LWT - Food Science and Technology 44 (2011) 1473e1476

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LWT - Food Science and Technology

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Acrylamide reduction in potato chips by using commercial asparaginase

in combination with conventional blanching
Franco Pedreschi a, *, Salomé Mariotti a, Kit Granby b, Jørgen Risum c
a
Department of Chemical Engineering and Bioprocesses, Pontificia Universidad Católica de Chile, Av. Vicuña Mackenna 4860, Macul, Santiago, Chile
b
The National Food Institute, The Technical University of Denmark, Moerkhoej Bygade 19, 2860 Soeborg, Denmark
c
BioCentrum-DTU, The Technical University of Denmark, Søltofts Plads, Building 221, DK-2800 Kgs, Lingby, Denmark

a r t i c l e i n f o a b s t r a c t

Article history: In this research acrylamide reduction in potato chips was investigated in relation to blanching and
Received 18 March 2010 asparaginase immersion treatments before final frying. Potatoes slices (Verdi variety, diameter: 40 mm,
Received in revised form thickness: 2.0 mm) were fried at 170
C for 5 min (final moisture content of w2.0 g/100 g). Prior to frying,
27 January 2011
potato slices were treated in one of the following ways: (i) Rinsing in distilled water (control I); (ii)
Accepted 1 February 2011
Rinsing in distilled water plus blanching in hot water at 85
C for 3.5 min; (iii) Rinsing in distilled water
plus immersion in an asparaginase solution (10000 ASNU/L) at 50
C for 20 min; (iv) Rinsing in distilled
Keywords:
water plus blanching in hot water at 85
C for 3.5 min plus immersion in an asparaginase solution
Potato chips
Blanching
(10000 ASNU/L) at 50
C for 20 min; (v) Rinsing in distilled water plus blanching in hot water at 85
C for
Acrylamide 3.5 min plus immersion in distilled water at 50
C for 20 min (control II). Blanching in hot water (ii) was
Asparaginase almost as effective as asparaginase potato immersion (iii) in order to diminish acrylamide formation in
Color potato chips (acrylamide reduction was w17% of the initial acrylamide concentration). When potato
slices were blanched before asparaginase immersion, the acrylamide content of the resultant potato
chips was reduced considerably by almost 90%. We have demonstrated that blanching of potato slices
plus asparaginase treatment is an effective combination for acrylamide mitigation during frying. It seems
to be that blanching provokes changes in the microstructure of potato tissue leading to an easier and
more effective diffusion of asparaginase.
Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction Acrylamide is considered as a “potential” carcinogen in humans

In April 2002, Swedish researchers sparked the world’s atten-
tion when they presented preliminary findings of acrylamide in
some fried and baked foods, most notably potato chips and French
fries (Tareke, Rydberg, Karlsson, Eriksson, & Tornqvist, 2002). They
attributed this fact to the higher temperatures reached in Maillard
non-enzymatic browning reactions required for desirable color,
flavor and aroma production in those cooking operations. Acryl-
amide was formed in several starch-based products (e.g. French
fries and potato chips) heated above 120
C. These two products
exhibited relatively high values of acrylamide of 424 mg/kg and
1739 mg/kg, respectively. Additionally, the potential capability of
different potato varieties to form acrylamide during heat treatment
correlated well with the concentration of reducing sugars (e.g.
glucose and fructose) and asparagine present in the tubers (Zyzak
et al., 2003).
* Corresponding author. Tel.: þ56 2 3545803; fax: þ56 2 3541221.
E-mail address: fpedreschi@ing.puc.cl (F. Pedreschi).
due to its implication in cancer in rats. In addition, acrylamide is
present in high concentrations in several highly popular and of high
consumption foods. Thus, acrylamide monitoring and mitigation
are issues of major relevance (Rosén and Hellenäs, 2002). The
analytical methods for acrylamide determination are based on (i)
gas chromatography and mass spectrometry -GCeMS- (Tareke
et al., 2002) or (ii) liquid chromatography and tandem mass spec-
trometry -LCeMSeMS- (Rosén and Hellenäs, 2002). Recently, an
LCeMSeMS methodology with a detection limit for acrylamide of
20 mg/kg in the case of French fries analysis was developed. This
new methodology allows simultaneous analysis of acrylamide and
their precursors such as asparagine and glucose (Nielsen, Granby,
Hedegaard, & Skibstead, 2006).
Maillard reaction is responsible for the brown color and tasty
flavor of baked, fried and toasted foods. The Maillard reaction has
also been shown to be the major pathway for the formation of
acrylamide in heated food products (Mottram, Wedzicha, &
Dodson, 2002; Stadler et al., 2002). Acrylamide is formed in the
Maillard reaction. For instance certain starch-based food products
such as potato chips, French fries, bread and processed cereals

0023-6438/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.lwt.2011.02.004
1474 F. Pedreschi et al. / LWT - Food Science and Technology 44 (2011) 1473e1476
contained acrylamide when heated above 120
C (Tareke et al.,
2002). So, producing chips with low levels of acrylamide or with
trace levels but without compromising the sensorial properties of
the final product seems to be the major challenge (Zhang & Zhang,
2007). Most of the methods to mitigate the formation of acrylamide
seek to remove its precursors (glucose, fructose, asparagine) or to
inhibit or reduce the intensity of the Maillard reaction by different
process modifications.
Methods to reduce acrylamide mainly focus on the process
parameters (e.g. vacuum frying or conventional frying at low
temperatures). Post-frying techniques could also eventually be
implemented to decrease acrylamide formation. But the major
challenge is the reduction of acrylamide levels in fried potatoes
without compromising the sensorial properties of the final product
(Pedreschi, 2009). One alternative could be the use of asparaginase.
This enzyme hydrolyzes free asparagine to aspartic acid which is
not a precursor to form acrylamide (Amrein, Schöbächler, Escher,
and Amadó, 2004). Furthermore, the use of asparaginase on the
nutritional and sensorial properties of the heated products has no
detrimental effects (Hendriksen, Kornbrust, Østergaard, & Stringer,
2009).
Commercial asparaginase is produced from Aspergillus oryzae
based on cloning technology. This enzyme has an optimum pH of
6e7. Good enzymatic activity is obtained between the 5e8 pH
range. The 5e8 pH range may be suitable for the production of
potato-based products (French fries and chips). By reusing the
enzyme immersion solutions the costs for the enzyme can be
reduced and the added protein content to the final product will be
kept low and thus will not affect the overall quality of the process
wastewater (Pedreschi, Kaack, & Granby, 2008).
Dried potato powder treated with L-asparaginase presented
significantly lower amounts of acrylamide formation 90e97%
(Ciesarova, Kiss, & Boegl, 2006). Additionally, some authors have
reported that French fries treated with asparaginase presented
a reduced content of acrylamide of 60% (Pedreschi et al., 2008).
Dough based applications with a reduction in final acrylamide
contents of 34%e92% when asparaginase was employed has been
reported by Hendriksen et al. (2009). Enzyme dose, dough resting
time and water content were identified as critical parameters.
There is need of approaches that mitigate acrylamide formation
in heated food products. Thus, the objective of this work was to
study the effect of blanching in combination with commercial
asparaginase to mitigate acrylamide formation during frying.
2. Materials & methods
2.1. Materials
Potatoes tubers (Verdi variety; moisture content of 72 g/100 g)
and palm oil (Fritex, Århus Oil, Denmark) were the raw materials.
Potatoes used in this research were grown in Denmark and were
stored at 8
C and 95 g/100 g of relative humidity. Then, they were
washed and peeled in an industrial peeler (model M591E4, IMC,
England). Slices (thickness of 2.0 mm) were cut from the pith of the
parenchymatous region of potato tubers using an electric slicing
machine (model EAS65, Berkel, USA). A circular cutting mold was
used to provide chips with a diameter of 40 mm.
2.2. Pre-treaments
Potato slices were rinsed immediately after cutting for 1 min in
distilled water to eliminate some starch material adhering to the
surface prior to frying. The following pre-treatments were applied
before frying:
A: Raw potato slices rinsed in water distilled without any
treatment (control I).
B: Blanched slices were considered those slices immersed in hot
distilled water at 85
C for 3.5 min in a ratio of potato to water
(g/g) of w0.5.
C:Rawslicesimmersedina10000ASNU/Lasparaginasesolutionat
50
C for 20 min (ratio of potato to enzyme solution e g/g e
ofw0.5).
D: Blanched slices in hot water at 85
C for 3.5 min and then,
immersed in a 10000 ASNU/L asparaginase solution at 50
C for
20 min (ratio of potato to enzyme solution e g/g e of w0.5).
E: Blanched slices in hot water at 85
C for 3.5 min and then,
immersed in distilled water at 50
C for 20 min (ratio of potato
to enzyme solution e g/g e of w0.5) e Control II.
An asparaginase solution containing 10000 ASNU/L (1 ASNU being
defined as the amount of asparaginase that produces 1 mmol of
ammonia per minute under the conditions of the assay
(pH ¼ 7  0.005; 37  0.5
C)) was prepared from commercially
available AcrilawayÒ (Novozymes A/S, Bagsvaerd, Denmark). Finally,
raw or pre-treated potato slices were deep-fried at 170  1
C for 5 min
(until reach a final moisture content of w2.0 g/100 g) in an electrical
fryer (Fritel 2505, Belgium). The experiments were made in duplicate.
2.3. Asparagine, glucose and acrylamide determination
For the determination of asparagine and glucose in the samples
before frying the liquid-chromatographyetandem mass spec-
trometry method described by Nielsen et al. (2006) was used with
slight modifications. Potato chips were homogenized using a mixer
(model 4169/4297, Braun AG, Kronberg, Germany). To an aliquot
homogenate of 0.3 g, internal standards such as 15N2-asparagine
and 13-D-glucose and 30 ml of boiled deionized water, were added.
The sample was extracted by a homogenizer (model Ultra Turrax
T25, Janke and Kunkel, Germany) at 10,000e12,000 rpm for 2 min.
The sample was centrifuged at 500g for 20 min (Heraeaus Mul-
tifuge, Osterode, Germany) and an aliquot of 4 ml was transferred to
Eppendorf vials and frozen to 18
C for at least 30 min and
subsequently centrifugated in an Eppendorf centrifuge at 12,100g
for 10 min (Minispin Centrifuge, Eppendorf Ag, Hamburg,
Germany) The sample thawed during centrifugation and starch
precipitated from the supernatant at this low temperature. The SPE
(Solid Phase Extraction) cleanup was performed by an automated
sampler (Gilson Aspec Xl, Gilson Company Inc., Ohio, USA) using
LiChroLut Rp-C18 SPE-cartridges (500 mg) from Merck (Darmstadt,
Germany). The eluate was transferred to Miniprep PTFE filter HPLC
vials with a pore diameter of 0.45 mm (Whatman Inc., USA).
For acrylamide analysis, acrylamide (2-propene amide) [CAS No.
79-06-1] (>99.5%) was obtained from SigmaeAldrich (St. Louis,
MO, US). Labeled D3-acrylamide (>98%) was from Polymer Source
Inc. (Dorval, Quebec Canada). The acrylamide analysis was per-
formed on 3 g aliquots of homogenized fried potato samples, added
internal standard 200 ml, 10 mg/ml D3-acrylamide and 30 ml of
deionized water. The extraction procedure was similar to the
asparagine analyses. However the cleanup was made on 300 mg
Isolute Multimode SPE-columns (IST). The LC system consisted of
a liquid chromatograph (model HP1100, Agilent Technologies,
USA). Separation was performed with 0.1 g/100 g formic acid in
water on a Hypercarb column (dimensions 2.1 mm  100 mm,
particle size 5 mm).
The MSeMS detection was performed on a Quattro Ultima triple
quadropole instrument from Micromass (UK). The source was kept
at 120
C and the desolvation temperature was 400
C. Nitrogen
was used as cone and desolvation gas with flow rates of 150 and
500 l/h, respectively. Argon was used as collision gas and kept at
 F. Pedreschi et al. / LWT - Food Science and Technology 44 (2011) 1473e1476
2500
2000
Acr yl am i de content ( g/ kg)
1500
1000
500
0
A B C D E
Pre-treatments
Fig. 1. Acrylamide content of potato slices treated with commercial asparaginase. A.
Raw potato slices (control I); B. Blanched potato slices at 85
C for 3.5 min; C. Raw
potatoes slices immersed in a 10000 ASNU/L asparaginase solution at 50
C for 20 min;
D. Blanched potato slices at 85
C for 3.5 min plus immersion in a 10000 ASNU/L
asparaginase solution at 50
C for 20 min; E. Blanched potato slices at 85
C for 3.5 min
plus immersion in distilled water at 50
C for 20 min (control II). All experiments were
done in duplicate.
a pressure 0.24 Pa. Detection was performed by multiple reactions
monitoring (MRM). Acrylamide and glucose were detected in
positive ion mode, while asparagine was detected in negative ion
mode. Quantification of the fragmentations was done using Mas-
sLynx software version 4.0 including QuanLynx.
2.4. Color determination
Potato chip color was measured using a colorimeter (model
ChromaMeter CR 200b, Minolta, USA) attached to a data-processor
DP-100 using the Commission Internationale d’Eclairage (CIE) Lab
L*, a* and b* color space. Calibration was performed against a white
standard tile using illuminant “C”. Triplicate reading were carried
out at 20
C on both sides of the chip and the mean value was
recorded. L*, a* and b* represent.
2.5. Statistical analysis
Analysis of variance was carried out using Statgraphic Statistical
Package (Version 4, Statistical Graphics Corporation, Rockville, USA)
including multiple range tests (P > 0.05) for separation of least
square means. Experiments were run in duplicate.
3. Results and discussion
3.1. Acrylamide content in pre-treated potato slices after frying
The control I potatoes sample (control I) contained 2047 mg/kg of
acrylamide (Fig. 1). Blanching resulted in almost 17%(350 mg/kg) less
acrylamide formation in potato slices after frying (Fig. 1B). This fact
has been explained previously by some authors since not only
reducing sugars but also asparagine (both acrylamide precursors)
are leached out during blanching (Pedreschi, Kaack, & Granby,
2004). When raw potato slices were immersed in the asparaginase
solution, the acrylamide content diminished in almost 15% (300 mg/
kg) (Fig. 1C). This reduction could be attributed principally to
a reduction of asparagine in the more external layers of the potato
chips that could be reached by the enzyme. These results indicated
that blanching treatment and the asparaginase immersion (without
previous blanching) are equivalent in diminishing acrylamide
formation in potato slices during frying. On the other hand, it is
worth to note that when blanched potatoes are immersed in
1475
asparaginase solution, the acrylamide content diminished dramat-
ically from 2047 mg/kg to 158 mg/kg. Surprisingly, a combined
thermal treatment of potato slices (blanching plus immersion in
distilled water) accounted for 62% of the total acrylamide reduction.
This result indicates the net effect of the asparaginase treatment on
acrylamide reduction in potato chips corresponds to the 75% of the
total acrylamide reduction (1536 mg/kg). The blanching will not only
facilitate enzyme diffusion but also facilitate asparagine to move
toward the surface. Heat treatment like blanching during potato
processing causes a change in the microstructure of potato strips
(e.g. the starch swells, cell wall degradation), that may further
improve the diffusion of asparagine toward the asparaginase in the
surrounding solution.
(Pedreschi et al., 2004) Apparently blanching produces micro-
structural changes in potato tissue which facilitates interaction
between asparagine and asparaginase. On the other hand, when
asparaginase gets in contact directly on raw potato slices, potato
microstructure makes the enzyme diffusion in the tissue more
difficult.
The potential of potato varieties to form acrylamide during heat
treatment has been shown to correlate well with the concentra-
tions of glucose and asparagine in the tubers (Amrein, Bachmann,
Noti, Biedermann, FerrazBarboza, Biedermann-Brem, Grob, Keiser,
Realini, Escher and Amadó, 2003). The raw potatoes in this
research contained 4.325 g/kg of asparagine and 0.097 g/kg of
glucose, respectively. Fig. 2a shows that blanching (85
C, 3.5 min)
had a considerable effect in removing glucose (whalf of the total
reduction). All samples that underwent blanching treatment had
a very similar percentage of glucose reduction, as expected. On the
other hand, the asparaginase hydrolyzes asparagine to form
aspartic acid and ammonia. As stated by the manufacturer of the
a 0,12
0,10
Glucose content (g/ kg)
0,08
0,06
0,04
0,02
0,00
A B C D E
Pre-treatments
b 6,00
5,00
Asparagi ne content (g/ kg)
4,00
3,00
2,00
1,00
0,00
A B C D E
Pre-treatments
Fig. 2. Effect of asparaginase on the content of glucose (a) and asparagine (b) of potato
slices before frying. Samples AeE as in Fig. 1. All experiments were done in duplicate.
1476 F. Pedreschi et al. / LWT - Food Science and Technology 44 (2011) 1473e1476
80
70
60
50
Col or com ponent
40
30
20
10
0
-10 A B C D
-20
Pre-treatments
Fig. 3. Color of potato slices treated with commercial asparaginase. Samples AeE as in
Fig. 1. L*: white; a*: gray and b*: black. All experiments were done in duplicate.
commercial enzyme, its asparaginase exhibits amidase but no
peptidase activity, so as no peptide bonds are hydrolyzed. Fig. 2b
shows that the blanching treatment reduced the asparagine
content by 0.43 g/100 g due to its leaching out from the potato
tissue to the blanching water. Additional heating of potato slices at
50
C for 20 min in distilled water after blanching reduced acryl-
amide content by an additional 44% (907 mg/kg) (sample E). Both
samples treated with asparaginase only and those which in addi-
tion were previously blanched, did not contain detectable amounts
of asparagine, suggesting a complete removal of this precursor by
the action of the enzyme. There is no coherence between treat-
ments B and C for asparagine and acrylamide content. Blanching
plus asparaginase immersion led to a complete removal of aspar-
agine yielding potato chips with significantly lower acrylamide
content.
The color of fried potato is the result of non-enzymatic
browning reactions (Maillard reactions) which depend on the
reducing sugar and asparagine content, and the temperature of
frying and the frying time. The effect of asparaginase treatment on
the color of potato chips is shown in Fig. 3. Values of L* a* and b*
components did not differ considerably among all the pre-treat-
ments tested. Potato chips previously blanched and then immersed
in distilled water at 50
C for 20 min behaved in the same way.
These results suggest that an asparaginase treatment did not lead to
less darker potato chips due to the reduction of asparagine.
4. Conclusions
Blanching improved the action of commercial asparaginase in
reducing asparagine concentration in potato pieces and leading to
potato chips with lower acrylamide contents. When asparaginase
acts directly on potato slices (without a blanching treatment
before), its effect in reducing acrylamide content in potato chips is
significantly lower. Asparaginase treatment did not affect signifi-
cantly the color of potato chips.
Acknowledgments
Authors acknowledge financial support from FONDECYT Project
N
1070031. Collaboration of Novozymes A/S, Kims A/S and Aarhus
E Oil (www.AAK.com) are highly appreciated for supplying us with
the asparaginase, the potatoes and the oil, respectively.
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