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ORIGINAL PAPER
Effects of roasting on phenolics composition and antioxidant
activity of peanut (Arachis hypogaea L.) kernel flour
Mar Mar Win •Azizah Abdul-Hamid •
Badlishah Sham Baharin •Farooq Anwar •
Nazamid Saari
Received: 4 January 2011 / Revised: 13 July 2011 / Accepted: 16 July 2011 / Published online: 9 August 2011
ÓSpringer-Verlag 2011
Abstract The effects of roasting on the phenolics com-
position and antioxidant activity of peanut (Arachis hypo-
gaea L.) kernel flour were appraised. Peanut kernel flour,
with and without skin, were roasted at 160 °C for 10, 20,
30, 40 and 50 min. The resultant changes in the antioxidant
activity of roasted peanut kernel flour were assessed by the
determinations of total phenolics, 1,1-diphenyl-2-picryl-
hydrazyl free radical-scavenging capacity, percent inhibi-
tion of linoleic acid oxidation and thiobarbituric acid test
and compared with those of unroasted kernel flour. It was
observed that roasting significantly (p\0.05) increased
the antioxidant activity of the peanut kernel flour. HPLC
analysis revealed the detection of three phenolic acids
(p-hydroxybenzoic, chlorogenic, p-coumaric), two flavo-
nols (quercetin, kaempferol), and a stilbene (resveratrol)
both in the roasted and unroasted samples. In peanut kernel
flour without skin, the contents of the phenolics increased
in the initial roasting phase, however, decreased gradually
in the later phase ([20 min of roasting time). In contrast,
over the course of heating, the amounts of phenolics were
noted to be slightly increased in the peanut kernel flour
with skin; the most significant (p\0.05) increase occurred
in the concentration of p-coumaric acid and quercetin at 30,
40, and 50 min of roasting. The results of this study reveal
that optimum roasting time should be sought to enhancing
the antioxidant capacity and phenolics concentration in
peanut kernel flour.
Keywords Peanut kernel Roasting time TPC
Colorimetric antioxidant assays Phenolics acids
Flavonols
Introduction
Peanut (Arachis hypogaea L.) is one of the major oilseed
crops cultivated in many regions across the world. It is
valued as an important food protein source in the devel-
oping and developed countries. Peanuts are not only used
as a source of edible oil but are also consumed directly or
incorporated into different foods such as snacks and con-
fectionary products for nutrition purposes. Epidemiological
studies suggest that consumption of peanut and peanut
based-products can protect against the incidence of coro-
nary heart diseases (CHD) by decreasing low-density
lipoprotein (LDL) cholesterol and reducing the risk of
development of type II diabetes as well as controlling
weight gain [1,2]. Nearly, 80% of the fat in peanut is in the
form of monounsaturates (oleic acid) that may help to
lowering LDL cholesterol level in blood. Oleic acid may
also play a key role in food-derived hormonal interaction in
the intestine, which boosts satiety between meals by
M. M. Win A. Abdul-Hamid (&)B. S. Baharin
F. Anwar N. Saari
Department of Food Science, Faculty of Food Science
and Technology, Universiti Putra Malaysia,
UPM 43400, Serdang, Selangor, Malaysia
e-mail: azizah@food.upm.edu.my
M. M. Win
e-mail: marwin807@gmail.com
B. S. Baharin
e-mail: badli@putra.upm.edu.my
F. Anwar
e-mail: fqanwar@yahoo.com
N. Saari
e-mail: nazamid@putra.upm.edu.my
F. Anwar
Department of Chemistry and Biochemistry, University of
Agriculture Faisalabad, Faisalabad 38040, Pakistan
123
Eur Food Res Technol (2011) 233:599–608
DOI 10.1007/s00217-011-1544-3
prolonging the feeling of fullness, therefore, contributing to
weight management [3,4].
Peanut kernels are typically considered as a good source
of antioxidant components and phytosterols and contain
about 50% lipid, 25% protein, and 16% carbohydrate
making them a nutritious alternative to meat products. A
number of phenolics such as hydroxybenzoic acid, ferulic
acid, coumaric acid, resveratrol, flavonoids (catechin and
procyanidins), and flavonols (quercetin and kaempferol)
have been identified in peanuts kernels along with con-
siderable amount of total tocopherols (80–140 mg/kg). In
addition to its desirable fatty acids profile, the purported
health benefits associated with consumption of peanut
kernel are mainly attributed to these bioactive compounds
[5,6]. A dry powder (peanut flour), obtained after partial
extraction of oil, from the roasted peanut kernel, is com-
mercially used as additive to increase the protein content of
various food commodities including baked goods, sauces,
dressing, etc. Some recent studies also report the rheolog-
ical, foaming, emulsifying, and water holding properties of
peanut flour [7,8]. Peanut skin (testa or seed coat),
although a potential source of plophenols, especially pro-
anthocyanidins and condensed tannins, is sometime con-
sumed along with the peanut-derived foods; however, it is
mostly discarded as an agro-waste during peanut process-
ing [9,10].
Roasting is an important step in peanut processing
industry as it is used to enhance the flavor, color, texture,
and overall palatability of the end-user products. Mature
peanut kernels are commonly dry roasted at 160 °C for
20–30 min to prepare roasted, salted peanuts [11]. How-
ever, actual roasting intensities may depend upon the
required characteristic, flavor, and applications. During the
process of roasting, some chemical changes may occur in
which sugars can condense with free amino acids, peptides,
or proteins leading to the formation of brown Maillard
reaction products with potential antioxidant activity [12].
In addition to free forms, plants also contain considerable
amounts of bound form antioxidant phenolics, it is there-
fore perceived that some processing methods might be
employed to break these covalently bounded polymeric
compounds to liberate into free forms so as to enhancing
their antioxidant capacity [13]. For instance, previously, it
has been reported that heat treatment liberated the low-
molecular weight compounds into their free forms and
hence increased the antioxidant capacity of peanut skin,
hulls, and kernels [10,14,15].
As far as we know, there have been no earlier reports yet
available studying the influence of roasting times on the
phenolics composition and antioxidant activity of peanut
kernel flour, with and without skin. Such investigations are
important to devise an optimum roasting time offering
peanut products with better antioxidant attributes. The main
objective of this research, therefore, was to elucidate the
effect of different roasting times on the phenolics compo-
sition and antioxidant attributes of two different forms of
peanut kernel flour (with and without skin) leading to
exploring their potential uses for functional foods.
Materials and methods
Reagents
Trifluoroacetic acid (TFA), standards of phenolic acids
(p-hydroxybenzoic acid, chlorogenic acid, p-coumaric
acid, ferulic acid, and gallic acid), flavonoids (epicatechin,
dihydroquercetin, luteolin, and kaempferol) stilbene (trans-
resveratrol), butylated hydroxyanisole (BHA), and
a-Tocopherol, 2,2-Diphenyl-1-picryhydrzyl radical (DPPH)
were from Sigma Chemical Company (St. Louis, MO, USA)
and ferrous chloride, 2,4,6-tripyridyl-s-triazine, linoleic acid,
thiobarbituric acid, ferric chloride, and tween 20 were
obtained from Fisher Scientific (Ottawa, ON, Canada). All
other reagents used were of analytical grade from Fisher
Scientific.
Sample preparation and roasting
Raw, peanut pods (Virginia spreading type variety) were
obtained from an Agricultural Farm in Nay-Pyi-Taw
Township, Myanmar. Three different peanut raw samples,
harvested from different agriculture plots, were assayed.
The selection of the present peanut variety was based upon
its high yield and productivity and popularity among local
consumers. The pods (3 kg) were manually dehulled and
the kernels recovered roasted in an electric oven (Memm-
ert, UL40, Germany) equipped with an air circulation
system. The roasting temperature was set at 160 °C fol-
lowing the method of Damame et al. [11] and maintained
for 2 h to reach equilibrium before used. The roasting times
were increased gradually from 10 to 20, 30, 40, and
50 min. After cooling to room temperature, the skin was
removed from an appropriate amount/batch of the kernels,
producing kernels with and without skin. Triplicate treat-
ments were applied for roasting process. The roasted pea-
nut kernels with and without skin were ground separately
using a commercial grinder (Pensonic, Malaysia) and then
sieved to get fine power (peanut flour). Peanut flour (10 g)
was defatted with n-hexane (100 mL) using a water bath
shaker for 8 h at 45 °C. After that, the defatted samples
were ambient dried, packed in amber bottles, and preserved
at -20 °C until used for antioxidants extraction and HPLC
analysis. Control samples (unroasted samples) of peanut
flour with and without skins were also defatted and kept at
the same conditions.
600 Eur Food Res Technol (2011) 233:599–608
123
Extraction of antioxidant components
Extraction of antioxidant components was carried out
according to method of Chukwumah et al. [16] with slight
modifications. Each of the defatted ground kernel sample
(10 g) was extracted with pure methanol (100 mL) using a
water bath shaker for 2 h at room temperature (28 °C). The
suspension was filtered through a Whatman No. 1 filter
paper, and the residue obtained was re-extracted twice,
with additional 100 mL of methanol. The filtrates were
pooled and the solvent distilled off under reduced pressure
using a rotary evaporator at 40 °C. The crude concentrated
extracts were used for the following antioxidant activity-
related experiments.
Total phenolic contents
The amounts of total phenolics in the roasted and unroasted
peanut kernel flour with and without skin were determined
using Folin–Ciocalteau procedure as described by [17].
Methanol extract (0.4 mL) of each sample (1 mg/mL
concentration) was mixed with 2 mL of the Folin–Ciocal-
teau reagent. After 5 min, 1.6 mL of sodium carbonate
solution (75%) was added to maintain basic condition for
the reaction to occur between phenolic components and
Foiln reagent. The reaction mixture was mixed well using
vortex machine and then incubated for 30 min at room
temperature (28 ±1°C) in the darkness. The absorbance
of the reaction mixture was then noted at 750 nm using a
UV–visible spectrophotometer (Shimadzu, Japan). The
standard calibration curve was prepared using gallic acid
standard solutions of known concentrations (0.02, 0.04,
0.06, 0.08, 0.10, and 0.20 mg/mL), and the amounts of
total phenolics were calculated as gallic acid equivalent
mg/g of dry sample (mg GAE/g dry sample).
Free radical-scavenging capacity (DPPH)
Antioxidant activity of methanol extracts from roasted and
unroasted peanut kernel flour samples was measured using
2,2-diphenyl-1-picrylhdrazyl (DPPH) according to the
method as described by Brand-Willian et al. [18], with
minor modifications. The extracts (0.5 mL) of varying
concentration were mixed with 2.5 mL of freshly prepared
DPPH solution (25 mg/L). The solution was then incubated
in the darkness at room temperature for 30 min, and the
decrease in absorbance was noted at 515 nm using a UV–
visible Spectrophotometer (Shimadzu, Japan). For blank,
pure methanol was used in place of the sample. For posi-
tive controls, 0.1 mg/mL of each a-tocopherol and BHA
(reference antioxidants) were employed. The percent
DPPH free radical scavenged by each sample extract was
calculated by the following equation:
%DPPH ¼A0A1
A0100
A
0
absorbance of DPPH solution without sample/standard
solution, and A
1
absorbance of the sample/standard solu-
tion at 30 min reaction.
Inhibition of linoleic acid peroxidation
Methanol extracts of roasted and unroasted peanut kernel
flour were tested for their antioxidant activity by measuring
the inhibition of linoleic acid peroxidation following a
previously reported method of Yen and Hsieh [19]. Test
sample 0.5 mL (1 mg/mL in absolute ethanol) was mixed
with 2.5 mL of linoleic acid emulsion (0.02 M, pH 7.0) in
phosphate buffer. This emulsion was prepared by mixing
and homogenizing 0.280 g of linoleic acid, 0.280 g of
tween 20 as emulsifier, and 50 mL of phosphate buffer
solution. The resulting reaction mixture was subjected to
incubation at 37 °C for 96 h. The magnitude of linoleic
acid oxidation was determined by the peroxide value fol-
lowing a colorimetric method as described by Yen et al.
[20]. Briefly, to 0.1 mL sample solution, 4.7 mL of ethanol
(75%), 0.1 mL of ammonium thiocyanate (30%), and fer-
rous chloride (0.1 mL, 0.02 M in 3.5% HCl) were added
sequentially. After 3 min of reaction, the absorbance of the
resulting reaction mixture was read at 500 nm using a
spectrophotometer (Shimadzu, Japan). The degree of oxi-
dation was measured after every 24 h until a day after the
absorbance of the control reached its maximum. A control
contained all reagents without the sample extract was
prepared simultaneously. Besides, BHA, a-tocopherol, and
quercetin were used as positive controls (1 mg/mL each
compound). The percent inhibition of linoleic acid oxida-
tion was calculated as the following equation:
Inhibition of linoleic acid oxidation %ðÞ¼
1Absorbance at 500 nm in the presence of sample 96 h
Absorbance at 500 nm in the absence of sample 96 h
100
Thiobarbituric acid method
The antioxidant activity of the extracts from roasted and
unroasted peanut kernel flour was also measured using thio-
barbituric acid (TBA) method as described by Ottolenghi [21]
and Kikuzakiand Nakatani [22]. The same reaction mixture as
employed for measurement of percent inhibition of linoleic
acid oxidation test at the final day of the experiment was used
for TBA test. Briefly, to 1 mL of assay mixture, 2 mL of 20%
trichloroacetic acid (TCA) solution and 2 mL of TBA solution
were added. The reaction mixture was then incubated in a
boiling water bath for 10 min. After cooling to room
Eur Food Res Technol (2011) 233:599–608 601
123
temperature, the solution was centrifuged at 9009gfor
20 min, and the absorbance of the supernatant was measured
at 532 nm using a spectrophotometer (Shimadzu, Japan).
HPLC analysis of phenolic compounds
The analysis of selected phenolic acids (p-hydroxybenzoic,
caffeic, chlorogenic, p-coumaric, and ferulic), flavonoids
(epicatechin, quercetin, kaempferol, and luteolin), and
stilbene (trans-resveratrol) was based on the fact they are
commonly found in peanuts. The analysis was performed
by HPLC on the acid-hydrolyzed samples according to the
method as described by Wang et al. [23], with slight
modifications. Briefly, roasted and unroasted kernel flour
(0.5 g) was mixed with 10 mL of 80% methanol and 1 mL
of 1.2 M hydrochloric acid and hydrolyzed by incubation
in a water bath at 80 °C for 2 h. After hydrolysis, it was
cooled at room temperature and centrifuged at 1,2009gfor
5 min. The supernatant was filtered using a 0.45 lm nylon
membrane filter, prior to an HPLC analysis.
A Waters HPLC system equipped with Waters 2487 Dual
Wavelength Absorbance Detector, Waters 600 Pump and
controlled by Waters Empower2 software (Waters, Milford,
MA) was used. The separation of the phenolics was carried
out on Waters reverse-phase (RP) Symmetry C
18
column
(150 93.9 mm, 5 lm) operated at room temperature. The
mobile phase consisted of TFA in deionized water (pH 2.5)
as solvent A and absolute methanol (99.99%) as solvent B.
The gradient conditions used were as follows: 100–50%
solvent A (0–20 min), 50–40% solvent A (20–30 min), and
40–100% solvent A (30–40 min). The mobile phase flow
rate was set at 1.0 mL/min, and a 20 lL sample volume was
injected. The detection of the phenolic was monitored at
280 nm because this wavelength is near the maximum
absorbance wavelength for hydroxybenzoic acids
(270–280 nm), hydroxycinnamic acids (290–300 nm), and
some flavonoids (250–270 nm, 330–350 nm) [24]. The
phenolic standard solutions were prepared by dissolving
respective pure compounds in absolute methanol at various
concentrations (0.005, 0.01, 0.02, 0.05, 0.1, and 0.2 mg/mL)
and injected into the HPLC system to construct the cali-
bration curves for each standard compound. Identification of
the unknown phenolics was based on matching their reten-
tion times with those of pure standards of phenolics. Peak
areas, based on external standard calibration curves, were
used for quantification purposes. The amounts were
expressed as lg/g defatted sample.
Statistical analysis
Three different peanut samples were analyzed individually
in triplicate. All experiments were performed in triplicates.
The data were expressed as means ±standard deviations
of triplicate determinations (n=393). One-way analysis
of variance (ANOVA) and Duncan’s multiple range test
(DMRT) were carried out to assess the significance of the
differences between means (p\0.05) using SAS System
for Windows Version 7 (SAS Institute Inc., Cary, NC,
USA).
Results and discussions
Total phenolic contents
It is well known that there is a positive association between
consumption of plant foods rich in phenolic antioxidants
and health. Thermal processing is reported to significantly
alter the phytochemical and functional composition in
legumes including peanuts [16,25]. Chukwumah et al. [16]
investigated the effects of different processing techniques
such as boiling, oil-, and dry-roasting on the phenolics
composition and antioxidant activity of peanuts. In the
present study, we investigated the effects of different
roasting times on the total phenolic contents (TPC) of
peanut kernel flour with and without skin. Results of the
study showed that TPC of peanut kernel flour with and
without skin increased significantly (p\0.05) as function
of roasting times (Fig. 1). The amount of TP in the roasted
peanut kernel without skin significantly (p\0.05)
increased from 0.94 mg GAE/g in unroasted control to
1.61, 1.83, 2.16, and 2.04 mg GAE/g in the samples
roasted for 20, 30, 40, and 50 min at 160 °C, respectively.
However, TPC of roasted sample without skin were almost
unaffected during the initial stage (10 min) of roasting. In
agreement with our present finding, some previous studies
also revealed that initial stage of heating or roasting
Fig. 1 Effect of roasting times on total phenolic contents (TPC)of
peanut kernel flour with and without skin. Values with same small
letters (a,b,c) are not significantly different (p\0.05), among
different roasting times. Values with same capital letters (A,B) are not
significantly different (p\0.05) between the samples
602 Eur Food Res Technol (2011) 233:599–608
123
process did not exert significant effect on the TPC of
buckwheat and apricot kernel [26,27].
For peanut kernel flour with skin, TPC increased sig-
nificantly (p\0.05) as roasting times increased and
maximum values, 4.08 and 3.86 mg GAE/g, were obtained
for 40 and 50 min roasted samples, respectively. It can be
seen that peanut kernel flour with skin contained signifi-
cantly higher amount of total phenolics than peanut flour
without skin. A higher phenolic content in peanut kernel
flour with skin might be ascribed to the presence of certain
phenolics such as proanthocyanidins (condensed tannins)
in the skin. According to Karcheshy and Hemingway [28],
peanut skin was found to be a rich source of proanthocy-
anidins that contributed 17% by weight of the skin. Of the
identified proanthocyanidins, about 50% were low-molec-
ular weight oligomers [28].
It can be expected that bound form phenolics with larger
molecular weight, both in peanut kernel flour with and
without skin, might have been liberated into simple free
forms by heat treatment leading to enhancing over all TPC
of the samples. Several studies reported that heat treatment
is effective toward increasing the total phenolic content in
different foods such as dry beans [29], carob powder [30],
vegetables [31], and grape seeds [32]. Boateng et al. [29]
explained that disruption of the cell wall through heating or
by the breakdown of insoluble phenolic compounds as
function of thermal treatments could lead to better
extractability of phenolic compounds in dry beans. How-
ever, Lee et al. [14] revealed that simple heat treatment did
not liberate covalently bound phenolic compounds from
rice hull while far-infrared treatment broke the esterified
phenolic bonds. This indicates that an effective processing
method for liberation of such bound plant phenolics into
simpler forms may differ from species to species [33]. In
addition, the increase in total phenolics of peanut kernel
flour in this study may also be linked to the development of
Maillard reaction products that are reported to be formed
during roasting process. Yu et al. [10] investigated that
Maillard reaction products might lead to increase the
amounts of total phenolics or phenolic-like complexes that
further contribute to higher absorbance readings measured
by Folin assay.
DPPH free radical-scavenging activity
The antioxidant activity of methanol extracts from roasted
peanut kernel flour with and without skin, supported with
two positive controls, i.e., BHA and a-tocopherol, was
determined by measuring their DPPH
radical-scavenging
capacity (Fig. 2). DPPH is a stable free radical that has
ability to accept an electron or hydrogen from antioxidant
compounds and is then converted to a DPPH stable mol-
ecule. The loss of DPPH radical is examined by the
decrease in the magnitude of absorbance of the antioxidant
solution at 515 nm. In the present experiment, DPPH free
radical-scavenging activity of the extracts from peanut
kernel flour without skin linearly increased as function of
roasting times. The extracts, from 20, 30, 40, and 50-min
roasted samples of peanut flour without skin, showed sig-
nificantly (p\0.05) higher DPPH radical-scavenging
activity (68.93, 71.70, 77.26, and 79.57%, respectively)
than 10-min roasted (51.22%) and unroasted samples
(48.09%). The roasting time was found to be a critical
factor to determine the overall antioxidant activity of the
peanut kernel flour without skin. The highest antioxidant
activity, in terms of DPPH free radical-scavenging capac-
ity, was exhibited between 20 and 50 min of roasting. On
the other hand, the peanut kernel flour with skin, when
roasted for 10–50 min, also showed good scavenging
activity (86.96–88.61%), relative to that of unroasted
sample (75.15%). The present scavenging ability values of
roasted kernel flour with skin were comparable to that of
positive control a-tocopherol (89.00%).
The potential health benefits of plant phenolics are
mainly due to their free radical-scavenging activities
through donating a hydrogen atom and or an electron from
an aromatic hydroxyl group to free radicals [34]. In the
present study, an enhancement in the radical-scavenging
activity, observed in both the roasted peanut kernel flour
with and without skin, might be attributed to their better
ability to release some bound antioxidant phenolic com-
pounds to act as free radical scavengers, from the cell
matrix upon roasting [35]. On the other hand, other phe-
nomenon may also involve, for example, better solubility
of non-phenolic compounds (such as Maillard reaction
products) following the thermal treatments, which may
further enhance the free radical-scavenging properties of
processed foods [35,36]. Jeong et al. [37] also investigated
Fig. 2 Effect of roasting times on DPPH radical-scavenging activity
of peanut kernel flour with and without skin. Values with same small
letters (a,b,c) are not significantly different (p\0.05), among
different roasting times. Values with same capital letters (A,B) are not
significantly different (p\0.05) between the samples
Eur Food Res Technol (2011) 233:599–608 603
123
that roasting increased the radical-scavenging activity of
sesame meal extracts and a positive linear correlation
between melanoidin content and antioxidant activity of the
roasted coffee brew fractions was established [38]. In our
study, the increase in the radical-scavenging activity of
peanut kernel flour, in relation to increasing roasting times,
is in good agreement with the previous studies on apricot
kernels [27] and okra seed flour [39].
Antioxidant activity in linoleic acid peroxidation
system
The effects of roasting treatments on antioxidant activity of
peanut kernel flour were also assessed by measuring per-
cent inhibition of linoleic acid oxidation using a colori-
metric method (Fig. 3). The peroxyl radicals, formed in the
initiation step of oxidation as result of abstraction of
hydrogen from a fatty acid and subsequent oxygen
involvement, are good oxidizing agents. These peroxyl
radicals oxidize ferric thiocyanate to ferrous thiocyanate
yielding a colored complex, the intensity of which is
measured at 500 nm and can be used as a basis for the
measurement of peroxide level formed during the initial
stage of lipid oxidation [40]. At a concentration of 500 lg
in the final reaction mixture, the extracts from samples of
roasted peanut kernel flour with skin inhibited
63.03–69.33% peroxidation of linoleic acid as against
26.86–56.29% for peanut kernel flour without skin,
wherein, the roasting effects were followed in a time-
dependent manner.
Interestingly, both raw (unroasted) and roasted peanut
kernel flour with skin exhibited higher lipid peroxidation
inhibition activity (65.91 and 63.03–69.33%, respectively),
almost comparable to those of quercetin (65.71%), and
tocopherol (73.05%) but lower than that of BHA (89.30%).
An increase in the peroxidation inhibition magnitude of the
extracts, from peanut kernel flour with skin, might be due
to higher amounts of polyphenols present in peanut skin
that contributed to inhibit the accumulation of oxidative
products. Generally, it is believed that outer layers of seed
such as peel, shell, and hull contain higher amounts of
polyphenolic compounds to protect the seed from oxidative
damage [41]. Similarly, as our present findings, the seed
coat extracts of red and black bean, containing higher
amount of phenolic substances, exhibited a stronger anti-
oxidant activity against lipid peroxidation [42]. Nonethe-
less, the stability of antioxidants of roasted samples might
be due to the formation of Maillard reaction products.
During heat treatment, the formation of the Maillard
reaction products is believed to be responsible for
increasing the overall antioxidant capacity of the foods.
Maillard pathway can produce several antioxidant com-
pounds with strong reducing power such as reductones and
amino-reductone that can inhibit low-density lipoprotein
(LDL) oxidation, and the concentrations of Maillard type
polymers are increased with increasing heat treatment [43].
Antioxidant activity in TBA method
During the oxidation process, peroxides are gradually
decomposed to lower molecular compounds, mainly mal-
ondialdehyde (MDA) [44] and their relative concentrations
are measured by TBA method. Malonaldehydes, one of the
lipid oxidation products can react with free amino group of
proteins, phospholipid, and nucleic acids leading to struc-
tural modification, which induce dysfunction of immune
systems [41]. At low pH and high temperature, MDA binds
TBA to form red complex and the absorbance can be
measured at 532 nm on the final day of the incubation
period (1 day after the control reached maximum absor-
bance in inhibition of linoleic peroxidation assay).
Antioxidant activity (as measured by TBA test, the data
given as absorbance values) of roasted peanut kernel flour
with and without skin is displayed in Fig. 4. In this method,
the lower the absorbance values the higher the antioxidant
activity. Interestingly, also in this assay, the antioxidant
activity of roasted peanut flour without skin was found to
be higher than those of unroasted sample and the control.
However, the present values were found to be significantly
(p\0.05) lower than those of positive controls (quercetin,
tocopherol, and BHA). These results also support that the
Maillard reaction products, probably formed due to roast-
ing of peanut kernel, are able to decrease the lipid oxida-
tion rate revealing antioxidant potential. The effect of
Maillard reaction products on lipid oxidation in preheated
model systems has been studied by Mastrocola and Munari
[45]. In their studies, the simultaneous induction of the
Fig. 3 Effect of roasting times on antioxidant activity of peanut flour
with and without skin as measured by percent inhibition of linoleic
acid peroxidation. Values with same small letters (a,b,c) are not
significantly different (p\0.05), among different roasting times.
Values with same capital letters (A,B) are not significantly different
(p\0.05) between the samples
604 Eur Food Res Technol (2011) 233:599–608
123
Maillard reaction in heated lipid fractions models greatly
affected the development of lipid oxidation by slowing the
reaction and increasing the antioxidant activity of the sys-
tem. Also in this assay, the antioxidant activity of roasted
and unroasted peanut flour with skin was not significantly
(p\0.05) different from those of quercetin and tocopherol
but lower than that of BHA. These results correlated well
with those obtained previously using the linoleic acid
emulsion method. It is well known that phenolic compounds
act as hydrogen donors and reduce the extent of formation
of hydroperoxides and lipid oxidation [46].
Individual phenolics composition measured by HPLC
Based on our preliminary trials, we observed that a number
of complex and unidentifiable peaks appeared in non-
hydrolyzed samples analyzed, which might be due to
detection of bound (esterified form) phenolics in conjunc-
tion with carbohydrates and other related moieties. HPLC
analysis of the hydrolyzed samples is the most recom-
mended and appreciating approach. Therefore, for sim-
plicity and clarity of the analysis, we used the hydrolyzed
samples for detection of target phenolic compounds. The
differences between control samples (non-hydrolyzed) and
hydrolyzed samples referring to polyphenols were consid-
ered during the quantification of phenolics final amounts in
the samples tested. HPLC chromatograms obtained for the
separation of standard phenolic compounds and the phe-
nolic compounds in roasted peanut kernel flour with skin
are given as Figs. 5and 6, respectively.
In the present study, three phenolic acids (p-hydroxy-
benzoic, chlorogenic, and p-coumaric), two flavonols
(quercetin and kaempferol), and stilbene (resveratrol) were
mainly detected in the tested samples as measured by RP-
HPLC. The data generated showing the effects of different
roasting times that affected the individual phenolic com-
pounds of peanut flour, with and without skin, are sum-
marized in Table 1. The results indicated that the contents
of phenolic acids, flavonols, and resveratrol in roasted
peanut kernel flour without skin were gradually increased
up to 20 min of roasting. Thus, in case of peanut kernel
flour without skin, after 20-min roasting, the concentration
of p-hydroxybenzoic acid increased from 133.49
to 146.00 lg/g, chlorogenic acid 32.01–37.16 lg/g and
p-coumaric acid 73.08–81.88 lg/g, respectively. Our
finding are in agreement with the work of Talcott et al. [15]
who reported that roasting increased the concentration of
the predominant antioxidant phenolic acid (p-coumaric
acid) in peanut that might be attributed to heat-catalyzed
hydrolytic reactions of its native esterified or bound forms
liberating into free forms. Naturally, polyphenols occur as
in both free and bound forms. Some processing methods
Fig. 4 Effect of roasting times on antioxidant activity of peanut
kernel flour with and without skin measured by TBA method. Values
with same small letters (a,b,c) are not significantly different
(p\0.05), among different roasting times. Values with same capital
letters (A,B) are not significantly different (p\0.05) between the
samples
Fig. 5 HPLC chromatogram showing the separation of standard
phenolic compounds (1) gallic acid, (2)p-hydroxybenzoic acid, (3)
chlorgenic acid, (4) caffeic acid, (5) epicatechin, (6)p-coumaric acid,
(7) ferulic acid, (8) resveratrol, (9) quercetin, (10) daidzin, (11)
luteolin, and (12) kaempferol
Fig. 6 A typical HPLC chromatogram showing the separation of
phenolic compounds in roasted peanut kernel flour with skin (2)
p-hydroxybenzoic acid, (3) chlorogenic acid, (6)p-coumaric acid, (8)
resveratrol, (9) quercetin, and (12) kaempferol
Eur Food Res Technol (2011) 233:599–608 605
123
such as roasting have been shown to increase the poly-
phenolic content of foods probably due to releasing of
bound form antioxidants into free forms and coupled with
formation of Millard reaction products due to roasting
effects. Dabrowski and Sosulski [47] studied free and hy-
drolysable polyphenols in ten oil seeds including peanuts
wherein esterified forms of p-coumaric, ferulic, and caffeic
acids were detected in defatted peanut flour. It has been
revealed that the degree of roasting influenced the quality
and antioxidant properties of peanut kernels, creating a
complex environment for peanut quality assessment [15].
Flavonols (e.g., quercetin and kaempferol) are one of the
important classes of flavonoids that are secondary metab-
olites in plants and have beneficial effects for human health
because of their antioxidant, antiproliferative, and anti-
carcinogenic properties [23]. Recently, Wang et al. [23]
reported that peanut kernels contained trace amount of
kaempferol and higher amounts of quercetin than other
legumes. The presently determined amount of quercetin
(104.46 lg/g) in unroasted peanut kernel flour without skin
and 121.47 lg/g in unroasted peanut kernel with skin were
found to be lower than those reported by Wang et al. [23];
however, kaempferol content (1.56 and 2.31 lg/g, respec-
tively) was comparable with that of Wang et al. [23]. In
their report, peanut kernels (including skin) contained
higher amount of quercetin (133–289 lg/g) and its con-
centration mainly depended on kernel seed-coat color,
whereas kaempferol was 1.92–4.66 lg/g.
In the present study, the concentration of flavonols in
peanut kernel flour without skin was also affected as result
of roasting. The increase in concentration of quercetin from
104.46 to 133.00 lg/g and kaempferol from 1.56 to
2.68 lg/g as result of 20-min roasting may be linked to the
breakdown of flavonol glycosidic bonds to respective
aglycons under the thermal treatment. Zill-e-Huma et al.
[48] reported a similar phenomenon where, thermal treat-
ment led to the degradation of the quercetin glucosides and
increased the concentration of free quercetin aglycone in
microwave heated onion. It was also established that
roasting caused a slight increase, from 0.11 to 0.13 lg/g, in
the content of resveratrol. In contrary to our result, Sander
et al. [49] reported that roasted Virginia and Spanish pea-
nuts (176 °C) contained less amount of resveratrol than
that of unroasted ones. The differences in data obtained in
our study in comparison to those of Sander et al. [49] may
be probably due to different roasting temperatures
employed (160 °C vs. 176 °C).
On the other hand, in the present analysis, prolong
roasting ([20 min) slowly decreased the contents of
phenolics in peanut kernel flour without skin while a sig-
nificant (p\0.05) decline in the amounts occurred at
50 min roasting. It is understandable that practically up to
certain temperatures, the concentration of phenolic com-
ponents may increase due to improved cell wall rupturing,
or due to other favorable reactions, leading to their higher
recovery into the solvents; however, a prolonged heating
may decrease the concentration of naturally occurring
polyphenolics in food products [50]. A similar study on the
effect of different roasting process on carob powder indi-
cated that phenolic compounds were decreased after
75 min roasting at temperatures of 135, 150, and 165 °C
[31]. The present results indicate that an appropriate
Table 1 Phenolic compounds (lg/g) analyzed by HPLC in roasted peanut kernel flour with and without skin
Samples Phenolic acids Flavonols Stilbene
p-Hydroxybenzoic Chlorogenic p-Coumaric Quercetin Kaempferol Resveratrol
Kernel flour without skin (min)
0 133.49 ±12.91
abA
32.01 ±3.49
aA
73.38 ±2.98
aA
104.46 ±6.27
bB
1.56 ±0.27
bB
0.11 ±0.01
abA
10 141.00 ±1.00
aA
34.00 ±1.00
aA
76.00 ±4.00
aA
110.00 ±3.00
bB
2.60 ±0.50
aA
0.12 ±0.03
abA
20 146.00 ±6.00
aA
37.16 ±2.12
aA
81.88 ±5.54
aA
133.00 ±6.00
aB
2.68 ±0.10
aA
0.13 ±0.00
aA
30 141.50 ±3.50
aA
33.79 ±1.22
aA
75.79 ±4.33
aB
134.41 ±4.86
aB
2.37 ±0.09
aA
0.08 ±0.00
bB
40 132.50 ±7.50
abA
35.27 ±2.25
aB
59.44 ±6.67
bcB
128.50 ±1.50
aB
1.44 ±0.05
bcB
0.08 ±0.00
bB
50 112.80 ±4.80
bB
23.04 ±0.22
bB
51.62 ±3.55
cB
81.56 ±3.44
cB
0.96 ±0.10
cB
0.03 ±0.00
cB
Kernel flour with skin (min)
0 131.30 ±5.99
aA
32.60 ±2.80
bA
61.86 ±6.79
bA
121.47 ±7.71
cA
2.31 ±0.37
abA
0.13 ±0.01
abA
10 136.48 ±0.30
aA
38.15 ±1.5
abA
60.09 ±9.07
bA
136.37 ±3.71
cA
2.70 ±0.27
abA
0.14 ±0.01
abA
20 137.11 ±1.62
aA
34.32 ±1.16
bA
60.09 ±1.17
bB
164.99 ±4.69
bA
2.19 ±0.15
bA
0.14 ±0.01
abA
30 136.47 ±2.39
aA
35.63 ±1.28
abA
93.62 ±6.46
aA
168.70 ±0.83
abA
2.99 ±0.25
aA
0.13 ±0.01
abA
40 143.74 ±5.96
aA
43.17 ±4.11
aA
92.40 ±1.65
aA
178.05 ±1.11
aA
2.85 ±0.04
abA
0.14 ±0.01
abA
50 144.06 ±1.62
aA
44.42 ±3.99
aA
92.83 ±2.62
aA
171.31 ±5.96
aA
2.14 ±0.20
bA
0.16 ±0.00
aA
Values with same small letters (a, b, c) are not significantly (p\0.05) different among different roasting times. Values with same capital letters
(A, B) are not significantly (p\0.05) different between the samples
606 Eur Food Res Technol (2011) 233:599–608
123
roasting time (\20 min) should be employed to enhance
and/or retain optimum amount of phenolic compounds in
peanut flour without skin.
Interestingly, in case of roasted peanut kernel flour with
skin, the concentrations of phenolics, especially p-coumaric
acid and flavonol (quercetin), were linearly increased as
function of roasting times. As evident in Table 1, the ori-
ginal contents of p-coumaric acid (61.86 lg/g) and quer-
cetin (121.47 lg/g) were notably (p\0.05) enhanced after
30, 40, and 50 min roasting to levels as high as 93.62, 92.40,
and 92.83 lg/g and 168.70, 178.05, and 171.31 lg/g,
respectively. Improvement in phenolics contents and anti-
oxidant activity of peanut during roasting can be supported
from the literature. During heating process, compounds
with free amino groups such as lysine can undergo a
sequence of complex reactions with carbonyl compounds to
produce intermediate Maillard reaction products such as
furans and its derivatives like tetrahydrofuran (THF), mel-
anoidins, pyrroles, pyrazines, and other heterocyclic com-
pounds that not only impart color, flavor, and aroma to the
heated end-use products but also contribute to enhance the
antioxidant activity [16,51,52]. Melanoidins with high-
molecular weight are also one of the end products of
Maillard reaction and might have some antioxidant activity
[52]. However, Yanagimoto et al. [53] reported that pyra-
zine formed during roasting of coffee had no antioxidant
activity whereas furans and pyrroles exhibited minor anti-
oxidant activity. As peanut contain considerable amounts of
carbohydrates and amino acids, it is expected that Millard
reaction derived products might have been the major con-
tributor to enhancing the antioxidant attributes of peanut
during roasting. Therefore, it is possible to say that while
peanuts kernel were roasted with their skin intact, these
Maillard reaction products may interact with the mono-
meric and oligomeric proanthocyanidins present in peanut
skin generating some new antioxidant compounds [28,54].
It was reported that various oligomeric bridged com-
pounds were formed from the reaction between cyanidin-3-
O-glucoside and Maillard reaction products such as furfural
compounds that may play a major role in the flavanol
polymerization process [55]. Yu et al. [56] investigated that
proanthocyanidin (trimers and tetramers) content of peanut
skin decreased after roasting whereas its monomers content
increased. Saffan [57] also revealed that heat-stressed
peanut seedlings at 40 °C exhibited significantly
(p\0.05) higher polyphenolic contents (phenolic acids
and flavonoids) than those of the control. The other pos-
sible reason is that proanthocyanidin compounds most
likely condensed tannins, in peanut skin, have degraded
into simpler phenolics during heat treatment leading to
increasing the overall contents of phenolics [58]. Similar
phenomenon was reported by Rakic et al. [59] where, the
contents of gallic acid increased significantly following
thermal treatment of oak acorns, whereas those of hydro-
lysable tannins degraded resulting in an increase of simple
phenolics such as gallic acid. Additionally, the significant
increase in p-coumaric and quercetin contents at 30, 40,
and 50 min roasting times in this work may also be
explained either as a result of the breakdown of the
molecular structures containing phenolic groups or by the
thermal stability of these compounds.
Overall, in this research, the increase in the antioxidant
capacity of roasted peanut flour, with and without skin, can
be likely linked to better release of some antioxidant
phenolics such as phenolic acids, flavonols, and degrada-
tion of tannins to simple phenolics as well as due to the
contribution of Maillard reaction products following
roasting process. Obviously, a mixture of such compounds
might have contributed to enhanced antioxidant activity.
Conclusion
The results of this study revealed that roasting has signif-
icantly affected the antioxidant activity and the phenolic
composition of peanut flour, with and without skin. Inter-
estingly, the antioxidant activity and amounts of phenolic
compounds in peanut kernel flour with skin were appre-
ciably increased even up to 50-min roasting; however, the
longer roasting time ([20 min) resulted in the degradation
of phenolic compounds in peanut flour without skin.
Therefore, we suggest that proper roasting timing should be
taken into account to retain and/or enhance the natural
antioxidant phenolics of peanut flour with or without skin.
Based on the high phenolics concentration and superior
antioxidant capacity, the roasted peanut flour with skin can
be recommended for uses as a potential source of valuable
functional bioactives. Since peanut skin is often treated as
an agro-waste, it can be explored as a cheap and renewable
raw material for isolation of antioxidant compounds for
protecting other lipid containing food products, especially
the vegetable oils as well as an ingredient of functional
foods and nutraceuticals. Furthermore, an in-depth study on
structural elucidation of some novel antioxidants formed
during peanut roasting process is highly recommended.
Acknowledgments The authors are highly thankful to Oil Crop
Development Project (UTF/MYA/006) in Myanmar initiated by the
Ministry of Agriculture and Irrigation, Myanmar and FAO for
financing this study and the Universiti Putra Malaysia for providing us
for laboratory facilities.
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