Evaluation of the Methods for Determination of the Free Radical Scavenging Activity by DPPH 11

Bulgarian Journal of Agricultural Science, 17 (No 1) 2011, 11-24

Agricultural Academy

EVALUATION OF THE METHODS FOR DETERMINATION

OF THE FREE RADICAL SCAVENGING ACTIVITY BY DPPH
G. MARINOVA* and V. BATCHVAROV
Institute of Cryobiology and Food Technologies, BG – 1407 Sofia, Bulgaria

Abstract
MARINOVA, G. and V. BATCHVAROV, 2011. Evaluation of the methods for determination of the free
radical scavenging activity by DPPH. Bulg. J. Agric. Sci., 17: 11-24

The survey of the methods for determination of free radical scavenging activity by DPPH has been done.
The differences between methods conditions and their evaluation are presented. It was determined the effect
of methods conditions by ruggedness testing of methods. It was specified that the accuracy of the method for
determination of free radical scavenging activity is effected by the solvent used (ethanol or methanol) and the
sample/reagent DPPH volume ratio. The coefficient of variation of the method with ethanol is twice lower that
the respective one determined with using of methanol. The calibration curves with ascorbic acid (Vitamin C) and
α-Tocopherol (Vitamin E) and solvent ethanol and methanol were plotted. They are characterized with very high
regression coefficients. Based on the analysis and evaluation of the methods, the results of ruggedness testing
of methods, coefficient of variations of determination with solvent ethanol and methanol and recommendations
of some authors it was proposed modification of the method for determination of free radical scavenging activ-
ity of beer and beverages with DPPH. The modification of the method includes: 0.06 mM solution of DPPH in
ethanol, reaction mixture 1.5 ml diluted sample and 1.5 ml DPPH solution, 30 minutes time of reaction in dark,
measurement of absorbance at 517 nm, presentation of the results as equivalent of Vitamin C antioxidant activity.
It was investigated the effect of malt and hops on the antioxidant activity of wort and beer. It was established that
the main free radical scavenging activity of beer is attributed by the malt used. The hopping increases addition-
ally the values of the parameter. During the different stages of the brewing process the free radical scavenging
activity is changed. The differences between the free radical scavenging activity of laboratory and production
beers indicated the very important role of raw materials and technology used. The free radical scavenging activ-
ity of beers determined by ethanol is higher (an average 8,2 - 38.9 % for the used beer samples) than the values
obtained by solvent methanol.

Key words: method DPPH, modification of method, ethanol, methanol, malt, beer

Abreviations: DPPH - 2,2-Diphenyl-1-picrylhydrazyl, BHA – Butylated xydroxy anisole, BHT – Bu-

tylated hydroxy toluene, EVCAA – equivalent vitamin C antioxidant activity, EVEAA - equivalent
vitamin E antioxidant activity, FRSA – free radical scavenging activity, EBC – European Brewery
Convention

Corresponding author: ibhi@speedbg.net; ibhi@prolink.bg

12 G. Marinova* and V. Batchvarov

Introduction or free radical scavenging activity (Kumazawa

and Nakayama, 2001; Okawa et al., 2001; Pavlov
There is great number of methods for determi- et al., 2002; Yang et al., 2004; Bankeblia, 2005;
nation of antioxidant capacity of foods and bever- Kaukovirta-Norja et al., 2005; Kitao et al., 2005;
ages based on different principles: peroxyl radical De et al., 2007; Marxen et al., 2007; Aghar and
scavenging (Oxygen Radical Absorbance capacity, Masood, 2008; Chen et al., 2009; Fukushima et al.,
ORAC); Total Radical-trapping Antioxidant Power 2009; Tabart et al., 2009; Gülcin et al., 2010; Ren
(TRAP); metal reducing power (Ferric Reducing et al., 2010; Uddin et al., 2010), the antioxidant
Antioxidant Power, FRAP); Cupric Reducing activity (Schlesier et al., 2002; Molyneux, 2003;
Antioxidant Power (CUPRAC); hydroxyl radical Potter at al., 2007; Butkhup and Samappito, 2008;
scavenging (deoxiribose assay); organic radical Lachman et al., 2008; Belisario-Sanchez et al.,
scavenging (2,2-Azino-bis(3-ethylbenz-thiaz- 2009; Hua et al., 2009; Amit et al., 2010; Shikanga
oline-6-sulfonic acid, ABTS); 2,2-Diphenyl-1- et al., 2010), the radical scavenging capacity or free
picrylhydrazyl, DPPH); quantification of the prod- radical scavenging capacity (Sanchez-Moreno et
ucts formed during the lipid peroxidation (Thio- al., 1999; Dragović-Uzelac et al., 2007; Ivanov,
barbituric Acid Reactive Substances, TRAPS); 2007; Mihalev et al., 2007; Ting et al., 2008; Roy
Low-density Lipoproteins (LDLs) oxidation, etc. et al., 2010), the radical scavenging assay or free
(Pérez-Jiménez and Saura-Calixto, 2008). The radical scavenging assay/method (Ismail and
most widely-used procedures for measurement Hong, 2002; Liebenberg, 2004; Othman et al.,
of antioxidant capacity are FRAP, ABTS, TEAC 2005; Wang and Li, 2007; Morais et al., 2009),
(Trolox equivalent antioxidant capacity) , DPPH the antiradical activity (Brand-Williams et al.,
and ORAC (Pérez-Jiménez et al., 2008). 2005; Sroka and Cisowski, 2005; Stoilova et al.,
The DPPH method is rapid, simple, accurate 2005; Lahman et al., 2006), the antioxidant capac-
and inexpensive assay for measuring the abil- ity (Freitas et al., 2006; Dvořáková et al., 2008;
ity of different compounds to act as free radical Perez-Jimenez et al., 2008), the DPPH scavenging
scavengers or hydrogen donors, and to evaluate amount (Jing et al., 2008a; Jing et al., 2008b), the
the antioxidant activity of foods and beverages total antioxidant activity (Tarozzi et al., 2004;
(Prakesh, 2001). The DPPH method is described Singh et al., 2008), the DPPH method/assay
as a simple, rapid and convenient method inde- (Prakash, 2001; Kamkar et al., 2010), the DPPH
pendant of sample polarity for screening of many scavenging assay (Gupta et al., 2007), the DPPH
samples for radical scavenging activity (Marxen test/method (Kwon et al., 2003), the DPPH radi-
et al., 2007). The method DPPH is widely used for cal scavenging effect (Kim et al., 2002), the radi-
measurement of free radical scavenging ability of cal activity (Paulová et al., 2004), the free radical
antioxidants (Perez-Jimenez and Saura-Calixto, scavenging method (Qian and Nihorimbere, 2004),
2008; Perez-Jimenez et al., 2008). For determi- the decoloration of DPPH radical (Silva, 2004),
nation of radical scavenging activity of different the antioxidant content (Miller et al., 2000). The
foods, beverages and substrates were elaborated a most used names of the method DPPH are the
great variety of methods with utilisation of DPPH free radical scavenging activity and antioxidant
(1,1-Diphenyl-2-picrylhydrazyl). They are based activity. The most correct name, which described
on the original methods of Blois (1958) and Brand- the mechanism of the reaction, is the first one. Not
Williams et al. (1995). The great diversity of meth- only the name but the conditions of the described
ods and modifications is evident from its different in the literature methods are very different. The
names. It is known many methods using DPPH for substantial differences are in sample preparation,
determination of: the radical scavenging activity extraction of antioxidants (solvent, temperature,
Evaluation of the Methods for Determination of the Free Radical Scavenging Activity by DPPH 13

etc.), selection of end-points and expression of 2010; Shikanga et al., 2010) 0.06 mM (Qian and
results. That means that the comparison between Nihorimbere, 2004; Lachman et al., 2006; Ivanov,
the values reported by different laboratories can be 2007; Mihalev et al., 2007) 0.05 mM (Ismail and
quite difficult (Pérez-Jiménez et al., 2008). Hong, 2002; Silva, 2004; Kitao et al., 2005; Oth-
The goal of this investigation is critical analysis man et al., 2007) and 0.09 mM (Liebenberg, 2004;
and evaluation of the used methods for determi- Tarozi et al., 2004; Sroka and Cisowski, 2005).
nation of the free radical scavenging activity and The DPPH concentration differences lead to the
suitable modification of the method DPPH for ap- very substantial distinctions in the ratio between
plication in beer, wine, tee and others beverages. volumes of sample and reagent. In literature could
be found ratios from 3:1 (Gupta et al., 2007; Gülcin
Literature Review of the et al., 2010) tо 1:600 (Lachman et al., 2006). Al-
methods DPPH most every method used own volume ratio sample/
reagent. The ratio 1:1 were used by five methods
Antioxidant compounds may be water-soluble, (Ismail and Hong, 2002; Molyneux, 2003; Kitao et
lipid-soluble, and insoluble or bound to cell walls al., 2005; Othman et al., 2007; Singh et al., 2008)
(Prakash, 2001). The most utilised solvents for the ration 1:7,5 were used by two methods (Ivanov,
determination of the radical scavenging activity 2007; Mihalev et al., 2007), and the ratio 3:1 were
by DPPH are methanol and ethanol. Methanol as used by another two methods (Gupta et al., 2007;
a solvent is used by Miller at al. (2000); Prakash Gülcin et al., 2010).
(2001); Kim et al. (2002); Molyneux (2003); The duration of the reaction of radical scaveng-
Tarozzi et al. (2004); Qian and Nihorimbere ing activity between DPPH solutions and sample
(2004); Benkeblia (2005); Kaukovirta-Norja et al. varied from 1 minute (Sroka and Cisowski, 2005)
(2005); Sroka and Cisovski (2005); Lachman et al. to 240 minutes (Miller et al., 2000; Prakash,
(2006); Gupta et al. (2007); Ivanov (2007); Marxen 2001). Different authors used 5 min (Lahman et
et al. (2007); Mihalev et al. (2007); Butkhup and al., 2006; Tabart et al., 2009), 10 min (Lahman et
Samappito (2008); Dvořáková et al. (2008); Lach- al., 2008), 15 min (Pavlov et al. 2002), 20 min
man et al. (2008); Pérez-Jiménez et al. (2008); (Ismail and Hong, 2002; Kitao et al., 2005; Othman
Singh et al. (2008); Tabart et al. (2009); Kamkar et et al., 2005; Ivanov, 2007; Mihalev et al., 2007;
al. (2010); Shikanga et al. (2010), while Ethanol as Singh et al., 2008), 30 min (Kim et al., 2002; Kwon
a solvent is used by Pavlov et al. (2002); Kwon et et al., 2003; Molyneux, 2003; Tarozzi et al., 2004;
al. (2003); Molyneux (2003); Liebenberg (2004); Yang et al., 2004; Stoilova et al., 2005; Gupta et
Paulová et al. (2004); Silva (2004); Yang et al. al., 2007; Wang and Li, 2007; Gülcin et al., 2010;
(2004); Kitao et al. (2005); Othman et al. (2005); Kamkar et al., 2010), 60 min (Liebenberg, 2004;
Stoilova et al. (2005); Agshar and Masood (2008) De et al., 2007), 90 min (Asghar and Masood,
and Gülcin et al. (2010). It is evident that 22 cited 2008) and 120 min (Dvořáková et al., 2008). The
methods used methanol, while 12 prepared the most frequently used duration of the reaction is
DPPH solutions and samples with ethanol. 30 minutes (10 references) and 20 minutes (6
The concentration of the DPPH working solu- references).
tion in discussed methods ranges in a wide lim- The determination of radical scavenging activ-
its: from 0.05 mM to 1.5 M (Kim et al., 2002). ity by DPPH is effectuated under different wave
Relatively often are used the concentration of 0.10 lengths. The absorbances of the assays were mea-
mM (Miller et al., 2000; Kwon et al., 2003; Gupta sured between 492 nm (Shikanga et al., 2010) and
et al., 2007; Asghar and Masood, 2008; Singh 540 nm (Liebenberg, 2004). The wavelength 515
et al., 2008; Kamkar et al., 2010; Gülcin et al., nm were used by Brand-Williams et al. (1995);
14 G. Marinova* and V. Batchvarov

Miller et al. (2000); Molyneaux (2003); He and Ni- by the authors for calculation of the radical scav-
horimbere (2004); Benkeblia (2005); Kaukovirta- enging activity by DPPH or inhibition of DPPH
Norja et al. (2005); Sroka and Cisowski (2005); which are presented below (in %):
Lahman et al. (2006); Ivanov (2007); Mihalev et
al. (2007); Dvořáková et al. (2008); Lahman et A = Acontrol - Asample
al. (2008); Pérez-Jiménez et al. (2008), 516 nm Acontrol x 100;
by Molyneaux (2003); Kaukovirta-Norja et al.
(2005), 517 nm by Prakash (2001); Ismail and (Bankeblia, 2005; Kitao et al., 2005; Moly-
Hong (2002); Pavlov et al. (2002); Kwon et al. neaux, 2003; Pavlov et al, 2002; He and Nihorim-
(2003); Molyneaux (2003); Silva (2004); Yang et bere, 2004; Singh et al., 2008; Wang and Li, 2007;
al. (2004); Kitao et al. (2005); Kaukovirta-Norja Kamkar et al., 2010)
et al. (2005); Othman et al. (2005); Paulová et al.
(2005); Gupta et al. (2007); Wang and Li (2007); B = (1 - Asample )
Asghar and Masood (2008); Singh et al. (2008); Acontrol x 100;
Tabart et al. (2009); Kamkar et al. (2010); Gül-
cin et al. (2010), 518 nm by Molyneaux (2003); (Lahman et al., 2008; Lahman et al., 2006; Oth-
Stoilova et al. (2005), 520 nm by Kim et al. (2002); man et al., 2005; Gülcin et al., 2010)
Molyneaux (2003) and 525 nm by Tarozzi et al.
(2004). It is evident that the most utilised wave C = [1 - Asample - Ablank ]
lengths for measurement of absorbance are 517 nm Acontrol x 100);
(18 references) and 515 nm (13 references).
The radical scavenging activity could be calcu- (Stoilova et al., 2005; Yang et al., 2004)
lated by using different standard solutions. The lit-
erature survey indicated that 5 standards were used D = 1 - Asample
for expression of the results. Vitamin C (Ascorbic Acontrol x 100;
acid) is used by Kwon et al. (2003); Molyneaux
(2003); Liebenberg (2004); Paulová et al. (2004); (Ismail and Hong, 2002)
Tarozzi et al. (2004); Othman et al. (2005); Lah-
man et al. (2006); Wang and Li (2007); Asghar and E = Asample
Masood (2008); Lahman et al. (2008); Shikanga Acontrol x 100; (Liebenberg, 2004)
et al. (2010). Trolox is selected by Miller et al.
(2000); Prakash (2001); Liebenberg (2004); Pau- F = Acontrol - Asample
lová et al. (2004); Silva (2004); Dragović-Uzelac (Sroka and Cisowski, 2005)
et al. (2007); Ivanov (2007); Mihalev et al. (2007);
Tabart et al. (2009). Vitamin E (α-Tocopherol) Some authors express the results as EC50 (ef-
is used by Ismail and Hong (2002); Molyneaux ficient concentration value) – concentration of the
(2003); Silva (2004); Gupta et al. (2007); Marxen substrate that causes 50 % loss of the DPPH activ-
et al. (2007); Asghar and Masood (2008). Rarely ity (colour) (Kim et al., 2002; Kwon et al., 2003;
are used BHT (Ismail and Hong, 2002; Asghar Liebenberg, 2004; Asghar and Masood, 2008;
and Masood, 2008) and BHA (Singh et al., 2008). Marxen et al., 2007; Prakash, 2001; Kaukovirta-
The most frequently used standards according the Norja et al., 2005; Sanchez-Moreno et al., 1999;
literature are ascorbic acid (11 references), Trolox Pérez-Jiménez et al., 2008; Amit et al., 2010). Ad-
(9 references) and α-tocopherol (6 references). ditional results as time taken to reach the steady
Last but not least different equations are used state at EC50 (tEC50) and antiradical efficiency (AE
Evaluation of the Methods for Determination of the Free Radical Scavenging Activity by DPPH 15

= 1/ EC50 tEC50) were proposed by Pérez-Jiménez yeasts – middle fermenting strain Saccaromyces
et al. (2008). carlsbergensis. The main fermentation was for
Molyneux (2003) discussed profound the vari- three days at 15oC and four days at 10oC. The aging
ous methods using DPPH. He recommended for of beer was for two weeks at 4oC. Beer in bottles
increasing the accuracy of the method for 1-cm was purchased from the supermarket.
pathlength spectrophotometric cuvettes with All measurements of free radical scavenging
a maximum working volume 4 ml to use 2 ml activity were performed in triplicate and standard
DPPH solution and 2 ml sample (ratio 1:1), sol- deviation was calculated.
vent methanol or ethanol (not water or acetone),
concentration of the DPPH solution in the range Results and Discussion
50 to 100 μM, reaction time 30 min and suitable
standards or ”positive controls” as ascorbic acid Determination of the methods conditions
(Vitamin C) and α-tocopherol (Vitamin E). Ac- (ruggedness testing of methods)
cording him the EC50 has the drawback that the Evaluation of the methods and modifications for
higher the antioxidant activity, the lower is the determination of the radical scavenging activity by
value of parameter. DPPH shows that the main factors influenced the
reproducibility are the solvent, duration of the re-
Materials and Methods action, sample to reagent ratio and the wave length
for absorbance measurement of the decolouration
The DPPH (1,1-Diphenyl-2-picrylhydrazyl) of the reaction mixture. The DPPH solution was
was obtain from Sigma-Aldrich Chemie GmbH, prepared by dissolving 0.0024 g DPPH in 100 ml
Germany; the L-(+)-Ascorbic acid and DL- methanol or ethanol (0.06 mM). On the base of
alpha-Tocopherol from Dr. Ehrenstorfer GmbH, the most frequent use of the mentioned conditions,
Germany; the methanol p.a. and ethanol p.a. from presented by the cited authors were chosen two
Merck Chemicals, Germany. possibilities. It was used the testing of the analyti-
The congress wart was produced on the auto- cal procedure for ruggedness (ruggedness testing
matic laboratory device „Bender and Hobein”. of methods) according to Analytica EBC (1998).
The malts used (from barley and wheat) were crop The ruggedness testing determined the method’s
2009. The worts were hopped by two doses hops conditions, which render substantial effect on
pellets – from bitter variety Magnum and from the accuracy. The chosen factors are presented in
aromatic variety Spalt Select in ratio 70:30 on Table 1.
the base of total 70 mg/l alpha-bitter acids. The The effect of the identified variables on the
boiling of worts were 90 minutes under reflux. method results is assessed using experimental
For the main fermentation were used dry brewing designs referred as two level factorial and frac-
Table 1
Factors, effected the method of determination of radical scavenging activity
       
Factor Name - +

A Solvent Methanol Ethanol

B Duration of reaction 20 min 30 min
C Sample/Reagent Ratio 0.2 ml/1.5 ml 1.5 ml/1.5 ml
D Wave length 515 nm 517 nm
16 G. Marinova* and V. Batchvarov

tional factorial designs. Eight experimental trails Sm = ± 0.01522; EA = 0.02855;

are carried out in duplicate in a randomised man- EB = 0.007; EC = 0.206; ED = 0.005
ner at specific combinations of factor levels. The
two level factorial design is described in terms of The effects of the main factors, the confidence
analysis of variance (ANOVA) and this is used to limits and the confidence intervals are presented
interpret the results of the trial and to examine the in Table 3.
effects of variables. The results of the experimental The results in Table 3 indicated that the factors
design are presented in Table 2. solvent and sample/reagent DPPH ratio influenced
The following calculation estimated the effect significantly the accuracy of the method. The other
of each factor. There were calculated the Total factors duration of the reaction and wave length
(T), the differences (D), the sum of the squares include 0 for the confidence interval with 95 %
(∑D2), experimental error variance (Se2), main confidence, which means that they did not effected
effects error variance (Sm2), standard error of the statistically the accuracy of the method.
main effects (Sm), main effects EA, EB, EC and ED,
confidence limits and confidence intervals. Determination of the effect of the DPPH
and samples solvent
∑D2 = 0.002796; Se2 = 0.00017475; The antioxidant activity is usually measured
Sm2 = 0.0000436875; in food extracts obtained with different aqueous-
Table 2
Results of the trials for determination of the effect of the factors
                 
Number Factor A Factor B Factor C Factor D Trial 1 Trial 2 T D

1 Methanol 20 min 0.2 ml/1.5 ml 515 nm 0.313 0.318 0.631 - 0.005

2 Ethanol 20 min 0.2 ml/1.5 ml 517 nm 0.656 0.696 1.352 - 0.040
3 Methanol 30 min 0.2 ml/1.5 ml 517 nm 0.305 0.306 0.611 - 0.001
4 Ethanol 30 min 0.2 ml/1.5 ml 515 nm 0.653 0.678 1.331 - 0.025
5 Methanol 20 min 1.5 ml/1.5 ml 517 nm 0.186 0.186 0.372 0
6 Ethanol 20 min 1.5 ml/1.5 ml 515 nm 0.381 0.393 0.774 - 0.012
7 Methanol 30 min 1.5 ml/1.5 ml 515 nm 0.173 0.172 0.345 0.001
8 Ethanol 30 min 1.5 ml/1.5 ml 517 nm 0.403 0.383 0.786 0.020

Table 3
Effects of the factors (conditions) of the method for determination of the radical scavenging activity
         
Main Confidence
Factor Confidence Interval Effect
effect Limit
A Solvent 0.2855 ± 0.01522 from 0.27026 to 0.30074 Yes
B Duration of the reaction 0.007 ± 0.01522 from – 0.02224 to 0,00824 No
C Sample/Reagent ratio – 0.206 ± 0.01522 from – 0.19076 to – 0.22124 Yes
D Wave length 0.005 ± 0.01522 from – 0.01024 to 0.02024 No
Evaluation of the Methods for Determination of the Free Radical Scavenging Activity by DPPH 17

Table 4
Radical scavenging activity and variation coefficient for the solvent ethanol and methanol
     
Value EVCAA, mmol/l ethanol EVCAA, mmol/l, methanol
Maximum, mmol/l 1013.78 900.16
Minimum, mmol/l 902.16 724.10
Average, mmol/l 955.3133 818.5107
Standard deviation, mmol/l 28.85177 48.98722
Coeficient of variation, % ± 3.02 ± 5.98
organic solvents (methanol, ethanol, acetone, determination of the radical scavenging activity
chloroform etc.). There is not ideal solvent that by DPPH and solvent ethanol is twice lower that
would be entirely satisfactory for extraction of the method with utilisation of the methanol as a
the total antioxidants, present in foods, especially solvent. That means that the reproducibility with
those associated with complex carbohydrates and ethanol is two times better than with methanol;
proteins (Pérez-Jiménez et al., 2008). - The results obtained for the radical scavenging
For determination of the solvent effect it was activity by DPPH and ethanol is higher than the
measured 15 times the radical scavenging activ- results obtained by methanol (an average 16.7 %
ity by DPPH (prepared in ethanol or methanol) for the used beer sample). This probably is due to
of the same beer sample, diluted by methanol or the better extraction by ethanol of the substances,
ethanol. The results were obtained by calibration which possessed antioxidant properties;
graphs and the modification of the method DPPH - The ethanol is natural component of the beer
described below. The results were expressed as and wine, which means better solvent for these
EVCAA (equivalent vitamin C antioxidant activ- samples than methanol;
ity) and as EVEAA (equivalent vitamin E anti-
oxidant activity). Based on the results obtained Calibration curves for determination of
were calculated the coefficient of variation for radical scavenging activity by DPPH
the solvents ethanol and methanol. The results for The most useful standard for preparation of
radical scavenging activity, expressed as mmol/l calibration graphs is ascorbic acid (Vitamin C).
EVCAA are presented in Table 4. Relatively often for the same purposes is used by
The results from Table 4 could be summarized different authors α-tocopherol (Vitamin E). The
as follows: standard solutions were prepared by methanol and
- The coefficient of variation of the method for ethanol. The calibration graphs were obtained by

Table 5
Characteristics of the calibration curves
       
Equivalent Solvent R2 y = ax + b

Vitamin C Ethanol 0.9957 y = 159.06(Acontrol - Asample) + 6.0687

Vitamin C Methanol 0.9958 y = 152.72(Acontrol - Asample) + 2.5337
Vitamin E Ethanol 0.9995 y = 88.15(Acontrol - Asample) - 1.3005
Vitamin E Methanol 0.9997 y = 80.776(Acontrol - Asample) - 0.7636
18 G. Marinova* and V. Batchvarov

the modification of the method DPPH described Determination

below. They are presented on Table 5. The linear Sample: Add 1.5 ml diluted sample and 1.5
dependences were characterized by very high ml DPPH solution in test tube, stopper with a
values of the regression coefficients. They are glass ball and mix well. Store 30 min in dark
used for expression of the results as EVCAA and and determine the absorbance at 517 nm against
as EVEAA. diluted blank.
Control: Add 1.5 ml diluted blank and 1.5 ml
Modification of the method for DPPH solution in test tube and mix well. Store 30
determination of free radical min in dark and determine the absorbance at 517
scavenging activity in beer by DPPH nm against diluted blank.
Based on the analyses and evaluation of the de-
scribed methods, the results of the ruggedness test Calculation of the results
for determination of the conditions of the method, The results are calculated by calibration curves,
coefficients of variation and recommendations of prepared with Ascorbic acid (Vitamin C) – (EV-
some authors was elaborated modification of the CAA, equivalent vitamin C antioxidant activ-
method for determination of free radical scaveng- ity) and α-Tocopherol (Vitamin E) – (EVEAA,
ing activity by DPPH in beer and beverages. equivalent vitamin E antioxidant activity) rendered
Diluted sample: Dilute 13.3 ml degassed beer, in account the dilution factor and expressed in
attemperate to 20оС to 100 ml with water, attem- mmol/l. For the beverages, produced on the plant
perate to 20оС in volumetric flask and mix well. basis as beer, wine, tea, fruit and vegetable juices
Pipette 2.5 ml diluted beer in volumetric flask 25 it is advisable to express the results in equivalent
ml and adds 20 ml ethanol. Allow to stand 20 min Vitamin C. The results could be expressed so as
to 20oC and fill up the flask to 25 ml with attemper- % free radical scavenging activity (FRSA) with
ated ethanol and mix well. Transfer the content to DPPH or as % inhibition of the free radical with
fluted filter. Filtered diluted sample store at 20оС DPPH accords the described formula A and C
before using. respectively.
Diluted blank: Dilute 2.5 ml water with ethanol
in volumetric flask 25 ml. Attemperate 20 min to Investigation on the effect of malt and hops
20оС. If is necessary filter through fluted filter. on the antioxidant activity of wart
DPPH solution: Dilute 0.0024 g DPPH in and beer
100 ml ethanol (0.06 mM). Attemperate 20 min It was investigated the effect of malt and hops
to 20оС. Prepare fresh every day. on the antioxidant activity – free radical scaveng-

Table 6
Free radical scavenging activity of laboratory sweet wort, hopped wort, young beer and final beer produced
from malted barley
         
Sample EVCAA, mmol/l EVEAA, mmol/l FRSA, % Inhibition, %

Sweet wort 542.93 ± 11.70 181.71 ± 6.18 9.13 ± 0.24 8.50 ± 0.24
Hopped wort 609.27 ± 15.31 216.81 ± 8.00 10.49 ± 0.32 9.86 ± 0.31
Young beer 573.55 ± 4.42 197.71 ± 2.34 9.75 ± 0.09 9.13 ± 0.08
Final beer 591.41 ± 4.42 207.36 ± 2.34 11.53 ± 0.10 10.81 ± 0.10
Evaluation of the Methods for Determination of the Free Radical Scavenging Activity by DPPH 19

Table 7
Free radical scavenging activity of laboratory sweet wort, hopped wort, young beer and final produced
from malted wheat
         
Sample EVCAA, mmol/l EVEAA, mmol/l FRSA, % Inhibition, %

Sweet wort 571.00 ± 7.66 196.56 ±4.05 9.70 ± 0.16 9.08 ± 0.16
Hopped wort 685.82 ± 7.66 257.29 ± 4.05 12.05 ± 0.16 11.42 ± 0.16
Young beer 599.07 ± 4.42 211.41 ± 2.94 10.28 ± 0.09 9.65 ± 0.09
Final beer 688.37 ± 4.42 258.64 ± 2.39 13.79 ± 0.10 13.07 ± 0.10

ing activity of wort and beer. In Table 6 and Table the increasing of polyphenols and other antioxi-
7 are presented the results for free radical scaveng- dants content due to the hopping. After the main
ing activity of worts, young beers and final beers fermentation the free radical scavenging activity
prepared from malted barley and malted wheat. of young beer lightly decreased respectively by
The utilized solvent was methanol. The results are 5.9 % for this produced from the malted barley
expressed as EVCAA, EVEAA, FRSA according and by 12.6 % for this produced from the malted
formula A and as inhibition according formula C, wheat, expressed as EVCAA. These reductions
render an account of the dilution factors. probably are result of the certain oxidation of the
The results in Table 6 and Table 7 indicated antioxidants of young beer. In final beer, as result
that the sweet wort has relatively high free radi- of the reducing activity of brewing yeast during
cal scavenging activity. The sweet wort produced the aging and limited oxygen access, the beer an-
from the malted wheat presented higher antioxi- tioxidant activity is again higher. During all of the
dant activity. This is due very likely to the higher production stages the antioxidant content of wort
antioxidant content in malted wheat and weaker and beer from malted wheat is higher than from
oxidation in the course of brewing process. It is the malted barley one.
evident that the antioxidant activity of wort and
beer is mainly effected by the free radical scaveng- Investigation of the free radical
ing activity of the malt. The hops doses increase scavenging activity of different beers
the antioxidant activity of hopped wort produced Table 8 and Table 9 presented the free radical
from malted wheat additionally by 20.1 %, and scavenging activity of the production final beer,
of hopped wort produced from malted barley by brewed from malted barley and malted wheat. For
12.2 %, expressed as EVCAA. These raise in the dilution of the beer sample and DPPH solution
antioxidant activity is directly connected with preparation were used methanol and ethanol. The

Table 8
Free radical scavenging activity of laboratory final beer produced from malted barley and malted wheat
(using methanol as a solvent)
         
Sample EVCAA, mmol/l EVEAA, mmol/l FRSA, % Inhibition, %

Malted barley 586.31 ± 15.31 204.66 ± 8.10 11.41 ± 0.36 10.70 ± 0.36
Malted wheat 688.37 ± 15.94 258.64 ± 8.43 13.78 ± 0.37 13.07 ± 0.37
20 G. Marinova* and V. Batchvarov

Table 9
Free radical scavenging activity of laboratory final beer produced from malted barley and malted wheat
(using ethanol as a solvent)
         

Sample EVCAA, mmol/l EVEAA, mmol/l FRSA, % Inhibition, %

Malted barley 814.46 ± 7.97 217.61 ± 4.42 13.12 ± 0.19 12.17 ± 0.19
Malted wheat 883.55 ± 4.60 258.89 ± 2.55 14.77 ± 0.11 13.74 ± 0.12

Table 10
Free radical scavenging activity determined with methanol of the ordinary pilsner beer from aging tanks
of pub brewery
         
Sample EVCAA, mmol/l EVEAA, mmol/l FRSA, % Inhibition., %

Aging tank 1 1037.96 ± 7.66 443.54 ± 4.05 19.25 ± 0.16 18.62 ± 0,16
Aging tank.3 989.48 ± 4.42 417.90 ± 2.33 18.26 ± 0.09 17.63 ± 0.09
Table 11
Free radical scavening activity determined with ethanol of the laboratory final beer, produced from malted
barley from different regions of country, crop 2008
         
Sample EVCAA, mmol/l EVEAA, mmol/l FRSA, % Inhibition., %

Region Lom 1446.98 ± 4.60 568.13 ± 2.55 28,72 ± 0,11 28,35 ± 0,12
Region V. Tarnovo 1481.18 ± 7,97 588.75 ± 4,42 29.64 ± 0,20 29.25 ± 0,20
Region St. Zagora 1436.34 ± 7.98 562.24 ± 4.42 28.46 ± 0.20 28.06,2 ± 0.20
Mix of the regions 1462.92 ±4.60 576.97 ± 2.55 29.12 ± 0.12 28.72 ± 0.11
results obtained confirmed again that the antioxi- tivity of laboratory beers, produced from malted
dant activity determined with ethanol solution is barleys from different regions of the country. They
higher than this obtained with solvent methanol (an showed very high (above 1400 mmol/l equivalent
average 28.4 % - 38.9 % for the used beer sam- vit. C) and very similar values of the free radical
ples). Similar to the results of Table 6 and Table 7, scavenging activity.
the free radical scavenging activity is again higher Table 12 and Table 13 presented the free radical
for the beer, produced from malted wheat. scavenging activity of two beers from the super-
The free radical scavenging activity of ordi- market. They showed very big differences in the
nary pilsner beer, produced in pub brewery was free radical scavenging activity. The Almus beer
determined. The solvent used was methanol. The had with 52% higher free radical scavenging ac-
results are presented in Table 10. The free radical tivity than Zagorka beer, expressed as EVCAA.
scavenging activity of beer was relatively high, These results indicated the very important role
about 1000 mmol/l EVCAA. of the raw materials and technology used for the
Table 11 presented free radical scavenging ac- beer production. The results obtained confirmed
Evaluation of the Methods for Determination of the Free Radical Scavenging Activity by DPPH 21

Table 12
Free radical scavenging activity of beers from supermarket (using methanol as a solvent)
         
EVCAA, EVEAA,
Sample FRSA, % Inhibition, %
mmol/l mmol/l
Almus, glass bottle 0.5 l, shelf life
1298.23 ± 7.66 581.21 ± 4.05 49.43 ± 0.29 43.47 ± 0.29
16.07.2009
Zagorka, glass bottle 0.5 l shelf life
851.69 ± 4.42 345.02 ± 2.34 15.84 ± 0.10 15.67 ± 0.10
25.01.2010
Table 13
Free radical scavenging activity of beers from supermarket (using ethanol as a solvent)
         
Sample EVCAA, mmol/l EVEAA, mmol/l FRSA, % Inhibition, %

Almus, glass bottle 0.5 l, shelf

1404.45 ± 7.07 544.57 ± 4.42 46.24 ± 0.27 37.10 ± 0.27
life 16.07.2009
Zagorka, glass bottle 0.5 l shelf
942.02 ± 7.96 288.29 ± 4.42 14.92 ± 0.19 14.73 ± 0.19
life 25.01.2010
again that the antioxidant activity determined with of beer and beverages with DPPH. The values of
ethanol solution is higher than this obtained with free radical scavenging activity of beer determined
solvent methanol (an average 8.2 % - 10.6 % for by using of ethanol are higher and more precise
the used beer samples). than the respective ones determined by methanol.
The antioxidant activity of the beer is attributed
Conclusions mainly by the raw materials, especially malt and
the technology used.
The literature review of the methods for de-
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Received July, 2, 2010; accepted for printing January, 10, 2011.