LWT - Food Science and Technology 55 (2014) 397e402

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LWT - Food Science and Technology

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Changes in S-allyl cysteine contents and physicochemical properties

of black garlic during heat treatment
Sang Eun Bae a, Seung Yong Cho b, **,1, Yong Duk Won c, Seon Ha Lee d, Hyun Jin Park a, e, *,1
a
School of Life Science and Biotechnology, Korea University, Seoul, Republic of Korea
b
Institute of Life Sciences and Biotechnology, Korea University, Seoul, Republic of Korea
c
Uiseong Black Garlic Farming Association, Gyeongsangbuk-do, Republic of Korea
d
College of Health and Welfare, Kyungwoon University, Gyeongsangbuk-do, Republic of Korea
e
Department of Packaging Science, Clemson University, Clemson, SC 29634-0370, USA

a r t i c l e i n f o a b s t r a c t

Article history: This study aimed to investigate the effects of temperature on the black garlic manufacturing process. The
Received 14 September 2011 moisture content, pH, browning intensity, S-allyl cysteine (SAC) content and antioxidant activity,
Received in revised form including DPPH radical scavenging activity and reducing power, were determined. The moisture content
25 March 2013
of garlic gradually decreased throughout the heating process. The rate of moisture removal was higher at
Accepted 4 May 2013
high temperatures compared with low temperatures. The pH also decreased more signicantly in garlic
heated at high temperatures. The browning intensity increased with increasing temperature. The SAC
Keywords:
contents of black garlic were signicantly different according to heating temperature; the garlic samples
Black garlic
Heat-treated garlic
heated at a low temperature had a higher SAC contents. Antioxidant activity, as determined by the DPPH
S-Allyl cysteine radical scavenging activity and reducing power, increased when the garlic was exposed to higher
Antioxidant activity temperatures.
2013 Elsevier Ltd. All rights reserved.

1. Introduction tendency to cause stomach upset. Therefore, in recent years,

Garlic (Allium sativum L.) is widely used as a seasoning for food
and has long been used as a medicinal agent for the treatment of
multiple human diseases and disorders. Recently, garlic has
attracted much research interest due to its benecial health effects,
which include antioxidant activity (Gorinstein et al., 2006), anti-
microbial activity (Rees, Minney, Plummer, Slater, & Skyrme, 1993)
and anticancer activity (Durak, Yilmaz, Devrim, Perk, & Kamaz,
2003). Accordingly, garlic has become one of the most wide-
spread disease-preventing foods (Block, 1992). Although garlic has
many active components that contribute to its health benets,
including allicin and its derivatives, consumption of unprocessed
raw garlic is limited due to its characteristic odor, taste and
* Corresponding author. School of Life Sciences and Biotechnology, Korea Uni-
versity, 1,5-Ka, Anam-Dong, Sungbuk-gu, Seoul 136-701, Republic of Korea.
Tel.: 82 2 3290 3450; fax: 82 2 953 5892.
** Corresponding author. Institute of Life Sciences and Biotechnology, School of
Life Sciences and Biotechnology, Korea University, 1,5-Ka, Anam-Dong, Sungbuk-gu,
Seoul 136-701, Republic of Korea. Tel.: 82 2 3290 4282; fax: 82 2 953 5892.
E-mail addresses: sycho@korea.ac.kr (S.Y. Cho), hjpark@korea.ac.kr (H.J. Park).
1
Hyun Jin Park and Seung Yong Cho contributed equally to this research as co-
corresponding authors.
various processing methods such as heat treatment, aging and
fermentation have been used to eliminate the offensive odor and
improve the palatability of garlic.
Heat treatment is the most widely used processing method for
removing the unpleasant taste and odor of garlic. When garlic
undergoes heat treatment, various physicochemical changes occur,
including changes in avor, color and nutrient content. Particularly,
heat treatment leads to non-enzymatic browning reactions such as
the Maillard reaction, caramelization and the chemical oxidation of
phenols. A number of non-enzymatic browning reactions are
associated with the formation of compounds with strong antioxi-
dant properties (Legault, Hendel, & Talburt, 1954; Manzocco,
Calligaris, Mastrocola, Nicoli, & Lerici, 2000; Osada & Shibamoto,
2006; Peleg, Mannheim, & Berk, 1970; Yanagimoto, Lee, Ochi, &
Shibamoto, 2002; Yilmaz & Toledo, 2005).
Black garlic is a type of heat-treated garlic that is generally
produced by heating whole bulbs of raw garlic at high temperature
under controlled humidity for more than 1 month. Many studies
have demonstrated the health benets of black garlic. It was re-
ported that black garlic has stronger antioxidant activity compared
with raw garlic and may have greater efcacy for preventing
metabolic diseases and alcoholic hepatotoxicity (Ide & Lau, 1999;
Ide, Matsuura, & Itakura, 1996).

0023-6438/$ e see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.lwt.2013.05.006
398 S.E. Bae et al. / LWT - Food Science and Technology 55 (2014) 397e402
During the heat treatment process, the raw garlic turns black in
color, and the texture of the nal product is sticky and jelly-like, with
a sweet and sour avor. Black garlic has been readily adopted as an
ingredient in processed food materials, such as beverages, candy and
ice cream, due to its sweet and syrupy taste (Shin et al., 2008).
An increase in S-allyl cysteine (SAC) content is also an important
change that occurs during the black garlic manufacturing process.
Generally, raw garlic contains 20e30 mg/g of SAC (Kodera et al.,
2002), and the amount of SAC in black garlic is ve to six times
higher than that in raw garlic (Bae, Cho, Won, Lee, & Park, 2012;
Wang et al., 2010). SAC is formed by the enzymatic hydrolysis of
g-glutamyl-S-allyl cysteine (GSAC) by g-glutamyl transpeptidase
(g-GTP, EC 2.3.2.2) (Kodera et al., 2002). SAC is one of the major
sulfur-containing amino acid compounds that is thought to be
responsible for the benecial effects of garlic, which include anti-
oxidant, anticancer, antihepatopathic and neurotrophic activities
(Kodera et al., 2002).
The estimated market value of black garlic is approximately
94 million US dollars in Korea, and it has rapidly increased in popu-
larity over the last few years in Korea as well as in America. However,
quality factors of black garlic products have not yet been established,
and there is no standardized method for manufacturing black garlic.
Accordingly, black garlic is made by a variety of methods, and most of
them are proprietary, which has resulted in the production of black
garlic with variable quality. The absence of a quality index or stan-
dardized methods of black garlic production could be an obstacle to
the growth of the black garlic industry. Therefore, it is necessary to
establish a quality factor for black garlic based on its major benecial
compounds and functional properties to ensure the continuous
growth of the black garlic industry.
There is a remarkable increase in SAC and browning along with
an associated increase in antioxidant activity during the black garlic
manufacturing process. Indeed, these are the most critical quality
factors of black garlic, as it was originally used for health promo-
tion. However, little information is available regarding the hun-
dreds of browning products, and many parameters can inuence
the formation of various chemical species, which exert different
antioxidant properties (Manzocco et al., 2000). Therefore, the use of
browning products as a quality factor for black garlic would be
inappropriate. SAC is stable throughout a range of pH and tem-
perature conditions (Peng et al., 1996), and critically, the amount of
SAC in black garlic is notably higher than that in raw garlic.
Accordingly, SAC and the associated antioxidant activity could be
used as quality factors for black garlic products.
The activity of g-GTP, which governs the formation of SAC, is
affected by temperature (Hanum, Sinha, & Cash, 1995). Therefore,
temperature during the black garlic manufacturing process is
considered to be one of the most important factors affecting the
quality of black garlic.
In this study, we investigated the effects of manufacturing
temperature on the properties of black garlic by analyzing the
amount of SAC and antioxidant activity to assess whether tem-
perature can be used to control the quality indicators of black garlic.
2. Materials and methods
2.1. Samples
Garlic (A. sativum L.) was purchased from Uiseong, Korea. Black
garlic was produced via heat treatment of raw garlic bulbs in a
thermo-hydrostat chamber (TH-G-800, Jeio-Tech, Seoul, Korea) set
at varying temperatures (40, 55, 70 and 85  C) with 70% relative
humidity (RH) for 45 days. Samples were removed after 1, 3, 5, 10,
15, 30 and 45 days of heat treatment. The garlic samples were
stored in a freezer at 4  C prior to analysis.
2.2. Measurement of moisture content, browning intensity and pH
The moisture content of heated garlic samples was determined
by measuring the weight loss using a moisture analyzer after slicing
and drying the bulbs (105  C, MX-50, A&D Company, Tokyo, Japan;
Ichikawa, Ide, & Ono, 2006).
To measure pH and browning intensity, 10 g of a heated garlic
sample was blended in 100 mL of distilled water (Milli-Q, Millipore,
Bedford, MA, USA). The pH of the heated garlic sample was then
measured using a pH meter (420A, Thermo Orion, Beverly, MA,
USA), and the browning intensity was measured spectrophoto-
metrically at an absorbance of 420 nm (Optizen 3220UV, Mecasys,
Daejeon, Korea; Lee, Lee, Do, & Shim, 1998). Heated garlic samples
were diluted with distilled water to obtain a range of absorbance
signals.
2.3. Determination of S-allyl cysteine
The heated garlic sample (10 g) was chopped in 70 mL of
distilled water. This mixture was then ltered, and the ltrates
were processed using an AccQ-Fluor Reagent Kit (Waters, Milford,
MA, USA). Derivatization was performed by adding 100 mL of AccQ-
Tag Fluor borate buffer and 20 mL of reconstituted AccQ-Fluor
reagent (in 10 mmol/L acetonitrile) to a 20-mL aliquot of the garlic
sample. The reaction mixture was incubated for 15 min at 55  C in a
shaking water bath. Aliquots (10 mL) of the samples were nally
subjected to HPLC with uorescence detection (474 scanning
uorescence detector; Waters, Milford, MA, USA).
Separation of the analyte was carried out using an AccQ-Tag C18
column (150 mm  3.9 mm ID, 4 mm; Waters, Milford, MA, USA) at a
ow rate of 1.0 mL/min. The solvent system consisted of two elu-
ents: Eluent A was composed of 100 mL/L AccQ-Tag eluent A
(containing 140 mmol/L sodium acetate, 17 mmol/L triethylamine
and 1 mmol/L calcium disodium EDTA [pH 5.05]), and eluent B was
composed of acetonitrile:water (3:2, v/v). A gradient elution was
employed starting with 95:5 A:B (v/v) and ending with 0:100 A:B
(v/v) (Bae et al., 2012).
2.4. Antioxidant activity
2.4.1. DPPH radical scavenging assay
The free radical scavenging activity of the heated garlic sample
was measured via the 1,1-diphenyl-2-picryl-hydrazil (DPPH)
radical scavenging assay using the method of Blois (1958) with
some modications. Briey, a 1 mmol/L solution of DPPH was
prepared in ethanol, and 2 mL of this solution was added to 2 mL of
the garlic sample extract at various concentrations (in distilled
water). The mixtures were shaken vigorously and were then
allowed to stand at room temperature for 30 min, after which their
absorbance was measured at 517 nm using a spectrophotometer
(Optizen 3220UV). Ascorbic acid was used as a positive control.
DPPH solution that did not contain a garlic sample was used as a
control. The DPPH radical scavenging activity was expressed as:
DPPH radical scavenging activity%
1  absorbance of sample=absorbance of control  100
2.4.2. Reducing power assay
The reducing power of the heated garlic sample was determined
according to the method of Oyaizu (1986), with some modications.
Various concentrations of heated garlic sample extracts in phos-
phate buffer (0.2 mol/L, pH 6.6) were added to potassium ferricya-
nide (1 mL, 10 mg/mL), after which the mixture was incubated at
 S.E. Bae et al. / LWT - Food Science and Technology 55 (2014) 397e402 399

50  C for 20 min. Trichloroacetic acid solution (2.5 mL, 100 mg/mL)

was added to the mixture, which was then centrifuged at 650  g for
10 min. The supernatant was then mixed with ferric chloride (1 mL,
1 mg/mL) for 10 min, after which the absorbance was measured at
700 nm. Reaction mixtures with higher absorbance values had
increased reducing power. Ascorbic acid was used as a positive
control.

2.5. Statistical analysis

The data are presented as the means  standard deviation (SD)

of ve independent experiments. The data were analyzed by two-
way ANOVA followed by Duncans multiple-range test using SAS
(version 9.0; SAS Inc., Cary, NC, USA). Differences were considered
statistically signicant at P < 0.05 for all tests.

3. Results and discussion

3.1. Moisture content, browning intensity and pH

Changes in the moisture content of black garlic due to heat

treatment at various temperatures during the manufacturing pro-
cess are shown in Fig. 1. The moisture content of raw garlic was
66.1 g/100 g. During the black garlic manufacturing process, the
moisture content decreased continuously over time. The moisture
content of the heated garlic samples decreased to 45.3e53.4 g/
100 g over a period of 45 days. Lower moisture contents were noted
at higher temperatures.
Changes in browning intensity and pH at various temperatures
during the black garlic manufacturing process are shown in Fig. 2.
In general, the black color development in garlic is associated with a
non-enzymatic browning reaction and is greatly dependent on
heating temperature (Rapusas & Driscoll, 1995; Samaniego-
Esguerra, Boag, & Robertson, 1991). The browning intensity of
heated garlic samples increased with increasing temperature
(Benzing-Purdie, Ripmeester, & Ratcliffe, 1985), and the rate of in-
crease in browning intensity was faster at a higher heating
temperatures.
The browning intensity of garlic samples heated at 40  C and
85  C for 45 days was 0.69 and 2.05, respectively. The browning
intensity of garlic sample heated at 85  C rapidly increased
during the early stages of heat treatment and reached a plateau
after 15 days. Changes in the browning intensity of garlic samples
Fig. 1. Changes in the moisture content of heated garlic samples after 45 days of heat
treatment at various temperatures during the black garlic manufacturing process. The
experiment was repeated ve times.
Fig. 2. Changes in the browning intensity and pH of heated garlic samples after 45
days of heat treatment at various temperatures during the black garlic manufacturing
process (a: pH, b: browning intensity). The experiment was repeated ve times.
heated at 40e70  C were less profound than those of garlic
samples heated at 85  C. Under these temperature conditions,
relatively little color change was observed during the early stages
of processing. However, over 45 days of heat treatment, the
browning intensity continuously increased. In the garlic sample
heated at 70  C, the browning intensity reached a similar level as
that in the garlic sample heated at 85  C in the nal stages of heat
treatment.
Similar results for browning intensity have also been reported in
studies of red ginseng. The rate of formation of browning products
in red ginseng is positively associated with processing temperature,
with specic Q10 values. Studies have shown that when the tem-
perature increased, the browning product formation also
increased; however, the initial induction period decreased (Choi,
Kim, Sung, & Hong, 1981; Kim & Kim, 1990).
These results could be attributed to the inuence of water ac-
tivity (aw); the rate of the browning reaction is known to reach a
maximum at aw values in the range of 0.5e0.7 (Kaanane & Labuza,
1989; Labuza & Saltmarch, 1981). The 70% RH and time required for
producing black garlic in the present study might have created a
situation in which the aw of the heated garlic sample reached a state
of equilibrium with the RH in the chamber in which the black garlic
was produced. This aw condition is thought to facilitate the
browning reaction in heated garlic samples.
The browning reaction is also affected by the moisture content
of the garlic sample (Kim, Jo, Kwon, & Park, 1992). The water con-
tent appeared to affect the activation energy of non-enzymatic
400 S.E. Bae et al. / LWT - Food Science and Technology 55 (2014) 397e402

browning, and higher water contents decreased the temperature Table 1

dependence of browning. Heat treatment in the black garlic Changes in the S-allyl cysteine (SAC) content (mg/g dry weight) of heated garlic
samples during the black garlic manufacturing process at various temperatures.
manufacturing process increased the rate of moisture removal.
Therefore garlic heated at high temperatures is thought to require a Time (days) Temperature ( C)
relatively higher activation energy for browning based on lower 40 55 70 85
moisture content. aH aH aH
0 19.61  0.35 19.61  0.35 19.61  0.35 19.61  0.35aH
A signicant decrease (P < 0.05) in pH was also observed with the 1 43.09  0.69aG 36.29  1.00bG 34.22  0.64cG 28.78  1.64dG
progression of the heat treatment process. The pH of raw garlic was 3 62.73  0.61aF
52.61  0.95 bF
46.40  0.75cF 40.72  0.51dF
6.42, whereas after heat treatment, the pH values of the garlic 5 78.56  0.44aE 62.30  1.71bE 57.01  0.94cE 49.39  1.25dE
96.97  0.33aD 78.58  1.09bD 70.72  1.72cD 63.31  1.02dD
samples heated at 40  C and 85  C for 5 days were 5.94 and 4.09, 10
15 108.03  1.27aC 92.83  1.08bC 80.91  1.29cC 71.36  1.47dC
respectively. Thereafter, the pH decreased gradually with time, 30 120.58  0.63aB 103.27  1.77bB 93.37  1.35cB 81.04  1.08dB
reaching 5.00 and 3.05 after heating at 40  C and 85  C, respectively, 45 124.67  1.61aA 113.25  1.82bA 113.25  1.82cA 85.46  0.81dA
for 45 days. These results can be attributed to the dependence of pH The values were measured in mg/g dry weigh.
on the temperature in the early stages of heat treatment. The experiment was repeated ve times, and all values are means  standard de-
Similar results were also observed in the red ginseng viation.
aed
manufacturing process. The pH decreased with the formation of Means followed by different superscripts in the same row are signicantly
different at P < 0.05.
browning products during the manufacturing process, and the AeH
Means followed by different superscripts in the same column are signicantly
decrease in pH progressed as the heating temperature increased different at P < 0.05.
(Choi et al., 1981; Lee et al., 1998).
The pH decrease in the heated garlic sample was, in part, asso-
ciated with the production of browning materials upon heat sample heated at low temperature. As a result, the garlic sample
treatment during the black garlic manufacturing process. The for- heated at 40  C had a higher SAC content than that heated at 85  C.
mation of carboxylic acids (which are produced by the oxidation of However, g-GTP is known to be completely inactivated within
the aldehyde group in aldohexose, acidic compounds and decreases 30 min at temperatures over 75  C (Dos Anjos, Machdo, Ferro, &
in basic amino acids by combining with sugar) has been reported to Bogin, 1998; Lorenzen, Martin, Clawin-Rdecker, Barth, &
be responsible for the decrease in pH in the browning reactions Knappstein, 2010; McKellar, 1996; Zehetner, Bareuther, Henle, &
(Choi et al., 1981; Kim, Do, & Oh, 1981). Klostermeyer, 1995). Based on these studies, high temperature
The pH is closely related to the growth of microorganism in conditions (85  C in this study) contributed to the increase in SAC
food. Below a pH of approximately 4.2, most microorganisms that content during the early stages of the black garlic manufacturing
cause food poisoning are well controlled; however, lactic acid process; thereafter, changes in SAC content could have been caused
bacteria and many species of yeasts and molds grow well at pH by other unidentied factors. For example, SAC formation by the
levels below 4.2. In addition, bacterial spores are killed by heat reduction of alliin has been shown in some studies, although the
more rapidly at acidic pH values than at neutral pH (Rahman, 2007, mechanism of SAC formation by hydrolysis of GSAC is strongly
chap. 12). In the garlic sample heated at 85  C, the pH decreased to supported by most researchers (Egen-Schwind, Eckard, & Kemper,
below 4. Therefore, black garlic samples heated to 85  C may be 1992; Jandke & Spiteller, 1987; Koch & Lawson, 1996; Kodera
more biologically stable with respect to microbial growth. How- et al., 2002; de Rooij, Boogaard, Rijksen, Commandeur, &
ever, this was not the case for the garlic sample heated at 40  C due Vermeulen, 1996).
to its high pH.
3.3. Antioxidant activity
3.2. Change in S-allyl cysteine content
The DPPH radical scavenging activity of heated garlic samples is
Changes in the amount of SAC at various temperatures in the shown in Table 2. The DPPH radical scavenging activity indicates
black garlic manufacturing process are shown in Table 1. The the ability of an antioxidant compound to donate electrons or
amount of SAC increased signicantly as the heat treatment process hydrogen, thereby converting DPPH into a more stable molecule
progressed, and garlic samples heated at low temperatures had with a reduced absorbance (Brand-Williams, Cuvelier, & Berset,
higher amounts of SAC than those heated at higher temperatures.
The SAC content of garlic samples heated at 40  C for 45 days was
Table 2
124.67 mg/g, whereas that of garlic samples heated at 85  C was
Changes in DPPH radical scavenging ability (%) of heated garlic samples during the
only 85.46 mg/g. black garlic manufacturing process at various temperatures.
The highest SAC production at 40  C in the present study was
Time (days) Temperature ( C)
associated with g-GTP activity. As mentioned above, the activity of
g-GTP is affected by the processing temperature and is attributed to 40 55 70 85
the formation of SAC. The reported optimum temperature for g-GTP 0 6.21  0.99aF 6.21  0.99aG 6.21  0.99aG 6.21  0.99aH
activity is 40  C (Hanum et al., 1995), which is in agreement with 1 7.35  0.14cF 6.87  0.48cG 8.33  0.31bF 9.06  0.55aG
the results of this study. These ndings are similar to those reported 3 8.89  0.74cE 8.72  0.76cF 11.34  1.30bE 15.57  0.80aF
5 10.86  0.50dD 11.45  0.65cE 16.00  0.61bD 20.04  0.98aE
in the study by Park et al. (2010), in which it was suggested that the 10 12.75  0.98dC 16.33  1.30cD 20.50  1.25bC 29.00  1.15aD
change in the SAC content of garlic is affected by aging temperature 15 17.41  1.33dB 20.71  1.42cC 25.43  1.70bB 35.14  0.76aC
and time and that increased SAC production was observed under 30 21.52  1.10dA 27.12  0.86cB 32.88  1.47bA 42.88  1.59aB
heat treatment at 40  C when compared with 30  C and 50  C. 45 22.50  1.05dA 30.34  1.43cA 33.93  1.15bA 44.77  1.22aA
The formation of SAC is also affected by the water-facilitated The values were measured in %.
reaction between GSAC and g-GTP (Koch & Lawson, 1996). Water The experiment was repeated ve times, and all values are means  standard de-
in garlic aids the g-GTP reaction, aiding hydrolysis, and an abun- viation.
aed
Means followed by different superscripts in the same row are signicantly
dance of water supports the transformation of GSAC into SAC. Garlic different at P < 0.05.
samples heated at higher temperatures showed higher water loss, AeH
Means followed by different superscripts in the same column are signicantly
which caused the g-GTP reaction to be less active than that in the different at P < 0.05.
 S.E. Bae et al. / LWT - Food Science and Technology 55 (2014) 397e402 401

1995; Guerard & Suyama-Martinez, 2003). The DPPH radical scav-
enging ability of the heated garlic samples was signicantly higher
than that of the raw garlic samples. Moreover, the radical scav-
enging ability increased with increasing temperature from 40 to
85  C (P < 0.05). The free radical scavenging activity of raw garlic
was 6.21%; this activity increased to 44.77% upon heating at 85  C
for 45 days, whereas that of the garlic sample heated at 40  C was
increased to only 22.50%.
Woo et al. (2007) investigated the effect of garlic aroma extract
on electron donating ability to DPPH radicals at various tempera-
tures. The electron donating ability (%) of the garlic aroma extract
gradually increased with increasing temperature from 100 to
120  C. This result suggests that the electron donating ability (%) is
lower at low temperatures and higher at high temperatures.
In general, the DPPH radical scavenging activity was concomi-
tant with reducing power. The reduction of the Fe3/ferricyanide
complex to the ferrous form occurs in the presence of antioxidants,
which provide the reducing power. Changes in reducing power at
various temperatures in the black garlic manufacturing process are
shown in Table 3. The reducing power of the heated garlic samples
increased signicantly with temperature and time (P < 0.05) and
was highest at high temperatures after 45 days of heat treatment.
The reducing power of raw garlic was 0.08; this value increased to
1.90 and 3.13 after 45 days of heat treatment at 40  C and 85  C,
respectively.
The representative antioxidant materials in garlic are phenolic
compounds, including avonoids and sulfur compounds such
as diallyl sulde, trisuldes and SAC (Leelarungrayub,
Rattanapanone, Chanarat, & Gebicki, 2006). Signicantly, SAC
has been reported to have antioxidant activity and is thought to be
responsible for the antioxidant activity of heated garlic samples by
inhibiting free radicals (Borek, 2001; Imai, Ide, Moriguchi,
Matsuura, & Itakura, 1994). However, the temperatures resulting
in the highest SAC content and antioxidant activity, including
DPPH radical scavenging activity and reducing power, did not
match. In this study, SAC content increased to a much greater
extent in garlic samples heated at low temperatures; however,
DPPH radical scavenging activity and reducing power largely
increased in garlic samples heated at high temperatures. There-
fore, although SAC is a compound with strong antioxidant prop-
erties, the increase in SAC at high temperatures was not strong
enough to account for the high antioxidant activity of garlic
samples heated at these temperatures.
Other active constituents may contribute to the antioxidant
activity of the heated garlic samples. Many studies have indicated
that the presence of browning products is related to increases in
antioxidant activity, and browning products have been shown to
exert antioxidant action by breaking the free radical chain through
the donation of hydrogen atoms (Eichner, 1981; Manzocco et al.,
2000). A positive and highly signicant relationship between to-
tal phenolics and antioxidant activity in plant products has also
been previously demonstrated (Stratil, Klejdus, & Kubn, 2006).
In this study, the browning intensity gradually increased during
the black garlic manufacturing process, and it exhibited a trend
similar to that of DPPH radical scavenging activity and reducing
power of garlic samples heated at various temperatures. In addi-
tion, an increase in the contents of total polyphenols and avonoids
in black garlic compared with those in raw garlic has also been
reported. Moreover, all these properties were enhanced in garlic
samples heated at high temperatures (Nencini, Menchiari, Franchi,
& Micheli, 2011; Park, Park, & Park, 2009; Sato, Kohno, Hamano, &
Niwano, 2006; Shin et al., 2008).
The results of this study suggest that changes in antioxidant
activity, including DPPH radical scavenging activity and reducing
power at various temperatures, in the black garlic manufacturing
process are well correlated with all of which are responsible for the
antioxidant activity of heated garlic samples. Moreover, the tem-
perature utilized in the black garlic manufacturing process was
shown to be an important factor affecting the antioxidant activity of
heated garlic samples.
4. Conclusion
The effects of temperature on the moisture content, pH,
browning intensity, SAC content and resultant antioxidant activity
in the black garlic manufacturing process were determined. The
moisture content of raw garlic was 66.10 g/100 g and gradually
decreased with temperature throughout heating process over 45
days of heat treatment. In garlic samples heated at higher tem-
peratures, the browning intensity also increased. The pH decreased
more signicantly at higher temperatures during 45 days of heat
treatment. The SAC content increased to a greater extent at rela-
tively low temperatures. These results are related to the activity of
g-GTP on temperature. On the contrary, antioxidant activity based
on DPPH radical scavenging activity and reducing power increased
more at higher temperatures through the increased formation of
browning products, SAC, total phenols and total avonoids. These
results suggest that the antioxidant activity of black garlic is
signicantly associated with multiple compounds.

Acknowledgments
Table 3
Changes in the reducing power (O.D. value) of heated garlic samples during the black This study was supported by a grant from the Uiseong Black
garlic manufacturing process at various temperatures.
Garlic Farming Association of Korea and Korea University.
Time (days) Temperature ( C)

40 55 70 85 References
0 0.08  0.01aE 0.08  0.01aE 0.08  0.01aG 0.08  0.01aF
Bae, S. E., Cho, S. Y., Won, Y. D., Lee, S. H., & Park, H. J. (2012). A comparative study of
1 0.09  0.01bE 0.08  0.01bE 0.09  0.01bFG 0.11  0.01aF
the different analytical methods for analysis of S-allyl cysteine in black garlic by
3 0.08  0.01dE 0.12  0.01cE 0.16  0.01bF 0.36  0.01aE
HPLC. LWT e Food Science and Technology, 46, 532e535.
5 0.11  0.01dE 0.16  0.01cE 0.32  0.01bE 0.40  0.01aE
Benzing-Purdie, L. M., Ripmeester, J. A., & Ratcliffe, C. I. (1985). Effects of temper-
10 0.27  0.01dD 0.32  0.05cD 0.57  0.01bD 0.87  0.02aD ature on Maillard reaction products. Journal of Agricultural and Food Chemistry,
15 0.74  0.02dC 0.92  0.02cC 1.19  0.03bC 1.34  0.05aC 33, 31e33.
30 1.53  0.03cB 1.66  0.10cB 2.05  0.09bB 2.38  0.13aB Block, E. (1992). The organosulfur chemistry of the genus Allium e implications for
45 1.90  0.12dA 2.24  0.13cA 2.72  0.14bA 3.13  0.26aA the organic chemistry of sulfur. Angewandte Chemie International Edition in
English, 31, 1135e1178.
The values were given as O.D. value.
Blois, M. S. (1958). Antioxidant determination by the use of a stable free radical.
The experiment was repeated ve times, and all values are means  standard de- Nature, 181, 1199e1200.
viation. Borek, C. (2001). Antioxidant health effects of aged garlic extract. Journal of Nutri-
aed
Means followed by different superscripts in the same row are signicantly tion, 131, 1010Se1015S.
different at P < 0.05. Brand-Williams, W., Cuvelier, M. E., & Berset, C. (1995). Use of a free radical method
AeH
Means followed by different superscripts in the same column are signicantly to evaluate antioxidant activity. Lebensmittel-Wissenschaft und Technologie, 28,
different at P < 0.05. 25e30.
402 S.E. Bae et al. / LWT - Food Science and Technology 55 (2014) 397e402
Choi, J. H., Kim, W. J., Sung, H. S., & Hong, S. K. (1981). Quality changes in red ginseng
extract during high temperature storage. Journal of Korean Agricultural Chemical
Society, 24, 166e174.
de Rooij, B. M., Boogaard, P. J., Rijksen, D. A., Commandeur, J. N., & Vermeulen, N. P.
(1996). Urinary excretion of N-acetyl-S-allyl-l-cysteine upon garlic consump-
tion by human volunteers. Archives Toxicology, 70, 635e639.
Dos Anjos, F., Machdo, A., Ferro, C., & Bogin, E. (1998). g-Glutamyl trans-
peptidase as a marker for pasteurization of raw milk. Journal of Food
Protection, 61, 1057e1059.
Durak, I., Yilmaz, E., Devrim, E., Perk, H., & Kamaz, M. (2003). Consumption of
aqueous garlic extract leads to signicant improvement in patients with benign
prostate hyperplasia and prostate cancer. Nutrition Research, 23, 199e204.
Egen-Schwind, C., Eckard, R., & Kemper, F. H. (1992). Metabolism of garlic constit-
uents in the isolated perfused rat liver. Planta Medica, 58, 301e305.
Eichner, K. (1981). Antioxidant effect of Maillard reaction intermediates. Progress in
Food and Nutrition Science, 5, 441e451.
Gorinstein, S., Leontowicz, M., Leontowicz, H., Najman, K., Na-miesnik, J., Park, Y. S.,
et al. (2006). Supplementation of garlic lowers lipids and increases antioxidant
capacity in plasma of rats. Nutrition Research, 26, 362e368.
Guerard, F., & Suyama-Martinez, M. T. (2003). Antioxidant effect of protein hydro-
lysates in the reaction with glucose. Journal of the American Oil Chemists Society,
80, 467e470.
Hanum, T., Sinha, N. K., & Cash, J. N. (1995). Characteristics of g-glutamyl trans-
peptidase and alliinase of onion and their effects on the enhancement of py-
ruvate formation in onion macerates. Journal of Food Biochemistry, 19, 51e65.
Ichikawa, M., Ide, N., & Ono, K. (2006). Changes in organosulfur compounds in
garlic cloves during storage. Journal of Agricultural and Food Chemistry, 54,
4849e4854.
Ide, N., & Lau, B. H. (1999). Aged garlic extract attenuates intracellular oxidative
stress. Phytomedicine, 6, 125e131.
Ide, N., Matsuura, H., & Itakura, Y. (1996). Scavenging effect of aged garlic
extract and its constituents on active oxygen species. Phytotherapy Research, 10,
340e341.
Imai, J., Ide, S., Moriguchi, T., Matsuura, H., & Itakura, Y. (1994). Antioxidant and
radical scavenging effects of aged garlic extract and its constituents. Planta
Medica, 60, 417e420.
Jandke, J., & Spiteller, G. (1987). Unusual conjugates in biological proles originating
from consumption of onions and garlic. Journal of Chromatography B: Biomedical
Sciences and Applications, 421, 1e8.
Kaanane, A., & Labuza, T. P. (1989). The Maillard reaction in foods. Progress in Clinical
& Biological Research, 304, 301e327.
Kim, H. K., Jo, K. S., Kwon, D. Y., & Park, M. H. (1992). Effects of drying temperature
and sulting on the qualities of dried garlic slices. Journal of Korean Agricultural
Chemical Society, 35, 6e9.
Kim, M. H., & Kim, B. Y. (1990). Development of optimum processing conditions in
air dried garlics using response surface methodology. Journal of Food Science
and Nutrition, 19, 234e238.
Kim, S. D., Do, J. H., & Oh, H. I. (1981). Antioxidant activity of Panax ginseng
browning products. Journal of Korean Agricultural Chemical Society, 24, 161e166.
Koch, H. P., & Lawson, L. D. (1996). Garlic: The science and therapeutic application of
Allium sativum L. and relative species (pp. 135e212). Willinam & Wikins.
Kodera, Y., Suzuki, A., Imada, O., Kasuga, S., Sumioka, I., Kanezawa, A., et al.
(2002). Physical, chemical and biological properties of S-allylcysteine, an
amino acid derived from garlic. Journal of Agricultural and Food Chemistry, 30,
622e632.
Labuza, T. P., & Saltmarch, M. (1981). The nonenzymatic browning reaction as affected
by water in foods. Water activity: Inuences on food quality (pp. 605e650). New
York: Academic Press.
Lee, J. W., Lee, S. K., Do, J. H., & Shim, K. H. (1998). Characteristics of the water
soluble browning reaction of Korean red ginseng as affected by heating treat-
ment. Journal of Ginseng Research, 22, 193e199.
Leelarungrayub, N., Rattanapanone, V., Chanarat, N., & Gebicki, J. M. (2006).
Quantitative evaluation of the antioxidant properties of garlic and shallot
preparations. Nutrition, 22, 266e274.
Legault, R. R., Hendel, C. E., & Talburt, W. F. (1954). Retention of quality in dehy-
drated vegetables through in package desiccation. Food Technology, 8, 143e149.
Lorenzen, P. C., Martin, D., Clawin-Rdecker, I., Barth, K., & Knappstein, K. (2010).
Activities of alkaline phosphatase, gamma-glutamyl transferase and lactoper-
oxidase in cow, sheep and goats milk in relation to heat treatment. Small
Ruminant Research, 89, 18e23.
Manzocco, L., Calligaris, S., Mastrocola, D., Nicoli, M. C., & Lerici, C. R. (2000). Review
of non-enzymatic browning and antioxidant capacity in processed foods. Trends
in Food Science & Technology, 11, 340e346.
McKellar, R. C. (1996). Inuence of ice-cream mix components on the thermal
stability of bovine milk g-glutamyl transpeptidase and Listeria innocua. Inter-
national Dairy Journal, 6, 1181e1189.
Nencini, C., Menchiari, A., Franchi, G. G., & Micheli, L. (2011). In vitro antioxidant
activity of aged extracts of some Italian Allium species. Plant Foods for Human
Nutrition, 66, 11e16.
Osada, Y., & Shibamoto, T. (2006). Antioxidative activity of volatile extracts from
Maillard model system. Food Chemistry, 98, 522e528.
Oyaizu, M. (1986). Studies on products of browning reactions: antioxidative activ-
ities of products of browning reaction prepared from glucosamine. Japanese
Journal of Nutrition, 44, 307e315.
Park, J. H., Park, Y. K., & Park, E. (2009). Antioxidative and antigenotoxic effects of
garlic (Allium sativum L.) prepared by different processing methods. Plant Foods
for Human Nutrition, 64, 244e249.
Park, S. H., Kim, S. H., Kim, H. S., Jung, Y. K., Kim, Y. R., Lee, H. Y., et al. (2010).
Research of S-allyl-(L)-cysteine content changes in aged garlic. In Proceedings of
the 2010 ASABE annual international meeting (pp. 4506e4512). Pittsburgh, USA.
Peleg, Y., Mannheim, C. H., & Berk, Z. (1970). Changes in quality of dehydrated
kibbled onions during storage. Journal of Food Science, 35, 513e517.
Peng, J. P., Chen, H., Qiao, Y. Q., Ma, L. R., Narui, T., Suzuki, H., et al. (1996). Two new
steroidal saponins from Allium sativum and their inhibitory effects on blood
coagulability. Acta Pharmaceutica Sinica, 31, 607e612.
Rahman, M. S. (2007). Handbook of food preservation (2nd ed.). New York: CRC Press.
Rapusas, R. S., & Driscoll, R. H. (1995). Kinetics of non-enzymatic browning in onion
slices during isothermal heating. Journal of Food Engineering, 24, 417e429.
Rees, L. P., Minney, S. F., Plummer, N. T., Slater, J. H., & Skyrme, D. A. (1993).
A quantitative assessment of the antimicrobial activity of garlic (Allium sat-
ivum). World Journal of Microbiology and Biotechnology, 9, 303e307.
Samaniego-Esguerra, C. M., Boag, I. F., & Robertson, G. L. (1991). Kinetics of quality
deterioration in dried onions and green beans as a function of temperature and
water activity. Lebensmittel-Wissenshaft und Technologie, 24, 53e58.
Sato, E., Kohno, M., Hamano, H., & Niwano, Y. (2006). Increased antioxidative po-
tency of garlic by spontaneous short-term fermentation. Plant Foods for Human
Nutrition, 61, 157e160.
Shin, J. H., Choi, D. J., Lee, S. J., Cha, J. Y., Kim, J. G., & Sung, N. J. (2008). Changes of
physicochemical components and antioxidant activity of garlic during its pro-
cessing. Journal of Life Science, 18, 1123e1131.
Stratil, P., Klejdus, B., & Kubn, V. (2006). Determination of total content of
phenolic compounds and their antioxidant activity in vegetables e evaluation
of spectrophotometric methods. Journal of Agricultural and Food Chemistry, 54,
607e616.
Wang, D., Feng, Y., Liu, J., Yan, J., Wang, M., Sasaki, J., et al. (2010). Black garlic (Allium
sativum) extracts enhance the immune system. Medicinal and Aromatic Plant
Science and Biotechnology, 4, 37e40.
Woo, K. S., Yoon, H. S., Lee, Y. R., Lee, J. S., Kim, D. J., Hong, J. T., et al. (2007).
Characteristics and antioxidative activity of volatile compounds in heated garlic
(Allium sativum). Food Science and Biotechnology, 16, 822e827.
Yanagimoto, K., Lee, K. G., Ochi, H., & Shibamoto, T. (2002). Antioxidative activity of
heterocyclic compounds formed in Maillard reaction products. International
Congress Series, 1245, 335e340.
Yilmaz, Y., & Toledo, R. (2005). Antioxidant activity of water-soluble Maillard re-
action products. Food Chemistry, 93, 273e278.
Zehetner, G., Bareuther, C., Henle, T., & Klostermeyer, H. (1995). Inactivation kinetics
of g-glutamyl-transferase during the heating of milk. Zeitschrift fr
Lebensmittel-Untersuchung und eForschung, 201, 336e338.