Effect of heat treatment on the antioxidant capacity of garlic

Maejo Int. J. Sci. Technol. 2009, 3(01), 60-70
Maejo International
Journal of Science and Technology
ISSN 1905-7873
Available online at www.mijst.mju.ac.th
Full Paper
Effect of heat treatment on the antioxidant capacity of garlic
Wiwat Wangcharoen* and Wallaya Morasuk
Department of Food Technology, Faculty of Engineering and Agricultural Industry, Maejo University,
Chiangmai 50290, Thailand
*Corresponding author, e-mail: wiwat@mju.ac.th
Received: 31 July 2008 / Accepted: 15 January 2009 / Published: 28 January 2009

Abstract: The determination of the antioxidant capacity of dry and wet-heated garlic, at 70,
100, and 121 ?C by 3 different methods, namely ferric reducing antioxidant power (FRAP)
assay, improved ABTS radical cation decolourization assay, and DPPH free radical
scavenging activity, together with the determination of total phenolic content and formation
of browning pigments of the same materials was carried out. The result showed that the
antioxidant capacity of heated garlic was decreased by the decomposition of some phenolic
and sulfur-containing compounds. However, when browning pigments developed, the
antioxidant capacity of the heated brown garlic increased with the degree of browning,
provided that it was not too dark. In addition, this study showed that ABTS and FRAP
assay were better methods for expressing the antioxidant capacity of garlic due to its total
phenolic content, although FRAP and DPPH assay were better if the antioxidant capacity of
garlic was mainly caused by browning pigments.
Key words: garlic, antioxidant capacity, heat treatment

Introduction
Garlic (Allium sativum Linn.) is widely used as a food ingredient and a medicinal plant in many
countries, especially in Asia. It is also a recommended Thai medicinal plant for the primary health care
system [1]. Epidemiologic studies show an inverse correlation between garlic consumption and
progression of cardiovascular disease. Garlic has been shown to inhibit enzymes involved in lipid
synthesis, decrease platelet aggregation, prevent lipid peroxidation of oxidised erythrocytes and LDL,

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increase antioxidant status, and inhibit angiotension-converting enzyme in the body [2-3]. It also
prevents DNA damage in essential hypertension [4].
Evidence from several investigations suggests that the biological and medicinal functions, such as
antimicrobial, hypolipidemic, antioxidant, and antithrombotic properties that have been attributed to
garlic are related to a variety of sulphur-containing compounds, including volatiles such as allicin, non-
volatile water-soluble sulphur compounds such as S-allyl cysteine, and lipid-soluble sulphur compounds
such as diallyl sulphide and diallyl disulphide [5-9]. Allicin is formed when alliin, a sulphur-containing
amino acid, comes into contact with the enzyme alliinase when raw garlic is chopped, crushed, or
chewed. Allicin scavenges hydroxyl radicals (OH?), and prevents the lipid peroxidation of liver
homogenate in a concentration-dependent manner [10]. The antioxidant activity in the liposome
system of diallyl sulphide, diallyl disulphide, S-ethyl cysteine, and N-acetyl cysteine derived from garlic
was demonstrated, but this activity was lost when the temperature reached 65 ?C [11].
Fresh, dried and fried garlic are used as food ingredients or seasonings in Thai cuisine. During the
cooking or heating process, non-enzymatic browning reactions including Maillard reaction,
caramelisation, and chemical oxidation of phenols occur. The antioxidant activity of the products from
Maillard reaction [12-14] and caramelisation [15] have been reported. On the other hand, the decrease
in antioxidant capacity of boiled garlic at 100 ?C has been found [16-17].
In this present work we report the determination of the antioxidant capacity of garlic during the
drying and wet heating process at 70, 100, and 121 ?C, which are representatives of three heating
conditions, i.e. heating below boiling point of water or pasteurisation, heating at boiling point of water
or sterilisation of high acid and acid food (pH < 4.5), and heating above boiling point of water or
sterilisation of low acid food (pH > 4.5). The determination was done by three different methods, viz.
ferric reducing antioxidant power (FRAP) assay, improved ABTS radical cation decolourization assay,
and DPPH free radical scavenging assay. Total phenolic content and formation of browning pigments
(absorbance at 420 nm) were also determined. The correlations of antioxidant capacity (%) with total
phenolic content (%), and of antioxidant capacity (%) with absorbance at 420 nm were then analysed.


Materials and Methods
Chemicals
Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) was purchased from Aldrich.
TPTZ (2,4,6-tripyridyl-s-triazine) and DPPH (2,2-diphenyl-1-picrylhydrazyl) were purchased from
Sigma. ABTS [2,2’-azinobis (3-ethylbenzothiazoline-6-sulphonic acid)], Folin-Ciocalteau phenol
reagent, ferric chloride, ferrous sulphate, gallic acid, glacial acetic acid, hydrochloric acid, sodium
acetate, potassium persulphate, sodium carbonate, and vitamin C were purchased from Fluka. All
chemicals were of analytical grade.

Sample preparation
Fresh garlic was purchased from a local market and edible portion was homogenised with a blender.
Sample tubes (25 x 150 cm) containing 2 g each of blended garlic were dried in a hot air oven at 70,
100, or 121 ?C. For wet heating, 10 ml of deionised water was added to the blended garlic in each

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Maejo Int. J. Sci. Technol. 2009, 3(01), 60-70


sample tube before heating in a water bath at 70 or 100 ?C, or in an autoclave at 121 ?C. A sample tube
was collected 10 times during a maximum of 10 hours of heating period. Sample extraction method of
Leong and Shui [18] was modified. Ten ml of deionised water were added to the collected dried
sample tube. (For wet heating, deionised water had been added before heating.) The extraction was
done by vortex mixing for 1 min. The mixture was then filtered through a Whatman filter paper no. 1.
The filtrate was adjusted to 10 ml by deionised water and this extract was used for all assays. An extract
of blended fresh garlic was prepared for comparison and the weight change during the drying process
also was recorded.

Ferric reducing antioxidant power (FRAP) assay
FRAP, a method for measuring total reducing power of electron-donating substances, was applied
according to Benzie and Strain [19]. Briefly, 6 ml of working FRAP reagent (0.1 M acetate buffer :
0.02 M FeCl3 : 0.01 M TPTZ = 10 : 1 : 1) prepared daily were mixed with 20 ?l of extract sample. The
absorbance at 593 nm was recorded after a 30-min incubation at 37 °C. FRAP values were obtained by
comparing with standard curves created by Fe2+ (0 - 14 µg), Trolox (0 - 35 µg) and vitamin C (0 - 15
µg), and reported as mg Fe2+, Trolox and vitamin C equivalent per gram of sample (dry weight).

ABTS radical cation decolourization assay
The method of Re et al. [20], based on the ability of antioxidant molecules to quench the long-lived
ABTS radical cation (ABTS?+), was modified. ABTS?+ was produced by reacting 7 mM ABTS stock
solution with 2.45 mM potassium persulphate (final concentration) and allowing the mixture to stand in
the dark at room temperature for 12 - 16 hours before use. The ABTS?+ solution was diluted with
deionised water and 95 % ethanol (1 : 1) to an absorbance of 0.70 (+ 0.02) at 734 nm. Twenty
microlitres of the extract were mixed with 6 ml of the diluted ABTS?+ solution. The decrease of
absorbance was recorded at 1 min after mixing. Trolox (0 - 30 µg) and vitamin C (0 - 20 µg) were
used as standards, and the results were reported as mg Trolox and vitamin C equivalent per gram of
sample (dry weight).

DPPH free radical scavenging activity
The method of Brand-Williams et al. [21], based on the reduction of DPPH radical solution in the
presence of hydrogen donating antioxidants, was used with some modification. DPPH radical solution
(0.8 mM) in 95% ethanol was prepared. One thousand microlitres of the extract were diluted to 5.4 ml
using deionised water and 95 % ethanol (1 : 1) before 0.6 ml DPPH radical solution was added and the
mixture shaken vigorously. The decrease of absorbance was recorded at 1 min after mixing. Trolox
(0 - 50 µg) and vitamin C (0 - 40 µg) were used as standards, and the results were reported as mg
Trolox and vitamin C equivalent per gram of sample (dry weight).

Total phenolic content (TPC)
The Folin-Ciocalteau micro method of Waterhouse [22] was used. Sixty microlitres of the extract
were diluted with deionised water to 4.8 ml, and 300 ?l of Folin-Ciocalteau reagent were added and the
mixture shaken. After 8 min, 900 ?l of 20% sodium carbonate were added and mixed. The solution

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Maejo Int. J. Sci. Technol. 2009, 3(01), 60-70


was left at 40 °C for 30 min before the absorbance at 765 nm was read. Gallic acid (0 - 50 µg) was used
as standard, and the results were reported as mg gallic acid equivalent per gram of sample (dry weight).

Formation of browning pigments
Formation of browning pigments was determined by the official method [23]. The absorbance of
extracts was measured at 420 nm.

Calculation and statistical analysis
The values of FRAP, ABTS, DPPH, and TPC (mg standard equivalent per gram of sample (dry
weight)) were calculated using the equation below:



Values of FRAP, ABTS, DPPH, and
[(SA - BA) / (Slope)] [10 / U]
=
TPC (mg standard equivalent per gram of
[2] [1-MC][1,000]
sample (dry weight))


where: SA
= Sample absorbance for FRAP value and TPC or absorbance decrease of
sample for ABTS and DPPH values
BA
= Blank (no extract) absorbance for FRAP value and TPC or absorbance
decrease of blank for ABTS and DPPH values (extract was substituted by
deionised water for blank)

Slope
= Slope of standard curve


[10 / U] = Total volume of extract (10 ml) / Used volume of extract (ml)

[2]
= Weight of used sample (g)

MC
= % Moisture content / 100
[1,000] = Factor for changing µg to mg

Changes of antioxidant capacity, TPC, and weight (%) were calculated using the equation below:


Changes of antioxidant capacity, TPC,
Value of collected sample x 100

=
and weight (%)
Value of blended fresh garlic

Each experiment was performed in triplicate and was conducted on separate purchased samples
(triple measurements for each purchased sample). The bivariate correlations between changes of
antioxidant capacity and total phenolic content, and between changes of antioxidant capacity and
absorbance at 420 nm were analysed.

Results and Discussion
In this experiment, water was used as extracting solvent since about 97% of garlic components are
water-soluble [6], and some previous work also confirmed that water is a good solvent for measuring
antioxidant capacity and TPC of garlic extract [16,24], although a better result with hexane has been
obtained by Leelarungrayub et al. [25]. Antioxidant capacity and TPC of aqueous fresh garlic extract
are shown in Table 1. Values of antioxidant capacity and TPC of aqueous garlic extract have been

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Maejo Int. J. Sci. Technol. 2009, 3(01), 60-70


reported by several workers but they are difficult to compare because of the differences in sample
sources, sample preparations, methodology details, standards used, among others. For example,
Gorinstein et al. [16] reported ABTS = 26.1 + 2.4 ?mol Trolox equivalent per gram of sample (dry
weight), and TPC = 11.42 + 1.45 mg gallic acid equivalent per gram of sample (dry weight). Jastrzebski
et al. [17] reported FRAP = 3,400 + 270 mmol Trolox equivalent per 100 grams of sample (fresh
weight), DPPH = 69 + 5.1 % inhibition, and TPC = 49.3 + 3.1 mg gallic acid equivalent per 100 grams
of sample (fresh weight). Wangcharoen and Morasuk [24] reported FRAP, ABTS and DPPH = 0.14 +
0.04, 1.06 + 0.11 and 0.16 + 0.03 mg vitamin C equivalent per gram of sample (fresh weight)
respectively, and TPC = 0.41 + 0.10 gallic acid equivalent per gram of sample (fresh weight).
Leelarungrayub et al. [25] found ABTS = 1462 + 22 mg extract equivalent to 1 mg of Trolox, and TPC
? 3,800 mg gallic acid equivalent per kg of dry weight of extract. Holvorsen et al. [26] found FRAP =
0.21 – 0.24 mmol FeSO4.7H2O equivalent per 100 grams of fresh weight of edible portion, and Nuutila
et al. [27] found DPPH = 62.1, 60.9, and 43 % inhibition, and TPC = 75 + 8.8, 115 + 12.9, and 95 +
7.8 mg gallic acid equivalent per kg of freeze-dried samples of Finnish organic garlic, Finnish garlic, and
Hungarian garlic, respectively.

Table 1. Antioxidant capacity by various methods and total phenolic content (TPC) of aqueous extract
of fresh garlic samples (mg standard equivalent per gram of sample (dry weight))

Standard
FRAP
ABTS
DPPH
TPC
Fe2+
0.56 + 0.15



Trolox
1.13 + 0.31
5.17 + 0.54
0.57 + 0.08

Vitamin C
0.44 + 0.12
3.41 + 0.35
0.48 + 0.07

Gallic acid



1.29 + 0.19

Changes of antioxidant capacity (%), TPC (%), weight (%), and 420 nm absorbance of the aqueous
extract of garlic samples during heating process are shown in Figures 1 and 2. In the case of drying
(Figure 1), the antioxidant capacity values and TPC tended to decrease with drying time when garlic
samples were dried at 70 ?C, except the DPPH value, which seemed to increase at 10 hours. The
weight of garlic samples rapidly decreased in the first three hours and was steady after 4 hours, whilst
the absorbance at 420 nm evidently decreased in the first and the second hour and gradually increased
after the third hour. At 100 and 121 ?C, all antioxidant capacity values and TPC decreased and reached
the lowest point at 2.5 and 1.5 hours respectively, before increasing when the drying time was further
increased. The weight of garlic extracts rapidly decreased in the first hour and was constant after that

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Maejo Int. J. Sci. Technol. 2009, 3(01), 60-70


Drying at 70 'C
Drying at 70 'C
250
100
0.900
FRAP
Weight (% )
90
0.800
TPC (% )
)
ABTS

200
80
C
0.700
P

(
%
DPPH
Absorbance at 420 nm
m
70
i
t
y
t
;

T
0.600

n
c
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0
2
a 150
t
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60
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0.100
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0.000
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1
2
3
4
5
6
7
8
9
10
0
1
2
3
4
5
6
7
8
9
10
Drying tim e (Hour)
Drying time (Hour)


Drying at 100 'C
Drying at 100 'C
100
0.900
250
Weight (% )
FRAP
90
0.800
)

TPC (% )
80
200
ABTS
C
0.700
P

(
%
DPPH
Absorbance at 420 nm
m
70
i
t
y
t
;

T
0.600

n
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0
0
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1
2
3
4
5
6
7
8
9
10
0
1
2
3
4
5
6
7
8
9
10
Drying tim e (Hour)
Drying time (Hour)


Drying at 121 'C
Drying at 121 'C
100
0.900
250
90
W eight (% )
FRAP
0.800
)

80
TPC (%)
200
ABTS
C
0.700
P

(
%
DPPH
m
70
Absorbance at 420 nm
t
;

T

n
i
t
y
0.600
n
0
c
2
a
60
t
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150
p
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0.400
n
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100
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W
T
10
0.100
0
0
0.000
0
1
2
3
4
5
6
7
8
9
10
0
1
2
3
4
5
6
7
8
9
10
Drying time (Hour)
Drying time (Hour)



Figure 1. Antioxidant capacity, total phenolic content, and absorbance at 420 nm of aqueous extracts
of garlic samples, together with weight of garlic samples during drying process at 70, 100, and 121 ?C

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66
Maejo Int. J. Sci. Technol. 2009, 3(01), 60-70


time, whilst the absorbance at 420 nm decreased to a minimum at about 0.5 hour before it evidently
increased after that.
In the case of wet heating (Figure 2) at 70 ?C, FRAP and ABTS values tended to decrease for 70
min before slightly increasing after that time, whilst DPPH value slowly increased until about 30 min,
after which it slowly decreased and started to increase again after 70 min. At 100 ?C, ABTS value
steadily decreased, whilst FRAP and DPPH values seemed to increase for about 20 min before
decreasing steadily for FRAP and with fluctuation for DPPH. This result agrees with that of Gorinstein
et al. [16], who reported 13 – 17 % decrease of ABTS assay for boiled garlic at 100 ?C for 20 min, and
also agrees with that of Jastrzebski et al. [17], who showed a significant decrease of FRAP and DPPH
assay of boiled garlic at 100 ?C for 40 and 60 min. On the other hand, at 121 ?C of wet heating, DPPH
value increased rapidly, whilst FRAP and ABTS values also did albeit less so and only after an initial
decrease.
The decrease of antioxidant capacity of garlic extract upon heating was most probably due to the
decomposition of some phenolic compounds and also some sulphur-containing compounds such as
diallyl sulphide, diallyl disulphide, S-ethyl cysteine, and N-acetyl cysteine, which would be lost when the
temperature reached 65 ?C [11]. However, when browning pigments were formed, the antioxidant
capacity of the heated brown garlic was regained as a result of certain compounds, including phenolic
compounds, being created during the browning reactions [12-15]. The browner garlic, with higher
value of absorbance at 420 nm, expressed higher antioxidant capacity, but at the end of the heating
period at 121 ?C, the antioxidant capacity started to decrease (Figures 1 and 2). This shows that the
antioxidant capacity of the heated brown garlic would be dropped if it was heated for a long time so
that its colour was too dark.
From the results of bivariate correlation, the correlation coefficient (a number between -1 and 1
which measures the degree to which two variables are linearly related) of changes of antioxidant
capacity (%) with TPC showed that ABTS values were higher correlated with TPC in all cases of
drying process, followed by FRAP and DPPH values respectively. In the case of wet heating process,
FRAP values seemed to be higher correlated with TPC than ABTS and DPPH values respectively. The
bivariate correlation of changes of antioxidant capacity (%) with absorbance at 420 nm showed that
FRAP and DPPH values were highly correlated with browning pigment formation in drying and wet
heating process at 121 ?C (Table 2). These differences might be due to the fact that ABTS and DPPH
method involve free radicals reacting with phenolic and browning pigment compounds, while for FRAP
assay, it is a method for measuring total reducing power of electron donating substances, which is not
directly related to free radical reactions and not as specific as ABTS and DPPH assay.

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Maejo Int. J. Sci. Technol. 2009, 3(01), 60-70


Wet heating at 70 'C
Wet heating at 70 'C
100
0.700
180
FRAP
90
0.600
160
)
ABTS






80
C
P

(
%
DPPH
m
70
0.500
t
;
T

n
i
t
y 140
n
0
c
t
e
2
a
60
n
)
0.400
p
o
t

4
a

c
50

a
120
e
t

c
l
i
c




(
%
c
n
o
0.300
n
a
40
n
a
i
d
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r
b
100
x
h
30
o
0.200
s
l

p
b
t
i
o
A
n
t
a
20
a
TPC (% )
80
A
o
0.100
T
10
Absorbance at 420 nm
0
0.000
60
0
10
20
30
40
50
60
70
80
90
100
0
10 20 30 40 50 60 70 80 90 100
Heating time (Min)
He ating time (Min)


Wet heating at 100 'C
Wet heating at 100 'C
100
0.700
180
FRAP
90
0.600
160
)
ABTS
80






C

(
%
DPPH
P
m
70
0.500

n
i
t
y 140
t
;
T
0
c
n
2
a
60
t
e
0.400
p
n
)
t

4
a
o
50

a
120

c
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t

c


(
%
c
n
l
i
c
0.300
n
a
a
40
o
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d
n
r
b
100
e
x
30
o
h
0.200
s
b
t
i
o
l

p
A
n
20
t
a
TPC (%)
80
A
o
T
0.100
10
Absorbance at 420 nm
0
0.000
60
0
10
20
30
40
50
60
70
80
90
100
0
10 20 30 40 50 60 70 80 90 100
Heating time (Min)
Hea ting tim e (Min)


Wet heating at 121 'C
Wet heating at 121 'C
100
0.700
180
FRAP
90
0.600
160
)
ABTS
80






C

(
%
DPPH
P
m
70
0.500

n
i
t
y 140
t
;
T
0
c
n
2
a
60
t
e
0.400
p
n
)
t

4
a
o
50

a
120

c
e
t

c


(
%
c
n
l
i
c
0.300
n
a
40
o
a
i
d
n
r
b
100
e
x
30
o
h
0.200
s
b
t
i
o
l

p
TPC (%)
A
n
20
t
a
80
A
o
Absorbance at 420 nm
0.100
T
10
0
0.000
60
0
10
20
30
40
50
60
70
80
90
100
0
10 20
30
40
50 60
70
80 90 100
Hea ting time (Min)
Hea ting tim e (Min)



Figure 2. Antioxidant capacity, total phenolic content, and absorbance at 420 nm of aqueous extracts
of garlic samples during wet heating process at 70, 100, and 121 ?C

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Maejo Int. J. Sci. Technol. 2009, 3(01), 60-70


Table 2. Correlation coefficients of changes of antioxidant capacity with total phenolic content (TPC)
and with absorbance at 420 nm of aqueous extract of garlic samples during heating processes

Drying process

FRAP
ABTS
DPPH
70 °C
Total phenolic content (TPC)
0.891
0.919
0.636
100 °C

0.799
0.941
0.616
121 °C

0.597
0.961
0.436
70 °C
Absorbance at 420 nm
-0.114
0.050
-0.151
100 °C

0.090
0.100
0.377
121 °C

0.757
0.419
0.834
Wet heating process



70 °C
Total phenolic content (TPC)
0.801
0.489
0.313
100 °C

0.768
0.893
0.243
121 °C

0.374
0.271
-0.153
70 °C
Absorbance at 420 nm
0.156
0.637
-0.131
100 °C

-0.275
-0.017
-0.315
121 °C

0.847
-0.306
0.774


Conclusions
This work has shown that the antioxidant capacity of garlic is decreased by both drying and wet
heating. However, if browning pigments are developed from non-enzymatic browning reactions, the
brown garlic will regain its antioxidant capacity, which may increase to an appreciably higher level than
the starting value depending on the degree of browning. This expresses the high antioxidant capacity of
heated brown garlic, which is normally used as an ingredient in many Thai food recipes.

Acknowledgement
This work was a part of a research project supported by a grant from Office of Agricultural Research
and Extension at Maejo University, Thailand.

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