Study on reduction of acrylamide in fried bread sticks by addition of antioxidant of bamboo leaves and extract of green tea

131 Asia Pac J Clin Nutr 2007;16 (Suppl 1):131-136

Original Article

Study on reduction of acrylamide in fried bread sticks
by addition of antioxidant of bamboo leaves and extract
of green tea

Yu Zhang BSc and Ying Zhang PhD

Department of Food Science and Nutrition, School of Biosystems Engineering and Food Science, Zhejiang
University, Hangzhou, China


This paper investigated the efficiency of antioxidant of bamboo leaves (AOB) and extract of green tea (EGT) on
the reduction of acrylamide in fried bread sticks and summarized the optimal levels of two additives. Seven ex-
perimental groups including a control group were organized for both of additives. Fried bread sticks were made
via traditional processing technology. The flour was mixed with different levels (0.002–4.9 g/kg flour) of AOB
and EGT, respectively. The acrylamide level in fried bread sticks was determined by liquid chromatography tan-
dem mass spectrometry (LC-MS/MS). The sensory evaluation was performed in double blind manner. Results
showed that nearly 82.9% and 72.5% of acrylamide were reduced when the AOB and EGT addition levels were 1
and 0.1 g/kg, respectively. The elevated inhibitory effects of AOB and EGT on the acrylamide formation were
achieved with an increase of additive levels unless the spiking levels of AOB and EGT were greater than 1 and
0.1 g/kg, respectively. Sensory evaluation results showed that the flavor and texture of fried bread sticks proc-
essed by AOB and EGT had no significant difference compared to normal food matrixes (p>0.05) when both
AOB and EGT addition levels were no more than 1 g/kg. The present study indicated that both AOB and EGT
could significantly reduce the acrylamide content generated in fried bread sticks and keep original flavor and
crispness of fried bread sticks. This study could be regarded as an important contribution on the reduction of
acrylamide by natural antioxidants.

Key Words: acrylamide, fried bread sticks, reduction, antioxidant of bamboo leaves, extract of green tea



Introduction
Schiff base,10 decarboxylated Amadori products,11 acrylic
Acrylamide, a potential genetic and reproductive toxin, was acid,12 and acrolein,13 which play key roles in the final
detected in carbohydrate-rich fried or baked food samples generation of acrylamide.
by the research groups from Swedish National Food Ad-
Based on current knowledge, acrylamide may be re-
ministration (SNFA) and University of Stockholm in 2002.1 duced under the following situations: (i) Some key inter-
Such novel finding has attracted wide attention throughout mediates are eliminated under the change of reaction condi-
the world. Earlier toxicological studies demonstrated that tions; (ii) Other vinylogous compounds instead of acryla-
acrylamide is carcinogenic to experimental rats and mice, mide are formed; (iii) Some key pathways such as the
causing tumors at multiple sites in both species.2,3 The formation of Schiff base, Strecker type degradation, N-
International Agency for Research on Cancer (IARC) has glucoside pathway and ?-elimination reaction of the decar-
therefore classified it as “potential carcinogenic to hu-
boxylated Amadori compounds are blocked.10–11,14 During
mans”.4 However, epidemiologic studies of possible health these years, various effective methods for the reduction of
effects from exposures to acrylamide have not produced acrylamide in the actual food matrixes have been found
consistent evidence of increased cancer risk, in either such as prevention of reducing sugar liberation during the
occupationally exposed workers or the general populations storage period of food materials,15 change of heat process-
of several countries.5,6 Nevertheless, considering the ac-
ing methods,16 optimization of suitable cultivar and storage
knowledged neurotoxicity of acrylamide, recent investiga-
temperature of food materials,17 fermentation,18 modifica-
tion of considerable acrylamide levels in fried foods still tion of pH,19 reduction of ammonium bicarbonate,20 addi-
evoked an international health alarm. After the discovery of tion of competing amino acids,21 etc.
acrylamide formation in Maillard reaction,7,8 several hy-

potheses on its formation mechanisms were recommended
at very early stages of investigations. Initially, mechanistic
studies mainly focused on the acrylamide formation in Corresponding Author: Dr. Ying Zhang, Department of Food
vegetable oils or lipids since the problem primarily encom-
Science and Nutrition, Zhejiang University, 268 Kaixuan Road,
passed carbohydrate-rich foods that are fried or baked. To Hangzhou 310029, China
date, researches demonstrated some important intermedi-
Tel: +86 571 8697 1388; Fax: +86 571 8604 9803
ates including 3-aminopropionamide,9 decarboxylated Email: y_zhang@zju.edu.cn

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Y Zhang and Y Zhang 132
However, few studies reported the effective way to reduce
dreds of millions of people consume fried bread sticks as
acrylamide by the addition of food antioxidants. For in-
their breakfast in everyday life, so it is very indispensable
stance, a weak reduction effect on the acrylamide forma-
to find a feasible way to reduce acrylamide levels in this
tion was found via the addition of ascorbic acid in a po-
product.
tato-based model.22 Furthermore, nearly 50% reduction of

acrylamide occurred after the addition of a flavonoid
The aims of this study were
spice mix.23
(i) to investigate the effect of AOB and EGT on the re-
Antioxidant of bamboo leaves (AOB), a pale brown
duction of acrylamide in fried bread sticks; (ii) to summa-
powder extracted form bamboo leaves, was capable of
rize the optimal levels of two additives, which can be ap-
blocking chain reactions of lipid autooxidation, chelating
plied in this product.
metal ions of transient state, scavenging nitrite com-

pounds and blocking the synthetic reaction of nitrosamine
Materials and methods
reported by our previous study.24 Moreover, AOB was
Materials
testified to be a strong antioxidant activity and inhibitory
The wheat flour, yeast and baking soda used for the
effect on transition metal ion and free radical induced
preparation of fried bread sticks were purchased from a
deterioration of macromolecules in vitro.25 In addition,
local supermarket in Hangzhou (April, 2005). AOB was
AOB was allowed to be added into puffed foods, meat
prepared from the bamboo leaves of Phyllostachys nigra
products, fried foods and edible oils authorized by Minis-
var. henonis identified by Research Institute of Subtropi-
try of Health, P. R. China. The main functional compo-
cal Forestry of the Chinese Academy of Forestry (Hang-
nents in AOB are flavonoids, lactones and phenolic acids
zhou, China). Briefly, fresh bamboo leaves were collected
while flavone C-glucosides are a group of representative
during the autumn season in Anji district (Zhejiang, China)
flavonoids in AOB.26 On the other hand, extract of green
and air dried. The coarse powder of bamboo leaves was
tea (EGT) is also demonstrated as a strong antioxidant
obtained by crashing into the size of 20–40 mesh and 10 g
applied in many food matrixes.27 It is well known that
powder was extracted with the time of 1 h by 100 ml 30%
four kinds of flavanols, i.e. epicatechin (EC), epicatechin
(v/v) ethanol aqueous solution using the hot reflux
gallate (ECG), epigallocatechin (EGC), and epigallocate-
method. The filtrate was then isolated by membrane filtra-
chin gallate (EGCG) are the main components in EGT.
tion to remove macro- and micro-molecular components
AOB and EGT have been both approved as two kinds of
such as polysaccharides and minerals. Finally, AOB was
food antioxidants in Chinese national standards (i.e. GB-
obtained after concentrating in vacuum and spray drying.
2760). Meanwhile, the safety of both natural antioxidants
EGT was a gift from Professor Xianqiang Yang in De-
was systematically demonstrated in previous studies.28,29
partment of Tea Science (Zhejiang University, Hangzhou,
Addition of edible plant extracts in various products to
China). The total content of four main flavanols (EC,
exert their special effects is a potential technique for re-
ECG, EGC and EGCG) was approximately 98%.
duction of acrylamide in corresponding foods.

Fried bread sticks, a kind of all-time favorite cereal
Chemicals
breakfast food for Chinese and Asian people, have similar
Acrylamide (99%) and 13C3-labelled acrylamide (isotopic
processing style with lots of Western fried foods. Studies
purity 99%) were purchased from Sigma-Aldrich (St.
conducted so far indicated that moderate protein and high
Louis, MO, USA) and Cambridge Isotope Laboratories
carbohydrate foods such as potatoes developed substan-
(Andover, MA, USA), respectively. Formic acid (96%)
tially higher levels of acrylamide under heating condi-
was obtained from Tedia (Fairfield, OH, USA) while
tions.7,8 Therefore, fried bread sticks probably have high
methanol (HPLC-grade) was purchased from Merck
risk exposure of acrylamide under heating conditions.
(Whitehouse Station, NJ, USA). All of other solvents and
However, to our best knowledge, few published papers in
chemicals used for the determination of acrylamide were
peer-review journals reported the acrylamide content in
of analytical grade.
fried bread sticks. China has a huge population and hun-

) 250
)
g
300
g
k
k
g/
A
?
g/ ?
(
l
B
l ( 250
A
200
E
ve
leve
le
e
e
F
200
i
d
B
B
150
C
m
lamid
la
r
y
r
y 150
c
C
C
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A 100
A
D
D
100
D
50
E
50
AOB addition
EGT addition
level (g/kg)
level (g/kg)
0
0
0
0.002
0.01
0.1
1
2.5
4.9
0
0.002
0.01
0.1
1
2.5
4.9


Figure 1A Figure 1B

Figure 1. The relationship between acrylamide levels and different conditions of (A) AOB and (B) EGT treatments in fried bread sticks (n =
6). Error bars designate standard deviation (SD) and different letters indicate significant differences via Duncan’s multiple comparison test
(p<0.05). AOB, antioxidant of bamboo leaves; EGT, extract of green tea.

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133 Reduction of acrylamide in fried bread sticks

Table 1. The sensory evaluation results of fried bread sticks with AOB treatments in different groups (n = 10)†


Addition level of AOB (g/kg)
Control
0.002
0.01
0.1
1
2.5
4.9
Color
Mean
score
4.7 4.4 4.1 4.3 4.2 4.1 ND
p
value
– 0.193 0.081 0.269 0.052 0.051 ND
Flavor
Mean
score
4.1 3.5 3.6 3.5 3.6 2.5 ND
p
value
– 0.313 0.138 0.217 0.212 0.013* ND
Texture
Mean
score
4.0 3.8 3.6 3.4 3.3 3.2 ND
p value
–
0.591
0.373
0.111
0.132
0.003**
ND

† ND, the fried bread sticks treated with 4.9 g/kg of AOB were not evaluated because their sensory attributes were thoroughly unac-
ceptable. Full score: 5.0; * p<0.05, ** p<0.01.
Preparation of fried bread sticks
Sensory evaluation and statistical analysis
The preparation of fried bread sticks was conducted in the
Ten randomized volunteers were invited to score samples
training kitchen of a restaurant according to the traditional
from the control and test groups in terms of color, flavor,
technology and previous publication30 with some modifi-
texture and overall acceptability. Samples in each test
cations. As for the AOB and EGT test groups, a sequence
group via coating with the different level of AOB or EGT
of addition levels, i.e. 0.002, 0.01, 0.1, 1, 2.5 and 4.9 g/kg
were compared with samples in the control group. The
of AOB and EGT were mixed with the wheat flour in
sensory analysis was performed in double blind manner in
advance. Therefore, AOB and EGT spiked wheat flour
order to eliminate the effect of subjective prejudice. Ex-
were used in corresponding AOB and EGT test groups
perimental data from acrylamide analysis were shown as
respectively while non-spiked wheat flour was used in the
mean ± SD while statistical analysis was performed by
control group. Then, a piece of dough in each group was
Duncan’s multiple comparison tests and paired Student’s
prepared from selected wheat flour, yeast, baking soda
t-test to determine the significance differences for treat-
and salt. The prepared dough was then mixed with water
ment means of acrylamide formation and sensory estima-
and set for about 8 h. After yeast fermentation, the dough
tion results in different treatments, respectively.
was stretched on a flat board and cut into 2×15 cm strips.

Two of the strips were combined and stretched to about
Results
25 cm long. About 15 of such combined strips were
The correlation between acrylamide contents and differ-
dipped into a wok containing heated vegetable oil main-
ent addition treatments of AOB and EGT in fried bread
tained at 180±3 ºC and deep-fried to generate the product.
sticks was shown in Figure 1. Results indicated that fried
The oil temperature was monitored by a hand-held ther-
bread sticks with 0.002, 0.01, 0.1, 1, 2.5 and 4.9 g/kg
mocouple probe. After a certain period, final products
wheat flour of AOB treatments induced 8.5%, 38.4%,
were retrieved and set aside to cool and drain away the
66.4%, 82.9%, 63.0% and 29.9% reduction of acrylamide
excess oil before packing for subsequent laboratory test-
generation, respectively. Similarly, samples with the same
ing. All of fried products were then submitted for quanti-
addition levels of EGT treatments incurred 25.1%, 45.7%,
tative analysis of acrylamide. The experiments in each
72.5%, 46.1%, 25.3% and 6.0% reduction of acrylamide
test group and control group were performed in sextuple
formation. The acrylamide contents in all of AOB and
repeats (n = 6).
EGT treated groups were significantly difference from

that of the control group (p<0.01). Results of Duncan’s
Determination of acrylamide by LC-MS/MS
multiple comparison tests were also shown in Figure 1
The quantitative analysis of acrylamide was based on the
and p<0.05 was regarded as the mark of significant dif-
pretreatment of selected final products and performed by
ference.
liquid chromatography tandem mass spectrometry (LC-
The sensory evaluation results of fried bread sticks
MS/MS) with electrospray positive ionization (ESI+) us-
treated with AOB and EGT in different groups were
ing a Micromass Quattro Ultima mass spectrometer cou-
shown in Table 1 and 2, respectively. The flavor and tex-
pled to Waters 2695 HPLC chromatograph. The separa-
ture of fried bread sticks processed by AOB and EGT
tion of acrylamide was achieved with an Atlantis dC18
treatments had no significant difference compared to
column (150 mm × 2.1 mm, 5 ?m; Waters, Milford, MA,
normal food matrixes (p>0.05) when both of AOB and
USA) according to the optimized LC and MS/MS condi-
EGT addition levels were no more than 1 g/kg. The color
tions from our previous published methods.31 The acryla-
of samples could be regarded as no significant change
mide levels in selective samples were quantified by the
(p>0.05) when the AOB and EGT treatment levels ranged
internal standard (13C3-acrylamide) method. The limit of
0.002–2.5 g/kg and 0.002–1 g/kg, respectively. However,
quantification (LOQ) for acrylamide (10 ?g/kg) was esti-
the color of samples in the 2.5 g/kg of EGT treatment
mated by the MassLynx v4.0 software (Micromass, Man-
group showed great discrepancy compared to the control
chester, Lancashire, UK).
(p<0.01). Meanwhile, the sensory attributes of samples

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Y Zhang and Y Zhang 134
Table 2. The sensory evaluation results of fried bread sticks with EGT treatments in different groups (n = 10)†


Addition level of EGT (g/kg)
Control
0.002
0.01
0.1
1
2.5
4.9
Color
Mean
score
4.9 4.6 4.4 4.6 4.4 2.7 ND
p
value
– 0.081 0.096 0.193 0.052 0.002** ND
Flavor
Mean
score
4.5 4.0 4.0 3.7 3.0 2.3 ND
p
value
– 0.397 0.138 0.070 0.138 0.003** ND
Texture
Mean
score
4.4 3.7 3.7 3.7 3.8 2.0 ND
p
value
– 0.066 0.088 0.132 0.111 0.001** ND

† ND, the fried bread sticks treated with 4.9 g/kg of EGT were not evaluated because their sensory attributes were thoroughly unaccept-
able. Full score: 5.0; ** p<0.01.

with 4.9 g/kg of AOB and EGT treatments could not be
In the present study, a significant reduction effect of
acceptable.
two antioxidants on the acrylamide formation was found.

Results of acrylamide contents in fried bread sticks
Discussion
showed the opposite concentration-dependent relation-
In the present study, a less content of acrylamide (nearly
ships in different ranges of AOB and EGT treatments (Fig.
200 ?g/kg) in fried bread sticks than other deep-fried ce-
1). Such reverse tendency on the reduction of acrylamide
real-based foods has been found. Addition of AOB and
may relate to the inherent property of these two antioxi-
EGT greatly reduced the acrylamide content in the pre-
dants and the antioxidant activity of food matrixes, which
sent food matrixes. Especially the acrylamide level in
is so-called “antioxidant paradox”. In other words, it does
fried bread sticks could be reduced below 50 ?g/kg under
not mean that an inevitable forward or backward relation-
the optimal addition level of AOB or EGT. Such reduc-
ship presents between the reduction efficiency of acryla-
tion phenomenon may be due to the fermentation with
mide and the antioxidant activity of deep-frying systems.
yeast before deep-frying and the use of antioxidants.
First, previous study indicated the enhancement of
The extensive fermentation with yeast may be one of
acrylamide level dramatically with an increase of the an-
possible ways to reduce acrylamide content in products of
tioxidant activity of frying foods. Summa et al.34 found a
the present work. Free asparagine and sugars are both
direct correlation between the concentration of acryla-
important precursors for acrylamide formation in cereal-
mide and the antioxidant activity in self-prepared cookies
based products. In general, most of the asparagine was
and demonstrated that the combined conditions including
utilized after 2 h of fermentation with yeast. Sourdough
long baking time, high protein content in samples and low
fermentation, on the other hand, did not reduce the con-
moisture could simultaneously increase the acrylamide
tent of free asparagine as efficiently but had a strong
level and the antioxidant activity. Second, the acrylamide
negative impact on asparagine utilization by yeast. Such
content could be reduced via the effect of antioxidants on
results indicated that this type of fermentation may result
preventing the occurrence of acrolein reaction pathway.
in breads with higher acrylamide content than in breads
Actually, the possible effect of lipids used for deep-frying
fermented with yeast only. Compared with short fermen-
systems in acrylamide formation has induced an intensive
tation time (30 min), longer fermentation (6 h) reduced
controversy. Acrolein reaction pathway initially origi-
acrylamide content in bread made with whole grain wheat
nates from the degradation of lipids and promotes the
87%. For breads made with rye bran, the corresponding
oxidation of fatty acids or glycerol. It is well known that
reduction was 77%.32 Nevertheless, Baardseth et al.33
lipids heated at high temperature can lead to the formation
suggested that the lactic acid fermentation process of po-
of acrolein.35 Acrolein can further react via oxidation to
tato rods with Lactobacillus plantarum NC8 could also
generate acrylic acid or by formation of an intermediate
effectively reduce the acrylamide formation during pro-
acrylic radical. Both of the intermediates could then in-
duction of French fries. Surprisingly, the contents of sug-
duce acrylamide formation in the presence of a nitrogen
ars (i.e. glucose, fructose and sucrose) declined greatly
source under favorable reaction circumstance.13 Addition
while the content of free asparagine remained largely un-
of antioxidants could block the oxidation of acrolein to a
affected during fermentation. Such observations indicated
certain extent and further mitigate the generation of
that the reduction of acrylamide was due to decreasing
acrylamide. Therefore, the fact whether acrylamide could
level of sugars rather than reduction of free asparagine.
be reduced via addition of antioxidants should be con-
Unfortunately, an inevitable controversy about the effect
firmed by taking both of abovementioned factors into
of fermentation on the acrylamide generation made the
consideration and judging alternatively which factor plays
researchers feel confused which factor induces the reduc-
a predominant role in the food system. In fact, both reduc-
tion of acrylamide during the fermentation process, i.e.
tion and enhancement results of acrylamide formation via
via decreasing the content of free asparagine or sugars.
addition of different antioxidants were validated in differ-

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135 Reduction of acrylamide in fried bread sticks
ent published researches, which suggested the dual effects
5. Mucci LA, Sandin S, Bälter K, Adami H-O, Magnusson C,
of antioxidants on the generation of acrylamide.36 In the
Weiderpass E. Acrylamide intake and breast cancer risk in
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Many researches found effective ways to reduce the
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acrylamide content during heat processing but their sen-
Robert M-C, Riediker S. Acrylamide from Maillard reac-
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tion products. Nature 2002;419:449–450.
even not acceptable.37,38 For instance, the largest decrease
9. Granvogl M, Jezussek M, Koehler P, Schieberle P. Quanti-
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potato slices were soaked in acetic acid solution for 60
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(74%) was also observed after soaking of potato slices in
10. Zyzak DV, Sanders RA, Stojanovic M, Tallmadge DH,
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In the present study, experimental results showed that the
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chromatographic investigation of acrylamide formation in
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Devaud S, Goldmann T, Hau J, Blank I. In-depth mecha-
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nistic study on the formation of acrylamide and other viny-
as an important contribution on the reduction of acryla-
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Chem 2004;52:5550–5558.
breakfast by addition of natural antioxidants. However,
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the mechanism of acrylamide reduction by AOB still re-
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?
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mains to be clarified and will be conducted in due course.
Eur Food Res Technol 2005;220:451–458.
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tween cooks and analysts. Eur Food Res Technol
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2003;217:185–194.
We gratefully acknowledged the financial support by National
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Natural Science Foundation Council of China (Project No.
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Provincial Center for Disease Prevention and Control for his
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guidance of LC-MS/MS analysis.
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and in French fries. J Food Sci 2003;68:1287–1290.
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