JOURNAL OF FUNCTIONAL FOODS 5 ( 2 0 1 3 ) 8 0 –8 6

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Comparison of phenolic acids and flavonoids in black garlic

at different thermal processing steps

Ji-Sang Kima,*, Ok-Ju Kanga, Oh-Cheon Gweonb

a
Department of Food and Nutritional Science, Kyung Nam University, Changwon 631-701, Republic of Korea
b
Department of Curinary Arts & Bakery, Namhae College, Namhae 668-801, Republic of Korea

A R T I C L E I N F O A B S T R A C T

Article history: The phenolic acid and the flavonoid constituents of garlic subjected to different thermal
Received 13 June 2012 processing steps were examined. Black garlic was produced in a ripening chamber by using
Received in revised form a programmed stepwise heating schedule, as follows. Step 1: 90 C and 100% RH for 34 h;
23 August 2012 Step 2: 60 C and 60% RH for 6 h; Step 3: 75 C and 70% RH for 48 h; Step 4: 70 C and 60%
Accepted 27 August 2012 RH for 60 h; Step 5: 65 C and 50% RH for 192 h. The results of the present investigations
Available online 29 September 2012 showed that thermal processing affected quantities of each phenolic acid and flavonoid
component. The total phenolic content (TPC) and total flavonoid content (TFC) of the garlic
Keywords: subjected to different thermal processing steps were higher than those of fresh garlic. In
Black garlic particular, the black garlic cloves ripened using Step 1 (BG1), black garlic cloves ripened
Phenolic acid using Step 2 (BG2), black garlic cloves ripened using Step 3 (BG3), and black garlic cloves rip-
Flavonoid
ened using Step 5 (BG5) samples exhibited levels of TPC that were higher than the TFC,
Thermal processing
while the TFC in the fresh garlic (FG) and black garlic cloves ripened using Step 4 (BG4) sam-
ples were higher than the TPCs. Hydroxycinnamic acid derivatives were found to be the
major phenolic acids of garlic at different processing steps. Among the four major flavo-
noid subgroups in garlic, flavanols were found at the highest concentrations followed by
flavanones and flavones, except in the FG sample.
 2012 Elsevier Ltd. All rights reserved.

1. Introduction Polyphenols are divided into several classes, according to

Polyphenols are the most abundant antioxidants in our diet
and are common constituents of foods of plant origin and
are widespread constituents of fruits, vegetables, cereals, ol-
ive, dry legumes, chocolate and beverages, such as tea, coffee,
cocoa and wine (Ferri & Grassi, 2010; Grassi et al., 2008, 2009).
Polyphenols comprise a wide variety of molecules that have a
polyphenol structure (i.e., several hydroxyl groups on one or
more aromatic rings), but also molecules with one phenol
ring, such as phenolic acids and phenolic alcohols.
the number of phenol rings that they contain and to the struc-
tural elements that bind these rings to one another. Phenolic
acids present in plants are hydroxylated derivatives of benzoic
and cinnamic acids (Herrmann, 1989; Shahidi & Naczk, 1995,
Chapter 5). Flavonoids are divided into many categories,
including flavonols, flavones, catechins, proanthocyanidins,
anthocyanidins and isoflavonoids (Havsteen, 1983; Shahidi &
Naczk, 1995, Chapter 5). Many of the flavonoids and related
compounds are known to possess strong antioxidative charac-
teristics (Dziedzic & Hudson, 1983) and widely investigated as

* Corresponding author. Address: Department of Food and Nutritional Science, Kyung Nam University, 85(Woryeong-dong) Munhwanam
11-gil, Masanhappo-gu, Changwon-si, Gyeongsangnam-do 631-701, Republic of Korea. Tel.: +82 55 249 2185; fax: +82 55 245 5001.
E-mail address: jisangkim@kyungnam.ac.kr (J.-S. Kim).
1756-4646/$ - see front matter  2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.jff.2012.08.006
 JOURNAL OF FUNCTIONAL FOODS
a new source of bioactive ingredient that can be incorporated
into foods in the development of functional foods.
Garlic (Allium sativum L., Alliaceae) has been playing one of
the most important dietary and medicinal roles in human
beings for centuries. It has been cultivated since ancient
times, used as a spice and flavouring and, due to its potential
benefits in preventive and curative medicine, has been used
in many cultures (Rivlin, 2001). Health properties of garlic de-
pend on its bioactive compounds, especially the organosul-
phur compounds, which are also responsible for its pungent
flavour. In addition to these compounds, garlic is also charac-
terized by phenolic compounds (Lanzotti, 2006), which have
interesting pharmacological properties and are present in rel-
atively high amounts. Moreover, some garlic products such as
aged garlic extract or black garlic have been found to contain
an increased level of polyphenols compared to raw garlic
(Nencini, Menchiari, Franchi, & Micheli, 2011; Park, Park, &
Park, 2009).
Black garlic is a processed garlic product that is prepared
by heat treatment of the raw garlic at high temperature under
controlled humidity for more than 1 month. Black garlic prod-
ucts have emerged as one of the fastest-growing health-ori-
ented food product in Korean market with the growing
awareness of the health benefits of garlic (Bae, Cho, Won,
Lee, & Park, 2012). Moreover, thermal processes are com-
monly used in food manufacturing. One of the important
objectives of thermal processes is to raise the sensory quality
of foods, their palatability and to extend the range of colours,
tastes, aromas and textures in food (Capuano & Fogliano,
2011). In addition, heating processes lead to the formation
of biological compounds that are not originally present in
food. However, influences of thermal processes on the con-
centration of individual flavonoids and phenolic acids in gar-
lic are unknown. Therefore, the objective of the present study
was to measure the content of phenolic compounds (total
amount as well as individual flavonoids and phenolic acids)
in garlic and to analyze the influence of thermal processes
on garlic.
2. Materials and methods
2.1. Chemicals and reagents
Gallic acid, p-hydroxybenzoic acid, chlorogenic acid, catechin,
caffeic acid, epicatechin, epigallocatechin gallate, p-coumaric
acid, ferulic acid, m-coumaric acid, o-coumaric acid, querci-
trin, myricetin, resveratrol, morin, quercetin, naringenin, api-
genin, vanillic acid, kaempferol and formic acid were
purchased from Sigma Chemical Co. (St. Louis, MO, USA).
Acetonitrile and HPLC-grade water were purchased from J.T.
Baker (Phillipsburg, NJ, USA). All other chemicals and solvents
were of analytical reagent grade.
2.2. Sample preparation
Garlic was cultivated in Namhae-gun, Korea. Fresh garlic
bulbs were purchased from the Namhae Bomulsum agricul-
tural association (Namhae, Korea) in 2011. Black garlic was
produced in a ripening chamber (MBGAM-1500, Minyoung,
5 (2 0 13 ) 8 0–86 81
Co. Ltd., Korea), without removing the outer layers, by using
a programmed stepwise heating schedule, as follows. Step
1: 90 C and 100% RH for 34 h; Step 2: 60 C and 60% RH for
6 h; Step 3: 75 C and 70% RH for 48 h; Step 4: 70 C and 60%
RH for 60 h; Step 5: 65 C and 50% RH for 192 h. The samples
tested in this study were as follows: raw garlic cloves, black
garlic cloves at Step 1, black garlic cloves at Step 2, black garlic
cloves at Step 3, black garlic cloves at Step 4, and black garlic
cloves at Step 5. These samples were designated as FG, BG1,
BG2, BG3, BG4, and BG5, respectively. To prepare the garlic
powder, fresh garlic and black garlic cloves were peeled off.
They were frozen in liquid nitrogen, and immediately
freeze-dried. The resulting lyophilized garlic samples were
ground into a powder with a mortar and pestle. The resulting
powder was stored in sealed plastic bottles at 20 C until
analysis.
2.3. Analysis of total polyphenols
2.3.1. Extraction of polyphenols
The method described by the International Organization for
Standardization (ISO) 14502-1 was used (ISO, 2005). Briefly,
0.200 ± 0.001 g of each sample was weighed in an extraction
tube, and 5 mL of 70% methanol at 70 C was added. The ex-
tract was mixed and heated at 70 C for 10 min. After cooling
at room temperature, the extract was centrifuged at 7840g for
10 min. The supernatant was decanted in a graduated conical
tube. The extraction step was repeated third times. Both ex-
tracts were pooled and the volume adjusted to 10 mL with
cold 70% methanol. One millilitre of the extract was diluted
with water to 5 mL.
2.3.2. Determination of total polyphenol
The total polyphenol content (TPC) was determined spectro-
photometrically, using gallic acid as a standard, according to
the method described by the International Organization for
Standardization (ISO) 14502-1 (ISO, 2005). Briefly, 1.0 mL of
the diluted sample extract was transferred in duplicate to
separate tubes containing 5.0 mL of a 1/10 dilution of Folin–
Ciocalteu’s reagent in water. Then, 4.0 mL of a sodium car-
bonate solution (7.5%, w/v) was added. The tubes were then
allowed to stand at room temperature for 60 min before
absorbance at 765 nm was measured against water. All values
were expressed as mg gallic acid equivalents (GAE) per kg dry
matter of the garlic sample.
2.4. Determination of total flavonoid content
Total flavonoid content was determined using a colouri-
metric method described previously (Woisky & Salatino,
1998). Garlic powder (0.2 g) was added to 20 mL of 80%
methanol, extracted for 2 h at room temperature and cen-
trifuged at 18,000g for 15 min. The volume of the extract
was made up to 100 mL with 80% methanol. A portion of
0.5 mL was taken and 0.5 mL of 2% ethanolic solution of
AlCl3 was added to it. After 1 h at room temperature, the
absorbance was read at 420 nm. All values were expressed
as mg quercetin equivalents (QE) per kg dry matter of gar-
lic sample.
82 JOURNAL OF FUNCTIONAL FOODS
2.5. Analysis of phenolic acids
Total (free + bound) phenolic acids were determined by the
method of Mattila and Kumpulainen (2002) with modifica-
tions. Garlic powder (3 g) were weighed into a 200 mL glass
tube, and 35 mL of the mixture of methanol (containing 2 g/
L of butylated hydroxyanisole) and 10% acetic acid in the ratio
of 85:15 was added, and the mixture was homogenized. The
sample extract was then ultrasonicated for 30 min after addi-
tion of 15 mL of deionized water. The sample was then sub-
jected to alkaline hydrolysis with the addition of 60 mL of
deionized water containing 22 mM ethylenediaminetetraace-
tic acid (EDTA) and 2% ascorbic acid and 25 mL of 10 M NaOH.
This mixture was incubated at 30 C for 30 min. The solution
was then adjusted to 2 with 4 M HCl, and the phenolic acids
were extracted three times with 15 mL of ethyl acetate. Com-
bined extracts were filtered through anhydrous sodium sul-
phate, evaporated to dryness, and dissolved into 2 mL of
methanol. Finally, the sample was filtered and injected into
the HPLC.
The experiments were carried out using an Agilent 1260
infinity quaternary liquid chromatography (Hewlett Packard,
Wilmington, NC, USA) with a multiple wavelength detector,
MWD operating at 280 nm. Chromatographic separations
were performed on an Agilent zorbax rapid resolution high
definition SB-C18 column (2.1 · 100 mm I.D., 1.8 lm particle
size, Agilent Technologies, NC, USA). The column tempera-
ture was 30 C, and the flow rate was 0.3 mL/min. Eluents A
and B were used for gradient elution. Solution A was water
with 0.1% formic acid and solution B was acetonitrile with
0.1% formic acid. The following gradient was used: 0% B
(0 min), 5% (0–3.5 min), 15% (3.5–7.1 min), 40% (7.1–25 min),
40% (25–26 min), 100% (26–27 min), 100% (27–29 min) and 0%
(29–35 min). The data analysis was performed using Chemsta-
tion software (Hewlett Packard). The standards used were p-
hydroxybenzoic acid, gallic acid, chlorogenic acid, caffeic
acid, p-Coumaric acid, ferulic acid, m-Coumaric acid, o-Cou-
maric acid, and vanillic acid.
2.6. Analysis of flavonoids
Extracts were prepared according to the method of Hertog,
Hollman, and Venema (1992) with slight modifications.
Briefly, 15 mL of 62.5% aqueous methanol containing 2 g/L
tert-butylhydroquinone were added to 3 g of garlic powder.
Fifteen millilitres of 6 M HCl were then added, and the mix-
ture was refluxed at 90 C for 1 h. The extract was allowed
to cool, and the extract was then sonicated for 10 min, and
the flavonoids were extracted three times with 15 mL of ethyl
acetate. Combined extracts were filtered through anhydrous
sodium sulphate, evaporated to dryness, and dissolved into
2 mL of methanol. The experiments were carried out using
an Agilent 1260 infinity quaternary liquid chromatography
(Hewlett Packard) with a multiple wavelength detector,
MWD. Chromatographic separations were performed on an
Agilent zorbax rapid resolution high definition SB-C18 col-
umn (2.1 · 100 mm I.D., 1.8 lm particle size, Agilent Technol-
ogies). The column temperature was 30 C, and the flow rate
was 0.3 mL/min. Eluents A and B were used for the gradient
elution. Solution A was water with 0.1% formic acid and
5 ( 2 0 1 3 ) 8 0 –8 6
solution B was acetonitrile with 0.1% formic acid. The follow-
ing gradient was used: 0% B (0 min), 5% (0–3.5 min), 15% (3.5–
7.1 min), 40% (7.1–25 min), 40% (25–26 min), 100% (26–27 min),
100% (27–29 min) and 0% (29–35 min). Detection was per-
formed at a wavelength of 280 nm. The data analysis was per-
formed using Chemstation software (Hewlett Packard). The
standards used were catechin, epicatechin, epigallocatechin
gallate, quercitrin, myricetin, resveratrol, morin, quercetin,
naringenin, apigenin, and kaempferol.
2.7. Statistical analysis
All experimental data were analyzed by analysis of variance
(ANOVA) and significant differences among means from trip-
licate analysis at (P < 0.05) were determined by Duncan’s mul-
tiple range tests using the statistical analysis system (SPSS
17.0, IBM Inc., NY, USA).
3. Results and discussion
3.1. Total phenolic content (TPC) in garlic at different
thermal processing steps
The levels of phenolic compounds were expressed as gallic
acid equivalents (GAE), which corresponds to the mean re-
sponse of all the major phenolic compounds found in fruits
and vegetables (Georgé, Brat, Alter, & Amiot, 2005). The TPCs
in garlic at different thermal processing steps are shown in
Fig. 1. The average TPCs were 105.73, 412.36, 509.87, 696.66,
919.88, and 982.14 mg GAE/kg for the FG, BG1, BG2, BG3,
BG4, and BG5 samples, respectively. The TPC in the garlic
cloves subjected to different thermal processing steps was
significantly (P < 0.05) higher than that in fresh garlic. The
TPC was increased by about 4–10-fold in the black garlic
cloves compared with the fresh garlic. These results are con-
sistent with those obtained by Choi et al. (2008), who reported
that the concentration of the free polyphenolic and flavonoid
compounds in heated garlic was significantly higher than
those in fresh and steamed garlic. Guihua, Xingquian, Jiachu,
and Donghong (2007) also found that the heating process im-
prove phenolic content due to the cleaving of bound form (i.e.,
esterified and glycosylated), thus leading to the increase of
free forms. In addition, another probable reason for an in-
crease of the phenolic content in the heated sample is the de-
crease/inhibition of enzymatic oxidation involving the
antioxidant compounds in the raw plant material (Dewanto,
Wu, Adam, & Liu, 2002; Nicoli, Anes, Parpinel, & Franceschi,
1999). An increase in the TPC could be due to an increase in
the levels of complex polyphenols from the later phase of
the browning reaction as suggested by Robards, Prenzler,
Tucker, Swatsitang, and Glover (1999).
3.2. Total flavonoid content (TFC) in garlic at different
thermal processing steps
Flavonoids are complex structures belong to the polyphenol
distribution and have many functions in plants; these are
defensive compounds used against insects and several patho-
gens and are ideal natural antioxidants (Franco, Sineiro, &
Núñez, 2007). However, although most antioxidant activities
 JOURNAL OF FUNCTIONAL FOODS
Fig. 1 – The total phenolic content in garlic at different thermal processing steps. All values were expressed as mg gallic acid
equivalent (GAE) per kg dry matter of garlic sample.
from plant sources are derived from phenolic-type com-
pounds (Chandrasekara & Shahidi, 2011; John & Shahidi,
2010), these effects do not always correlate with the presence
of large quantities of phenolics. Therefore, both sets of data
need to be examined to understand the total phenolic and fla-
vonoid contents.
The TFCs in garlic at different thermal processing steps are
shown in Fig. 2. Consistent with the results noted for the TPC,
the TFC varied significantly (P < 0.05) at each different process-
ing step. The average TFCs were 595.38, 646.99, 673.82, 741.95,
834.16, and 869.94 mg GAE/kg for the FG, BG1, BG2, BG3, BG4,
and BG5 samples, respectively. The TFC of garlic subjected to
different processing steps was significantly (P < 0.05) higher
than that of fresh garlic. The TFC was increased by about
1.1–1.5-fold in the black garlic compared with fresh garlic. In-
deed, the BG1, BG2, BG3, and BG5 samples exhibited levels of
TPCs that were higher than the TFC, while the TPCs in the
Fig. 2 – The total flavonoid content in garlic at different thermal processing steps. All values were expressed as mg quercetin
equivalent (QE) per kg dry matter of garlic sample.
5 (2 0 13 ) 8 0–86 83
FG and BG4 samples were higher than the TFCs. These results
were consistent with those obtained by Scalzo, Iannoccari,
Summa, Morelli, and Rapisarda (2004), who reported that ther-
mal treatment generally induced an increase in the main phe-
nolic substances of orange juice, such as the anthocyanins
and total cinnamates. Choi, Lee, Chun, Lee, and Lee (2006) sug-
gested that bound polyphenolic and flavonoid compounds
could be liberated by heat treatment. Kim et al. (2006) also re-
ported an increase in the TPC in heated grape seeds due to the
liberated phenolic compounds.
3.3. Change in the phenolic acid contents in garlic at
different thermal processing steps
Chemically, phenolic compounds (phenolics) can be defined
as substances with an aromatic ring bearing one or more hy-
droxy substituents, including their functional derivatives
84 JOURNAL OF FUNCTIONAL FOODS 5 ( 2 0 1 3 ) 8 0 –8 6

(Shahidi & Naczk, 1995, Chapter 5; Strack, 1997). Because of
the diversity and complexity of the natural mixtures of phe-
nolic compounds in hundreds of herb extracts, it is rather dif-
ficult to characterize every compound and to elucidate their
structures, but it is not difficult to identify the major groups
and important types of phenolic compounds. Many medicinal
herbs and spices have been studied and to some extent and
their phenolic chemistry is known (Cai, Luo, Sun, & Corke,
2004). HPLC methods for most phenolic compounds in plants
have been developed (Bouchet, Lévesque, & Pousset, 2000;
Sakakibara, Honda, Nakagawa, Ashida, & Kanazawa, 2003).
In particular, a library of the analytical characteristics of phe-
nolic profile established by Rauter et al. (2012) and Lin et al.
(2012) could provide important reference data (retention time,
UV and visible kmax, spectra shapes). The phenolic acid con-
stituents of thermally processed garlic quantified by HPLC
by using nine standard phenolic acids are shown in Table 1.
The average total phenolic acid contents were 17.86 ± 0.07,
83.76 ± 0.17, 98.62 ± 0.26, 139.02 ± 0.17, 102.74 ± 0.25, and
88.52 ± 0.16 mg/kg for the FG, BG1, BG2, BG3, BG4, and BG5
samples, respectively. The total phenolic acid content was in-
creased by about 4.6–7.8-fold in the black garlic compared
with that in fresh garlic. Moreover, hydroxycinnamic acid
derivatives were found to be the major phenolic acids in garlic
at different thermal processing steps. The sum of the six
hydroxycinnamic acids was the highest in the BG3 sample
(87.08 mg/kg, based on dry matter). The total phenolic acid
content of the BG3 sample was significantly higher than that
in the other sample (P < 0.05). According to Bunea et al. (2008),
the increased concentrations of the phenolic acids following
thermal treatment may be explained either by their superior
stability compared with other phenolics, or because they are
released better from the matrix as a result of the breakdown
of supramolecular structures containing the phenolic groups.
The results above indicate that phenolic acid constituents
were distributed in various amounts for each phenolic acid
by thermal processing. The variability of phenolic acid
constituents among garlic samples at different thermal pro-
cessing steps could be caused by the different biochemical
mechanisms in their synthesis during thermal processing.
Therefore, studies should be performed in future to clarify
the biochemical mechanisms in phenolic acid synthesis dur-
ing thermal processing.
3.4. Changes in the flavonoid contents in garlic at
different thermal processing steps
The flavonoid constituents of garlic at different thermal pro-
cessing steps that were quantified by HPLC using 11 standard
flavonoids are shown in Table 2. The total flavonoid content
was increased by about 1.1–3.5-fold in the black garlic com-
pared with the fresh garlic. The decreasing order of the total
flavonoid content per sample was BG4 > BG5 > BG3 > BG2 >
BG1 > FG. Among the four major subgroups of flavonoids in
garlic, the flavanol content was found at the highest concen-
tration followed by the flavanones and flavones, except in the
FG sample. The flavones were found at the highest concentra-
tions in the FG sample. Kaempferol and naringenin were not
detected in any of the samples. Miean and Mohamed (2001)
found relatively high concentrations of the myricetin, querce-
tin, and apigenin flavonoids in garlic. Gorinstein et al. (2008)
found only quercetin and kaempferol in onions and garlic.
In contrast, none of these flavonoids were found by Sultana
and Anwar (2008). Moreover, the difference between flavonoid
types following thermal treatment may be attributed to vari-
ations within each flavonoid because of the variation in the
number and arrangement of the hydroxyl groups, the most
commonly occurring being those with dihydroxylation in
the 3 0 and 4 0 positions (Rice-Evans et al., 1996). The flavonoids,
especially the flavan-3-ols catechin, epicatechin, gallocate-
chin, and epigallocatechin, are more thermostable (Bravo,
1998). In addition, studies have shown that thermally pro-
cessed foods, especially fruits and vegetables, exhibited high-
er biological activities because of various chemical changes

Table 1 – The phenolic acid constituents in garlic at different thermal processing steps.
Concentrations (mg/kg of garlic sample, dry matter basis)
FG BG1 BG2 BG3 BG4 BG5

Hydroxybenzoic acid derivatives (HBA)

p-Hydroxybenzoic acid – – – – – –
Gallic acid 2.06 ± 0.09f 27.36 ± 0.36c 35.09 ± 0.99b 45.53 ± 0.35a 23.50 ± 0.34d 18.65 ± 0.17e
Vanillic acid – 4.44 ± 0.04c 4.55 ± 0.05c 6.41 ± 0.17a 5.62 ± 0.09b 3.56 ± 0.29d

Hydroxycinnamic acid derivatives (HCA)

Chlorogenic acid – – 4.60 ± 0.05d 12.22 ± 0.12b 13.40 ± 0.15a 4.81 ± 0.20c
Caffeic acid 7.48 ± 0.23d 12.21 ± 0.17a 9.51 ± 0.26b 8.79 ± 0.13c 4.43 ± 0.30e 4.40 ± 0.11e
p-Coumaric acid 1.25 ± 0.03f 11.10 ± 0.16e 16.77 ± 0.11d 25.44 ± 0.10c 32.73 ± 0.34a 29.51 ± 0.15b
Ferulic acid 1.57 ± 0.00e 11.35 ± 0.20b 11.44 ± 0.08b 19.52 ± 0.13a 4.47 ± 0.26c 3.47 ± 0.06d
m-Coumaric acid 4.84 ± 0.04f 13.99 ± 0.15a 9.12 ± 0.24c 6.67 ± 0.20e 8.86 ± 0.07d 10.83 ± 0.10b
o-Coumaric acid 0.66 ± 0.03f 3.31 ± 0.08e 7.54 ± 0.32d 14.44 ± 0.17a 9.73 ± 0.42c 13.29 ± 0.16b
Total HBA 2.06 ± 0.09f 31.8 ± 0.20c 39.64 ± 0.52b 51.94 ± 0.26a 29.12 ± 0.22d 22.21 ± 0.23e
Total HCA 15.80 ± 0.08f 51.96 ± 0.15e 58.98 ± 0.18d 87.08 ± 0.14a 73.62 ± 0.26b 66.31 ± 0.13c
Total phenolic acids 17.86 ± 0.07f 83.76 ± 0.17e 98.62 ± 0.26c 139.02 ± 0.17a 102.74 ± 0.25b 88.52 ± 0.16d
Values are mean ± SD (n = 5); –: not detected (limit of detection: 0.5 mg/kg).
FG, raw garlic cloves; BG1, black garlic cloves at Step 1; BG2, black garlic cloves at Step 2; BG3, black garlic cloves at Step 3; BG4, black garlic
cloves at Step 4; BG5, black garlic cloves at step 5.
a–f Different superscripts within a row indicate significant difference at P < 0.05 level.
 JOURNAL OF FUNCTIONAL FOODS 5 (2 0 13 ) 8 0–86 85

Table 2 – The flavonoid constituents in garlic at different thermal processing steps.

Concentrations (mg/kg of garlic sample, dry matter basis)
FG BG1 BG2 BG3 BG4 BG5

Flavanols
Catechin 9.26 ± 0.24e 10.34 ± 0.17c 10.49 ± 0.16c 9.70 ± 0.12d 14.41 ± 0.33b 17.51 ± 0.33a
Epicatechin 2.14 ± 0.13f 14.31 ± 0.51d 19.06 ± 0.37c 22.42 ± 0.37b 38.32 ± 1.28a 10.51 ± 0.11e
Epigallocatechin gallate 1.03 ± 0.10d,e 2.12 ± 0.06d 0.61 ± 0.01e 9.21 ± 0.23c 23.12 ± 1.42a 17.71 ± 1.56b

Flavones
Quercitrin 14.52 ± 0.23a 1.22 ± 0.17e 3.11 ± 0.17c 2.29 ± 0.14d 3.31 ± 0.29c 9.89 ± 0.48b
Apigenin – – – – – –

Flavonols
Myricetin 1.06 ± 0.04f 1.46 ± 0.10e 2.00 ± 0.08c 1.63 ± 0.03d 6.19 ± 0.04a 3.19 ± 0.14b
Resveratrol 0.61 ± 0.06c – 0.70 ± 0.06c – 6.42 ± 0.22a 1.42 ± 0.03b
Morin 1.33 ± 0.06e – 2.28 ± 0.14d 4.81 ± 0.42c 4.62 ± 0.38a 7.74 ± 0.44b
Quercetin – 2.54 ± 0.06c 1.70 ± 0.09e 4.71 ± 0.16b 7.31 ± 0.18a 2.13 ± 0.08d
Kaempferol – – – – – –

Flavanones
Naringenin – – – – – –
Total flavanols 12.43 ± 0.16f 26.77 ± 0.25e 30.16 ± 0.18d 41.33 ± 0.24c 75.85 ± 1.01a 45.73 ± 0.67b
Total flavones 14.52 ± 0.23a 1.22 ± 0.17e 3.11 ± 0.17c 2.29 ± 0.14d 3.31 ± 0.29c 9.89 ± 0.48b
Total flavonols 3.01 ± 0.05f 4.01 ± 0.08e 6.68 ± 0.09d 11.15 ± 0.20c 24.54 ± 0.21a 14.48 ± 0.17b
Total flavanones – – – – – –
Total flavonoids 29.95 ± 0.12f 31.99 ± 0.18e 39.95 ± 0.14d 54.77 ± 0.21c 103.70 ± 0.52a 70.10 ± 0.40b
Values are mean ± SD (n = 5); –: not detected (limit of detection: 0.5 mg/kg).
FG, raw garlic cloves; BG1, black garlic cloves at Step 1; BG2, black garlic cloves at Step 2; BG3, black garlic cloves at Step 3; BG4, black garlic
cloves at Step 4; BG5, black garlic cloves at Step 5.
a–f Different superscripts within a row indicate significant difference at P < 0.05 level.

that occur during heat treatment (Dewanto et al., 2002; Kim
et al., 2000). In general, the processing of vegetables resulted
in the breakdown of the cellulose structure of the plant cell
and thus improved the availability of bioactive compounds
(Van het Hof et al., 2000).
4. Conclusion
Acknowledgments
This research was supported by Basic Science Research Pro-
gram through the National Research Foundation of Korea
(NRF) funded by the Ministry of Education, Science and Tech-
nology (2011-0014738).

This study has provided information on the phenolic acid and R E F E R E N C E S

the flavonoid constituents of garlic subjected to different
thermal processing steps. The results of the present investi-
gations showed that the thermal processing affected the total
phenolic and flavonoid contents of the garlic. Moreover, this
study also showed the variable quantities for each phenolic
acid and flavonoid component. The TPC and TFC of the garlic
subjected to different thermal processing steps were higher
than those of fresh garlic. In particular, the BG1, BG2, BG3,
and BG5 samples exhibited levels of TPC that were higher
than the TFC, while the TFC in the FG and BG4 samples were
higher than the TPCs. The hydroxycinnamic acid derivatives
were found to be the major phenolic acids of garlic at the dif-
ferent processing steps. Among the four major flavonoid sub-
groups in garlic, the flavanols were found at the highest
concentrations followed by the flavanones and flavones, ex-
cept in the FG sample. Therefore, this study could be useful
for understanding the variability of the phenolic acid and fla-
vonoid constituents during thermal processing and might be
extended to investigate the biochemical mechanisms under-
lying the synthesis of phenolic acid and the flavonoids.
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