3. FOOD ChEMISTRy & bIOTEChNOLOGy 3.1. Lectures

Chem. Listy, 102, s265–s1311 (2008) Food Chemistry & Biotechnology
P64 DETERMINATION OF SELECTED
ANTIOxIDANT ENZyMES IN bARLEy AND
MALT
SIMOnA MACUCHOVá a , REnATA MIKULíKOVá a ,
IVAnA MáROVá b and SYLVIE BěLáKOVá a
a Research Institute of Brewing and Malting, Plc., Mostecká 7,
614 00 Brno, Czech Republic,
b Institute of Food Chemistry and Biotechnology, Faculty of
Chemistry, University of Technology Brno
Purkyňova 118, 612 00 Brno, Czech Republic,
macuchova@brno.beerresearch.cz
Introduction
Presence of Reactive Oxygen Species (ROS) negatively
affects the sensory stability of beer, thus, final quality of beer
is related to its antioxidant activity. Antioxidant content in
beer is substantially dependent on anti-oxidative activity of
the primary raw materials – barley and malt. In barley antioxidants
prevent the undesirable and generally negative changes
of grain quality during the cropping and stocking. Generally,
antioxidants can act against ROS by radical trapping,
by inhibition of their formation or by decreasing of their effects.
Complex antioxidant system acts as a well-coordinated
complex of low molecular weight- and enzyme antioxidants.
In barley antioxidant enzymes are represented mainly by
superoxide dismutase, catalase and peroxidase.
Superoxide dismutase (SOD) is the main antioxidative
enzyme in plant cells, where it stands for catalyse of dismutation
of superoxide to dioxygen and hydrogen peroxide. This
hydrogen peroxide is subsequently decomposited to dioxygen
and water; catalases and peroxidases stand in this reaction
as catalyzators.
In this work the influence of selected biological and chemical
factors on superoxide dismutase and catalase activity
in barley and malt were studied. As the part of the work was
the optimalization of enzyme activity determination methods
associated.
Methods
S a m p l e s o f B a r l e y a n d M a l t
The enzyme activities were studied in two sets
of 24 samples (6 barley varieties – Bojos, Jersey, Malz, Radegast,
Sebastian and Tolar, cultivated on 4 localities – Branišovice,
Hrubčice, Lednice and Krásné Údolí): the first set
treated by standard spraying, the second was not treated. The
malts originated from these samples were analysed using the
same methods as for barley grain analysis.
D e t e r m i n a t i o n o f S u p e r o x i d e
D i s m u t a s e
The modified method using the diagnostic kit RAnSOD
(Randox Laboratories, UK) was used for superoxide dismutase
activity determination. This method employs xanthine
oxidase to generate superoxide radicals, which react with
2-(4-iodophenyl)-3-(4-nitrophenol)-5-phenyltetrazolium
s716
chloride forming a formazan dye. The superoxide dismutase
activity is then calculated by the degree of this reaction’s
inhibition.
Because the original method was developed for superoxide
dismutase determination from human blood, the modification
for SOD analysis in barley and malt consists in extract
preparation from smashed and homogenized samples (Belcredi
at al., 2006): The sample is grinded and compounded
in distilled water at 45 °C. The mixture is compounded for
15 minutes in the tub at the same temperature of water bath.
After half-hour chilling the mixture is filtered. The filtrate
is analysed for SOD activity according to the RAnSOD kit
manual.
D e t e r m i n a t i o n o f C a t a l a s e
The most frequently used method for catalase activity
determination (Bergmeyer, 1970) modified by Góth (1991)
was used in this work. Góth’s modification is based on fact,
that the catalytic action of catalase could be stopped by addition
of ammonium molybdate forming stable coloured complex
with hydrogen peroxide.
Grinded sample is mixed with phosphate buffer at
pH = 7.0 and than is this mixture compounded for one hour
at the temperature of 5 °C. After subsequent centrifuging
is supernatant filtered and used for analysis: hydrogen peroxide
(at 12 mmol dm –3 concentration) is added to specimen
and after 1.5 minutes is also added ammonium molybdate
(at 16.2 mmol d m–3 concentration). The absorbance of formed
yellow complex is measured at 375 nm. The concentration of
remaining hydrogen peroxide is observed at calibrating curve.
Calculated amount of used hydrogen peroxide (in mmol min –1 )
agree with catalase activity (in U-units).
Results
SOD activity determination in chosen samples of barley
and malts shows significant differences. The highest values
of SOD activity were obtained in Bojos variety samples, the
lowest in Radegast variety; concerning locality differences
the highest values were founded in Branišovice.
Activity of catalase is corresponding with SOD activity
– also these values were the highest namely for Bojos variety
Table I
SOD and catalase activity (U g –1 of dry matter) in barley and
malt, depending on the localities
Locality Treatment SOD activity Catalase activity
Barley Malt Barley Malt
Branišovice non-treated 120 143 114 166
treated 128 149 115 148
Hrubčice non-treated 108 129 116 118
treated 116 118 116 120
Krásné non-treated 112 109 78 92
Údolí treated 97 106 71 85
Lednice non-treated 90 119 86 105
treated 86 117 90 138