Food Chemistry 157 (2014) 457–463

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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

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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 FeCl3
6H2O (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 FeSO4
7H2O 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% AlCl3
6H2O 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).

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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-

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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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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

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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.

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