Food Chemistry 157 (2014) 457–463
Contents lists available at ScienceDirect
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
Effects of different treatments on the antioxidant properties
and phenolic compounds of rice bran and rice husk
Pitchaporn Wanyo a, Naret Meeso b, Sirithon Siriamornpun a,⇑
a
Research Unit of Process and Product Development of Functional Foods, Department of Food Technology and Nutrition, Faculty of Technology, Mahasarakham University,
Kuntarawichai, Mahasarakham 44150, Thailand
b
Research Unit of Drying Technology for Agricultural Products, Faculty of Engineering, Mahasarakham University, Kuntarawichai, Mahasarakham 44150, Thailand
a r t i c l e
i n f o
Article history:
Received 16 December 2013
Received in revised form 11 February 2014
Accepted 15 February 2014
Available online 25 February 2014
Keywords:
Enzymatic
Drying
FIR
Phenolic compounds
Rice bran
Rice husk
a b s t r a c t
We investigated the changes of antioxidant activity and bioactive compounds in bran, rice husk and
ground rice husk after three different treatments, namely hot-air, far-infrared radiation (FIR), and cellulase, compared with raw samples. Overall, FIR-treated group showed a higher DPPH radical scavenging
activities, ferric reducing antioxidant power (FRAP), and total phenolic content (TPC) than did hot-air
and cellulase treatments for all samples. A signiﬁcant increase in a- and c-tocopherols was found in
FIR irradiated rice bran compared to raw bran, while a- and c-tocopherols in hot-air and cellulase treated
rice bran were remained unchanged. Cellulase signiﬁcantly increased the amount of vanillic acid; however a dramatic decrease of ferulic acid was observed. The contents of c-oryzanol in cellulase treated
ground rice husk were signiﬁcantly increased. Decreasing particle size in the husk was found to work
positively for enhancing antioxidant activities, c-oryzanol and phenolic compounds.
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
Rice is a staple food in many parts of the world. Rice is the main
agricultural product produced in Thailand and exported to other
countries. Approximately 21–26 million tons of rice are annually
produced (OAE, 2003). Cereal grains, especially rice, contain special
phenolic acids (such as ferulic acid, p-coumaric and diferulate) that
are not present in signiﬁcant quantities in fruit and vegetables
(Adom & Liu, 2002). Most of these compounds are found in different parts of cereal grains, particularly in distinct fractions from
milling the grains (Onyeneho & Hettiarachchy, 1992). Rice milling
waste is the by-product obtained from small-scale rice mills. The
rice milling waste fractions obtained contains husks, bran, polishing and small quantity of broken rice (Onyeneho & Hettiarachchy,
1992) and are cheap as they are discarded as waste. The commercial rice-milling process leads to products with low-value fractions,
such as husk and bran. Rice bran is a rich source of oryzanols or
steryl ferulate esters (Norton, 1995). In addition, rice bran is a
potential source of tocopherols, tocotrienols and phenolic compounds which have shown antioxidant activity (Nicolosi, Rogers,
Ausman, & Orthoefer 1994). Because rice husks are inedible, they
are used in various non-food applications, such as low-value waste
⇑ Corresponding author. Tel.: +66 857474136; fax: +66 43754086.
E-mail address: [email protected] (S. Siriamornpun).
http://dx.doi.org/10.1016/j.foodchem.2014.02.061
0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.
materials. However, rice husks offer the valuable nutritional
advantage that they contain an antioxidant-defence system to protect the rice seed from oxidative stress (Ramarathnam, Osawa,
Namiki, & Kawakishi 1988). However, most antioxidative phenolic
compounds in plants are found in a form of – covalently bound
with insoluble polymer (Niwa & Miyachi, 1986). Therefore it is necessary to ﬁnd an effective processing method to release those compounds. Several methods such as heat treatment, far-infrared (FIR)
radiation, and enzymatic treatment have been studied to liberate
and activate low molecular weight natural antioxidants for various
agricultural products such as barley (Duh, Yen, Yen, & Chang, 2001)
and rice hulls (Lee et al., 2003).Therefore, the aim of the present
study was to assess the inﬂuence of different treatments on the
antioxidant properties and phenolic compounds from rice
by-products, namely rice bran and husk.
2. Materials and methods
2.1. Sample preparation
Paddy-rice samples (KDML 105 variety) were obtained from
northeastern Thailand. These grains were milled to separate the
husks from the brown rice. Then the brown rice was polished to
obtain the bran. The husks were ground and passed through a
500 lm sieve screen, and was called ground rice husk. Moisture
458
P. Wanyo et al. / Food Chemistry 157 (2014) 457–463
was determined by drying at 110 °C to constant mass. This and all
other analyses were performed using triplicate samples and analytical results were expressed on a dry matter basis. The samples
were stored at 20 °C prior to analysis.
2.2. Pretreatment process
2.2.1. Hot-air and FIR treatment
Samples were subject to two different treatments, i.e., hot-air
and FIR. For each treatment, 100 g of raw samples were used.
The protocols used in this present study were selected from the
optimal conditions of each method, which were preliminarily studied in our lab. In hot-air treatment, the sample was treated by hotair drying machine at 120 °C for 30 min using hot-air oven (UFE
600, Memmert, Memmert Company, Germany). In FIR treatment,
the sample was FIR-irradiated in the FIR dryer at FIR intensity of
2 kW/m2 (FIR energy irradiated per FIR heater surface area). Drying
temperature was set at 40 °C and drying time of 2 h.
2.2.2. Enzyme aided
Two grams of samples were incubated with the cellulase mixture. Enzyme hydrolysis experiments were conducted at the conditions of pH and temperature advised by the enzyme manufacture
(pH = 5 and T = 50 °C) and reactions were run for 24 h. After the
enzymatic treatment, the proper solvent was added and extraction
was carried out at the extraction conditions described.
2.3. Chemicals and reagents
The compounds 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,4,6tripiridyl-s-triazine (TPTZ), Folin–Ciocalteu’s reagent, standards of
phenolic compounds (gallic acid (GA), protocatechuic acid (PCCA),
p-hydroxybenzoic acid (p-OH), vanilic acid (VA), chorogenic acid
(ChA), caffeic acid (CFA), syringic acid (SyA), p-coumaric acid
(p-CA), ferulic acid (FA), sinapic acid (SNA), rutin, myricetin,
quercetin, apigenin and kaempferol were obtained from Fluka
(Neu-Ulm, Germany). Oryzanol (Food Grade, 99.9% purity) was
obtained from Tsuno Rice Fine Chemicals Co., Ltd. (Wakayama,
Japan). The acetic acid, methanol, acetonitrile and other solvents
and reagents used in the HPLC analysis were purchased from
Merck (Darmstadt, Germany). All chemicals and reagents used in
the study were of analytical grade.
2.4. Assessment of antioxidant activity
2.4.1. Sample extraction
The extracts prepared from rice bran and rice husk were made
by 70 °C of distilled water for 2 h in a thermostated water-bath
(UMAX, UM-SW 50L). The ratio between sample and extraction
medium was 1:10 (w/v). The mixtures were then ﬁltered through
ﬁlter paper (Whatman No. 1) and the ﬁltrate used for analyzing
antioxidant activity in vitro. All analyses were performed in
triplicate.
2.4.2. DPPH radical scavenging activity
The 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) scavenging
activity of the extracts was measured as described by Butsat and
Siriamornpun (2010) with some modiﬁcations. Brieﬂy, sample
extract (0.1 mL) was mixed with 1.9 mL of a 0.1 mM DPPH in
ethanol. The mixture was vortexed (1 min), left to stand at room
temperature in dark (30 min) and then the absorbance of this
solution was read at 517 nm. The percent inhibition activity was
calculated as [(Ao Ae)/Ao] 100 (Ao = absorbance without
extract; Ae = absorbance with extract).
2.4.3. Ferric reducing/antioxidant power (FRAP) assay
The FRAP assay is a method of measuring the ability of reductants (antioxidants) to reduce Fe3+–Fe2+. The formation of blue coloured Fe2+–TPTZ complex (Fe2+ tripyridyltriazine) increases the
absorbance at 593 nm. The method of Kubola and Siriamornpun
(2008) was used with some modiﬁcations. The FRAP reagent was
freshly prepared by mixing 100 mL of acetate buffer (300 mM,
pH 3.6), 10 mL TPTZ solution (10 mM TPTZ in 40 mM/HCl), 10 mL
FeCl36H2O (20 nM) in a ratio of 10:1:1 and 12 mL distilled water,
at 37 °C. To perform the assay, 1.8 mL of FRAP reagent, 180 lL
Milli-Q water and 60 lL sample, standard or blank were then
added to the same test tubes, and incubated at 37 °C for 4 min;
absorbance was measured at 593 nm, using the FRAP working
solution as a blank. The reading of relative absorbance should be
within the range 0–2.0; otherwise, the sample should be diluted.
In the FRAP assay, the antioxidant potential of sample was determined from a standard curve plotted using the FeSO47H2O linear
regression equation to calculate the FRAP values of the sample.
2.5. Determination of total ﬂavonoid content
Total ﬂavonoid content (TFC) was determined using colorimetric method described by Abu Bakar, Mohamed, Rahmat, and Fry
(2009) with slight modiﬁcation. Brieﬂy, 0.5 mL of the extract was
mixed with 2.25 mL of distilled water in a test tube followed by
addition of 0.15 mL of 5% NaNO2 solution. After 6 min, 0.3 mL of
a 10% AlCl36H2O solution was added and allowed to stand for another 5 min before 1.0 mL of 1 M NaOH was added. The mixture
was mixed by vortex mixer. The absorbance was measured immediately at 510 nm using spectrophotometer. Results were expressed as mg rutin equivalents in 1 g of dried sample (mg RE/g).
2.6. Extraction and determination of c-oryzanol and tocopherols
contents
One-step equilibrium direct-solvent extraction was conducted
by the method of Butsat and Siriamornpun (2010) with some modiﬁcations. Each sample (1 g) was extracted with acetone at a ratio
of 1:10 w/v, vortexed at maximum speed for 1 min then centrifuged at 2500 rpm for 20 min, after which the solvent was removed. The residual was further extracted twice, and the
supernatants were combined before evaporating them to dryness
under nitrogen gas. The determinations were made in triplicate.
The contents of c-oryzanol and tocopherols were determined
using HPLC. The crude extracts were dissolved in the mobile phase
and ﬁltered through a 0.45 lm pore size syringe-driven ﬁlter. The
RP-HPLC system (Shimadzu) consisted of an auto sampler and column oven equipped with Inertsil ODS (4.6 mm 250 mm, 5 lm)
with mobile phase of acetonitrile/methanol (25:75, v/v), ﬂow rate
1.5 mL/min and photodiode-array detector at 292 nm for the analysis of tocopherols and at 325 nm for the analysis of c-oryzanol.
Calibration curves were constructed with the external standards.
2.7. Identiﬁcation and quantiﬁcation of phenolic compounds
2.7.1. Determination of total phenolic content
The total phenolics content (TPC) was determined using the Folin–Ciocalteu reagent as followed by Abu Bakar et al. (2009).
Brieﬂy, 300 lL of extract was mixed with 2.25 mL of Folin–Ciocalteu reagent (previously diluted 10-fold with distilled water) and
allowed to stand at room temperature for 5 min; 2.25 mL of sodium carbonate (60 g/L) solution was added to the mixture. After
90 min at room temperature, absorbance was measured at
725 nm using a spectrophotometer. Results were expressed as
mg gallic acid equivalents in 1 g of dried sample (mg GAE/g).
P. Wanyo et al. / Food Chemistry 157 (2014) 457–463
2.7.2. HPLC-DAD system for analysis of phenolic compounds
HPLC analysis was performed using Shimadzu LC-20AC pumps,
SPD-M20A Diode-array detection; chromatographic separations
were performed on a LUNA C-18 column (4.6 250 mm i.d.,
5 lm). The composition of solvents and gradient elution conditions
were described previously by Uzelac, Pospisil, Levaj, and Delonga
(2005). The solvent system used was a gradient of mobile phase
A containing 3% acetic acid in water; solution B contained a mixture of 3% acetic acid, 25% acetonitrile and 72% water. The following gradient was used: 0–40 min, from 100% A to 30% A – 70% B
with a ﬂow rate 1 mL/min; 40–45 min, from 30% A – 70% B to
20% A – 80% B with a ﬂow rate 1 mL/min; 45–55 min, from 20%
A – 80% B to 15% A – 85% B with a ﬂow rate 1.2 mL/min; 55–
57 min, from 15% A – 85% B to 10% A – 90% B with a ﬂow rate
1.2 mL/min; 57–75 min 10% A – 90% B with a ﬂow rate 1.2 mL/
min. Operating conditions were as follows: column temperature,
40 °C, injection volume, 20 lL, UV-Diode Array detection at at
280 nm (hydroxybenzoic acids), 320 nm (hydroxycinnamic acids)
and 370 nm (ﬂavonols) at a ﬂow-rate of 0.8 mL/min. Spectra were
recorded from 200 to 600 nm. Phenolic compounds in the samples
were identiﬁed by comparing their relative retention times and UV
spectra with those of standard compounds and were detected
using an external standard method.
2.8. Statistical analyses
Analysis of variance (ANOVA) was performed in a completely
randomized design, using Duncan’s Multiple Range Test. All determinations were done at least in triplicate and all were averaged.
The conﬁdence limits used in this study were based on 95%
(p < 0.05).
3. Results and discussion
3.1. Antioxidant properties, total phenolic and total ﬂavonoid content
The DPPH radical scavenging, and FRAP assays were used to
evaluate the antioxidant capacities of rice bran and rice husk samples. DPPH is a stable free-radical compound widely used to test
the free-radical scavenging ability of various samples (Sakanaka,
Tachibana, & Okada, 2005). However, in vitro assays such as DPPH
method does not reﬂect real antioxidant activity in complex reaction although it is a radical. Therefore the cellular model systems
should be further studied. The FRAP assay measures the reducing
potential of an antioxidant reacting with a ferric tripyridyltriazine
(Fe3+–TPTZ) complex to produce a coloured ferrous tripyridyltriazine (Benzie & Strain, 1996). Generally, the reducing properties
are associated with the presence of compounds which exert their
action by breaking the free-radical chain by donating a hydrogen
atom. The reduction of the Fe3+–TPTZ complex to a blue-coloured
Fe2+–TPTZ occurs at low pH (Benzie & Strain, 1996). Changes in
antioxidant activity of rice bran and rice husk as affected by different treatments are presented in Table 1. DPPH of rice bran and
ground rice husk also increased with FIR irradiation. After FIR radiated, the percent inhibition of DPPH from rice bran and ground rice
husk increased from 88% to 92% and 91% to 93%, respectively. However, hot-air and cellulase did not affect radical scavenging activity
of rice bran and rice husk extracts. These indicate that the increase
was not induced by heat or enzymatic but by FIR radiation. FIR
radiation caused an increase of reducing power (FRAP) in all samples followed by hot-air drying, cellulase treated. Overall, we found
that the DPPH radical scavenging activity and FRAP values were
not signiﬁcantly affected by hot-air and cellulase aided treatment
in rice bran. This indicated that the increase was induced by the
FIR treatment, thereby supporting a previous study that FIR radia-
459
tion increases the antioxidant activity of mulberry leaves (Wanyo,
Siriamornpuna, & Meeso, 2011), kaprow leaves (Raksakantong,
Siriamornpun, Ratseewo, & Meeso, 2011) and rice hull extracts
(Lee et al., 2003). FIR may have the capability to cleave covalent
bonds and liberate antioxidants such as ﬂavonoids, carotene,
tannin, ascorbate, ﬂavoprotein or polyphenols from repeating polymers (Niwa, Kanoh, Kasama, & Neigishi, 1988), hence increases
antioxidant activities.
Phenolic compounds are widely distributed in fruits and vegetables (Li, Smith, & Hossain, 2006), they have received considerable
attention. Their potential antioxidant activities and free-radical
scavenging abilities have potentially beneﬁcial implications in human health (Li et al., 2006). TPC was determined in comparison
with standard gallic acid and the results were expressed in terms
of mg GAE/g dry sample. FIR radiation provided the highest values
of TPC among samples studied including untreated samples
(Table 1). These increases in phenolics due to FIR heat could break
the covalent bonds of polymerized polyphenols subsequently
transform high molecular phenolics to low molecular phenolics
(Niwa & Miyachi, 1986). This type of increment in phenolic compounds was also observed in previous research of Kim, Kim, Jeong,
Jo, and Lee (2006) and our studies (Raksakantong et al., 2011; Siriamornpun, Kaisoon, & Meeso, 2012; Wanyo et al., 2011). Kim et al.
(2006) reported that the contents of catechins including epicatechin, epicatechin gallate, epigallocatechin, and epigallocatechin
gallate, were increased by FIR irradiation during the manufacturing
process of green tea. Similarly, we found that TPC was increased by
the combined FIR–HA treatment in mulberry leaves (75%) (Wanyo
et al., 2011), marigold ﬂower (8%) (Siriamornpun et al., 2012) and
kaprow leaves (5%) (Raksakantong et al., 2011). Moreover, the
changes of polyphenols content was also observed in FIR treated
defatted soybean and grape berries, which showed almost 2 and
3 folds higher polyphenol content than untreated samples (Rim,
Jung, Kim, & Lee, 2005). TPC of all samples was not signiﬁcantly affected by hot-air. The total phenolic contents of cellulase-treated
rice husk and ground rice husk were signiﬁcantly (p < 0.05) increased, while that of rice bran was decreased, compared with
the control (raw bran).
Flavonoids are the most common and widely distributed group
of plant phenolic compounds that are characterized by a benzo-ypyrone structure, which is ubiquitous in fruits and vegetables (Abu
Baker et al., 2009). Total ﬂavonoid can be determined in the sample
extracts by reaction with sodium nitrite, followed by the development of coloured ﬂavonoid–aluminium complex formation using
aluminium chloride which can be monitored spectrophotometrically at 510 nm (Abu Baker et al., 2009). The hot-air dried of all
samples contained the lowest amount of TFC as compared to the
raw and other treated samples. The total ﬂavonoid contents of
hot-air and FIR treated rice bran, rice husk and ground rice husk
were signiﬁcantly decreased, while that of cellulase-treated was
unchanged, compared with the control of each sample (Table 1).
A relatively high increase of TPC (32%) and TFC (29%) in ground
rice husk could be caused by the reduction of the particle size. The
positive effects of reducing the particle size have been reported to
provide very positive results for extracting phenolic compounds
with good antioxidant properties in previous studies, such as for
black currant pomace (Landbo & Meyer, 2001) and black cohosh
(Mukhopadhyay, Luthria, & Robbins, 2006). As a consequence,
the smaller the particle size of rice husks, the higher the extraction
efﬁciency and thus the higher the DPPH radical scavenging activity,
FRAP value and total phenolic content of the resulting extract.
However, positive effects of reducing the particle size are not always obvious, as a reduction of the particle size can obstruct the
solvent access to all the exposed surface area of the solid and release the phenol compounds (Pinelo et al., 2007). The increases
in phenolic content of the extracts from cellulase-treated rice husk
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P. Wanyo et al. / Food Chemistry 157 (2014) 457–463
Table 1
Effect of different treatments on antioxidant activity of rice bran and rice husk.
Sample
DPPH (% inhibition)
FRAP (lmol FeSO4/g DW)
TPC (mg GAE/g DW)
TFC (mg RE/g DW)
Rice bran
Raw
Hot-air
FIR
cellulase
87.93 ± 0.05c
87.23 ± 0.60c
92.21 ± 0.69a
88.39 ± 0.87c
28.57 ± 0.36b
28.87 ± 0.12b
34.41 ± 0.13a
28.67 ± 0.15b
3.52 ± 0.06b
3.58 ± 0.03b
4.05 ± 0.03a
3.05 ± 0.03d
3.88 ± 0.09a
3.08 ± 0.10c
3.59 ± 0.16b
3.72 ± 0.10ab
Rice husk
Raw
Hot-air
FIR
Cellulase
87.10 ± 0.38c
74.29 ± 1.34c
87.75 ± 0.87c
87.44 ± 0.55c
13.52 ± 0.13h
14.07 ± 0.12g
18.28 ± 0.37f
13.75 ± 0.15gh
1.12 ± 0.01h
1.13 ± 0.02h
2.20 ± 0.04e
1.24 ± 0.02g
2.66 ± 0.11d
2.05 ± 0.05f
2.29 ± 0.05e
2.52 ± 0.03d
Ground rice husk
Raw
Hot-air
FIR
Cellulase
90.86 ± 0.82b
90.01 ± 0.51b
92.64 ± 0.74a
90.53 ± 0.92b
19.66 ± 0.16e
20.25 ± 0.31d
21.73 ± 0.14c
19.80 ± 0.16e
1.68 ± 0.00f
1.70 ± 0.02f
3.14 ± 0.05c
2.21 ± 0.05e
3.76 ± 0.29 ab
3.09 ± 0.08c
3.55 ± 0.10b
3.74 ± 0.08ab
Values are expressed as mean ± standard deviation (n = 3). Means with different letters in the same column were signiﬁcantly different at the level p < 0.05.
and ground rice husk were not responsible for the antioxidant
capacity of the samples as determined by the DPPH radical scavenging activity methods and the FRAP assay. Our result was also
in agreement with Yu et al. (2002) and Othman, Ismail, Ghani,
and Adenan (2007), who found no correlation between scavenging
activity and TPC. In contrast, a study by Kubola and Siriamornpun
(2008) found correlations between TPC and antioxidant activity as
determined by DPPH and FRAP assay in bitter gourd fractions.
Enzyme aided extraction has been reported to have both
adverse and favourable effects on bioactive compounds. Increases
in antioxidant activity by enzyme aided extraction have been
reported by many studies, mostly in plants (Li et al., 2006). The
increase in the release of polyphenols by the enzyme preparations
suggests that these enzymes may contain activities that directly
promote selective release of antioxidant phenols or modify
released phenols to more potent antioxidant compounds. Aside
from the favourable effects of enzyme aided extraction on bioactive compounds, there was a decrease in the anthocyanin content
in fruit juice (Chaovanalikit et al., 2012).
The possible explanation for an increase of percent recovery
with the use of enzymes is that the enzymes disrupt the integrity
of the cell walls; as a result, the extraction is more efﬁcient. However, when cells are disrupted, the protein may be released along
with the phenolic compounds, which are known to form complex
compounds (Li et al., 2006).
Far infrared radiation has been proved to increase the total
polyphenolic content and antioxidant activity in extract of natural
products (Lee et al., 2003; Niwa & Miyachi, 1986). Consistent with
this, the present study showed that total phenolic content and
antioxidant activity in the extracts from rice by-product were increased signiﬁcantly by FIR. Therefore, FIR of rice by-product represents a more efﬁcient treatment than other treatment, in terms
of the effect on total phenolic content and antioxidant activity of
extracts from rice bran and rice husk.
3.2. Percentage losses or gains for antioxidant activity, total phenolic,
and total ﬂavonoid content
Percentage losses or gains for DPPH, FRAP, TPC and TFC of rice
bran, rice husk and ground rice husk by different treatments compared to raw samples are shown in Table 1. The increased values of
TPC and FRAP as affected by FIR accounted for approximately 15%
and 20% in rice bran, 96% and 35% in rice husk, and 87% and 10% in
ground rice husk. While both TPC and FRAP values were increased
less than 10% by hot air drying method. In addition, TPC was
increased approximately 10% in rice husk and 30% in ground rice
husk. In contrast, TPC was decreased by cellulase treatment in rice
bran.
In case of DPPH radical scavenging activity, there were
increases in DPPH radical scavenging activity FIR and cellulasetreated rice bran (FIR: 4.87%, cellulase: 0.52%), rice husk (FIR:
0.75%, cellulase: 0.39%) and ground rice husk (FIR: 1.96%), respectively, while DPPH decreased rice bran (0.80%), rice husk (0.41%)
and ground rice husk (0.94%) of hot-air treated and (0.36%) of cellulase-treated ground rice husk. For the TFC, all samples indicate
(rice bran, rice husk and ground rice husk) decreased dramatically,
with losses of hot-air: 20.53%, 22.80% and 17.74%, FIR: 7.53%,
13.78% and 5.43%, and cellulase: 3.98%, 5.28% and 0.51%, respectively. FIR rays, deﬁned as electromagnetic waves (3–1000 lm)
are known to release and activate low molecular weight natural
antioxidants from the plant materials (Lee et al., 2003). FIR rays
are biologically active and transfer heat to the center of materials
evenly without decomposing the molecules on material’s surface
(Niwa et al., 1988). FIR may capable to cleave covalent bonds and
release some bound- antioxidants such as ﬂavonoids, carotene,
tannin, ascorbate, ﬂavoprotein, or polyphenols from polymers
(Niwa & Miyachi, 1986). Effects of processing methods on bioactive
compounds of plant samples could vary from no change to significant losses, or even enhancement in bioactive compounds and
antioxidant activities (Raksakantong et al., 2011; Wanyo et al.,
2011).
3.3. c-Oryzanol and tocopherols
The effects of treatment on the c-oryzanol and tocopherols contents of rice bran and rice husk extracts are shown in Table 2. For
extracts of all samples, the amount of c-oryzanol ranged from
0.566 mg/g in hot-air treated rice husk to 5.701 mg/g in raw rice
bran. The bran fraction was the best source of c-oryzanol (5.28–
5.70 mg/g), followed by ground rice husk (0.90–0.99 mg/g) and rice
husk (0.56–0.57 mg/g), respectively. All samples indicate
decreased slightly, with losses of FIR and hot-air treatment, respectively. It was observed that the contents of c-oryzanol were not
signiﬁcantly (p < 0.05) increased by cellulase treatment in all samples except for ground rice husk. Similar to TPC, an increase of coryzanol in ground rice husk may have resulted from the reduction
of particle size. This is in agreement with the report by Pinelo et al
(2007). They found that reducing the sample size could increase
the extraction of certain bioactive compounds.
The three forms of a-,c- and d-tocopherols were detected in rice
bran. The a- and c-tocopherols of different treatments indicated
that the FIR irradiated had the greatest content of rice bran
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P. Wanyo et al. / Food Chemistry 157 (2014) 457–463
Table 2
Effect of different treatments on c-oryzanol and tocopherols of rice fractions of rice bran and rice husk.
Sample
c-Oryzanol (mg/g)
a-Tocopherols (lg/g)
c-Tocopherols (lg/g)
d-Tocopherols (lg/g)
Rice bran
Raw
Hot-air
FIR
Cellulase
5.701 ± 0.022a
5.281 ± 0.018c
5.612 ± 0.006b
5.698 ± 0.012a
82.15 ± 2.84b
63.50 ± 2.56c
95.78 ± 3.81a
83.42 ± 5.26b
5.04 ± 0.02b
5.03 ± 0.07b
5.14 ± 0.09a
5.04 ± 0.03b
Nd
Nd
7.84 ± 0.12
Nd
Rice husk
Raw
Hot-air
FIR
Cellulase
0.570 ± 0.008g
0.566 ± 0.001g
0.567 ± 0.011g
0.573 ± 0.005g
Nd
Nd
Nd
Nd
Nd
Nd
Nd
Nd
Nd
Nd
Nd
Nd
Ground rice husk
Raw
Hot-air
FIR
Cellulase
0.979 ± 0.003e
0.898 ± 0.014f
0.966 ± 0.011e
0.992 ± 0.001d
Nd
Nd
Nd
Nd
Nd
Nd
Nd
Nd
Nd
Nd
Nd
Nd
Values are expressed as mean ± standard deviation (n = 3). Means with different letters in the same column were signiﬁcantly different at the level p < 0.05.
(95.78 and 5.14 lg/g DW), followed by cellulase treated bran
(83.42 and 5.04 lg/g DW), raw rice bran (82.15 and 5.04 lg/
g DW), and then hot-air treatment (63.50 and 5.03 lg/g DW),
respectively. In addition, we found that the presence of the two
forms was not signiﬁcantly affected by cellulase. Surprisingly, dtocopherols were only detected in FIR irradiated rice bran (Table 2).
Tocopherols, collectively known as vitamin E, are important dietary antioxidants (Traber, 2006). They are a family of phenolic
compounds; each contains a chromanol ring system and a 16-carbon side-chain. There are four forms of tocopherols namely a-, b-,
c-, or d-tocopherol, depending upon the number and position of
methyl groups on the chromanol ring. Among those, the d-tocopherol is more active than a- or c-tocopherol in inhibiting tumour
growth, however the serum and tumour levels of d-tocopherol
were much lower than levels of the other tocopherols (Li et al.,
2011). Food processing may improve the properties of naturally
occurring antioxidants or induce the formation of new compounds
with antioxidant properties, so that the overall antioxidant activity
increases or remains unchanged (Tomaino et al., 2005). Many antioxidant phenolic compounds in plants are most frequently present
in a covalently bound form with insoluble polymers (Niwa &
Miyachi, 1986). If this bonding is not strong FIR treatment could
liberate and activate low-molecular-weighted natural antioxidants
in plants (Lee et al., 2003). As can be observed, the c-oryzanol content of extracts was enhanced concurrently with decreasing particle size regardless of the use of enzyme treatment. Presumably,
when the particle size is reduced, the accessible surface for the
extractive solvent and the enzyme attack is increased, resulting
in the observed c-oryzanol content in ground rice husk. This
increase of the surface area induces an increase of the amount of
potential enzyme-binding sites that should lead, under the conditions of this study, to improved cellulose hydrolysis yields (Jacquet,
Vanderghem, Danthine, Blecker, & Paquot, 2013). The diffusion to
the surface of solid is reported to be one of the major limiting steps
in solid/liquid extraction (Vandenburg et al., 1997). The intra-particle diffusion resistance is generally lower for smaller particles
because of their shorter diffusion path. Moreover, smaller particles
would provide greater surface area and their cell walls could have
been broken to a greater degree during grinding (Jeng, Lai, Kao, Wu,
& Sung, 2013). Therefore, the greater recovery of c-oryzanol from
ground rice husk is expected.
3.4. Identiﬁcation and quantiﬁcation of phenolic compounds
Phenolic compounds are the most active antioxidant derivatives
in plants which mostly found in the outer layers of plants, such as
the peel, shell and hull, contain large amounts of polyphenolic
compounds to protect the inner components (Bors, Michel, &
Stettmaier, 2001). A number of the phenolic acids are linked to various cell-wall components, such as arabinoxylans and proteins
(Hartley, Morrison, Himmelsbach, & Borneman, 1990). They are
known to be good natural antioxidants which not only their ability
to donate hydrogen or electrons but also they are stable radical
intermediates (Maillard, Soum, Boivia, & Berset, 1996).
RP-HPLC analysis was used to identify the phenolic compounds
of rice bran and rice husk extracts, by comparison with standard
compounds. Phenolic acids are hydroxylated derivatives of hydrobenzoic and hydrocinnamic, which often occur in plants as esters,
glycosides and bound complexes (Germano et al., 2006). In the rice
bran and rice husk analysed, it was possible to identify 4 hydroxybenzoic acid (HBA): gallic acid, protocatechuic acid, p-hydroxybenzoic acid, vanilic acid and 6 hydrocinnamic acid (HCA): chorogenic
acid, caffeic acid, syringic acid, p-coumaric acid, ferulic acid, and
sinapic acid. The distribution of phenolic acids in all samples is presented in Table 3. The main phenolic acids found in all samples
were ferulic, protocatechuic, gallic and vanillic acids. Comparing
the phenolic acids of all samples, the rice bran contained the highest levels of protocatechuic acid, with concentrations from 12.7
to17.8 lg/g. In rice husk and ground rice husk, no p-hydroxybenzoic acid or syringic acid were detected. In the rice bran and rice
husk, the total content of the HCA group was higher than the total
content of the HBA group. We found that HCA group was ranged
from 25 to 33 lg/g in rice bran, 19 to 31 lg/g in rice husk and 24
to 34 lg/g in ground rice husk, while HBA group was ranged from
22 to 30 lg/g in rice bran, 11 to 17 lg/g in rice husk and 17 to
26 lg/g in ground rice husk, respectively. The total content of the
HCA group of different treatments indicated that the FIR irradiated
samples had the greatest content of rice bran, rice husk and ground
rice husk (32.95, 30.75 and 45.13 lg/g DW), followed by hot-air
(32.97, 24.72 and 33.62 lg/g DW), raw samples (27.50, 22.26 and
29.00 lg/g DW), and then cellulase treated (24.87, 19.00 and
23.56 lg/g DW), respectively. For the total content of the HBA
group of different treatments indicated that the FIR irradiated
had the greatest content of rice bran (30.75 lg/g DW), followed
by hot-air (25.78 lg/g DW), cellulase treated (22.73 lg/g DW),
and then raw rice bran (22.34 lg/g DW), respectively. FIR had
higher content of HBA on rice husk and ground rice husk (16.67
and 26.07 lg/g DW), than did cellulase treatment (11.51 and
20.68 lg/g DW), raw samples (11.31 and 19.59 lg/g DW), and
hot-air treatment (11.08 and 17.27 lg/g DW), respectively. Greater
amounts of all phenolic acids were found in treated samples compared to raw samples, except for ferulic and gallic acids, which
462
P. Wanyo et al. / Food Chemistry 157 (2014) 457–463
Table 3
Effect of different treatments on phenolic acids of rice bran and rice husk (lg/g dry weight).
Sample
Hydrobenzoic acids
GA
Hydrocinnamic acids
Total
PCCA
pOH
VA
ChA
CFA
SyA
p-CA
FA
SNA
Rice bran
Raw
Hot-air
FIR
Cellulase
5.47 ± 0.41d
5.77 ± 0.23d
7.76 ± 0.52c
5.50 ± 0.04d
12.69 ± 0.48c
16.04 ± 0.47b
17.82 ± 0.78a
12.86 ± 1.32c
Nd
Nd
Nd
Nd
4.18 ± 0.02fg
3.97 ± 0.02g
5.18 ± 0.07d
4.37 ± 0.10f
3.02 ± 0.09c
4.90 ± 0.15a
3.22 ± 0.04bc
3.03 ± 0.04c
3.43 ± 0.03b
3.51 ± 0.02b
3.63 ± 0.05b
3.44 ± 0.03b
3.18 ± 0.05c
4.58 ± 0.17a
3.68 ± 0.13b
3.24 ± 0.03c
3.05 ± 0.02c
3.12 ± 0.02b
3.13 ± 0.04b
3.06 ± 0.01c
11.04 ± 0.02de
12.63 ± 0.08cd
14.84 ± 2.72b
8.31 ± 0.79fg
3.77 ± 0.02g
4.22 ± 0.05f
4.44 ± 0.13e
3.80 ± 0.04g
49.84 ± 0.90de
58.75 ± 0.64c
63.70 ± 3.17b
47.59 ± 1.07e
Rice husk
raw
Hot-air
FIR
Cellulase
3.78 ± 0.07e
3.81 ± 0.01e
5.88 ± 0.04d
3.75 ± 0.06e
3.40 ± 0.01e
3.66 ± 0.02e
5.58 ± 0.27d
3.47 ± 0.02e
Nd
Nd
Nd
Nd
4.13 ± 0.03fg
3.61 ± 0.08h
5.21 ± 0.35d
4.29 ± 0.01fg
2.82 ± 0.03c
3.56 ± 0.56b
3.19 ± 0.55bc
2.84 ± 0.05c
3.46 ± 0.03b
3.51 ± 0.04b
6.00 ± 0.19a
3.51 ± 0.02b
Nd
Nd
Nd
Nd
1.52 ± 0.01d
1.53 ± 0.01d
1.55 ± 0.01d
1.52 ± 0.01d
9.92 ± 0.16ef
11.49 ± 1.00de
15.18 ± 1.19b
6.54 ± 0.80g
4.53 ± 0.10e
4.62 ± 0.08d
4.83 ± 0.08c
4.59 ± 0.06e
33.57 ± 0.17g
35.80 ± 0.60g
47.42 ± 2.33e
30.51 ± 0.81h
Ground rice husk
Raw
8.72 ± 0.07b
Hot-air
8.85 ± 0.31b
FIR
10.05 ± 0.76a
Cellulase
8.67 ± 0.23b
3.48 ± 0.02e
3.65 ± 0.03e
6.39 ± 0.01d
3.61 ± 0.02e
Nd
Nd
Nd
Nd
7.39 ± 0.33c
4.78 ± 0.02e
9.62 ± 0.04a
8.40 ± 0.02b
3.22 ± 0.06bc
5.23 ± 0.10a
3.59 ± 0.06b
3.22 ± 0.10bc
3.49 ± 0.03b
3.56 ± 0.05b
6.19 ± 0.33a
3.52 ± 0.04b
Nd
Nd
Nd
Nd
3.13 ± 0.03b
3.13 ± 0.03b
3.22 ± 0.02a
3.15 ± 0.02b
13.72 ± 0.24bc
15.29 ± 0.74b
25.64 ± 0.87a
8.18 ± 0.75fg
5.43 ± 0.07b
6.42 ± 0.10a
6.50 ± 0.12a
5.48 ± 0.15b
48.58 ± 0.51de
50.90 ± 1.17d
71.20 ± 1.65a
43.21 ± 0.81f
Values are expressed as mean ± standard deviation (n = 3). Means with different letters in the same column were signiﬁcantly different at the level p < 0.05.
were found in lower amounts in cellulase treatment. As can be
seen from Table 3, FIR was the only treatment caused an increase
of HBA and HBC contents while other two treatments brought
about both favourable and adverse effects. Interestingly, it was
observed that cellulase resulted in an increase of total HBA in all
kinds of samples however it gave adverse results for HBC. In our
present study, we found that the results between raw materials
and control (pH 5.0 at 50 °C for 24 h) were not signiﬁcantly different (p < 0.05) (the data not shown). This may have been due to the
conversion of ferulic acid into vanillic acid and protocatechuic acid
(Samejima, Tatarazako, Arakawa, Saburi, & Yoshimoto, 1987). The
catabolism of these compounds is an important aspect for the mineralization of plant wastes because they are released during the
breakdown of lignin and cell wall materials by white-rot fungi.
Moreover, there is a growing interest in the potential use of ferulic
acid as a feedstock for biocatalytic conversion into other valuable
molecules such as styrenes, polymers, epoxydes, alkylbenzenes,
vanillin and vanillic acid derivatives, guaiacol, cathecol, and protocatechuic-acid-related cathecols (Rosazza, 1995). For hot air drying, HBA was increased in rice bran but that was decreased in
ground rice husk while HBA of rice husk remained unchanged.
The HBAs (gallic acid, protocatechuic acid and vanillic acid) and
HCAs (caffeic acid, p-coumaric acid, ferulic acid and sinapic acid)
showed the highest content in all samples with FIR. Only two
HCAs, namely chlorogenic and syringic acids, were found to be
higher in the samples with hot-air treatment, compared to those
by other treatments. This is in agreement with our previous study
(Wanyo et al., 2011). We found that the total content of phenolic
compounds increased in FIR–HA dried samples compared to those
of hot-air dried mulberry leaf tea, except for chlorogenic and syringic acids, which were found in greater amounts in hot-air dried
commercial tea. The mechanism of phenolic acid can be proposed
that similar to phenomenal in TPC, FIR may capable to cleave covalent bonds and release some bound- antioxidants such as ﬂavonoids, carotene, tannin, ascorbate, ﬂavoprotein, or polyphenols
from polymers (Niwa & Miyachi, 1986).
For ﬂavonoids, it was possible to identify 5 ﬂavonids (rutin,
myricetin, quercetin, apigenin and kaempferol). In rice bran were
only detected kaempferol, and rice husk and ground rice husk were
only detected myricetin (Table 4). Decreases of kaempferol content
in FIR (15%) and hot-air treated rice bran (17%) were observed,
while it was not signiﬁcantly affected by cellulase. For the rice husk
and ground rice husk with different treatment, myricetin content
decreased slightly, with losses of 9% and 7% in hot-air, 6% and 5%
in FIR, and 1% in cellulase treatment, respectively. However, the
slight difference may be due to the thermal degradation of these
Table 4
Effect of different treatments on ﬂavonoids of rice bran and rice husk (lg/g dry weight).
Sample
Flavonoid contents (lg/g DW)
Total
Rutin
Myricetin
Quercetin
Apigenin
Kaempferol
Rice bran
Raw
Hot-air
FIR
Cellulase
Nd
Nd
Nd
Nd
Nd
Nd
Nd
Nd
Nd
Nd
Nd
Nd
Nd
Nd
Nd
Nd
2.08 ± 0.02a
1.73 ± 0.02c
1.77 ± 0.01b
2.07 ± 0.02a
Rice husk
Raw
Hot-air
FIR
cellulase
Nd
Nd
Nd
Nd
25.87 ± 0.10e
23.62 ± 0.08h
24.34 ± 0.05g
25.69 ± 0.02f
Nd
Nd
Nd
Nd
Nd
Nd
Nd
Nd
Nd
Nd
Nd
Nd
25.87 ± 0.10e
23.62 ± 0.08h
24.34 ± 0.05g
25.69 ± 0.02f
Ground rice husk
Raw
Hot-air
FIR
Cellulase
Nd
Nd
Nd
Nd
32.62 ± 0.10a
30.25 ± 0.06d
30.92 ± 0.10c
32.26 ± 0.05b
Nd
Nd
Nd
Nd
Nd
Nd
Nd
Nd
Nd
Nd
Nd
Nd
32.62 ± 0.10a
30.25 ± 0.06d
30.92 ± 0.10c
32.26 ± 0.05b
2.08 ± 0.02a
1.73 ± 0.02c
1.77 ± 0.01b
2.07 ± 0.02a
Values are expressed as mean ± standard deviation (n = 3). Means with different letters in the same column were signiﬁcantly different at the level p < 0.05.
P. Wanyo et al. / Food Chemistry 157 (2014) 457–463
compounds. Treatment processes lead also to ﬂavonoid degradation (Irina & Mohamed, 2012). The proportion lost depends on
the treatment method.
4. Conclusion
The present study has demonstrated that different treatments
resulted in both favourable and adverse effects on quantity and
composition of bioactive compounds of rice by-products. Overall,
the rice by-product samples with FIR radiated had higher content
of bioactive compounds such as tocopherols phenolic acids and
antioxidant activities, compared with hot air dried and cellulase
treated samples as well as with raw samples. According to the
results from our present study, FIR should be considered as suitable treatment for rice by-product with respect to preserving its
bioactive compounds and, antioxidant properties. The present
study has provided useful information on treatment optimization
of bioactive compounds from rice by-products for further food
applications.
Acknowledgement
This research has supported by The Royal Golden Jubilee Ph.D.
Program (RGJ) under The Thailand Research Fund (TRF).
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