Food Chemistry 402 (2023) 134224

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Effectiveness of asparaginase on reducing acrylamide formation in bakery

products according to their dough type and properties
Selahattin Gazi a, Neslihan Göncüoğlu Taş a, Ahmet Görgülü b, Vural Gökmen a, *
a
Food Quality and Safety (FoQuS) Research Group, Department of Food Engineering, Hacettepe University, 06800 Beytepe, Ankara, Turkey
b
Eti Gıda Sanayi ve Ticaret A.Ş. Research and Development Center, Eskişehir, Turkey

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

Keywords: Effect of asparaginase was tested in different biscuit and cracker doughs, and wafer batter by changing pro­
Acrylamide cessing conditions such as enzyme dosage, dough resting time and temperature, mixing speed and time, and
Asparagine mixing procedure of the recipe components. Acrylamide reductions achieved were 96, 80, 54% in rotary cut
Asparaginase
biscuits, crackers and wire cut cookies, respectively due to high water activity of their dough. Asparaginase did
Dough types
Bakery products
not affect surface color or spread ratio. There was a positive correlation between asparagine content and
acrylamide formation except for rotary molded biscuit doughs. No significant decrease was found in rotary
molded biscuits because of low water (water activity of 0.70) and high fat content. This indicated that water
activity of dough is an important factor for the effectiveness of asparaginase treatment. The results suggested that
water activity value exceeding 0.75 is needed in the dough to effectively reduce asparagine, so acrylamide in
bakery products.

1. Introduction
Acrylamide is a genotoxic, carcinogenic, and mutagenic compound
formed by the interaction of reducing sugars and asparagine in foods
during heat treatment (Mottram, Wedzicha, & Dodson, 2002; Nem­
atollahi, Mollakhalili, & Mousavi, 2021). In 1994, acrylamide was added
to the list of probable human carcinogens (Class 2A) by the International
Agency for Research on Cancer (International Agency for Research on
Cancer, 1994). In 2002, the Swedish National Food Administration
declared acrylamide to be a toxic heat treatment contaminant originated
from food (Swedish National Food Administration, 2002). Intense
acrylamide formation was reported in commonly consumed foods high
in asparagine and reducing sugar content, such as bakery products,
French fries and coffee (Gökmen & Şenyuva, 2006). The average daily
acrylamide intake determined by FAO/WHO was 0.2–1 mg/kg body
weight, however, the amount of acrylamide detected in processed food
products was much higher and far above the daily intake values (JECFA,
2007; Koszucka, Nowak, Nowak, & Motyl, 2020).
Bakery products are cereal-based, carbohydrate, lipid, and protein-
containing, heat-treated products that can be consumed quickly (Del­
cour & Hoseney, 2010). Dough types are divided into two according to
their method of preparation as hard and short dough. Products such as
hard biscuits have high water content in the dough, and their dough is an
example of hard dough. In these products, all ingredients are mixed at
the same time and protease enzyme is used to reduce the gluten devel­
opment (Manley, 2001). The short dough is obtained by creaming and
the ingredients are added sequentially to the mixture, and the role of fat
in dough formation is to inhibit gluten development by competing with
the flour added to the system for water (Delcour & Hoseney, 2010;
Manley, 2001). Wire cut dough and rotary dough are examples of short
dough and these products are easier to process mechanically than
products containing enhanced gluten (hard dough) and expand a little
during baking (Delcour & Hoseney, 2010; Manley, 2001). Today, in
addition to the rapidly increasing interest of consumers in healthy
nutrition, bakery products that allow fast consumption and have
attractive sensory properties cannot be abandoned (Friedman & Levin,
2008; Sarion, Codina, & Dabija, 2021). However, due to the reducing
sugar content and high amount of asparagine in cereals, intense acryl­
amide formation is inevitable in bakery products as a result of heat
treatment (Sarion, Codina, & Dabija, 2021). Excessive consumption of
bakery products threatens human health in terms of acrylamide expo­
sure, and there is no law that limits the amount of acrylamide in bakery
products (Sarion, Codina, & Dabija, 2021). However, a regulation
limiting the amount of acrylamide in foods is expected to be published in

* Corresponding author.
E-mail address: vgokmen@hacettepe.edu.tr (V. Gökmen).

https://doi.org/10.1016/j.foodchem.2022.134224
Received 3 April 2022; Received in revised form 1 September 2022; Accepted 10 September 2022
Available online 13 September 2022
0308-8146/© 2022 Elsevier Ltd. All rights reserved.
S. Gazi et al.
the near future, and for this purpose, acrylamide benchmark levels for
foods prepared by the European Union have been determined to assist
food manufacturers in mitigation of acrylamide (The European Com­
mission, 2017). According to this regulation, the benchmark level for
biscuits and wafers is 350 µg/kg, and the benchmark level for crackers
with the exception of potato-based crackers is 400 µg/kg (The European
Commission, 2017).
There are different strategies for mitigation of acrylamide in bakery
products. These are generally classified as agronomy, formulation, and
process-based strategies (Food Drink Europe, 2019; JECFA, 2007). In
agronomy-based strategies, the aim is generally to produce cereals with
low asparagine and reducing sugar content (Food Drink Europe, 2019).
Strategies related to formulation change are using refined flour instead
of whole grain flour, optimizing water and fat content, changing the
baking agents, adding organic acids, changing the type of sugar, adding
amino acids, adding cations, adding antioxidants and polyphenols
(Capuano et al., 2009; Capuano, Garofalo, Napolitano, Zielinski, &
Fogliano, 2010; Gökmen, Açar, Serpen, & Morales, 2008; Gökmen &
Şenyuva, 2006, 2007; Xu, Oruna-Concha, & Elmore, 2016; Zhang &
Zhang, 2008). Strategies depending on the changes in the process are
using low thermal load, using new heat treatment methods, and
fermentation (Anese, Sovrano, & Bortolomeazzi, 2007; Fredriksson,
Tallving, Rosén, & Åman, 2004; Palazoǧlu & Gökmen, 2008). Some of
these acrylamide reduction strategies cause significant losses in aroma,
taste, color, and texture of the products (Bartkiene et al., 2013; Masi,
Dinnella, Barnabà, Navarini, & Monteleone, 2013; Xu et al., 2016).
Usage of asparaginase stands out as an acrylamide reduction strat­
egy. L-asparaginase (E.C.3.5.1.1) is an enzyme that belongs to the amino
acid amidohydrolase enzyme class and classified as GRAS (Generally
Recognized as Safe) (Anese, Suman, & Nicoli, 2009). Asparaginase hy­
drolyzes asparagine to aspartic acid, thereby reduces acrylamide for­
mation very efficiently (Hendriksen, Kornbrust, Ostergaard, & Stringer,
2009). It has some advantages compared to other reduction strategies
because it could achieve very high (>99 %) acrylamide reductions
depending on the type of the product and it does not affect the sensory
quality of the product (Amrein, Schönbächler, Escher, & Amadò, 2004;
Kumar, Shimray, Indrani, & Manonmani, 2014; Pedreschi, Kaack, &
Granby, 2008; Zyzak et al., 2003).
The number of studies on the use of asparaginase enzyme in bakery
products is very limited. Effectiveness of asparaginase in dough could
depend on the dosage of asparaginase in dough, pH, and water activity
of dough as well as processing-related factors such as dough resting
temperature, time, dough mixing procedure etc. Therefore, this study
aims to investigate the effect of asparaginase enzyme for the mitigation
of acrylamide in different bakery products (rotary cut biscuits, rotary
molded biscuits, wire cut cookies, crackers, and wafers) by using
asparaginase enzyme in dough in different dosages and under different
dough processing conditions.
2. Materials and methods
2.1. Chemicals and consumables
The wheat flour, shortening, potassium metabisulfite, invert sugar,
powdered sugar, sodium hydroxide, ammonium bicarbonate, sodium
bicarbonate, sodium acid pyrophosphate, lecithin, NaCl, ethyl vanillin,
and protease enzyme used to prepare the dough were supplied by Eti
(Eskişehir, Turkey). Commercial asparaginase enzyme with an activity
of 3500 ASNU/g was obtained from Novozymes (Bagsvaerd, Denmark).
Acrylamide (99 %), asparagine (98 %), potassium hexacyanoferrate
(III), zinc sulfate, acetonitrile (gradient grade), were purchased from
Sigma (Deisenhofen, Germany). Deuterated acrylamide was obtained
from Toronto Research Chemicals Inc. (North York, Canada). Formic
acid (98 %) and methanol were purchased from Merck (Darmstadt,
Germany). The Carrez I and Carrez II solutions were obtained by dis­
solving 15 g potassium hexacyanoferrate (III) and 30 g zinc sulfate in
2
Food Chemistry 402 (2023) 134224
100 mL of deionized water, respectively. Syringe filters (nylon, 0.45 µm)
and pH calibration solutions at pH 4 and 7 were from IsoLab (Wertheim,
Germany). Oasis MCX cartridges were obtained from Waters (Millford,
MA, USA). The SeQuant ZIC-HILIC (150 × 4.6 mm, 3.5 μm) column was
purchased from Merck (Darrmstadt, Germany) and Atlantis C18 (150
mm × 4.6 mm, 5 μm) column was purchased from Waters (Millford, MA,
USA). Deionized water was used though out the experiments (0.55 μS/
cm).
2.2. Preparation of the dough and bakery products
The formulations of sweet/semi-sweet hard dough with low fat and
high water content (rotary cut biscuit doughs), soft doughs with low
water and high fat content (rotary molded biscuit doughs), and wire cut
cookie doughs were from a local company. Rotary cut crackers (pretzel
sticks) and wafer batter were prepared as in the method defined by
Manley (2001) with some modifications. The preparation of the dough
of the bakery products as well as their physical properties (initial pH and
water activity values) and their baking conditions were given in Fig. 1.
Rotary cut biscuit doughs was prepared according to all-in-one
mixing method and all ingredients (including the asparaginase
enzyme) mixed in the mixer at once (Kitchen aid artisan mixer, Benton
Harbor, MI, USA). The ingredients were as follows: wheat flour, short­
ening, powdered sugar, invert sugar, protease, sodium bicarbonate,
ammonium bicarbonate, sodium acid pyrophosphate, NaCl, ethyl
vanillin, and water. To investigate the effect of process parameters on
asparaginase activity, the following treatments were applied: increasing
the enzyme dosage (which was indicated shortly as ASNU in the appli­
cations stands for ASNU/kg flour), adding a dough resting period,
increasing the dough resting time (RT) and/or temperature, increasing
the mixing time (MT) and speed (MS), and adding shortening (S) to the
mixing medium after a period of mixing time. The details of the treat­
ments are given in Table 1. Rotary cut biscuit doughs were cut and
shaped with standard circular molds (thickness of 2 mm and diameter of
44.5 mm) and six pieces of biscuit dough were placed in a conventional
oven (Memmert UN 55, Schwabach, Germany) and baked at 230 ◦ C for
8.5 min.
Creaming mixing was used for the preparation of the rotary molded
biscuit dough. The ingredients were as follows: wheat flour, shortening,
powdered sugar, invert sugar, sodium bicarbonate, ammonium bicar­
bonate, sodium acid pyrophosphate, lecithin, NaCl, ethyl vanillin, and
water. Firstly, the cream was formed by mixing shortening and sugar
and then water-soluble ingredients were added. In the last step, flour
was added together with the asparaginase enzyme to make the dough.
Asparaginase treatments applied were increasing the enzyme dosage,
adding a dough resting period, increasing the dough resting time and/or
temperature, and changing the mixing procedure (MPC) of the in­
gredients (Table 1). To change the mixing procedure of the ingredients,
the water-soluble ingredient mixture and flour containing enzyme were
mixed first and then the prepared cream was added to make the dough.
In the application of 2000 ASNU + NaHCO3, sodium bicarbonate was
added with flour instead of with the water-soluble ingredients. A total of
6 pieces of rotary molded biscuit dough were shaped with standard
circular molds (thickness of 2 mm and diameter of 44.5 mm). They were
placed on the baking tray and baked at 210 ◦ C for 8.5 min.
The wire cut cookie dough was also prepared by the creaming
method. The ingredients were as follows: wheat flour, shortening,
powdered sugar, invert sugar, sodium bicarbonate, ammonium bicar­
bonate, sodium acid pyrophosphate, lecithin, NaCl, ethyl vanillin, and
water. Asparaginase enzyme was added to the doughs other than con­
trol. To increase the efficiency of the asparaginase, a dough resting
period was applied, the enzyme dosage, the dough resting time and/or
temperature was increased, and the mixing procedure of ingredients was
changed (Table 1). The mixing procedure change of the ingredients was
applied as in the case of rotary molded biscuit dough. A total of 6 pieces
of wire cut cookie dough was shaped with the same standard circular
S. Gazi et al.
Fig. 1. Preparation of the doughs of different bakery products and their baking conditions.
molds and baked at 210 ◦ C for 8 min.
All in one mixing method was used for the preparation of rotary cut
cracker dough. The ingredients were as follows: wheat flour, shortening,
ammonium bicarbonate, NaCl, potassium metabisulfite, and water
(Manley, 2001). The asparaginase treatments were increasing the
enzyme dosage, adding a dough resting period, increasing the dough
resting temperature (Table 1). The pasta production apparatus of the
mixer was used to prepare crackers which have 2 mm diameter and 80
mm length. A total of 24 rotary cut cracker dough was placed on the
baking tray and NaOH solution (1 %, w/v) was sprayed onto them. After
then, the rotary cut cracker doughs were baked at 210 ◦ C for 9 min.
Wafer batter was prepared by all in one mixing method by using
wheat flour, shortening, sodium bicarbonate, lecithin, NaCl, and water
(Manley, 2001). The asparaginase treatments were increasing the
enzyme dosage, adding a dough resting period, increasing the dough
resting time, and/or temperature (Table 1). The wafer batter was not
baked, only the dough was stored at − 40 ◦ C until the asparagine
analysis.
In treatments with a resting period, the dough was covered with
stretch film. A portion of the dough was immediately placed in the
freezer at − 40 ◦ C to inhibit asparaginase activity and stored until the
analysis. All baking experiments were performed twice. The bakery
products taken out of the oven were cooled at room temperature for half
an hour, packaged and stored at room temperature until the analysis.
2.3. Analysis of acrylamide
Acrylamide analysis of the bakery products was done with some
3
Food Chemistry 402 (2023) 134224
modifications after a triple-stage extraction previously described by
Gökmen, Morales, Ataç, Serpen, & Arribas-Lorenzo (2009). A 10 mM
formic acid solution containing 20 µg/kg of d3-acrylamide was prepared
as the extraction solution. In the first step of extraction, 0.5 mL Carrez I,
0.5 mL Carrez II, and 9 mL extraction solution were added onto 1 g
sample and they were mixed in a mixer for 4 min. Then, the supernatant
of the sample was removed and collected in a tube after the centrifu­
gation for 3 min at 6080 × g. In the second step of the extraction, 5 mL
extraction solution was added onto the pellet, and they were mixed for 4
min. Then, the supernatant was removed and collected in the tube after
the centrifugation at 6080 × g for 3 min. The third step of the extraction
had the same stages as the second step. All the supernatants combined in
the tube mixed for 2 min and then transferred to an Eppendorf tube to be
stored until the analysis at − 40 ◦ C. The Eppendorf tube was centrifuged
at 10000 × g for 3 min after thawing and the supernatant was passed
through a preconditioned Oasis MCX cartridge. The clear extracts
transferred into vials were analyzed by using Waters Acquity H Class
UPLC system (Millford, MA, the USA) connected to a triple quadrupole
mass detector with electrospray ionization. Atlantis T3 column (4.6
x150 mm, 3 µm) was used for the analysis. 10 mM formic acid in water
was used as the mobile phase in the system at a flow rate of 0.2 mL/min
at 25 ◦ C. The electrospray source had the following properties: capillary
voltage of 2.00 kV; cone voltage of 22 V; extractor voltage of 4 V; source
temperature of 120 ◦ C; desolvation temperature of 400 ◦ C; and des­
olvation gas (nitrogen) flow of 900 L/h. The flow rate of the collision gas
(argon) was set to 100 L/h. Quantification of acrylamide was performed
by using the fragmentation ion (m/z 55) of the precursor ions of m/z 72.
Quantification of d3-acrylamide was performed by using the
S. Gazi et al.
Table 1
Enzyme applications in rotary cut, rotary molded, wire cut, cracker and batter
type of doughs for bakery products.
Bakery Products ASNU Applications*
Rotary cut biscuits 1000 ASNU
2000 ASNU
3000 ASNU
1000 ASNU + 15 min RT
2000 ASNU + 15 min RT
3000 ASNU + 15 min RT
2000 ASNU + 30 min RT
2000 ASNU + 15 min × 37 ◦
2000 ASNU + MS
2000 ASNU + MT
2000 ASNU + MS × MT
2000 ASNU + S
Rotary molded biscuits 2000 ASNU
2000 ASNU + 15 min RT
2000 ASNU + 15 min × 37
◦
2000 ASNU + NaHCO3
2000 ASNU + MPC
5000 ASNU + 30 min RT
Wire cut cookies 1000 ASNU
2000 ASNU
3000 ASNU
5000 ASNU
7000 ASNU
9000 ASNU
12000 ASNU
1000 ASNU + 15 min RT
2000 ASNU + 15 min RT
2000 ASNU + 30 min RT
3000 ASNU + 15 min RT
5000 ASNU + 15 min RT
7000 ASNU + 15 min RT
9000 ASNU + 15 min RT
9000 ASNU + 15 min × 37 ◦
12000 ASNU + 15 min RT
2000 ASNU + MPC
5000 ASNU + MPC
9000 ASNU + MPC
Crackers 2000 ASNU
3000 ASNU
2000 ASNU + 15 min RT
3000 ASNU + 15 min RT
2000 ASNU + 15 min × 37 ◦
Wafers 1000 ASNU
2000 ASNU
1000 ASNU + 15 min RT
2000 ASNU + 15 min RT
2000 ASNU + 30 min RT
1000 ASNU + 15 min × 37 ◦
*ASNU: Amount of L-asparaginase that syntheses one micromole of ammonia per
minute under standard conditions per kg flour.
S: Shortening, RT: Resting Time, MS: Mixing Speed, MT: Mixing Time, MPC:
Mixing Procedure Change.
fragmentation ion (m/z 58) of the d3-acrylamide (m/z 75). The cali­
bration curve was prepared at the levels of 1, 2, 5, 10, 20 µg/L and the
extraction solvent was injected as blank. The results were reported as µg
acrylamide/kg sample.
2.4. Analysis of asparagine
Asparagine analysis in the dough samples was performed by some
modifications according to method previously described by Kocadağlı,
Özdemir, & Gökmen (2013). A triple-stage extraction procedure was
applied. An extraction solution containing 0.1 % formic acid in aceto­
nitrile:water (1:1, v/v) was prepared to both extract the free asparagine
from the matrix and to stop the asparaginase activity. In the first stage of
the extraction, 10 mL of extraction solution was added onto 1 g sample,
and they were mixed in a mixer for 20 min. Then, the supernatant ob­
tained after centrifugation at 6080 × g for 3 min was taken into another
Food Chemistry 402 (2023) 134224
tube and stored. In the second step of the extraction, 5 mL extraction
solution was added onto the pellet, and they were mixed for 5 min. The
supernatant was transferred to the tube after centrifugation at 6080 × g
for 3 min. The third stage of the extraction had the same steps as the
second stage, the supernatants were combined, mixed, and transferred
to an Eppendorf tube. Then, the Eppendorf tubes were centrifuged at
10000 × g for 3 min. After then, the samples were diluted 1:1 (v/v) with
acetonitrile to a final acetonitrile content of 75 % and centrifuged at
10000 × g for 3 min. The supernatant was passed through a 0.45 μm
nylon filter and taken into vials. The asparagine was analyzed by Waters
C RT Acquity H Class UPLC system (Millford, MA, the USA) coupled with a
triple quadrupole mass detector with electrospray ionization.
SeQuant® ZIC®-HILIC column (150 × 4.6 mm, 3.5 μm) was used for the
analysis. The mobile phases were 0.1 % formic acid in water (A) and 0.1
% formic acid in acetonitrile (B). A gradient elution program was
applied which was 20:80 (A:B) for 2 min, 50:50 (A:B) for 5 min, 20:80
C RT (A:B) for 3 min. The total run time was 10 min and the flow rate was 0.5
mL/min. The column temperature was 40 ◦ C. The electrospray source
had the following properties: capillary voltage of 3.5 kV; cone voltage of
20 V; extractor voltage of 3 V; source temperature of 120 ◦ C; desolvation
temperature of 370 ◦ C; and desolvation gas (nitrogen) flow of 900 L/h.
The quantification of asparagine was performed by using the fragmen­
tation of the precursor ion of m/z 133 to m/z 116. The asparagine
standards used for the calibration curve were prepared at the levels of
0.1, 0.2, 0.5, 1, 2 mg/L and the extraction solvent was injected as blank.
The results were given as mg asparagine/kg sample.
2.5. Measurement of spread ratio
The spread ratio was calculated as the ratio of the diameter to the
C RT height. The diameter and height of the samples were measured with a
digital caliper. Two samples were taken from each bakery product and
measurements were taken from 3 different points of the samples.
2.6. Measurement of color
C RT
Color measurements were performed by using image analysis pre­
viously described by Mogol & Gökmen (2014). The results were given as
L*a*b* values. Measurements were performed in two replicates.
2.7. Measurement of pH
C RT
A 10 mL portion of deionized water was added onto 1 g dough
sample, and it was mixed in the shaker (Heidolph, Schwabach, Ger­
many) for 15 min. After then, the sample was centrifuged in a centrifuge
(Hettich Universal 320, Massachusetts, USA) at 6080 × g for 3 min. pH
measurement was made from the supernatant by using a pH meter
(Mettler & Toledo S220-K SevenCompact, Greifensee, Switzerland).
Measurements were performed in two replicates.
2.8. Measurement of water activity
The dough was weighed as 1 g and loaded into the cell of a water
activity measuring device (Novasina LabTouch-aw meter, Lachen,
Switzerland). Measurements were performed at 27 ◦ C in two replicates.
2.9. Statistical analysis
All data were subjected to one way ANOVA analysis of variance and
Tukey’s test using SPSS version 22. statistical program (IBM, 2013) to
reveal the similarities and differences between samples in all experi­
mental results. Differences at p < 0.05 were considered as significant.
4
S. Gazi et al. Food Chemistry 402 (2023) 134224

3. Results and discussion
3.1. Effects of asparaginase on asparagine and acrylamide reductions
3.1.1. Rotary cut biscuit and dough
The acrylamide concentration (µg/kg sample) and acrylamide
reduction (%) in rotary cut biscuits (sweet/semi-sweet hard dough with
low fat and high water content) after enzyme treatments are given in
Table 2. After application of 1000, 2000, and 3000 ASNU/kg flour
asparaginase enzyme dosages to the rotary cut biscuit dough, 35 %, 65
%, and 80 % acrylamide reduction was achieved, respectively. As the
enzyme dosage applied to rotary cut biscuit dough was increased, the
formation of acrylamide was mitigated significantly (p < 0.05). Simi­
larly, asparagine reductions obtained were 68 %, 95 %, and 99 % after
1000 ASNU, 2000 ASNU, and 3000 ASNU applications, respectively.
More asparagine converted to aspartic acid when the dosage of aspar­
aginase increased as there was still asparagine in the dough. Therefore,
acrylamide reduction was more effective when 3000 ASNU/kg flour
enzyme dosage applied to the rotary cut biscuit dough.
Adding dough resting period after enzyme application to dough did
not make any significant change in the acrylamide except for 3000
ASNU + 15 min RT treatment (96 %) where significant changes were
observed (p < 0.05) compared to 3000 ASNU treatment (80 %). Addi­
tionally, dough resting time did not change the asparagine content of the
asparaginase applied rotary cut biscuit dough (p > 0.05). Although the
difference in the asparagine content of 3000 ASNU + 15 min RT and
3000 ASNU treatments was low, the difference in their acrylamide
content and acrylamide reduction was higher indicating that even small
amount of change in the asparagine content of the dough may result in
higher changes in acrylamide content of the biscuits. Therefore, it could
be said that adding a dough resting period after asparaginase application
could be necessary for enzyme to show its activity. It could be suggested
that acrylamide can be reduced in rotary cut biscuits by selecting
appropriate asparaginase dosage and resting the dough after enzyme
treatment. In a study conducted by Kukurová, Ciesarová, Mogol, Açar, &
Gökmen (2013), asparaginase enzyme (500 U/kg flour) was added to
cookie dough and the dough without resting and with 15 min resting
time was baked at 205 ◦ C for 11 and 15 min. They found that acrylamide
reduction was higher when the dough rested, compared to the cookies
prepared without resting the dough at both baking conditions.
To reveal the effect of prolonging the dough resting time on acryl­
amide reduction in rotary cut biscuits, the resting time of the dough was

Table 2
Acrylamide content (µg/kg sample) and acrylamide reduction (%) in biscuits and asparagine concentration (mg/kg dough) and asparagine reduction (%) in biscuit
dough after asparaginase enzyme applications.
Bakery Products ASNU Applications* Acrylamide (µg/kg) Acrylamide reduction (%) Asparagine (mg/kg) Asparagine reduction (%)

Rotary cut biscuit dough Control 1672 ± 74a – 122.7 ± 12a –

1000 ASNU 1086 ± 29b 35 40.2 ± 7.3b 68
2000 ASNU 588 ± 32de 65 6.6 ± 1.8c 95
3000 ASNU 330 ± 42f 80 1.5 ± 0.4c 99
1000 ASNU + 15 min RT 933 ± 2bc 44 19.2 ± 1.6bc 85
2000 ASNU + 15 min RT 398 ± 42ef 76 2.1 ± 0.3c 98
3000 ASNU + 15 min RT 67 ± 16 g 96 0.2 ± 0.3c 100
2000 ASNU + 30 min RT 404 ± 47ef 76 4.9 ± 0.3c 96
2000 ASNU + 15 min × 37 ◦ C RT 262 ± 103 fg 84 1.1 ± 0.6c 99
2000 ASNU + MS 790 ± 76 cd 53 10 ± 0.1c 92
2000 ASNU + MT 720 ± 16 cd 57 7.3 ± 2.7c 94
2000 ASNU + MS × MT 774 ± 126 cd 54 5.6 ± 1.3c 96
2000 ASNU + S 820 ± 14 cd 51 13.8 ± 2.5c 89

Rotary molded biscuit dough Control 423 ± 71a – 113.5 ± 2.3ab –

2000 ASNU 399 ± 22a 6 113.3 ± 4.5ab –
2000 ASNU + 15 min RT 438 ± 9a – 113.8 ± 2.1ab –
2000 ASNU + 15 min × 37 ◦ C RT 496 ± 5a – 103.0 ± 13.2abc 9
2000 ASNU + NaHCO3 479 ± 132a – 117.2 ± 5.5a –
2000 ASNU + MPC 412 ± 109a 3 87.6 ± 2.1c 23
5000 ASNU + 30 min RT 500 ± 75a – 90.4 ± 3.2bc 20

Wire cut cookie dough Control 884 ± 118ab – 129.3 ± 10.3a –

1000 ASNU 872 ± 31ab 1 116.2 ± 1.2ab 10
2000 ASNU 935 ± 127a – 105.6 ± 4.3abc 18
3000 ASNU 797 ± 26abc 10 102.0 ± 7.2abcd 21
5000 ASNU 616 ± 104bcdef 30 100.9 ± 12.7abcd 22
7000 ASNU 528 ± 36cdef 40 90.3 ± 4.4bcde 30
9000 ASNU 623 ± 73bcdef 30 87.0 ± 2.4bcde 33
12000 ASNU 557 ± 11cdef 37 90.6 ± 14.4bcde 30
1000 ASNU + 15 min RT 862 ± 130ab 2 116.9 ± 1.6ab 9
2000 ASNU + 15 min RT 774 ± 34abcd 12 101.9 ± 1.4abcd 21
2000 ASNU + 30 min RT 712 ± 0.1abcde 19 96.0 ± 0.9abcd 26
3000 ASNU + 15 min RT 657 ± 27bcdef 26 104.1 ± 9.8abc 19
5000 ASNU + 15 min RT 542 ± 29cdef 39 78.7 ± 13.7cde 39
7000 ASNU + 15 min RT 469 ± 38ef 47 73.3 ± 10.8cde 43
9000 ASNU + 15 min RT 480 ± 82ef 46 68.5 ± 3.2de 47
9000 ASNU + 15 min × 37 ◦ C RT 443 ± 50ef 50 57.2 ± 11.4e 56
12000 ASNU + 15 min RT 505 ± 9def 43 72.5 ± 8.6cde 44
2000 ASNU + MPC 552 ± 29cdef 38 104.9 ± 1.0abc 19
5000 ASNU + MPC 406 ± 20f 54 88.3 ± 12.1bcde 32
9000 ASNU + MPC 515 ± 8def 42 97.7 ± 4.9abcd 24

*ASNU: Amount of L-asparaginase that syntheses one micromole of ammonia per minute under standard conditions per kg flour.
S: Shortening, RT: Resting Time, MS: Mixing Speed, MT: Mixing Time, MPC: Mixing Procedure Change. Uppercase letter indicates the statistically significant dif­
ferences (p < 0.05) in the columns for each biscuit type according to Tukey’s test.

5
S. Gazi et al.
increased from 15 min to 30 min. No difference was found in the
acrylamide contents of the rotary cut biscuits after 2000 ASNU + 15 min
RT (76 %) and 2000 ASNU + 30 min RT (76 %) applications to the
dough (p > 0.05). There was also no significant change in their aspar­
agine content (p > 0.05). Increase in the dough resting time had no effect
on acrylamide mitigation as 15 min already enough for the interaction of
enzyme and substrate in the rotary cut biscuit dough.
To understand the effect of dough resting temperature on acrylamide
reduction, dough resting temperature was increased from 25 ◦ C to 37 ◦ C.
The acrylamide reduction after 2000 ASNU + 15 min RT (76 %) and
2000 ASNU + 15 min × 37 ◦ C RT (84 %) applications was not statisti­
cally significant (p > 0.05). In parallel to acrylamide reduction, there
was no statistically significant difference (p > 0.05) in their asparagine
contents.
To reveal the effect of increasing the dough mixing time and speed on
mitigation of acrylamide formation, 2000 ASNU + MS, 2000 ASNU +
MT, 2000 ASNU + MS × MT treatments were applied to dough. After
these applications the acrylamide content of the rotary cut biscuits
decreased by 53 %, 57 % and 54 %, respectively. No statistically sig­
nificant difference was found in the asparagine concentration of the
dough after 2000 ASNU + MS, 2000 ASNU + MT, 2000 ASNU + MS ×
MT treatments (p > 0.05) compared to 2000 ASNU application. Addi­
tionally, there was no significant difference in acrylamide content of the
biscuits after these applications and 2000 ASNU application (p > 0.05)
indicating that increase in mixing speed and/or time did not have a
reducing effect on acrylamide formation in rotary cut biscuits.
To address the question whether the enzyme and substrate interac­
tion would occur easier when the shortening was added to the matrix at
the final stage of mixing, the ingredients other than shortening was
mixed and the shortening was added at the 8th min of mixing period.
Acrylamide content of the rotary cut biscuits was not different from each
other after 2000 ASNU + S and 2000 ASNU applied to dough (p > 0.05).
The difference in the asparagine reduction of the dough after 2000
ASNU + S (89 %) and 2000 ASNU (95 %) treatments was also insig­
nificant (p > 0.05). Anese, Quarta, Peloux, & Calligaris (2011) reported
that the presence of fat in the food matrix limited the association be­
tween enzyme and substrate and using hydrogel instead of fat allowed
easier interaction of enzyme and substrate in the food matrix. This result
showed that, on contrary to expectations, the shortening did not have a
negative effect on enzyme and substrate interaction.
3.1.2. Rotary molded biscuit and dough
Acrylamide content of rotary molded biscuits and asparagine content
of rotary molded biscuit dough (soft doughs with low water and high fat
content) after asparaginase applications were presented in Table 2. No
significant decrease in acrylamide content of the rotary molded biscuits
was observed after any of the asparaginase applications compared to
control (p > 0.05). This could be attributed to the limited water content
of the rotary molded biscuit dough that did not let asparaginase to be
active (Fig. 1). Similarly, there was no statistically significant difference
in asparagine content of the dough after any of the applications except
for 2000 ASNU + MPC (p > 0.05). Even though a 23 % reduction in
asparagine was achieved in 2000 ASNU + MPC application, the effect of
the same application on mitigation of acrylamide formation was very
limited.
3.1.3. Wire cut cookie and dough
There was no significant change in acrylamide content of the wire cut
cookies when the dough was treated with asparaginase dosages up to
7000 ASNU/kg flour (p > 0.05) and the reduction was up to 40 %
(Table 2). After applying 12000 ASNU to the dough, the reduction of
acrylamide in wire cut cookies (37 %) was different from the control (p
< 0.05). There was no statistically significant difference in asparagine
concentration of the dough after enzyme application up to 12000 ASNU/
kg flour (p > 0.05). This indicated that the asparaginase enzyme was not
optimally active in the wire cut cookie dough matrix. Therefore, no
6
Food Chemistry 402 (2023) 134224
significant reduction of acrylamide in the wire cut cookies was observed
at low dosages and the reduction was limited at high dosages. In a study
by Huang, Liu, Sun, & Yan (2013), 0.5 U/g flour, 1 U/g flour, 2 U/g
flour, 10 U/g flour, and 100 U/g flour asparaginase enzyme dosage was
applied to short dough biscuit, and as the enzyme dosage increased,
more mitigation in acrylamide formation was achieved until a certain
enzyme dosage. While the highest acrylamide reduction was 90 % at 10
U/g flour dosage, the difference between the 100 U/g flour and 10 U/g
flour treatments was reported to be insignificant (p > 0.05).
There was no significant difference in acrylamide content of the wire
cut cookies whose dough was treated with asparaginase and have resting
time (15 min) at the same asparaginase dosage (p > 0.05). Additionally,
the difference between the asparagine concentration of the dough that
have a resting time (15 min) and with no resting time at the same dos­
ages was insignificant (p > 0.05). This can be explained by the fact that
the resting period was not effective in this biscuit type since the enzyme
could not work effectively enough.
The absence of a significant difference between 2000 ASNU + 30 min
RT (19 %) treatment and 2000 ASNU + 15 min RT (12 %) in their
acrylamide content (p > 0.05) showed that the increase in resting time
was not effective in reducing acrylamide formation. Consistent with the
acrylamide results, the difference in asparagine reduction in the dough
after 2000 ASNU + 30 min RT (26 %) and 2000 ASNU + 15 min RT (21
%) treatments was not significant (p > 0.05).
There was no significant difference in the acrylamide content of wire
cut cookies after 9000 ASNU + 15 min × 37 ◦ C treatment and 9000
ASNU + 15 min RT treatment applied to dough at room temperature (p
> 0.05). No significant difference was observed between the asparagine
reduction (56 %) of 9000 ASNU + 15 min × 37 ◦ C RT application and
the asparagine reduction (47 %) of 9000 ASNU + 15 min application (p
> 0.05). Hence, it could be said that increase in the resting temperature
of the wire cut cookie dough matrix did not lead to a decrease in
acrylamide.
To reveal the effect of mixing procedure, 2000 ASNU + MPC, 5000
ASNU + MPC, and 9000 ASNU + MPC were applied to wire cut cookie
dough and acrylamide reductions were 38 %, 54 %, and 42 % in wire cut
cookies, respectively. However, the difference between the acrylamide
content of these applications and those of the enzyme applications
whose mixing procedure was not modified was insignificant (p > 0.05).
It was concluded that addition of water-soluble ingredients and enzyme
containing flour first and then cream had no effect on acrylamide con­
tent compared to application of regular creaming method in wire cut
cookie dough.
3.1.4. Rotary cut crackers and dough
As a result of the asparaginase applications performed to observe the
effect of enzyme dosage to rotary cut cracker dough, significant acryl­
amide reductions from 45 % (2000 ASNU/kg flour) to 79 % (3000
ASNU/kg flour) were achieved (p < 0.05) in rotary cut crackers
(Table 3). This reduction indicated that the dosage of asparaginase had a
positive effect in mitigation of acrylamide in rotary cut crackers. In a
study conducted by Kumar et al. (2014), different dosages of aspar­
aginase (50 U, 100 U, 200 U, and 300 U) enzymes were applied to sweet
breads, whose composition was as simple as rotary cut crackers, and it
was revealed that the amount of acrylamide formed after cooking
decreased as the enzyme dosage increased. The highest percentage of
acrylamide reduction in both the crust and crumb of the sweet bread was
reported to be 97 % and 73 % when the highest dosage of 300 U applied,
respectively.
The difference in the asparagine reduction after applications with
resting period (2000 ASNU + 15 min RT (67 %) and 3000 ASNU + 15
min RT (80 %)) and the applications with no resting time (2000 ASNU
and 3000 ASNU) was insignificant (p > 0.05). 81 % and 93 % reductions
in asparagine concentrations of rotary cut cracker dough were achieved
after 2000 ASNU + 15 min RT and 3000 ASNU + 15 min RT treatments,
respectively (p > 0.05). The reason for the dough resting time to be
S. Gazi et al. Food Chemistry 402 (2023) 134224

Table 3
Acrylamide content (µg/kg sample) and acrylamide reduction (%) in rotary cut cracker and asparagine concentration (mg/kg dough) and asparagine reduction (%) in
rotary cut cracker dough and wafer batter after asparaginase enzyme applications.
Bakery Products ASNU Applications* Acrylamide (µg/kg) Acrylamide reduction (%) Asparagine (mg/kg) Asparagine reduction (%)

Rotary cut cracker dough Control 1377 ± 129a – 126.1 ± 8.2a –

2000 ASNU 757 ± 173b 45 26.2 ± 11.9b 79
3000 ASNU 288 ± 76c 79 12.0 ± 5.8b 90
2000 ASNU + 15 min RT 458 ± 122bc 67 24.1 ± 0.5b 81
3000 ASNU + 15 min RT 279 ± 45c 80 8.5 ± 1.5b 93
2000 ASNU + 15 min × 37 ◦ C RT 491 ± 72bc 64 16.2 ± 6.2b 87

Wafer batter Control – – 137.0 ± 5.9a –

1000 ASNU – – 4.7 ± 1.3b 97
2000 ASNU – – 6.4 ± 0.6b 95
1000 ASNU + 15 min RT – – 6.0 ± 0.3b 96
2000 ASNU + 15 min RT – – 5.6 ± 0.4b 96
2000 ASNU + 30 min RT – – 5.6 ± 1.0b 96
1000 ASNU + 15 min × 37 ◦ C RT – – 4.8 ± 0.1b 96

*ASNU: Amount of L-asparaginase that syntheses one micromole of ammonia per minute under standard conditions per kg flour.
RT: Resting Time. Uppercase letter indicates the statistically significant differences (p < 0.05) in the columns according to Tukey’s test.

ineffective in acrylamide reduction of rotary cut crackers might be the
inactivation of asparaginase enzyme due to 1 % NaOH application to the
surface of the dough.
There was no significant difference in the acrylamide content of the
rotary cut crackers after 2000 ASNU + 15 min and 2000 ASNU + 15 min
× 37 ◦ C RT treatments (p > 0.05). Similarly, the difference in asparagine
content of dough after 2000 ASNU + 15 min × 37 ◦ C RT (87 %) and
2000 ASNU + 15 min (81 %) treatments was not significant (p > 0.05)
which indicated that dough resting temperature did not affect aspar­
aginase activity in rotary cut cracker dough.
3.1.5. Wafer batter
Asparagine content of 1000 ASNU (97 %) and 2000 ASNU (95 %)
applications in wafer batters was not different from each other (p >
0.05) (Table 3). It was found that the increase in asparaginase dosage did
not affect the mitigation of asparagine because the asparaginase enzyme
converted the asparagine in the dough to aspartic acid even at a dose of
1000 ASNU/kg flour.
Asparagine reduction after 1000 ASNU + 15 min RT (96 %) and 2000
ASNU + 15 min RT (96 %) applications compared to the applications at
the same dosages without a resting period were not different from each
other (p > 0.05). Hence, it could be said that the dough resting period
did not affect asparagine reduction as the enzyme already reduced
asparagine at a high ratio without this period.
The difference in asparagine mitigation in the wafer batter after 2000
ASNU + 15 min RT (96 %) and 2000 ASNU + 30 min RT applications
(96 %) was insignificant (p > 0.05). It was shown that prolonging dough
resting time in wafer batter did not have a positive effect on asparagine
reduction. Moreover, increase in the dough resting temperature (1000
ASNU + 15 min and 1000 ASNU + 15 min × 37 ◦ C RT) did not affect
asparagine reduction in wafer batter.
3.2. Interrelation between asparagine and acrylamide contents
Gökmen, (2020), medium and high positive correlations (R2 = 0.45,
0.48, 0.90) were found between the asparagine content of biscuits ob­
tained from different cereal flours and the formation of acrylamide.
3.3. Effects of dough conditions on the effectiveness of asparaginase
3.3.1. Water activity
Water activity levels of dough types were given in Fig. 1. Wafer
batter had the highest water activity value (0.97 ± 0.01). The water
activity value of rotary cut cracker dough was 0.92 ± 0.01 and rotary
cut biscuit dough was 0.85 ± 0.02, respectively. The rotary molded
biscuit dough had the lowest water activity (0.70 ± 0.01) and there was
no significant difference in water activity values of the rotary molded
biscuit dough and the wire cut cookie dough (0.74 ± 0.01) (p > 0.05).
Asparaginase enzyme was most effectively active in the wafer batter
with the highest water activity value, resulting in a 97 % reduction in
asparagine even at the lowest enzyme dosage of 1000 ASNU/kg flour. It
was found that enzyme activity decreased as water activity decreased.
For instance, 30 % acrylamide reduction was achieved at 5000 ASNU/kg
flour dosage in wire cut cookies, while a 65 % reduction was achieved at
2000 ASNU/kg flour in rotary cut biscuits which has higher water ac­
tivity than wire cut cookies. In addition, the asparaginase enzyme that
did not have enough water for its activity in the rotary molded dough
could not provide any reduction in either asparagine or acrylamide.
Hendriksen et al. (2009) applied 1000 ASNU asparaginase to ginger
biscuits with different moisture contents. While a 90 % reduction in
acrylamide formation was reported to achieved in the sample with the
highest moisture content (19 %), the reduction in acrylamide was
limited to 19 % in the sample with the lowest moisture content (13 %).
The reason for this situation was explained by the authors that as the
decrease in the mobility of asparaginase enzyme in the matrix and the
difficulty in interacting with the enzyme and the substrate due to the low
water content.

According to the results obtained in enzymatic applications, the 3.3.2. pH

correlation coefficients (R2) between asparagine in the dough and
acrylamide in rotary cut biscuits, rotary molded biscuits, wire cut
cookies, and rotary cut crackers were found to be 0.74, 0.03, 0.64 and
0.91, respectively. This situation revealed that there was a high positive
interrelation between asparagine content and acrylamide in all products
except for the rotary molded dough biscuits where the enzyme was not
active during enzyme treatments of dough. Based on this positive
interrelation between asparagine and acrylamide, and when matrix ef­
fects were not considered, 97 % asparagine reduction in wafer batter
will be enough to mitigate acrylamide formation effectively also in
wafer. In the study conducted by Žilić, Aktağ, Dodig, Filipović, &
In the enzymatic treatment applied to rotary cut biscuit dough, ro­
tary molded biscuit dough, wire cut cookie dough, rotary cut cracker
dough, and wafer batter, pH values varied from 8.38 to 8.54, 7.54 to
8.01, 7.43 to 8.14, 7.86 to 7.98, 6.98 to 7.02, respectively (Table S1-S5).
The fact that there was no significant difference between the pH of the
enzymatic applications in these products in most of the applications
indicating that pH had no effect on the activity of asparaginase enzyme
in the studied bakery products.
In alkaline conditions (pH 8) the protonation of the amino group of
asparagine accelerates the formation of acrylamide by interacting more
with the carbonyl group (Rydberg et al., 2003). Additionally, the

7
S. Gazi et al.
optimum pH of the commercial asparaginase enzyme, also used in this
study, was reported to be 7 (Xu et al., 2016). Considering the bakery
products in this study, their pH seems favorable for acrylamide forma­
tion but not exactly at the optimum pH value of asparaginase. Therefore,
one could expect that the decrease in asparagine will be limited and,
accordingly, less decrease will be achieved in the amount of acrylamide.
However, on the contrary to this expectation, pH differences in different
product types did not affect acrylamide reduction.
The pH of the dough of rotary cut crackers and rotary cut biscuits
were slightly higher than the pH of the dough of rotary molded biscuits
and wire cut cookies. Normally, due to the above-mentioned reasons,
more acrylamide reduction is expected as the pH of the rotary molded
biscuit dough is lower. However, the high mitigation of acrylamide
formation in rotary cut crackers and rotary cut biscuits even in low
asparaginase dosages was related to water activity which was more
Fig. 2. Digital images of biscuits/cracker produced with the most effective enzyme applications in terms of asparagine and acrylamide mitigations for different
dough types.
8
Food Chemistry 402 (2023) 134224
effective on the activity of the asparaginase enzyme than pH in these
bakery product types.
3.4. Effect of asparaginase on spread ratio and color
After enzymatic treatments applied to rotary cut biscuit dough, ro­
tary molded biscuit dough, wire cut cookie dough and rotary cut cracker
dough, spread ratio values varied from 5.7 to 6.0, 6.1 to 7.2, 6.5 to 7.8,
35.9 to 36.1, respectively (Table S6-S9). Asparaginase applications did
not cause a significant difference in the spread ratio value of any bakery
product compared to the control samples in most of the applications.
This showed that the application of asparaginase enzyme did not have a
negative effect on the size of the bakery products. Similarly, L*a*b*
color values of bakery products showed no significant difference after
enzyme applications (Table S10-S13). The photos of the biscuits/
S. Gazi et al.
cracker were presented to compare the surface color of the control
samples and the samples that had the most acrylamide mitigation after
enzyme application (Fig. 2). In the control sample of the rotary cut
biscuits, the L*, a*, b* values were 71, 4.1, 42, respectively, and they
were 70, 4.4, 42 in the surface of the biscuit after the most effective
application (3000 ASNU + 15 min RT), respectively. The difference in
surface color values between control and after the most effective
application of rotary cut biscuit dough was insignificant (p > 0.05).
Additionally, no difference in L*a*b* color values of the control (70, 5.0,
41, respectively) and after the most effective enzyme application, which
was 5000 ASNU + MPC (66, 7.2, 43, respectively), was found for wire
cut cookies (p > 0.05). In the control sample of the rotary cut crackers,
the L*, a*, b* color values were 71, 1.3, 32, respectively. The L*a*b*
color values of the rotary cut crackers were 71, 0.5, 32, respectively after
the most effective application, which was 3000 ASNU + 15 min RT,
where no significant difference was found compared to control (p >
0.05). Accordingly, it was revealed that the application of asparaginase
enzyme did not have a negative effect on the surface color of bakery
products. In a study conducted by Anese et al. (2011), it was revealed
that there was no significant difference in color of 900 U/kg aspar­
aginase enzyme added sample and the sample without asparaginase
enzyme in short dough biscuits. Kukurová et al. (2013) prepared cookie
dough by using asparaginase enzyme at a dosage of 500 U/kg flour and
the dough was rested for 15 min, 30 min, and 60 min. They reported that
the L*a*b* values of the cookies were not different significantly
compared to control after baking (p > 0.05).
4. Conclusion
In asparaginase applications to reduce acrylamide in bakery prod­
ucts, the effects of process factors such as enzyme dosage, dough resting
period, dough resting time, dough resting temperature, adding fat to the
matrix at the last stage of mixing, dough mixing speed, dough mixing
time, and changes in mixing procedure were the parameters tested in
selected bakery products. As a result of asparaginase application, the
highest acrylamide reduction (96 %) was achieved in the rotary cut
biscuits, followed by rotary cut crackers (80 %), and wire cut cookies
(56 %). In general, acrylamide formation in tested bakery products was
positively correlated with asparagine content except for the rotary
molded biscuit. There was no reduction in acrylamide in rotary molded
biscuits under the tested conditions as rotary molded biscuit dough was
not a suitable medium for asparaginase to be active because of its
relatively low water activity. The results obtained in this study showed
for the first time that the water activity value of dough should be>0.70
for the asparaginase enzyme to be effective. It was observed that the
asparaginase enzyme was generally more effective in doughs with high
water activity. In the wafer batter with the highest water activity (0.97),
asparagine reduction>95 % occurred after all asparaginase treatments.
Moreover, asparaginase applications were found to have no negative
effect on the quality parameters such as surface color or spread ratio of
bakery products.
Overall, the results highlighted the importance of pH, and especially
water activity of the dough for an effective application of asparaginase
enzyme towards acrylamide mitigation in bakery products. Moreover,
addition of a resting period and prolonging dough resting time, by
themselves or in combinations were suggested to be applied to the
bakery products by considering the activity of enzyme. The results re­
ported here could help guiding the food industry on how to reduce
acrylamide by means of asparaginase enzyme below the benchmark
levels in bakery products to get prepared for the regulations limiting
acrylamide in the forthcoming period.
CRediT authorship contribution statement
Selahattin Gazi: Methodology, Formal analysis, Investigation, Data
curation, Software, Visualization, Writing - original draft. Neslihan
9
Food Chemistry 402 (2023) 134224
Göncüoğlu Taş: Conceptualization, Methodology, Validation, Investi­
gation, Writing - review & editing. Ahmet Görgülü: Methodology, Re­
sources, Writing - review & editing. Vural Gökmen: Conceptualization,
Methodology, Resources, Supervision, Writing - review & editing.
Declaration of Competing Interest
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.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.foodchem.2022.134224.
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