3. FOOD ChEMISTRy & bIOTEChNOLOGy 3.1. Lectures

Chem. Listy, 102, s265–s1311 (2008) Food Chemistry & Biotechnology
P18 AMINO ACIDS PROFILE OF SELECTED
whOLEGRAINS IMPORTANT TO
ACRyLAMIDE FORMATION IN CEREALbASED
PRODuCTS
ZUZAnA CIESAROVá a , KRISTínA KUKUROVá a ,
ALEnA BEDnáRIKOVá a , PETER HOZLáR b and
ľUBOMíR RUCKSCHLOSS b
a VÚP Food Research Institute, Priemyselná 4, 824 75 Bratislava,
Slovak Republic,
b Research and Breeding Station at Vígľaš-Pstruša, 962 12
Detva, Slovak Republic,
ciesarova@vup.sk
Introduction
Acrylamide as a suspected carcinogen attracts great
attention due to its widespread occurrence in many staple
foods of daily usage 1 as well as due to the recommendation of
the European Commission since 2005 to minimize its level 2 .
It is known that acrylamide arises from naturally occurred
compounds in plants such as reducing saccharides and amino
acids during the commonly used process of heat treatment
of foods 3,4 . Acrylamide is preferably formed from amino
acid L-asparagine, only in a less extent from aspartic acid,
glutamine, and glutamic acid in the presence of saccharides
during baking 5 . It is well established that the amount of reducing
sugars is more important than free asparagine for the
formation of acrylamide in potato-based products 6 . However,
in cereal foods including bread, the main determinant of acrylamide
formation during baking is free asparagine amount in
raw material and ingredients, in particular cereal flour.
The concentration of free asparagine have been studied
in different commercial milling fractions of wheat and rye 7 .
Whole grain flours showed higher amounts of asparagine (for
wheat and rye 0.5 g kg –1 and 1.1 g kg –1 , respectively) versus
sifted flours. Of the wheat fractions, wheat germs was
reported to have the highest level of asparagine (4.9 g kg –1 ).
However, agronomical factors (e.g. crop variety, climatic
conditions fertilizer regimes) may significantly impact the
amount of free asparagine in cereal crops 8 and consequently
the concentration of acrylamide in the final product. Regional
differences may account for levels that vary by more than
fivefold, and today the scientific data that may explain this
variability is lacking.
Based on this knowledge it can be said that in the cereal
sector the main way of acrylamide reduction is through
amino acids control responsible for acrylamide formation.
From the point of view of acrylamide formation, the selected
10 sorts of wholegrains appointed for human usage in bread
and cereal breakfast production which are bred in Slovakia
as well as 6 kinds of wheat flours from different milles in
Slovakia were assorted according to profile of amino acids
asparagine, aspartic acid, glutamine, and glutamic acid.
s613
Experimental
R a w M a t e r i a l s
Wheat grains of 5 varieties (PS-3/05, PS-11, PS-9/06,
PS-27/06, PS-51/06) and oat grains of 5 registrated varieties
(Vendelin, Valentin, Zvolen, Atego, Detvan) were obtained
from the Research and Breeding Station at Vígľaš-Pstruša.
Wheat flours originated from Slovak milles (PMD Bratislava,
Kolárovo, Sládkovičovo, Šurany) were purchased from local
markets.
R e a g e n t s
Asparagine (Asn) standard (99.5%) was supplied by
Fluka (Steinheim, Germany) and aspartic acid (Asp), glutamic
acid (Glu), glutamine (Gln) standards (99%) were
supplied by Merck (Darmstadt, Germany). D3-glutamic
acid (d3-Glu) standard (97%) was supplied by Cambridge
Isotope Laboratories (Andover, USA). Acetic acid (glacial)
was HPLC reagent grade and obtained from Fisher Scientific
(Loughborough, UK). Perfluorooctanoic acid (PFOA) (96%)
and HPLC gradient grade acetonitrile were obtained from
Sigma-Aldrich (Steinheim, Germany). Deionized water from
a PURITE Select system (Oxon, UK) was used for preparation
of amino acid and ion-pairing reagent solution.
I n s t r u m e n t a t i o n
The LC/ESI-MS-MS apparatus for quantification of
4 free amino acids were performed by Agilent 1200 HPLC
system (Waldbronn, Germany) consisting of a binary pump,
an autosampler and a temperature controlled column oven,
coupled to an Agilent 6410 Triple Quad detector equipped
with ESI interface. The analytical separation was performed
on a Purospher ® STAR RP-8ec column (150 mm × 4.6 mm,
3 µm) using isocratic mixture of 100 ml of acetonitrile and
900 ml of aqueous solution of PFOA (0.05mM) at a flow rate
0.5 ml min –1 at room temperature. All parameters of the ESI-
MS-MS system were based on in-source generation of the
protonated molecular ions of the 4 amino acids measured and
the internal standard (d3-Glu), as well as collision-induced
production of amino acid-specific fragment ions for multiple
reaction monitoring (MRM) experiments.
S a m p l e P r e p a r a t i o n
Stock solution of amino acids 1,000 µg ml –1 were prepared
by dissolving 25 mg of each in 25 ml of deionized water.
Working standards were prepared by diluting the stock solution
of amino acids to concentrations of 0.05–2.00 µg ml –1 .
Each working standard solutions consist of 0.5 µg ml –1 of
internal standard (d3-Glu). Finely ground or homogenized
sample (1 g) was extracted by 10 ml of 0.2mM acetic acid
and after mixing in a vortex mixer for 2 min the mixture was
centrifuged at 5,000 rpm for 10 min at –5 °C and filtered through
0.45 µm nylon syringe filter prior to LC/MS analysis 9 .
Results
In this study, 5 varieties of wheat grains, and 5 varieties
of oat grains as well as 6 kinds of wheat flours were used