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Food Chemistry 232 (2017) 67–78

Contents lists available at ScienceDirect

Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Phytochemical profiles and antioxidant activity of processed brown rice


products
Er Sheng Gong a,b, Shunjing Luo a, Tong Li b, Chengmei Liu a,⇑, Guowen Zhang a, Jun Chen a,
Zicong Zeng a, Rui Hai Liu b,⇑
a
State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, Jiangxi 330047, China
b
Department of Food Science, Cornell University, Ithaca, NY 14853, United States

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

Article history: The phytochemical profiles and antioxidant activity of free, soluble-conjugated, and bound fractions of
Received 23 October 2016 brown rice and its processed products (textured rice, cooked rice and rice noodle) were studied.
Received in revised form 10 March 2017 Nineteen phenolic acids were identified. Trans-ferulic acid was the most abundant monomeric phenolic
Accepted 28 March 2017
acid with trans-trans-8-O-40 diferulic acid being most abundant diferulic acid. Processing increased the
Available online 30 March 2017
content of free phenolic acids, but decreased the content of soluble-conjugated phenolic acids. The con-
tent of bound phenolic acids was increased by improved extrusion cooking technology and cooking, but
Chemical compounds studied in this article:
not affected by rice noodle extrusion. The total phenolic contents and antioxidant activities of free and
Trans-ferulic acid (PubChem CID: 445858)
Trans-p-coumaric acid (PubChem CID:
soluble-conjugated fractions were decreased after processing, whereas those of bound fraction were
637542) increased by improved extrusion cooking technology and cooking, but not affected by rice noodle extru-
Cis-ferulic acid (PubChem CID: 1548883) sion. Results indicated that whole foods designed for reducing chronic disease risk need to consider the
Trans-trans-8-O-40 diferulic acid (PubChem effects of processing on phytochemical profiles and antioxidant activity of whole grains.
CID: 101052653) Ó 2017 Elsevier Ltd. All rights reserved.
Cis-p-coumaric acid (PubChem CID:
1549106)
P-hydroxybenzoic acid (PubChem CID: 135)
Trans-sinapic acid (PubChem CID: 637775)
Chlorogenic acid (PubChem CID: 1794427)
Trans-trans-5-50 diferulic acid (PubChem
CID: 5281770)
Trans-trans-8-50 benzofuran diferulic acid
(PubChem CID: 10385446)

Keywords:
Phenolics
Whole grains
Antioxidant activity
Processing
Phytochemicals

1. Introduction reduced risk of chronic diseases, including cardiovascular disease,


type II diabetes, obesity and cancer (Jiang, Li, & Liu, 2016;
Epidemiological studies suggest that increased consumption of Okarter & Liu, 2010; Xi & Liu, 2016). The phytochemicals in whole
whole grains and whole grain products has been associated with grains are proposed to be partly responsible for the health benefits
of whole grains and whole grain products consumption (Liu, 2007;
Okarter & Liu, 2010). Rice (Oryza sativa L.) is the most important
⇑ Corresponding authors at: State Key Laboratory of Food Science and Technol- grain crop in Asia, and its processed products, such as cooked rice,
ogy, No. 235 Nanjing East Road, Nanchang University, Nanchang 330047, China rice noodle and textured rice, are staple foods in most populations.
(C. Liu). Department of Food Science, 245 Stocking Hall, Cornell University, Ithaca,
Whole grain rice is a rich source of phytochemicals with antioxi-
NY 14853-7201, USA (R.H. Liu).
dant activity (AOA), including phenolic acids (PAs), flavonoids,
E-mail addresses: gongersheng@163.com (E.S. Gong), luoshunjing@aliyun.com
(S. Luo), tl24@cornell.edu (T. Li), liuchengmei@aliyun.com (C. Liu), gwzhang@ncu. anthocyanins, proanthocyanidins, tocopherols, tocotrienols and
edu.cn (G. Zhang), chen-jun1986@hotmail.com (J. Chen), katzeng@163.com c-oryzanol (Okarter & Liu, 2010). PAs are the most common
(Z. Zeng), RL23@cornell.edu (R.H. Liu).

http://dx.doi.org/10.1016/j.foodchem.2017.03.148
0308-8146/Ó 2017 Elsevier Ltd. All rights reserved.
68 E.S. Gong et al. / Food Chemistry 232 (2017) 67–78

phenolic compounds found in rice (Liu, 2007). Phenolics exist as y-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), 2,4,6-tri
free, soluble-conjugated, and bound forms, with bound form being (2-pyridyl)-s-triazine (TPTZ), ascorbic acid, and 20 ,70 -dichlorofluor
predominant (Adom & Liu, 2002). The bound phenolics are linked escein-diacetate (DCFH-DA) were purchased from Sigma–Aldrich
to cell wall structural components, such as cellulose, lignin and (St. Louis, MO, USA). Acetone, ethyl acetate, methanol, hexane,
proteins (Acosta-Estrada, Gutierrez-Uribe, & Serna-Saldivar, 2014; potassium hydroxide (KOH), potassium phosphate monobasic,
Liu, 2007). Some monomeric and dimeric phenolics have been potassium phosphate dibasic, sodium carbonate, sodium hydrox-
detected and structurally identified in whole grains (Guo & Beta, ide (NaOH), potassium persulfate, hydrochloric acid (HCl), EDTA
2013; Qiu, Liu, & Beta, 2010). High concentrations of these pheno- disodium salt (Na2EDTA), ferric chloride, ferrous sulfate, high per-
lics are present in the germ and bran layers (Adom, Sorrells, & Liu, formance liquid chromatography (HPLC) grade acetonitrile and
2005). However, these bioactive phenolics are removed by polish- acetic acid were purchased from Xilong Chemical Co., Ltd. (Guang-
ing or milling. Whole grain rice with abundant phytochemicals dong, China). 2,20 -Azobis (2-amidinopropane) dihydrochloride
exhibit several unique bioactivities, such as antioxidant (Adom & (ABAP) was purchased from Wako Chemicals (Richmond, VA,
Liu, 2002), antimicrobial (Zhang, Ali, & Khan, 2014), antiviral USA). Mass spectrometry (MS) grade acetonitrile and formic acid
(Ray et al., 2013), anti-inflammatory (Niu et al., 2013), anticancer were purchased from Merck (Darmstadt, Germany).
and immunomodulatory activities (Hudson, Dinh, Kokubun,
Simmonds, & Gescher, 2000; Verschoyle et al., 2007), thus promot- 2.2. Brown rice sample and sample preparation
ing overall human health.
Since whole grains are thermally processed before consuming, A BR (Oryza sativa L.) cultivar (Sonjing 16) was used in this
it is important to understand the effect of processing on bioactive study. It was grown in Wuchang (Heilongjiang Province, China)
phenolics. A few researchers have done this work. In case of total during the 2014 growing season, and harvested in October. After
phenolic contents (TPCs) and AOAs of free fractions, most studies dehulled, BR was stored in sealed polyethylene containers at
found decreases after processing (Massaretto, Madureira Alves, 20 °C until use. For chemical determination, BR was ground by
Mussi de Mira, Carmona, & Lanfer Marquez, 2011; N’Dri et al., a DFY-200 mill (Linda Machinery, Zhejiang, China) and sieved
2013; Scaglioni, de Souza, Schmidt, & Badiale-Furlong, 2014; (100 mesh) to a uniform size, then stored in sealed polyethylene
Walter et al., 2013; Zaupa, Calani, Del Rio, Brighenti, & Pellegrini, containers at 20 °C.
2015), Dewanto, Wu, and Liu (2002) reported increases in sweet Cooked rice. BR grains were cooked using 400 ml of tap water to
corns, whereas de la Parra, Saldivar, & Liu (2007) observed both 200 g of rice. BR was cooked for 30 min in an electric pressure coo-
decreases and increases in corns. When it came to TPCs and AOAs ker (Medea MY-12SS509A, Guangdong, China). After cooling,
of bound fractions, a few studies found decrements after process- cooked rice was dried overnight in an oven at 45 °C, then ground
ing (de la Parra et al., 2007; Dewanto et al., 2002; N’Dri et al., to a fine powder by a DFY-200 mill and sieved (100 mesh) to uni-
2013; Scaglioni et al., 2014), Massaretto et al. (2011) observed form size. The cooking treatment was performed in triplicate. Sam-
increments, whereas Zaupa et al. (2015) reported decrements, ples were stored in sealed polyethylene containers at 20 °C.
increments, and no significant effects in different grain varieties. Texturized rice. Texturized rice was made using a single-screw
These processing effects depended on the processing methods extruder based on the method reported previously (Liu et al.,
applied, the type, and variety of whole grains analyzed. There is 2011). BR grains were soaked in excess tap water for 2 h, drained
no related report on the effect of processing on the TPCs and AOAs and then ground to a fine powder to pass through a 0.25 mm mesh
of soluble-conjugated fractions in these studies. Processing may screen in a hammer mill (Hongxing Machinery Limited Liability
cause depolymerisation of oligomers and high polymers into Company, Jiangxi, China). The moisture content of the rice flour
dimers and trimers, concomitant polymerisation reactions, and was measured by a halogen moisture analyzer HR83 (Mettler-
formation of strong complexes of the soluble phenolics with Toledo International Inc., Greifensee, Switzerland). In order to
macromolecules in a food matrix, which may reduce their solubil- obtain a total feed moisture content of 40%, 8 kg of the BR flour
ity (Massaretto et al., 2011), thus influencing health benefits of was blended with 2226 ml tap water in a mixer (Jinan Saixin
rice. Acosta-Estrada et al. (2014) reviewed that different forms Machinery Ltd., Shandong, China) for 20 min at a high speed
(free, soluble-conjugated, and bound) of phytochemicals are (365 rpm) to ensure homogeneity of the feeding material before
released and absorbed at different sites of the human gastrointesti- IECT. The temperature profiles in the feed, mix, screw conveyor,
nal tract, thus exhibiting different health benefits. To conclude, shearing compression metering, and die head zones were kept con-
however, limited data are available to understand the effect of pro- stant at 50, 65, 85, 120, and 95 °C, respectively. The feed and screw
cessing on the phytochemical profiles and AOA in free, soluble- speeds were 30 and 37.5 rpm, respectively. The rotary cut fre-
conjugated and bound fractions of brown rice (BR). quency was set as 35.5 Hz. After cooling, texturized rice was dried
The objectives of the present study were (1) to identify phenolic overnight in an oven at 45 °C, then ground to a fine powder by a
compounds in free, soluble-conjugated and bound fractions of BR; DFY-200 mill and sieved (100 mesh) to uniform size. The IECT
(2) to determine the effects of three processing methods of the sta- was performed in triplicate. Samples were stored in sealed poly-
ple foods, i.e. improved extrusion cooking technology (IECT), cook- ethylene containers at 20 °C until analysis.
ing, and rice noodle extrusion (RNE), on TPC and AOA; and (3) to Rice noodle. Rice noodle was made by a traditional rice noodle
determine the effect of processing on phenolic profiles of BR. machine. Briefly, 8 kg of BR grains were soaked in excess tap water
overnight. The soaked rice grains and proper water were passed
through an instant rice noodle machine (Shengdi Machinery Fac-
2. Materials and methods tory, Zhejing, China). The BR grains were extruded and milled at
high speed (800 rpm) in the noodle machine, resulting in heat gen-
2.1. Materials eration and flour steaming rapidly. Then the gelatinized BR flour
was passed through a 72-hole die to produce uniform strips. Long
Chlorogenic acid (CHA), p-hydroxybenzoic acid (p-HA), vanillic strips obtained from the cutting rolls of the rice noodle machine
acid (VA), trans-caffeic acid (trans-CFA), syringic acid (SYA), trans- were dried at room temperature for 24 h. The rice noodles were
p-coumaric acid (trans-p-COA), trans-ferulic acid (trans-FA), trans- ground to a fine powder by a DFY-200 mill and sieved (100 mesh)
sinapic acid (trans-SIA), gallic acid, Folin-Ciocalteu reagent, 2,20 -a to uniform size. The RNE treatment was performed in triplicate.
zino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 6-hydrox Samples were stored in sealed polyethylene containers at 20 °C.
E.S. Gong et al. / Food Chemistry 232 (2017) 67–78 69

2.3. Extraction of free phenolics reaction of 192 mg ABTS and 33 mg potassium persulfate in
50 ml of water. The mixture was stirred overnight in the dark,
Free phenolics were extracted using the method reported previ- afterwards, the solution was diluted with water up to a final absor-
ously by our laboratory (Adom & Liu, 2002; Zhang & Liu, 2015; bance of 0.70 ± 0.02 at 734 nm. 3920 ll of diluted ABTS+ solution
Zhang, Zhang, Zhang, & Liu, 2010). Briefly, 5 g of sample flour were was vortexed with 20 ll of sample extracts, then incubated at
blended with 30 ml of 80% chilled acetone for 10 min. After cen- 30 °C, and the absorbance was recorded at 734 nm after 10 min.
trifugation at 2500g for 10 min, the supernatant was removed Trolox was used to obtain the standard curve. The results were
and extraction was repeated two times. Supernatant was evapo- expressed as mg of Trolox equiv. (TE)/100 g DW. Data were
rated in a rotary evaporator to dryness at 45 °C and reconstituted reported as mean ± SD for three replications.
in 10 ml of deionized water to obtain soluble phenolics. Soluble
phenolic extract was acidized with concentrated HCl to pH 2. The
solution was extracted with hexane to remove lipids and then 2.8. Determination of ferric reducing antioxidant power (FRAP)
extracted with ethyl acetate for five times. The ethyl acetate frac-
tion was evaporated to dryness at 45 °C. Phenolic compounds were FRAP was estimated following the method described by Benzie
reconstituted in 5 ml of 80% methanol and labeled as free pheno- and Strain (1996). Briefly, the FRAP reagent was prepared by blend-
lics, then stored at 40 °C until use. All analyses were performed ing 2.5 ml of 10 mM TPTZ solution (in 40 mM HCl) plus 2.5 ml of
in triplicate. 12.024 mM ferric chloride and 25 ml of 300 mM acetate buffer
(pH 3.6) at 37 °C. Then, 2.4 ml of sample extract or standard solu-
2.4. Extraction of soluble-conjugated phenolics tion was mixed with 0.1 ml of FRAP reagent. The mixture was incu-
bated at 37 °C for 10 min and the absorbance was measured at
Soluble-conjugated phenolics were extracted using the method 593 nm. Ferrous sulfate solutions were used to perform the cali-
previously reported by our laboratory (Adom & Liu, 2002, 2005). bration curve. The results were expressed as mg of ferrous sulfate
Soluble-conjugated phenolics in the aqueous phase after free phe- equiv. (FE)/100 g DW. Data were reported as mean ± SD for three
nolics extraction were digested with 10 ml of 2 M NaOH for 1 h replications.
under nitrogen at room temperature. Then the solution was neu-
tralized with concentrated HCl to pH 2. The mixture was extracted
with hexane to remove lipids and then extracted with ethyl acetate 2.9. Determination of peroxyl radical scavenging capacity (PSC)
for five times. The ethyl acetate fraction was evaporated to dryness
at 45 °C. Phenolic compounds were reconstituted in 5 ml of 80% PSC was determined using the assay developed by our labora-
methanol and stored at 40 °C until use. All analyses were per- tory (Adom & Liu, 2005) with modification (Wang, Chen, Guo,
formed in triplicate. Abbasi, & Liu, 2016). Briefly, 100 ll of vitamin C standard solutions
or sample extracts were mixed with 100 ll of DCFH, which was
2.5. Extraction of bound phenolics prepared by hydrolyzing 2.48 mM DCFH-DA with 1.0 mM KOH to
remove the diacetate moiety. Then 50 ll of ABAP was added to ini-
Bound phenolics were extracted according to the method tiate the reaction at 37 °C. The fluorescence at 485 nm excitation
reported previously by our laboratory (Adom & Liu, 2002; was measured every 2 min in a run of 40 min after emission at
Kremer Faller, Fialho, & Liu, 2012). Briefly, 0.5 g of sample were 538 nm in a Fluoroskan Ascent fluorescent spectrophotometer
extracted thrice with 10 ml of 80% chilled acetone and the super- (Thermo Labsystems, MA, USA). Results were expressed as mg of
natant was discarded. The residue was digested with 10 ml of vitamin C equiv. (VCE)/100 g DW. Data are reported as mean ± SD
2 M NaOH under the same conditions as the soluble-conjugated for three replications.
phenolics. After acidification and centrifugation, the mixture was
extracted with hexane to remove lipids and then with ethyl acet-
ate. The ethyl acetate fraction was evaporated at 45 °C to dryness. 2.10. Identification of phenolics in brown rice by ultra-high
Phenolic compounds were reconstituted in 5 ml of 80% methanol performance liquid chromatography coupled with electrospray
and stored at 40 °C until use. All analyses were performed in ionization quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-
triplicate. MS)

2.6. Determination of total phenolic content The chromatographic separation was carried out on an Agilent
1290 infinity series UPLC system (Agilent Technologies, Santa
TPC was measured using the Folin-Ciocalteu colorimetric Clara, CA, USA) as reported previously (Li et al., 2016). The reversed
method reported by Singleton, Orthofer, and Lamuela-Raventos phase column was Eclipse XDB-C18 (4.6  150 mm, 5 lm). The
(1999) and modified in our laboratory (Adom & Liu, 2002; Zhu mobile phase was made up of 0.1% formic acid solution (A) and
et al., 2015). Sample extracts or standard solutions were treated acetonitrile (B). The solvent gradient elution was as follows:
with Folin-Ciocalteu reagent for 6 min. Then the mixture was alka- 0–3 min, 10% B; 3–15 min, 10–15% B; 15–20 min, 15–22% B;
linized with Na2CO3. The blue colour of the solution was developed 20–30 min, 22–35%; 30–50 min, 35–45% B; 50–60 min, 45–100%
for 90 min, and the absorbance was measured at 760 nm using a B; 60–65 min, 100–10% B. 10 ll of sample extract was injected
UV–VIS spectrophotometer (Purkinje General Instrument Co., and eluted at a flow rate of 0.6 ml/min. MS was performed on an
Ltd., Beijing, China). The measurement was compared to standard Agilent 6538 Q-TOF Micromass (Agilent Technologies, Santa Clara,
curve of gallic acid and expressed as mg of gallic acid equiv. CA, USA) equipped with an orthogonal electrospray ionization
(GAE)/100 g dry weight (DW). Data were reported as mean ± SD source operating in negative mode. The optimal values of the
for three replications. source parameters were as follows: ion source temperature,
150 °C; desolvation temperature, 325 °C; capillary voltage, 3500
2.7. Determination of trolox equivalent antioxidant capacity (TEAC) V; cone voltage, 35 V; cone gas (N2) flow rate, 50 L/h; desolvation
gas (N2) flow rate, 900 L/h. The collision energies were set at 10, 15,
TEAC was determined using the ABTS radical cation decoloriza- 20 and 25 V for monomeric PAs, 15, 20, 25, and 30 V for diferulic
tion assay reported by Re et al. (1999). ABTS+ was prepared by acids (DFAs), and the fragmentor voltage was set at 120 V.
70 E.S. Gong et al. / Food Chemistry 232 (2017) 67–78

2.11. Determination of phenolics in brown rice and processed brown trans-8-50 DFA, trans-trans-8-50 benzofuran DFA and trans-cis-8-50
rice products by reversed-phase high performance liquid benzofuran DFA displayed the same typical fragment ions at m/z
chromatography (RP-HPLC)

The chromatographic separation was carried out on an Agilent


1260 infinity series HPLC system (Agilent Technologies, Santa
Clara, CA, USA). The chromatographic column was a Waters Sun-
Fire C18 column (4.6  250 mm, 5 lm) (Massachusetts, USA). The
mobile phase for separation of monomeric PAs consisted of 1.3%
acetic acid solution (A) and acetonitrile (B). Separation of phenolic
monomers: 0–2 min, 10% B; 2–20 min, 10–13% B; 20–35 min,
13–20% B; 35–45 min, 20–35% B; 45–80 min, 35–45% B;
80–93 min, 45–100% B; 93–98 min, 100–8% B; 98–105 min, 8% B.
The mobile phase for separation of DFAs consisted of 0.1% acetic
acid solution (A) and acetonitrile (B). Separation of DFAs:
0–1 min, 10% B; 1–3 min, 10–11% B; 3–12 min, 11–15% B;
12–17 min, 15–18% B; 17–22 min, 18–20% B; 22–30 min, 20–30%
B; 30–55 min, 30–40% B; 55–58 min, 40–100% B; 58–65 min,
100–10% B. The injection volume of sample extract was 20 ll and
the flow rate was 0.8 ml/min. The determination of PAs was carried
out by area measurements at 280 nm for hydroxybenzoic acid
derivatives and 320 nm for hydroxycinnamic acid derivatives.
Calibration curves for the cis-isomers of hydroxycinnamic acid
derivatives were calculated using different fresh trans-
hydroxycinnamic acid derivatives solutions that were placed under
the UV lamp overnight to ensure different trans/cis transformation
ratios according to Hernanz et al. (2001). Trans-FA was used for
quantification of DFAs according to Chiremba, Taylor, Rooney,
and Beta (2012). The recoveries of PAs are shown in Supplemen-
tary Table 1.

2.12. Statistical analysis

Differences in mean values were determined by ANOVA fol-


lowed by Tukey’s tests at p < 0.05 significance level using SPSS ver-
sion 18.0 (SPSS Inc., USA). Multivariate analysis was performed
using the software XLSTAT 2016 (Addinsoft, NY, USA), where the
principal component analysis (PCA) was applied to the data set
after normalization by Pearson’s correlation matrix.

3. Results and discussion

3.1. Identification of phenolic compounds in brown rice

Twelve monomeric PAs were identified by UPLC-Q-TOF-MS


with authentic standards: CHA, p-HA, trans-CFA, VA, SYA, cis-CFA,
trans-p-COA, cis-p-COA, trans-SIA, trans-FA, cis-SIA, and cis-FA
(Supplementary Table 2).
By plotting the molecular ion at m/z 385 ([MH]) expected
from DFA, seven peaks (peaks 13–19) were detected. Their RP-
HPLC chromatogram are shown in Fig. 1E. After comparing RP-
HPLC elution sequences, MS spectra, MS/MS spectra, and UV spec-
tra (Supplementary Fig. 1) with the literature data (Dobberstein &
Bunzel, 2010; Guo & Beta, 2013), the seven peaks were identified
as: trans-trans-8-50 DFA, trans-trans-5-50 DFA, trans-cis-5-50 DFA,
trans-trans-8-O-40 DFA, trans-trans-8-50 benzofuran DFA, trans-cis-
8-50 benzofuran DFA, and trans-cis-8-O-40 DFA (Fig. 2). Trans-

"
Fig. 1. HPLC chromatogram (280 nm) of phenolic acids standards (A), free fraction
(B), soluble-conjugated fraction (C), bound fraction (D), and DFAs (E) of brown rice:
p-hydroxybenzoic acid (1), chlorogenic acid (2), vanillic acid (3), trans-caffeic acid
(4), syringic acid (5), cis-caffeic acid (6), trans-p-coumaric acid (7), cis-p-coumaric
acid (8), trans-ferulic acid (9), trans-sinapic acid (10), cis-ferulic acid (11), cis-sinapic
acid (12), trans-trans-8-50 DFA (13), trans-trans-5-50 DFA (14), trans-cis-5-50 DFA
(15), trans-trans-8-O-40 DFA (16), trans-trans-8-50 benzofuran DFA (17), trans-cis-8-
50 benzofuran DFA (18), and trans-cis-8-O-40 DFA (19).
E.S. Gong et al. / Food Chemistry 232 (2017) 67–78 71

Fig. 2. MS/MS spectra of seven DFAs acquired at a collision energy of 20 eV: trans-trans-8-50 DFA (13), trans-trans-5-50 DFA (14), trans-cis-5-50 DFA (15), trans-trans-8-O-40
DFA (16), trans-trans-8-50 benzofuran DFA (17), trans-cis-8-50 benzofuran DFA (18), and trans-cis-8-O-40 DFA (19).
72 E.S. Gong et al. / Food Chemistry 232 (2017) 67–78

326, 311, 297, 282, and 267. The further identification of these observed along with an increase in the content of bound-PAs after
three DFAs was achieved by comparing UV spectra and the reten- cooking. Scaglioni et al. (2014) showed that the contents of both
tion times in RP-HPLC with literature data (Dobberstein & Bunzel, free and bound PAs of rice were reduced after hydrothermal treat-
2010; Guo & Beta, 2013). The identification of trans-trans-8-O-40 ment. These processing effects depended on the processing meth-
DFA was confirmed by the UV spectrum of 8-O-40 DFA standard ods applied, the type, and variety of whole grains analyzed. To our
(Dobberstein & Bunzel, 2010) and its typical fragment ion at m/z knowledge, there was no information on the effect of processing on
193 (Guo & Beta, 2013). The fragments at m/z 178, 149, and 134 the soluble-conjugated-PAs profiles of whole grain rice. The reduc-
of trans-trans-8-O-40 DFA were attributed to losses of –CH3, – tions of soluble-conjugated-PAs are likely due to the fact that pro-
COOH, and –CH3 plus –COOH from the typical fragment ion at cessing promotes the formation of strong complexes of the
m/z 193, respectively. Trans-cis-8-O-40 DFA, which is the cis- soluble-conjugated-PAs with macromolecules in the BR matrix,
isomer of trans-trans-8-O-40 DFA, exhibited the same fragment ions which may reduce their solubility in extraction solvent, thus
as trans-trans-8-O-40 DFA. It eluted after trans-trans-8-O-40 DFA in decreased their determined contents (Walter et al., 2013).
RP-HPLC (Fig. 1E). Trans-trans-5-50 DFA displayed typical fragment In BR and processed BR products, bound-PAs were predominant
ions at m/z 385, 370, 341, 326, 282, and 267, which were similar to forms (88.4–94.1% of total), followed by soluble-conjugated
those reported by Guo and Beta (2013). What’s more, the UV spec- (4.9–10.5%) and free PAs (1.1–1.8%), which was consistent with
trum of trans-trans-5-50 DFA in this study matched that of the previous studies (Adom & Liu, 2002; Okarter, Liu, Sorrells, & Liu,
trans-trans-5-50 DFA standard reported by Dobberstein and 2010). As reported in whole grains previously (Okarter et al.,
Bunzel (2010). Trans-cis-5-50 DFA, which is the cis-isomer of 2010), the most representative PAs in free and bound fractions
trans-trans-5-50 DFA, exhibited the same fragment ions as trans- were FA isomers and trans-p-COA. However, in the soluble-
trans-5-50 DFA. It eluted after trans-trans-5-50 DFA in RP-HPLC conjugated fraction, the most representative PAs were FA isomers
(Fig. 1E). and trans-SIA, which was inconsistent with the previous results
Most of DFAs reported previously are trans-trans forms of Okarter et al. (2010). In addition, other PAs, i.e., cis-p-COA,
(Dobberstein & Bunzel, 2010; Guo & Beta, 2013; Qiu et al., 2010), CHA, cis-SIA, p-HA, SYA, VA, trans-CFA, and cis-CFA were also
and only the cis-isomers of 8-50 , 8-50 benzofuran and 5-50 DFA detected in significant or small quantities in three fractions. In
had been identified by Bunzel and Steinhart (2004). Other DFA most cases, long phenolic extraction procedures, exposure to air
cis-isomers have not been reported previously in whole grains. In and light, or application of hydrothermal treatment would result
the present study, we successfully detected and identified another in chemical isomerization of PAs. However, most previous studies
DFA cis-isomer, i.e., trans-cis-8-O-40 DFA (Fig. 2, 19). had failed to detect cis-isomers of PAs using HPLC (Chiremba et al.,
2012; Guo & Beta, 2013; Qiu et al., 2010). Therefore, PAs in whole
3.2. Contents of phenolic acids in brown rice and processed brown rice grains will be significantly underestimated without including the
products cis-isomers. In the present study, a gradient elution procedure of
105 min for separation of PAs using RP-HPLC was developed in
The contents of PAs in BR and processed BR products are given our laboratory. After applying it, the cis-isomers of four hydrox-
in Table 1. The total content of free-PAs of BR (4.29 lmol/100 g ycinnamic acid derivatives (FA, p-COA, SIA, and CFA) were success-
DW) was significantly increased after processing (p < 0.05). The fully separated (Fig. 1A–D). Cis-isomers of PAs were detected in
values for processed BR products ranged from 5.17 (textured rice) significant amounts when compared to their corresponding
to 6.83 lmol/100 g (rice noodle). Processing significantly trans-isomers (Table 1). The total contents of FA, p-COA, SIA, and
decreased the total content of soluble-conjugated-PAs CFA in BR and processed BR products would be significantly under-
(39.29 lmol/100 g). The values for processed BR products ranged estimated by 17.2–28.2%, 17.9–22.4%, 24.4–35.5%, and 19.5% with-
from 21.71 (textured rice) to 26.27 lmol/100 g (rice noodle). How- out including the cis-isomers. In addition, the contents of bound
ever, the total content of bound-PAs (316.52 lmol/100 g) was sig- cis-FA, cis-COA, and cis-SIA in cooked rice were higher than those
nificantly increased after IECT and cooking (p < 0.05), but not in textured rice and rice noodle (p < 0.05) (Table 1). It suggests that
significantly changed after RNE (p > 0.05). The values for processed the cooking treatment may be more effective in promoting the iso-
BR products ranged from 303.45 (rice noodle) to merization of these bound hydroxycinnamic acid derivatives.
407.59 lmol/100 g (textured rice). The total content of PAs Since ultraviolet absorbance of trans-SIA was much lower than
(360.10 lmol/100 g) was significantly increased after IECT and that of trans-FA at the same concentration (data not shown), and
cooking (p < 0.05), but not significantly changed after RNE the polarity of trans-SIA was closer to that of trans-FA in RP-
(p > 0.05). The values for processed BR products ranged from HPLC, previous studies did not detect it in whole grains
336.55 (rice noodle) to 434.47 lmol/100 g (textured rice). (Chiremba Rooney, & Beta, 2012; Fares et al., 2010; Scaglioni
To date, information on the effects of IECT, cooking, and RNE on et al., 2014). In this study, we successfully separated it from
the phytochemical profiles and AOA in free, soluble-conjugated trans-FA, and separated its cis-isomer from cis-FA in RP-HPLC
and bound fractions of BR was rare, only a few studies have inves- (Fig. 1A–D). In addition, the content of bound trans-SIA was dra-
tigated the effect of processing on the phenolic profiles of whole matically increased by 277.6–407.8% after processing, suggesting
grains. Zielinski, Kozlowska, and Lewczuk (2001) found increases that proper processing could be used to promote the release of
in the contents of free-PAs and bound-PAs of four grains (wheat, bound trans-SIA in whole grains.
barley, rye and oat) after processing. Dewanto et al. (2002) and DFAs were only detected in the bound fraction, with trans-
de la Parra, Saldivar, and Liu (2007) observed increases in the con- trans-8-O-40 , trans-trans-5-50 , trans-trans-8-50 benzofuran, and
tents of free and conjugated FA, and a decrease in the content of trans-trans-8-50 DFA being abundant DFAs, which was consistent
bound FA in corn after thermal processing. Fares, Platani, Baiano, with previous studies (Dobberstein & Bunzel, 2010; Guo & Beta,
and Menga (2010) analyzed the free and bound PAs in pasta, and 2013; Qiu et al., 2010). However, the other three DFAs, trans-cis-
found that cooking increased the content of bound-PAs, but did 5-50 , trans-cis-8-O-40 and trans–cis 8-50 benzofuran DFA were not
not affect the content of free-PAs. N’Dri et al. (2013) found that, reported in these studies. The most abundant DFA in BR and pro-
in sorghum, the content of free-PAs increased, whereas that of cessed BR products was trans-trans-8-O-40 DFA, followed by
bound-PAs decreased after cooking; in millet, cooking increased trans-trans-5-50 , trans-trans-8-50 benzofuran, trans-cis-5-50 , trans-
the content of free-PAs without affecting that of bound-PAs; cis-8-O-40 , trans-trans-8-50 and trans-cis-8-50 benzofuran DFA. The
whereas in fonio, a decrease in the content of free-PAs was sequence of the DFA contents was not the same in different whole
E.S. Gong et al. / Food Chemistry 232 (2017) 67–78 73

Table 1
The composition of phenolic acids in brown rice and processed brown rice products (lg/g DW).a

Phenolic acid Free Soluble-conjugated Bound Total


Trans-ferulic acid
Brown rice 3.04 ± 0.07c (0.9) 30.41 ± 0.75a (8.6) 320.70 ± 12.55bc (90.6) 354.15 ± 12.83bc
Textured rice 4.09 ± 0.07b (0.9) "34.6 13.19 ± 0.82c (3.0) ;56.6 417.59 ± 17.77a (96.0) "30.2 434.87 ± 18.56a "22.8
Cooked rice 2.38 ± 0.10d (0.7) ;21.7 12.25 ± 0.93c (3.4) ;59.7 346.74 ± 10.49b (96.0) "8.1 361.36 ± 9.82b "2.0
Rice noodle 5.22 ± 0.22a (1.6) "71.8 17.31 ± 0.58b (5.3) ;43.1 301.08 ± 10.36c (93.0) ;6.1 323.61 ± 9.88c ;8.6
Trans-p-coumaric acid
Brown rice 1.37 ± 0.12d (1.4) 7.29 ± 0.16a (7.5) 88.34 ± 5.15b (91.1) 97.01 ± 4.97b
Textured rice 1.78 ± 0.07c (1.6) "29.8 5.98 ± 0.30b (5.2) ;18.0 106.42 ± 6.14a (93.2) "20.5 114.18 ± 6.07a "17.7
Cooked rice 3.69 ± 0.13a (3.2) "168.8 7.40 ± 0.37a (6.3) "1.5 105.58 ± 4.76a (90.5) "19.5 116.67 ± 5.07a "20.3
Rice noodle 2.42 ± 0.17b (2.7) "76.4 7.14 ± 0.66a (7.8) ;2.0 81.60 ± 6.34b (89.5) ;7.6 91.16 ± 5.95b ;6.0
Cis-ferulic acid
Brown rice 0.97 ± 0.08b (1.1) 7.33 ± 0.27a (8.2) 80.66 ± 4.55b (90.7) 88.95 ± 4.69b
Textured rice 1.22 ± 0.09ab (1.3) "26.8 4.35 ± 0.28c (4.8) ;40.7 85.04 ± 8.07b (93.8) "5.4 90.62 ± 8.03b "1.9
Cooked rice 0.54 ± 0.07c (0.4) ;44.1 4.11 ± 0.20c (2.9) ;44.0 137.06 ± 11.20a (96.7) "69.9 141.70 ± 11.07a "59.3
Rice noodle 1.44 ± 0.23a (1.8) "48.8 5.23 ± 0.27b (6.6) ;28.6 72.04 ± 3.11b (91.5) ;10.7 78.71 ± 3.57b ;11.5
Trans-trans-8-O-40 DFAb
Brown rice NDc ND 33.68 ± 1.15b (100) 33.68 ± 1.15b
Textured rice ND ND 38.32 ± 1.93a (100) "13.8 38.32 ± 1.93a "13.8
Cooked rice ND ND 33.58 ± 1.21b (100) ;0.3 33.58 ± 1.21b ;0.3
Rice noodle ND ND 22.83 ± 1.25c (100) ;32.2 22.83 ± 1.25c ;32.2
Cis-p-coumaric acid
Brown rice 0.14 ± 0.02b (0.6) 1.08 ± 0.12b (4.6) 22.40 ± 1.17b (94.8) 23.62 ± 1.29b
Textured rice 0.15 ± 0.02b (0.6) "10.5 1.20 ± 0.06b (4.8) "10.7 23.60 ± 0.94b (94.6) "5.4 24.95 ± 1.01b "5.6
Cooked rice 0.37 ± 0.04a (1.1) "171.8 1.88 ± 0.11a (5.6) "73.7 31.40 ± 1.68a (93.3) "40.2 33.65 ± 1.58a "42.5
Rice noodle 0.19 ± 0.01b (0.8) "39.5 1.87 ± 0.09a (7.6) "73.3 22.54 ± 1.25b (91.6) "0.6 24.60 ± 1.21b "4.2
Trans-sinapic acid
Brown rice 0.33 ± 0.01c (1.5) 13.99 ± 0.28a (61.2) 8.55 ± 0.94c (37.4) 22.86 ± 0.74c
Textured rice 0.43 ± 0.03b (0.8) "30.3 8.92 ± 0.31b (16.9) ;36.2 43.40 ± 2.48a (82.3) "407.8 52.75 ± 2.69a "130.7
Cooked rice 0.41 ± 0.03b (1.0) "24.4 9.95 ± 0.63b (23.3) ;28.9 32.27 ± 1.69b (75.7) "277.6 42.63 ± 2.11b "86.5
Rice noodle 0.61 ± 0.03a (1.4) "83.3 9.29 ± 0.33b (21.8) ;33.6 32.61 ± 1.37b (76.7) "281.7 42.52 ± 1.70b "86.0
Chlorogenic acid
Brown rice ND 4.39 ± 0.36a (24.6) 13.45 ± 0.85b (75.4) 17.85 ± 1.03ab
Textured rice ND 2.97 ± 0.12b (15.9) ;32.3 15.70 ± 0.82a (84.1) "16.7 18.67 ± 0.72a "4.6
Cooked rice ND 2.62 ± 0.19b (13.5) ;40.3 16.77 ± 0.12a (86.5) "24.7 19.40 ± 0.26a "8.7
Rice noodle ND 2.79 ± 0.13b (17.1) ;36.5 13.57 ± 0.94b (82.9) "0.8 16.35 ± 0.81b ;8.4
Trans-trans-5-50 DFA
Brown rice ND ND 16.37 ± 0.23b (100) 16.37 ± 0.23b
Textured rice ND ND 19.58 ± 0.80a (100) "19.7 19.58 ± 0.80a "19.7
Cooked rice ND ND 15.07 ± 0.25c (100) ;7.9 15.07 ± 0.25c ;7.9
Rice noodle ND ND 12.54 ± 0.42d (100) ;23.4 12.54 ± 0.42d ;23.4
Trans-trans-8-50 benzofuran DFA
Brown rice ND ND 15.49 ± 0.77a (100) 15.49 ± 0.77a
Textured rice ND ND 17.13 ± 0.81a (100) "10.6 17.13 ± 0.81a "10.6
Cooked rice ND ND 8.76 ± 0.50c (100) ;43.4 8.76 ± 0.50c ;43.4
Rice noodle ND ND 10.67 ± 0.51b (100) ;31.1 10.67 ± 0.51b ;31.1
Cis-sinapic acid
Brown rice 0.19 ± 0.01b (1.5) 5.17 ± 0.19a (41.1) 7.23 ± 0.55c (57.4) 12.59 ± 0.70c
Textured rice 0.22 ± 0.03b (1.3) "14.5 3.57 ± 0.34c (21.0) ;31.1 13.22 ± 0.92b (77.8) "83.0 17.01 ± 0.97b "35.1
Cooked rice 0.24 ± 0.02b (1.1) "26.0 4.28 ± 0.30b (20.2) ;17.2 16.63 ± 1.04a (78.6) "130.1 21.15 ± 1.32a "68.0
Rice noodle 0.37 ± 0.02a (2.4) "93.3 3.42 ± 0.22c (22.1) ;33.9 11.68 ± 0.77b (75.5) "61.6 15.46 ± 0.87b "22.8
Trans-cis-5-50 DFA
Brown rice ND ND 11.09 ± 0.44a (100) 11.09 ± 0.44a
Textured rice ND ND 12.17 ± 0.91a (100) "9.7 12.17 ± 0.91a "9.7
Cooked rice ND ND 12.30 ± 0.71a (100) "10.9 12.30 ± 0.71a "10.9
Rice noodle ND ND 7.53 ± 0.52b (100) ;32.1 7.53 ± 0.52b ;32.1
Trans-cis-8-O-40 DFA
Brown rice ND ND 10.60 ± 0.89b (100) 10.60 ± 0.89b
Textured rice ND ND 14.44 ± 0.82a (100) "36.2 14.44 ± 0.82a "36.2
Cooked rice ND ND 5.70 ± 0.43c (100) ;46.2 5.70 ± 0.43c ;46.2
Rice noodle ND ND 2.21 ± 0.06d (100) ;79.1 2.21 ± 0.06d ;79.1
P-hydroxybenzoic acid
Brown rice 0.54 ± 0.05b (6.5) 4.85 ± 0.43a (57.5) 3.03 ± 0.16c (36.0) 8.43 ± 0.25a
Textured rice 0.53 ± 0.04b (5.5) ;2.9 1.57 ± 0.19c (16.4) ;67.6 7.51 ± 0.64a (78.1) "147.4 9.61 ± 0.85ab "14.0
Cooked rice 0.60 ± 0.06b (5.5) "10.3 2.04 ± 0.14bc (18.7) ;57.9 8.28 ± 0.77a (75.8) "173.0 10.92 ± 0.64a "29.6
Rice noodle 0.86 ± 0.07a (10.4) "58.5 2.39 ± 0.16b (28.9) ;50.6 5.03 ± 0.31b (60.7) "65.7 8.28 ± 0.51a ;1.7
Syringic acid
Brown rice 0.94 ± 0.02b (13.1) 1.85 ± 0.11a (25.8) 4.38 ± 0.30c (61.1) 7.17 ± 0.35b
Textured rice 0.64 ± 0.01d (8.8) ;31.3 0.78 ± 0.02d (10.6) ;57.8 5.92 ± 0.11b (80.6) "35.4 7.35 ± 0.11b "2.6

(continued on next page)


74 E.S. Gong et al. / Food Chemistry 232 (2017) 67–78

Table 1 (continued)

Phenolic acid Free Soluble-conjugated Bound Total


Cooked rice 2.01 ± 0.04a (18.8) "114.6 1.48 ± 0.10b (13.9) ;20.0 7.19 ± 0.71a (67.3) "64.3 10.68 ± 0.77a "49.1
Rice noodle 0.80 ± 0.05c (12.9) ;14.7 1.06 ± 0.04c (17.0) ;43.0 4.35 ± 0.16c (70.1) ;0.6 6.21 ± 0.21b ;13.4
Trans-trans-8-50 DFA
Brown rice ND ND 6.90 ± 0.43bc (100) 6.90 ± 0.43bc
Textured rice ND ND 8.03 ± 0.60b (100) "16.4 8.03 ± 0.60b "16.4
Cooked rice ND ND 11.08 ± 1.03a (100) "60.7 11.08 ± 1.03a "60.7
Rice noodle ND ND 6.14 ± 0.15c (100) ;11.0 6.14 ± 0.15c ;11.0
Trans-cis-8-50 benzofuran DFA
Brown rice ND ND 5.42 ± 0.41ab (100) 5.42 ± 0.41ab
Textured rice ND ND 5.24 ± 0.45ab (100) ;3.4 5.24 ± 0.45ab ;3.4
Cooked rice ND ND 5.99 ± 0.55a (100) "10.6 5.99 ± 0.55a "10.6
Rice noodle ND ND 4.37 ± 0.27b (100) ;19.4 4.37 ± 0.27b ;19.4
Vanillic acid
Brown rice 0.35 ± 0.02b (12.9) 0.89 ± 0.09a (32.7) 1.48 ± 0.12c (54.4) 2.72 ± 0.16c
Textured rice 0.43 ± 0.04b (12.7) "22.9 0.62 ± 0.02c (18.1) ;30.4 2.36 ± 0.08b (69.2) "59.7 3.41 ± 0.04b "25.5
Cooked rice 0.44 ± 0.05b (10.1) "24.7 0.61 ± 0.04c (14.0) ;31.8 3.29 ± 0.20a (75.9) "123.0 4.34 ± 0.25a "59.7
Rice noodle 0.59 ± 0.06a (18.4) "67.0 0.75 ± 0.01b (23.7) ;15.1 1.84 ± 0.17c (57.9) "24.6 3.18 ± 0.21bc "17.1
Trans-caffeic acid
Brown rice ND ND 0.70 ± 0.05a (100) 0.70 ± 0.05a
Textured rice ND ND 0.51 ± 0.02b (100) ;27.9 0.51 ± 0.02b ;27.9
Cooked rice ND ND 0.31 ± 0.02c (100) ;56.4 0.31 ± 0.02c ;56.4
Rice noodle ND ND 0.35 ± 0.01c (100) ;50.6 0.35 ± 0.01c ;50.6
Cis-caffeic acid
Brown rice ND ND 0.17 ± 0.02 (100) 0.17 ± 0.02
Textured rice ND ND ND ND
Cooked rice ND ND ND ND
Rice noodle ND ND ND ND
Total phenolic acids (lmol/100 g DW)d
Brown rice 4.29 ± 0.22c (1.2) 39.29 ± 1.39a (10.9) 316.52 ± 14.87b (87.9) 360.10 ± 16.48b
Textured rice 5.17 ± 0.21b (1.2) "20.5 21.71 ± 1.19c (5.0) ;52.4 407.59 ± 18.83a (93.8) "28.8 434.47 ± 18.24a "20.7
Cooked rice 5.98 ± 0.30b (1.4) "39.3 23.74 ± 1.45bc (5.6) ;48.7 394.83 ± 18.05a (93.0) "24.7 424.55 ± 19.78a "17.9
Rice noodle 6.83 ± 0.45a (2.0) "59.2 26.27 ± 1.26b (7.8) ;28.5 303.45 ± 13.37b (90.2) ;4.1 336.55 ± 15.06b ;6.5
a
Results are expressed as mean ± SD (n = 3). Percent contribution to total phenolic acid content is in parentheses. Numbers with (") or (;) indicate percentage increase or
decrease when compared to the control. Values of each column with no letters in common are significantly different (p < 0.05). bDFA: diferulic acid. cND = not detected. dTotal
amounts of phenolic acids in each fraction are expressed as lmol/100 g DW due to the differences in the molecule weights of phenolic acids.

grains (Dobberstein & Bunzel, 2010; Guo & Beta, 2013). The total 3.3. Total phenolic content
content of DFAs was slightly increased by 15.4% after IECT
(p < 0.05), whereas the content was not changed after cooking The free, soluble-conjugated, bound TPCs of BR and processed
(p > 0.05), but significantly decreased by 33.4% after RNE BR products are shown in Table 2. The free-TPC of BR (15.80 mg
(p < 0.05). To conclude, limited data were available on the effect GAE/100 g DW) significantly dropped after processing (p < 0.05).
of processing on the DFAs in whole grains. Zaupa et al. (2015) The values for processed BR products ranged from 11.15 (rice
found that two cooking techniques (boiling and risotto) decreased noodle) to 13.78 mg GAE/100 g (cooked rice). The soluble-
the contents of DFAs in red, black, and white rice, except that boil- conjugated-TPC (31.25 mg GAE/100 g) was significantly
ing did not affect those in white rice. decreased after processing (p < 0.05). The values for processed
Processed BR products had high contents of PAs BR products ranged from 9.72 (rice noodle) to 13.61 mg
(336.55–434.47 lmol/100 g DW) with bound-PAs being the GAE/100 g (cooked rice). The bound-TPC (85.24 mg GAE/100 g)
predominant forms (88.4–94.1% of total), followed by soluble- was significantly increased after IECT and cooking (p < 0.05),
conjugated (4.9–10.5%) and free PAs (1.1–1.8%). The potential but not affected after RNE (p > 0.05). The values for processed
health benefits of PAs are mostly related to their antioxidant activ- BR products ranged from 85.85 (rice noodle) to 106.41 mg
ity. Beyond the antioxidant activity, other biological activities of GAE/100 g (textured rice). The total-TPC (132.29 mg GAE/100 g)
PAs, such as prevention of cardiovascular diseases, anticancer was not significantly changed after IECT and cooking (p > 0.05),
activity, protection against side effects of chemotherapy, antimi- but significantly decreased after RNE (p < 0.05). The values for
crobial activity, anti-osteoclast activity, anti-inflammatory and processed BR products ranged from 106.72 (rice noodle) to
analgesic activities had also been reported (El-Seedi et al., 2012). 129.65 mg GAE/100 g (textured rice).

Table 2
Total phenolic contents (TPC) of brown rice and processed brown rice products.a

Sample TPC (mg gallic acid equiv./100 g DW)


Free Soluble-conjugated Bound Total
Brown rice 15.80 ± 0.63a (11.9) 31.25 ± 1.53a (23.6) 85.24 ± 4.06b (64.4) 132.29 ± 6.19a
Textured rice 11.71 ± 0.45c (9.1) ;25.9 11.14 ± 0.60c (8.6) ;64.3 106.41 ± 6.51a (82.3) "24.8 129.26 ± 7.48a ;2.3
Cooked rice 13.78 ± 0.48b (10.6) ;12.8 13.61 ± 0.59b (10.5) ;56.4 102.26 ± 4.25a (78.9) "20.0 129.65 ± 5.29a ;2.0
Rice noodle 11.15 ± 0.34c (10.4) ;29.4 9.72 ± 0.51c (9.1) ;68.9 85.85 ± 2.63b (80.4) "0.7 106.72 ± 3.48b ;19.3
a
Results are expressed as mean ± SD (n = 3). Percent contribution of each fraction to the total value is in parentheses. Numbers with (") or (;) indicate percentage increase or
decrease when compared to the control in each column. Values of each column with no letters in common are significantly different (p < 0.05).
E.S. Gong et al. / Food Chemistry 232 (2017) 67–78 75

The free-TPC of BR in the present study (13.77 mg GAE/100 g stomach and small intestine, whereas soluble-conjugated pheno-
FW) was lower than that of the rice (35.73 mg GAE/100 g FW) lics are released by mucosa cells cinnamoyl esterases and absorbed
reported by Adom and Liu (2002). It may be attributed to differ- in the small intestine, then these two forms would distribute
ences in the extraction method and varieties used. The Folin- throughout the body with health benefits, such as inhibition activ-
Ciocalteu reagent used for quantification of TPC can interact with ities against oxidation of LDL cholesterol and liposomes
a number of reducing nonphenolic substances, such as amino (Chandrasekara & Shahidi, 2011). Bound phenolics would survive
acids, certain reducing sugars, vitamin C, and other reducing com- stomach and intestinal digestion and reach the colon, thus exhibit-
pounds in crude extracts, and thus may lead to overestimation of ing health benefits in the prevention of colon cancer and other
TPC. Therefore, an additional ethyl acetate extraction (mainly for digestive cancers (Adom & Liu, 2002). In the present study, pro-
purifying phenolics) was applied to the crude extracts in the pre- cessed BR products contained significant amounts of free phenolics
sent study, but it was not included in their study. However, the (11.15–13.78 mg gallic acid equiv./100 g DW) and soluble-
bound-TPC of BR (74.26 mg GAE/100 g FW) was higher than that conjugated phenolics (9.72–13.61 mg gallic acid equiv./100 g
of rice (58.86 mg GAE/100 g FW) in their study due to varietal dif- DW), which may be released and absorbed in the stomach and
ference. Soluble-conjugated-TPC was not reported in their study. small intestine and exhibit inhibition activities against oxidation
The reductions in free-TPC of BR in the present study were congru- of LDL cholesterol and liposomes. Processed BR products had large
ent with the results of N’Dri et al. (2013), Massaretto et al. (2011) amounts of bound phenolics (85.85–106.41 mg gallic acid
and Scaglioni et al. (2014). To our knowledge, there are no reports equiv./100 g DW), which may be released by colon fermentation
on the effect of processing on the soluble-conjugated-TPC of rice. and exhibit health benefits in the prevention of cancer (Liu, 2007).
The decreases in soluble-conjugated-TPC is likely attributed to
the fact that processing can promote the formation of strong com- 3.4. Antioxidant activity
plexes of the soluble-conjugated phenolics with macromolecules
in the BR matrix. These decreases in soluble-conjugated-TPC were Three antioxidant assays (TEAC, FRAP, and PSC) were used to
consistent with the decreases in soluble-conjugated-PAs in BR. The determine the AOA in the present study. The free, soluble-
increases in bound-TPC of BR after IECT and cooking were in con- conjugated, bound AOAs of BR and processed BR products are
sistence with the result of Massaretto et al. (2011). These changes presented in Table 3. The free-TEAC of BR (30.05 TE/100 g DW)
in bound-TPCs were consistent with those of bound-PAs in BR. was significantly decreased after processing (p < 0.05). The values
However, RNE did not affect the bound-TPC of BR. It is likely due for processed BR products ranged from 18.75 (rice noodle) to
to that IECT and cooking might have allowed a more effective 23.53 mg TE/100 g (cooked rice). The soluble-conjugated-TEAC
extraction of bound phenolics under alkaline conditions through (62.61 TE/100 g) was significantly decreased after processing
destruction of the cell wall matrix, but RNE did not achieve that (p < 0.05). The values for processed BR products ranged from 35.36
because of its momentary treatment (10 s). (rice noodle) to 44.71 mg TE/100 g (cooked rice). However, bound-
Several studies on whole grain rice phenolics did not include TEAC (229.64 mg TE/100 g) was significantly increased after IECT
the determination of bound forms (Huang & Ng, 2012; Shao, and cooking (p < 0.05), but not significantly affected after RNE
Zhang, & Bao, 2011). Our laboratory developed a method to deter- (p > 0.05). Bound-TEACs of processed BR products ranged from
mine the complete phenolic profiles of whole grains, which 241.77 (rice noodle) to 280.97 mg TE/100 g (textured rice). The
included both free and bound forms (Adom & Liu, 2002). Whole total-TEAC of BR (322.31 mg TE/100 g) was not significantly affected
grain rice phenolics will be significantly underestimated without after processing (p > 0.05). The values of processed BR products ran-
accounting for bound forms. In the present study, the free and ged from 295.88 (rice noodle) to 340.87 mg TE/100 g (textured rice).
soluble-conjugated phenolics of BR and processed BR products The free-FRAP of BR (25.78 mg FE/100 g DW) significantly
contributed 9.1–11.9% and 8.6–23.6% to total-TPCs, respectively, dropped after processing (p < 0.05). The values for processed BR
while bound phenolics contributed 64.4–82.3%. Therefore, total- products ranged from 6.80 (rice noodle) to 9.24 mg FE/100 g
TPCs of BR and processed BR products would be underestimated (cooked rice). The soluble-conjugated-FRAP (38.34 mg FE/100 g)
by 64.4–82.3% without including bound phenolics. This is consis- was also significantly decreased after processing (p < 0.05). The
tence with the results reported by Adom and Liu (2002). values for processed BR products ranged from 7.77 (rice noodle)
As suggested by Adom and Liu (2002) and Acosta-Estrada et al. to 14.59 mg FE/100 g (cooked rice). The bound-FRAP (99.97 mg
(2014), free phenolics are rapidly released and absorbed in the FE/100 g) was significantly increased after IECT and cooking

Table 3
Antioxidant activities of brown rice and processed brown rice products.a

Free Soluble-conjugated Bound Total


Trolox equivalent antioxidant capacity (TEAC) (mg Trolox equiv./100 g DW)
Brown rice 30.05 ± 1.34a (9.3) 62.61 ± 2.12a (19.4) 229.64 ± 7.43c (71.2) 322.31 ± 8.30ab
Textured rice 19.05 ± 0.94c (5.6) ;36.6 40.85 ± 1.59b (12.0) ;34.8 280.97 ± 13.91a (82.4) "22.4 340.87 ± 16.45a "5.8
Cooked rice 23.53 ± 0.76b (7.1) ;21.7 44.71 ± 1.41b (13.4) ;28.6 264.48 ± 8.51ab (79.5) "15.2 332.73 ± 10.68a "3.2
Rice noodle 18.75 ± 0.82c (6.3) ;37.6 35.36 ± 1.05c (11.9) ;43.5 241.77 ± 7.04bc (81.7) "5.3 295.88 ± 8.91b ;8.2
Ferric reducing antioxidant power (FRAP) (mg ferrous sulfate equiv./100 g DW)
Brown rice 25.78 ± 0.81a (15.7) 38.34 ± 1.49a (23.4) 99.97 ± 3.55b (60.9) 164.08 ± 5.83a
Textured rice 7.16 ± 0.14c (5.1) ;72.2 10.48 ± 0.31c (7.5) ;72.7 122.70 ± 5.94a (87.4) "22.7 140.35 ± 6.39b ;14.5
Cooked rice 9.24 ± 0.21b (6.8) ;64.2 14.59 ± 0.43b (10.7) ;61.9 112.80 ± 3.93a (82.6) "12.8 136.63 ± 4.57b ;16.7
Rice noodle 6.80 ± 0.13c (6.0) ;73.6 7.77 ± 0.21d (6.8) ;79.7 98.97 ± 3.03b (87.2) ;1.0 113.53 ± 3.37c ;30.8
Peroxyl radical scavenging capacity (PSC) (mg vitamin C equiv./100 g DW)
Brown rice 5.03 ± 0.17a (13.5) 8.76 ± 0.28a (23.5) 23.45 ± 0.67b (63.0) 37.24 ± 0.84a
Textured rice 3.61 ± 0.09c (10.0) ;28.2 3.20 ± 0.07c (8.9) ;63.4 29.01 ± 1.34a (80.5) "23.7 36.02 ± 1.50a ;3.3
Cooked rice 4.18 ± 0.09b (11.8) ;16.9 4.11 ± 0.12b (11.6) ;53.1 27.12 ± 1.01a (76.6) "15.6 35.41 ± 1.23a ;4.9
Rice noodle 3.38 ± 0.10c (11.3) ;32.7 2.90 ± 0.05d (9.7) ;66.9 23.58 ± 0.48b (79.0) "0.5 29.86 ± 0.62c ;19.8
a
Results are expressed as mean ± SD (n = 3). Percent contribution of each fraction to total value is in parentheses. Numbers with (") or (;) indicate percentage increase or
decrease when compared to the control. Values of each column with no letters in common are significantly different (p < 0.05).
76 E.S. Gong et al. / Food Chemistry 232 (2017) 67–78

(p < 0.05), but not significantly affected after RNE (p > 0.05). The
values for processed BR products ranged from 98.97 (rice noodle)
to 122.70 mg FE/100 g (textured rice). The total-FRAP (164.08 mg
FE/100 g) was significantly decreased after processing (p < 0.05).
The values for processed BR products ranged from 113.53 (rice
noodle) to 140.35 mg FE/100 g (textured rice).
The free-PSC of BR (5.03 mg VCE/100 g DW) was significantly
decreased after processing (p < 0.05). The values for processed BR
products ranged from 3.38 (rice noodle) to 4.18 mg VCE/100 g
(cooked rice). The soluble-conjugated-PSC (8.76 mg VCE/100 g)
was also significantly decreased after processing (p < 0.05). The
values for processed BR products ranged from 2.90 (rice noodle)
to 4.11 mg VCE/100 g (cooked rice). The bound-PSC (23.45 mg
VCE/100 g) was significantly increased after IECT and cooking
(p < 0.05), but not significantly affected after RNE (p > 0.05). The
values for processed BR products ranged from 23.58 (rice noodle)
to 29.01 mg VCE/100 g (textured rice). The total-PSC (37.24 mg
VCE/100 g) was not significantly affected after IECT and cooking
(p > 0.05), but decreased after RNE (p < 0.05). The values for pro-
cessed BR products ranged from 29.86 (rice noodle) to 36.02 mg
VCE/100 g (textured rice).
Processing significantly affected AOA of BR, which was similar to
the effects on TPC. There are significant correlations between TPC and
TEAC (r = 0.995, p < 0.01), PA and TEAC (r = 0.995, p < 0.01), TPC and
FRAP (r = 0.996, p < 0.01), PA and FRAP (r = 0.984, p < 0.01), TPC and
PSC (r = 0.999, p < 0.01), PA and PSC (r = 0.992, p < 0.01), indicating
that phenolic compounds were the main components responsible
for the AOAs of BR and processed BR products. To date, limited
reports have studied the processing effects on AOA of rice. Previous
studies showed that processing can increase or decrease the AOA of
foods depending on the nature and molecular structure of the antiox-
idant compound, processing methods, food matrix, and the interac-
tion of antioxidants with other components (Fares et al., 2010;
Walter et al., 2013; Wu et al., 2004). The decreases of free-AOAs in
the present study were in agreement with the results of N’Dri et al.
(2013), who found that the free-AOAs (using TEAC assay) of three
African cereals (fonio, millet, and sorghum) were significantly
reduced by 90, 68, and 76% after cooking, respectively. However,
the increases in bound-AOAs were not congruent with those in their
study, where bound-AOAs were significantly decreased after cooking.
The decreases were mainly caused by the decreases of bound-TPCs in
their study. The changes in AOAs after processing in our study were
inconsistent with those reported by de la Parra et al. (2007), who
reported that free-AOAs (using PSC assay) of corns were increased,
and bound-AOAs were decreased after processing (thermal-alkaline
treatment). They suggested that it was probably attributed to the
increased soluble phenolics from bound phenolics. The inconsistency
is mainly caused by differences in the composition of whole grain and Fig. 3. Scores plot (A) and loading plot (B) of PCA of the three fractions of BR and
processing methods. To date, there is no report on the effect of pro- processed BR products. The labels of the samples BR, TR, CR, and RN refer to brown
rice, textured rice, cooked rice, and rice noodle, respectively; the labels of the
cessing on the soluble-conjugated-AOA of rice. The reductions in
fractions f, sc, and b refer to free, soluble-conjugated, and bound fractions,
soluble-conjugated-AOAs after processing were related to the respectively. The labels of evaluated parameters refer to Table 2, Table 3 and Fig. 1.
decreases in corresponding TPCs.
Oxidative stress is involved in aging and the pathology of a wide
range of chronic diseases (Ames & Gold, 1991). Dietary antioxi- noodle and textured rice, could be valuable as functional foods to
dants may combat oxidative stress in the body to maintain a bal- promote consumer health.
ance between oxidants and antioxidants, thus protecting the In rice milling, the bran layers and germ fraction removed dur-
body’s cells from damage caused by reactive oxygen species and ing polishing are high in fibres, vitamins, minerals, phenolics, and
free radicals. A diet rich in natural antioxidants, such as PAs, can other bioactive compounds, which are of a lot of health benefits
thus significantly increase the reactive antioxidant potential of (Okarter & Liu, 2010). Consuming whole grain rice, instead of pol-
the organism and thereby decrease the risk of oxidative stress- ished rice in diet, could maximally preserve the health benefits of
related diseases (Liu, 2007). In addition to antioxidant activity, a rice. Processing had significant effects on health benefits of rice. It
number of other bioactivities of rice have also been reported, such was found that some processing methods, such as germination
as antimicrobial, antiviral, anti-inflammatory, anticancer and (Patil & Khan, 2011) and fermentation (Liu et al., 2017), had poten-
immunomodulatory activities (Hudson et al., 2000; Niu et al., tial to enhance the health benefits of rice. The present study
2013; Ray et al., 2013; Verschoyle et al., 2007; Zhang et al., revealed that cooking and IECT could significantly increase the
2014). Therefore, brown rice products, such as cooked rice, rice content of bound phenolics of BR, thus may enhance health
E.S. Gong et al. / Food Chemistry 232 (2017) 67–78 77

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