on a Laboratory Mill 3100 (Perten Instruments, blueness, respectively. ΔE* measures overall color difference, derived
Huddinge, Sweden). Lastly, the milling fractions were amalgamated from square root of the sum of the a* square, b* square, and L square. It
with the flour streams. The extraction rate is a percentage that indicates is calculated using the formula given by Ari Akın et al. (2021):
how much flour can be extracted from a given amount of clean and √̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅
( )
conditioned grain. Based on this definition, the extraction rate was ΔE∗ = ΔL2 + Δa2 + Δb2 (3)
calculated.
2.9.3. Texture analysis
Front the center, a 2.54 cm slice was cut and used for texture anal­
2.6. Chemical analysis ysis. While adhering to AACCI Method 74–09.01, a texture analyzer was
used to determine firmness of the samples (Stable Microsystems, TA-XT
AACCI Method 44–40.01, 08–01.01, and 46–30.01 (Velp-NDA 701 plus, Godalming, Surrey, England) (AACC International, 2013).
Dumas Nitrogen Analyzer) were implemented to calculate the ash,
moisture, and protein totals of the whole wheat flour samples (AACC
International, 2013). Protein and ash values were conveyed on the
grounds of a dry basis.

3
P. Ari Akin et al.
2.10. Data analysis
The results were derived from utilizing an average ± standard de­
viation (SD) of two replicates, at a minimum. One-way variance analysis
(ANOVA) was conducted by Minitab 18 Statistical Software (Minitab
Inc., State College, PA, USA) with Tukey’s test atp < 0.05significance.
3. Results and discussion
3.1. Microbial analysis
Different ethanol concentrations in water were used to reduce the
microbial load of samples with low and high microbial loads. In Fig. 1,
the effect of different ethanol concentrations against the natural
microflora of Sample A and B has been presented. As can be seen in
Fig. 1, tempering sample A with 10, 20, and 30% ethanol resulted in
0.72, 1.02, and 1.98 log CFU/g reductions of APC while sample B
tempered with the same concentrations of ethanol resulted in 0.93, 1.80
and 2.59 log CFU/g reductions in APC, respectively. Similarly, sample A
tempered with 10, 20, and 30% ethanol resulted in 0.23, 1.49, and 2.84
log CFU/g reductions in YMC while tempering sample B with 10, 20, and
30% ethanol resulted in 0.42, 1.55, and 2.82 log CFU/g reductions in
YMC. In both samples with low (sample A) or relatively high microbial
load (sample B), a gradual decrease was observed in APC and YMC with
the use of 10, 20 and 30% ethanol. Furthermore, APC of sample A was
reduced from 5.02 ± 0.20 log CFU/g to below detectable levels with
50% ethanol and above. For ethanol concentrations of 50% and above,
no significant difference was observed in APC reduction, whereas a
decrease of 3.20, 3.20, 3.11, and 3.22 log units for 50, 70, 80, and 90%
ethanol was recorded, respectively. YMC in sample A was reduced from
5.96 ± 0.22 log CFU/g to below detectable levels at ethanol concen­
trations 50% while sample B with less than 50% ethanol resulted in 2.80
log CFU/g reductions in YMC. Similarly, 2.80, 2.73, 3.01, and 3.06 log
unit reductions in YMC were observed for samples tempered with 50%
ethanol and above which did not show any significant difference from
each other (p < 0.05). Tempering two wheat samples with 50% ethanol
caused the most significant reduction in microbial load. The microbial
reductions at ethanol levels above 50% are not significantly different (p
< 0.05). In general, a solution containing more than 70% ethanol so­
lution is required for microbial reduction in food applications. In this
study, however, the intended effect was reached using 50% ethanol
solution in wheat samples. The microbial reduction of wheat samples by
using 50% ethanol solution could be hypothesized that water in the
tempering solution is adsorbed by wheat faster than ethanol. Therefore,
water and ethanol diffusion coefficients of wheat kernels were calcu­
lated in the next phase of this research.
Fig. 1. The effect of different concentrations of ethanol against the natural microflora of wheat sample A and sample B (APC: Aerobic plate count, MYC: Yeasts and
molds). A: Wheat sample with lower microbial load, B: Wheat sample with high microbial load.
4
Journal of Cereal Science 111 (2023) 103673
Different chemicals are used to reduce the microbial load. Sabillón
et al. (2016b) conducted a study that was aimed to reduce the microbial
load by using saline and organic acid solutions during tempering. Lactic
acid (5%) and NaCl (1.0% wheat basis) reduced APC by 4.3 logs CFU/g.
In addition, İbanoǧlu (2001) investigated the effect of two different
levels (1.5 and 11.5 mg ozone/L) of ozonated water on the microbial
load of soft and hard wheat. Tempering soft wheat at 1.5 and 11.5 ppm
caused 1.2 and 2.1 logs CFU/g reductions in bacteria while tempering
hard wheat at 1.5 and 11.5 ppm caused 0.8 and 1.5 logs CFU/g re­
ductions in total bacteria. Moreover, 1.5 and 11.5 mg ozone/L caused
0.5 and 1.1 logs CFU/g reductions in mold and yeast count of soft wheat
while the same levels of ozone caused 0.4 and 1.0 logs CFU/g reductions
in mold and yeast count of hard wheat (İbanoǧlu, 2001). Compared to
the present study with recent studies, higher microbial reduction (~5.0
log CFU/g in APC and ~6.0 log CFU/g in YMC) in Sample A was ach­
ieved by using only ethanol. However, samples with higher microbial
load, sample B, slightly lower microbial reduction was observed. The
microbial reduction in APC and YMC in sample B are almost 3.0 log
CFU/g. One possible explanation for the difference in microbial reduc­
tion in samples A and B is ethanol does not kill bacterial spores (Centers
for Disease Control and Prevention, 2016). The exact mechanism behind
the difference of microbial reduction in sample A and B require more
research.
3.2. Soaking tests and calculation water and ethanol diffusion coefficients
of wheat kernels
To understand the change in a microbial reduction in wheat samples
by using different ethanol concentrations in water, the diffusion of pure
ethanol and water in the kernel were investigated. (The diffusion co­
efficients of water and ethanol were 0.07 × 10− 10 and 0.11 × 10− 10 m2/
s, respectively. The equilibrium water and ethanol contents of kernels
were determined as 0.821 g water/g dry wheat kernel and 0.042 g
ethanol/g dry wheat kernel, respectively. These results indicated that
wheat kernel can absorb water 19.6 times more than ethanol. Although
the differences between the diffusion coefficients of ethanol and water
are negligible, the differences in equilibrium moisture and ethanol
contents are significantly different (p < 0.05). As a result of the diffusion
of water into the wheat grain more than ethanol, the ethanol concen­
tration on the surface of the wheat grain remains constant over time,
while the water concentration gradually decreases. Therefore, 50:50
ethanol-water mixture showed antimicrobial activity against different
types of microorganisms as 70:30 ethanol-water mixture does. Thus, the
microbial load in the bran decreased. Similar results were found for
wheat straw and flour (Vareli et al., 1998).
P. Ari Akin et al. Journal of Cereal Science 111 (2023) 103673

3.3. Extraction rate and chemical composition Table 2

Farinograph and Mixolab characteristics of dough made with whole-wheat flour
As a result of the microbial analyses, it was determined that the samplesa.
lowest ethanol concentration at which the greatest reduction in micro­ Parameters Control Whole Whole Wheat Flour Obtained from
bial load of wheat was 50% (v/v). Therefore, the effects of solutions Wheat Flour Ethanol Tempered Wheat
containing 50% ethanol were examined. Experiments were conducted to Development Time 5.26 ± 0.05 b 13.40 ± 0.15 a
determine if 50:50 ethanol-water (v/v) mixture as a tempering agent (min)
affected the extraction rate and chemical composition of whole wheat Water Absorption 70.70 ± 0.14 a 66.80 ± 0.14 b
(%)b
flour. During milling, the extraction rate of the control was 71.1 ± 0.3%
Stability (min) 4.89 ± 0.47 b 7.37 ± 0.04 a
whereas that of tempered wheat with ethanol-water (50:50 (v/v)) Degree of Softening 72.00 ± 1.41 a 54.50 ± 2.12 b
mixture was 70.6 ± 0.4%, similar to the control (p > 0.05). This result (FU)c
showed that the ethanol-water mixture helped to separate bran and C1 (min) 4.65 ± 0.38 b 6.54 ± 0.16 a
endosperm of wheat grains as much as water could. Water Absorption 67.00 ± 0.00 a 66.00 ± 0.00 a
(%)
The effect of ethanol-water (50:50 (v/v)) tempering mixtures on the Stability (min) 8.00 ± 0.00 a 8.00 ± 0.00 a
composition of whole-wheat flour was also investigated. Table 1 illus­ C2 (Nm)d 0.53 ± 0.00 b 0.57 ± 0.00 a
trates the composition of whole wheat flour from water tempered wheat C3 (Nm)d 1.63 ± 0.00 a 1.72 ± 0.02 b
(control whole wheat flour) and ethanol-water mixtures (50:50 (v/v)) C4 (Nm)d 1.32 ± 0.00 a 1.45 ± 0.02 b
C5 (Nm)d 2.43 ± 0.03 a 2.35 ± 0.05 a
(referred to hereafter as “whole wheat flour obtained from ethanol
a 0.095 ± 0.004 a 0.090 ± 0.006 a
tempered wheat”. The control whole wheat flour had ash, moisture, and β 0.450 ± 0.079 a 0.526 ± 0.031 a
protein contents of 1.48, 16.00, 13.00%, respectively. The ash, protein, γ 0.105 ± 0.024 a 0.103 ± 0.007 a
and moisture contents of whole wheat flour obtained from ethanol a
Values represent mean ± standard deviation for duplicate determinations.
tempered wheat were 1.47, 16.60, 10.99%. Moreover, the control white Means in the same line with different letters indicate significant differences (p <
wheat flour had ash, moisture, and protein contents of 0.47, 13.50, 0.05).
13.20%, respectively. The ash, protein, and moisture contents of refined b
It was corrected for 500 FU.
wheat flour obtained from ethanol tempered wheat were 0.46, 13.30, c
FU: Farinograph Unit.
12.30%. As it can be seen in Table 1, no significant difference was found d
Nm: Newton-metre.
in the ash, and protein contents of refined and whole wheat flour which
obtain by tempering water and ethanol solution and. Only difference impacted on dough characteristics. Tempering wheat with ethanol-
was observed in the moisture content of whole and refined wheat flour. water mixture caused an increase in development time from 5.26 to
The differences in moisture contents between tempering samples with 13.40 min. However, tempering wheat grain with ethanol-water mixture
only water and ethanol solutions could be significant amount of alcohol- reduced the water absorption, causing a decrease from 70.70 to 66.80%.
infused water moves to the endosperm, some of it stays on the surface of Moreover, tempering wheat with ethanol-water mixtures (50:50 (v/v))
wheat grain or bran with the alcohol, which is evaporated along with the increased the dough stability as well as the degree of softening compared
alcohol during milling. This is supported by the fact that the numerical to the control dough. The ethanol-water mixture increased dough sta­
difference between the moisture content of whole wheat flour is higher bility from 4.89 to 7.37 min while decreasing the degree of softening
than that of white flour. Ibanoglu (2001) has found a similar result. from 72.00 to 54.50 FU. In general, longer development, higher stabil­
Tempering soft and hard wheat with ozonated water did not change the ity, and lower softening indicate strong flour. Ibanoglu (2001) also
extraction rates. The protein and ash content of refined flour from hard investigated the effect of tempering soft and hard wheat with ozonated
and soft wheat with ozone treatment were also not significantly different water on water absorption, development time, stability and degree of
(Ibanoglu, 2001). softening. No significant change was found in these parameters in dough
with refined flour obtained from soft and hard wheat tempered with
3.4. Dough properties ozonated water (Ibanoglu, 2001). The effect of tempering agent on the
rheology of dough with refined flour is easier to explain compared to
The Farinograph test was conducted to evaluate characteristics of whole wheat flour because whole wheat flour is a mixture of bran and
dough with control and whole wheat flour obtained from ethanol endosperm which contains both high levels of starch and protein.
tempered wheat. The data in Table 2 shows ethanol-water mixture Therefore, it is hard to say whether the change in Farinograph param­
eters comes from changes in the bran, starch, or protein components.
Table 1
To better understand the effect of ethanol tempering on the proteins
Characterization of refined and whole wheat flour samplesa. and starch in whole wheat flour under mechanical shear stress and
temperature constraint, the Mixolab test was done. The Mixolab analysis
Types of Flour Refined Wheat Flourb
can be divided into two parts. The first part (C1, C2, and a) shows
Moisture Protein (%) Ash (%) changes to the proteins of wheat flour during mixing while the later
(%)
mixing stages demonstrate the characteristics of starch. During the first
Control Refined Wheat Flour 13.50 ± 13.20 ± 0.47 ± stage of the test (0–8 min), the flour starts to hydrate with the combi­
0.03 a 0.06 a 0.01 a nation of stretching and alignment of the proteins which results in a
Refined Wheat Flour Obtained from 12.30 ± 13.30 ± 0.46 ±
Ethanol Tempered Wheat 0.07 b 0.06 a 0.02 a
three-dimensional viscoelastic structure (Liu et al., 2017).
Whole Wheat Flourb Table 2 shows that the dough development time was increased from
4.65 to 6.54 min in the whole wheat flour obtained from ethanol
Control Whole Wheat Flour 13.00 ± 16.00 ± 1.48 ±
0.02 a 0.01 a 0.01 a tempered wheat compared to the control. Similarly, the development
Whole Wheat Flour Obtained from 10.99 ± 16.60 ± 1.47 ± time of whole wheat flour obtained from ethanol tempered wheat was
Ethanol Tempered Wheat 0.01 b 0.02 a 0.01 a longer than that of control dough in the Farinograph test. Dough with
a
Values represent mean ± standard deviation for duplicate determinations. whole wheat flour obtained from ethanol tempered wheat had statisti­
Means in the same line with different letters indicate significant differences (p < cally lower water absorption (66%) compared to that of the control
0.05). (67%). Tempering wheat with dough with whole wheat flour obtained
b
Characterizations of whole wheat flour and refined flour were statistically from ethanol tempered wheat prolonged the dough development time
evaluated separately.

5
P. Ari Akin et al.
and reduced the water absorption. However, no significant difference
was found between the stability of control and dough with whole wheat
flour obtained from ethanol tempered wheat in the Mixolab test. More
research needs to be done to understand why ethanol-water mixture
tempering caused a longer dough development time and lower water
absorption.
In the Mixolab test, slope α is the indicator of protein weakening
speed under the heat. The slope α of both whole wheat flour was sta­
tistically the same. Also, dough stability time is mainly controlled by the
gluten strength of the flour as well as its resistance to kneading forces,
with a longer time indicating stronger gluten (Liu et al., 2017). No sig­
nificant difference was found between the stability of dough containing
whole wheat flour obtained from ethanol tempered wheat and control.
Based on the stability and slope α results, protein weakening speed and
gluten strength of both flours were not affected by ethanol-water
tempering. As a function of mechanical work and temperature, C2
measures protein weakness. In this research, the C2 value of the control
dough increased from 0.531 to 0.567 Nm. Szafranska (2015) found that
the C2 value was positively correlated with stability while the C2 value
was negatively correlated with the degree of softening in the Farino­
graph test. Similarly, in our research, the degree of softening reduced
while the stability of dough increased when wheat grains were tempered
with ethanol-water mixtures in the Farinograph test. This suggests
ethanol-water mixture as a tempering agent caused an increase in dough
strength.
The change of starch gelatinization (C3), hot gel stability (C4), and
starch retrogradation (C5) in the cooling phase had been observed in
control dough and dough with whole wheat flour obtained from ethanol
tempered wheat from 18 to 45 min of the test. C3 and C4 values of dough
with whole wheat flour obtained from ethanol tempered wheat had
statistically higher than the control dough, suggesting stronger gel for­
mation (Dubat, 2016; Liu et al., 2017). However, no significant differ­
ence was found in C5 values of control dough and dough with whole
Fig. 2. Pictures of bread made whole-wheat flour samples. A: Bread made with control whole-wheat flour, B: Bread made with whole wheat flour obtained from
ethanol tempered wheat.
6
Journal of Cereal Science 111 (2023) 103673
wheat flour obtained from ethanol tempered wheat. Starch retrograda­
tion reflects the staling rate of bakery products during storage. Based on
this result, products made with both flour could have the same staling
rate. Moreover, slopes β and γ are the indicators of starch gelatinization
and enzyme degradation speeds, respectively (Dubat, 2016). No signif­
icant difference was found slopes β and γ of control dough and dough
with whole wheat flour obtained from ethanol tempered wheat. It can be
concluded that the speeds of starch gelatinization and enzyme degra­
dation were not changed by ethanol-water mixtures tempering.
Taken together, the dough property measurements conducted in this
research show that in general, tempering wheat with ethanol and water
changed the rheology of dough as seen in Farinograph and Mixolab
characteristics by modifying protein and starch. However, more
research needs to be done to better understand how ethanol affects the
flour components.
3.5. Bread characteristics
3.5.1. Volume and specific volume
The volume and specific volume were performed to monitor the
changes in baking properties of whole wheat flour obtained from
ethanol tempered wheat. Bread loaves were presented in Fig. 2. Volume
and specific volume of loaves are shown in Table 3. The volume of the
control bread and the bread with whole wheat flour obtained from
ethanol tempered wheat were 618 and 617 cm3, respectively. The spe­
cific volume of the control bread and the bread with whole wheat flour
obtained from ethanol tempered wheat were 3.98 and 3.93 cm3/g. No
significant difference was found in volume and specific volume between
the control and the bread with whole wheat flour obtained from ethanol
tempered wheat. The same results were found by Sabillón et al. (2017)
for NaCl with organic acids (acetic or lactic acid). Tempering wheat with
NaCl with organic acids (acetic or lactic acid) did not change the volume
of whole wheat bread.
P. Ari Akin et al. Journal of Cereal Science 111 (2023) 103673

Table 3 Table 4
Volume, specific volume, firmness of bread made with whole wheat flour Color values of bread made with whole wheat flour samplesa.
samplesa. Treatment Flour
Parameters Control Whole Whole Wheat Flour Obtained from
L* a* b* ΔE
Wheat Flour Ethanol Tempered Wheat
Control whole wheat flour 85.58 ± 1.78 ± 10.51 ± 86.43 ±
Volume (cm3) 618 ± 5.77 a 616 ± 5.78 a
0.17 a 0.05 a 0.07 a 0.15 a
Specific Volume 3.98 ± 0.04 a 3.93 ± 0.04 a
Whole wheat flour obtained 85.66 ± 1.80 ± 10.64 ± 86.33 ±
(cm3/g)
from ethanol tempered 0.09 a 0.01 a 0.01 a 0.06 a
Firmness (g) 332.10 ± 2.30 a 333.16 ± 1.83 a
wheat
a
Values represent mean ± standard deviation for duplicate determinations. Crumb
Means in the same column with different letters indicate significant differences
Control whole wheat flour 58.35 ± 4.13 13.40 ± 60.95 ±
(p < 0.05). ±
0.17 a 0.04 a 0.02 a 0.18 a
Whole wheat flour obtained 58.40 ± 4.28 ± 13.34 ± 61.03 ±
3.5.2. Color and texture analysis from ethanol tempered 0.15 a 0.13 a 0.21 a 0.09 a
L*, a*, b* color parameters in flour, bread crumb, and bread crust has wheat
been shown in Table 4. No significant differences have been found in the Crust
color of flour, bread crumb, and bread crust of control loaves and loaves Control whole wheat flour 39.62 ± 8.33 ± 17.11 ± 40.42 ±
made with whole wheat flour obtained from ethanol tempered wheat. 0.38 a 0.14 a 0.07 a 0.27 a
Table 3 shows that control bread had higher crumb firmness compared Whole wheat flour obtained 39.07 ± 8.17 ± 17.16 ± 39.88 ±
to bread made with whole wheat flour obtained from ethanol tempered from ethanol tempered 0.28 a 0.14 a 0.04 a 0.29 a
wheat
wheat. L*, a*, b* color parameters in flour, bread crumb, and bread crust
a
of both samples were statistically the same. İbanoǧlu (2001) has found a Values represent mean ± standard deviation for duplicate determinations.
similar result. Tempering soft and hard wheat with ozonated water did Means in the same column with different letters indicate significant differences
not change the L*, a*, b* color parameters in flour. (p < 0.05).
The firmness value of control bread was 332.10 g while the firmness
of bread made with ethanol-water was 333.16 g. Based on the color and original draft. Şefika Evran: Investigation, Methodology, Writing -
texture results, ethanol-water mixture, as a tempering agent, did not original draft. Ismail Hakki Boyaci: Investigation, Methodology,
affect the color but firmness. One possible explanation for higher Writing - review & editing, Supervision.
hardness in bread with whole wheat flour obtained from ethanol
tempered wheat compared to the control is the diffusion of ethanol into
the wheat grain rather than water during the tempering process. Declaration of competing interest

4. Conclusion
Wheat-based products have recently been found to spread bacterial
disease therefore scientists in the food sector must take steps to enhance
wheat safety. A number of treatments, including ozone, heat, irradia­
tion, have been proposed as strategies to improve the microbiological
safety of wheat grains and flour. However, new flour decontamination
methods should continue to be explored, without compromising flour
functionality. In this research, the effects of tempering with different
concentrations (10–90% (v/v)) of ethanol on the microbial load and
quality properties of wheat flours were studied. Results showed that
50% ethanol as a tempering agent, was enough to reduce microbial load
to undetectable levels for wheat with low microbial load and to provide
almost 3.0 log CFU/g decrease for wheat with high microbial load. Since
water has a higher diffusion coefficient, it penetrates to wheat kernel
faster than ethanol, causing an increase in ethanol concentration of
wheat kernel surface.
As far as functionality of flour is concerned, tempering wheat with
50% ethanol resulted in longer dough development time, lower water
absorption, higher stability, and lower degree of softening compared to
control dough. According to the Mixolab parameters, C1, C2, C3, C4
values and stability were increased whereas slope α, β, γ, C5 values did
not have any significant change. In terms of bread characteristics, no
significant difference was found in firmness, specific volume, and vol­
ume. Similarly, the redness (a*), yellowness (b*), and darkness (L*) of
flour, crumb, and crust were statistically the same. Collectively, our
findings show that adding ethanol to water at the tempering step could
reduce the microbial load of wheat while preserving flour functional
properties. This could make wheat flour a ready-to-eat-product.
Credit author statement
Pervin Ari Akin: Investigation, Methodology, Writing - original
draft. Emine Kübra Tayyarcan: Investigation, Methodology, Writing -
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data availability
Data will be made available on request.
Acknowledgement
Emine Kubra Tayyarcan was supported by the 100/2000 doctoral
scholarship given by The Council of Higher Education Turkey and 2211/
A General Domestic Doctorate Scholarship Program given by The Sci­
entific and Technological Research Council of Turkey.
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