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Journal of Cereal Science xxx (2014) 1e6

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Journal of Cereal Science

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Hydrothermal and biotechnological treatments on nutraceuticalcontent and antioxidant activity of rice bran

P.M. Pradeep, A. Jayadeep*, Manisha Guha, Vasudeva SinghDepartment of Grain Science and Technology, CSIR e Central Food Technological Research Institute, Mysore 570 020, India

a r t i c l e i n f o

Article history:Received 3 June 2013Received in revised form21 December 2013Accepted 30 January 2014

Keywords:Rice branHydrothermal treatmentNutraceuticalsAntioxidant activity

Abbreviations: DPPHþ, 2, 2-diphenyl-1-picrylhydantioxidant power; HPLC, high performance liquid chradical absorbance capacity.* Corresponding author. Tel.: þ91 821 2510843; fax

E-mail addresses: jayadeep@cftri.res.in, jydpa@yah

http://dx.doi.org/10.1016/j.jcs.2014.01.0250733-5210/� 2014 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Pradeep, Pactivity of rice bran, Journal of Cereal Scienc

a b s t r a c t

The effect of processing such as steaming, germination and parboiling on nutraceuticals and in vitrobioactive properties of rice bran from three different rice varieties namely Jyothi (pigmented), IR64 andSona masuri (non-pigmented) were investigated. Within the varieties envisaged, pigmented Jyothi va-riety contained higher levels of vitamin E, soluble, bound and total polyphenol, flavanoids, free radicalscavenging activity and total antioxidant activity. Direct steam exposure of bran resulted in an increasein, ether extractives and oryzanol, as well as retention of all the vitamin E components, bound poly-phenols, flavonoids and decrease in soluble and total polyphenol content, free radical scavenging activityand total antioxidant activity compared to native. Parboiling as well as germination of paddy resulted inan increase, in the content of ether extractives and oryzanol, whereas other bioactive propertiesdecreased compared to native. Hence it may be concluded that bioactive components and antioxidantproperties were significantly higher in Jyothi bran compared to the other two paddy brans, and pro-cessing leads to changes in bioactive properties with maximum retention of bioactive components in thesteamed bran.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Rice production in India is an important part of the Indianeconomy. Rice is consumed as milled rice after removal of bran andembryo fractions by milling. Bran obtained during polishing ispartly used for oil extraction and the major portion is used as cattlefeed or remains unutilized. The nutritional value of rice bran isphenomenally superior to other bran and works naturally foroptimal health benefits. Rice bran is a rich source of fibre, protein,minerals, vitamins (Juliano, 1994), phytochemicals such as geory-zanol (a mixture of 10 ferulate esters of triterpene alcohol) (Zhiminet al., 2001), tocols (tocopherols and tocotrienols) and polyphenols(Aguilar-Garcia et al., 2007). These phytochemicals show antioxi-dant and free radical scavenging activity (Butsat and Siriamornpun,2010) and are associated with the cure of nerve imbalance,menopausal problems, serum hypercholesterolemia, coronaryheart disease and cancer (Cicero and Derosa, 2005).

razyl; FRAP, ferric reducingromatography; ORAC, oxygen

: þ91 821 2517233.oo.co.in (A. Jayadeep).

.M., et al., Hydrothermal and be (2014), http://dx.doi.org/10

Even though rice bran has been mainly used as animal feed, byadopting suitable technology it can be made edible for humanconsumption (Saunders, 1990). In addition to physical processing,biotechnological processing such as germination can also affect thequality of bran. Biochemical changes associated with the germi-nation has been reported to alter the nutraceutical properties in thebran containing brown rice (Jayadeep and Malleshi, 2011; Tianet al., 2004). Germinated rice bran showed significant improve-ment in g-aminobutyric acid (GABA), dietary fibre, ferulic acid,tocotrienols and g-oryzanol (Kayahara et al., 2000).

The limitation of using rice bran in food applications is largelydue to its deterioration by enzymatic activities, especially those oflipase and lipoxidase. Rice bran with extended storage stability canbe obtained by various heat treatments (Yan et al., 2003). Hydro-thermal treatment is reported to extend the shelf life of bran(Thanonkaew et al., 2012). Wet heating is effective in permanentlydenaturing lipases, and pressurized heating (autoclave) will reducethe heating time and so will reduce the destruction of bioactivecompounds in rice bran (Damayanthi, 2001).

Application of appropriate technologies, such as rice bran sta-bilization and biotechnological processing to enhance the quality ofbran can affect their bioactive properties which may further justifythe application of technological interventions as a future foodprocessing option. Hence the objective of our study mainly focused

iotechnological treatments on nutraceutical content and antioxidant.1016/j.jcs.2014.01.025

P.M. Pradeep et al. / Journal of Cereal Science xxx (2014) 1e62

on analysing the content of bioactive nutraceuticals and antioxi-dant properties of bran from pigmented and non-pigmented Indianrice varieties and also the effect of processing technologies on theircontent.

2. Materials and methods

2.1. Chemicals and reagents

All chemicals used were of analytical grade. Ferulic acid, DPPHþ,Tocopherols were procured from Sigma, USA and Tocovid capsule,Hovid Bhd, Malaysia, was used as a source of tocotrienols.

2.2. Raw materials

Three varieties of paddy namely IR64, Sona masuri (SM) (bothhaving non-pigmented bran) and Jyothi (having pigmented bran)were procured from APMC market, Mysore, India. All the varietiesof paddy were cleaned to remove dust and other extraneous ma-terials and stored at room temperature in plastic containers. Pro-cessing such as parboiling, steaming and germination were carriedout on a laboratory scale.

2.3. Processing parameters

All the three varieties of paddy were processed in 5 kg batches.

2.3.1. ParboilingAll the three varieties of paddy were soaked overnight and

steamed for 30min at atmospheric pressure and dried at 50 �C in anair drier for 3 h.

2.3.2. SteamingAll the three varieties of paddy were subjected to shelling and

milling to obtain bran. Further, the bran was subjected to opensteaming in an autoclave for 5 min and subsequently dried in airdrier at 50 �C for 3 h.

2.3.3. Germination and steamingEach of the paddy varieties were soaked overnight in adequate

water at ambient conditions, drained, spread on a jute blanket at athickness of 1 cm, covered with another wet jute blanket, sprinkledwith water 3 hourly and the germinated paddy collected after 24 h.It resulted in formation of only rudimentary rootlet and shoot.Germinated paddy was spread on trays, steamed for 2 min anddried in an air drier at 50 �C for 3 h.

2.3.4. Milling and polishingAll processed paddy samples and control (native) were shelled

in a rubber roll sheller and polished in McGill abrasion polisher toobtain bran of 5% degree of polish. The bran samples were sieved toget 710 microns through fractions and stored at �10 �C in a freezeruntil analysis.

2.4. Estimation of oryzanol

Petroleum ether (60e80 �C) extract was analysed for oryzanolcontent spectrophotometrically by scanning the wavelength in therange 220e420 nm, maximum absorbance was recorded at 314 nmand content calculated on the basis of the absorbance of 1% stan-dard oryzanol solution at 314 nm (Seetharamaiah, and Prabhakar,1986). Oryzanol content in oil obtained through defatting of ricebran was also analysed.

Please cite this article in press as: Pradeep, P.M., et al., Hydrothermal and bactivity of rice bran, Journal of Cereal Science (2014), http://dx.doi.org/10

2.5. HPLC analysis of vitamin E

Vitamin E (tocopherols and tocotrienols) content of the sampleextracted by methanol was quantified by a reverse phase HPLCmethod (Chen, and Bergman, 2005) using Shimadzu system withRF10A XL fluorescent detector, LC10AT pumps, System controllerSCL-10A and the chromatograms were recorded and processed byLC-10A class software. The extracts were separated on an AscentisExpress C18 column (4.6 � 150 mm, 2.7 um), SUPELCO, PA, USAusing a gradient solvent system consisting of acetonitrile, meth-anol, isopropanol and aqueous acetic acid as solvent A and aceto-nitrile, methanol and isopropanol as solvent B. Fluorescencedetector was set at excitation and emissionwavelengths of 298 and328 nm, respectively.

2.6. Extraction and estimation of soluble and bound polyphenols

For soluble polyphenol estimation, samples were extracted withmethanol and centrifuged, supernatant was filtered throughWhatman No.1 filter paper and the filtrate was stored in the freezeruntil analysis. The residue obtained was extracted with 1% HClmethanol and used for bound polyphenol estimation (Siwela et al.,2007) and analysed by Folin Ciocalteau’s reagent as per Singletonet al. (1995) at 760 nm using ferulic acid as standard.

2.7. Determination of total flavonoids

For flavonoid estimation, the hexane defatted sample wasextracted with 1% acidic methanol for 1 h at room temperature andanalysed by the AlCl3 reagent method (Bao et al., 2005). Totalflavonoid content was calculated using the standard catechin curveand expressed as milligrams equivalent of catechin per 100 g ofsample.

2.8. DPPHþ radical scavenging ability assay

Methanol soluble extract of the sample was mixed with DPPHþreagent and absorbance was read at 517 nm. DPPHþ reagent wasused as blank, and percentage reduction was monitored (Bondetet al., 1997). Catechin was used as the standard.

2.9. Determination of total antioxidant activity

Total antioxidant activities of samples were quantified inmethanol soluble extract using phosphomolybdenum reagent(Pilar et al., 1999). Results were calculated and expressed asequivalents using the molar extinction coefficient of a-tocopherol.

2.10. Statistical analysis

All determinations were made in triplicate and were reported asmean � SD values. Values were subjected to Student t-test to studythe level of significance at p < 0.05 (Snedecor, & Cochran, 1994).

3. Results and discussion

3.1. Oryzanol content (Table 1)

It was observed that, among the varieties analyzed, IR64 brancontained the highest oryzanol content whereas Jyothi containedthe lowest. Studies carried out in our laboratory has shown thatthere is not much difference in oryzanol content in de-husked riceof these two varieties. The lower content in Jyothi bran observed inthis experiment can also be due to the difference in the batch ofpaddy collected. Bergman and Xu (2003) reported differences in

iotechnological treatments on nutraceutical content and antioxidant.1016/j.jcs.2014.01.025

Fig. 1. HPLC Chromatograms of vitamin E components (a ¼ delta, b ¼ gama, c ¼ alphatocotrienol and d ¼ delta, e ¼ gama, f ¼ alpha tocopherols) in Jyothi bran A.) Raw(10 ul) B. Steamed (20 ul), C Parboiled (20 ul), and D. germinated & steamed (20 ul).

Table 1Fat, moisture and Oryzanol content in processed bran from different varieties (mg/100 g).

Sample Moisture Fat Oryzanol

IR64 Raw 13.1 � 0.35 22.8 � 0.30a 239 � 8.0a

Steaming 10.2 � 0.20 25.6 � 0.35b 278 � 1.0b

Parboiling 12.3 � 0.25 31.1 � 0.43b 360 � 5.0b

Germinated & steamed 11.5 � 0.30 28.8 � 0.40b 341 � 11.0b

SM Raw 16.7 � 0.35 20.5 � 0.40a 219 � 4.0a

Steaming 10.6 � 0.40 22.1 � 0.48b 308 � 3.0b

Parboiling 12.0 � 0.25 24.9 � 0.49b 377 � 1.0b

Germinated & steamed 13.7 � 0.30 24.8 � 0.39b 363 � 3.0b

Jyothi Raw 15.4 � 0.20 18.7 � 0.48a 168 � 1.0a

Steaming 11.5 � 0.21 20.4 � 0.43b 196 � 9.0b

Parboiling 13.1 � 0.35 24.3 � 0.31b 255 � 6.0b

Germinated & steamed 12.5 � 0.25 23.3 � 0.43b 226 � 6.0b

Values are mean � SD of three independent observations. a, b values with differentsuperscripts significantly different at p < 0.05 when raw of each variety comparedwith respective process.

P.M. Pradeep et al. / Journal of Cereal Science xxx (2014) 1e6 3

oryzanol content in varieties and attributed it to the genotype,growing environment and the solvent used for extraction.

Parboiled bran had the highest oryzanol content followed bygerminated and steamed bran, compared to native. This may be dueto the hydrothermal process induced changes in bran and conse-quent excess oil release during extraction. Increase in etherextraction by ohmic heat processing has been reported byLakkakula et al. (2004). Increase in oryzanol in brown rice on par-boiling has also been reported by Shobana et al. (2011). However,oryzanol content per total fat was higher in all our processes exceptsteaming. It may be due to the over extractability from the branmatrix due to the severity of the treatments. Thanonkaew et al.(2012) has reported the process induced disintegration of cellwall and consequent increase in fat soluble components. It may beconcluded that the amount of health beneficial oryzanol is eitherincreased or retained in spite of processing treatments to improvethe bran quality.

3.2. Vitamin E content

HPLC chromatographic analysis of vitamin E in Jyothi nativebran fractions comprised gama-tocotrienol (49%), alpha tocopherol(24%) and gama-tocopherol (18%) as the major components (Fig. 1).Contents of gama-tocotrienol, alpha-tocopherol, gama-tocopherol,total tocotrienol, total tocopherol and total vitamin E are given inTable 2.

Steaming did not affect the content of vitamin E componentssignificantly in the Jyothi bran. On further assessment of vitamin Eon a total fat basis (Fig. 2), it was observed that steaming has notinduced any significant loss of health beneficial vitamin E compo-nents except alpha-tocopherol which is known to be thermo labile.This indicates that steaming is an ideal method for stabilization andretention of these nutraceuticals. Zadernowski et al. (1999) alsoobserved that heat treatment (100 �C for 30 min) did not affectvitamin E in oat flour.

Parboiling resulted in an increase in gama-tocotrienol, totaltocotrienol and total vitamin E content by 17% in bran. There wassignificant loss of most of the vitamin E components on a fat basisexcept for an 18% increase in gamma and total tocotrienol whichcan be attributed to the increase in fat content in bran on parboiling(Sondi et al., 1980).

Major vitamin E components decreased both in bran as well asin oil during germination and steaming. Germination associatedutilization of vitamin E, as well as, disintegration of cell wall(Opassiri et al., 2010) and consequent exposure of vitamin E contentin oil to steam, may be the reason for the decrease.

Please cite this article in press as: Pradeep, P.M., et al., Hydrothermal and biotechnological treatments on nutraceutical content and antioxidantactivity of rice bran, Journal of Cereal Science (2014), http://dx.doi.org/10.1016/j.jcs.2014.01.025

Table 2Effect of processing on Vitamin E components in Jyothi bran.

Vitamin E(mg/g bran)

Raw Steamed Parboiled Germinated &steamed

Gama-tocotrienol

135.03 �5.9a

153.06 �14.35a

205.22 �11.69b

101.93 �13.0b

Totaltocotrienol

155.85 �6.07a

176.21 �17.61a

227.74 �12.07b

114.11 �14.72b

Gama-tocopherol

49.82 �2.92a

49.93 �4.00a

50.32 �0.95a

29.06 �3.52b

Alpha-tocopherol

65.6 �0.03a

65.49 �1.49a

38.82 �1.81b

13.23 �1.3b

Totaltocopherol

118.15 �3.18a

117.07 �6.86a

92.38 �0.81b

45.35 �2.42b

Totalvitamin E

274.03 �2.89a

293.22 �24.48a

320.12 �11.26b

159.49 �17.14b

Values are mean � SD of three independent observations. a, b values with differentsuperscripts significantly different at p < 0.05 when raw of each variety comparedwith respective process.

Table 3Polyphenols (mg Ferulic acid Eq:/100 g).

Sample Soluble Bound Total

IR64 Raw 554 � 11.0a 290 � 10.0a 844 � 5.0a

Steamed 370 � 15.0b 267 � 8.0a 637 � 10.0b

Parboiled 207 � 13.0b 125 � 12.0b 331 � 16.0b

Germinated &steamed

200 � 2.0b 135 � 16.0b 335 � 18.0b

SM Raw 571 � 31.0a 194 � 19.0a 765 � 46.0a

Steamed 344 � 3.0b 171 � 8.3b 515 � 10.0a

Parboiling 323 � 18.0b 122 � 2.0b 446 � 17.0b

Germinated &steamed

228 � 15.0b 119 � 5.0b 347 � 16.0b

Jyothi Raw 2569 � 65.0a 1194 � 72.0a 3762 � 68.0a

Steamed 1935 � 200.0b 1065 � 102.0a 3001 � 316.0b

Parboiled 281 � 5.0b 596 � 35.0b 877 � 30.0b

Germinated &steamed

871 � 13.0b 1033 � 73.0b 1904 � 74.0b

Values are mean � SD of three independent observations. a, b values with differentsuperscripts significantly different at p < 0.05 when raw of each variety comparedwith respective process.

P.M. Pradeep et al. / Journal of Cereal Science xxx (2014) 1e64

3.3. Soluble and bound polyphenol content (Table 3)

Soluble polyphenol in Jyothi bran (2569 mg FA Eq. (Ferulic acidEquivalent)/100 g) isw4 higher than in IR64 (554 mg FA Eq./100 g)and Sona masuri (571 mg FA Eq./100 g) bran. Physical processing ofbran by steaming resulted in 25e40% loss of soluble polyphenols,whereas parboiling of the paddy resulted in 43e63% loss in non-pigmented bran and 89% loss in pigmented bran. Germinationand steam stabilization resulted in 60e66% loss irrespective ofvariety.

Bound polyphenol in Jyothi bran (1194 mg FA Eq./100 g) iswfour times higher than in IR64 (290) and Sona masuri (194) bran.Content of bound polyphenol is wtwo times less than the solublepolyphenols. Physical processing of bran by steaming resulted in 8e12% loss only, whereas parboiling of paddy resulted in 37e57% lossin bound polyphenols in the bran varieties. Germination and steamstabilization resulted in 39e53% loss in IR64 & Sona masuriwhereas only 13% in Jyothi. Jyothi being high in polyphenols,germination induced liberation of bound polyphenols and conse-quent over extractability may be the reason for reduced loss.

Since pigmented bran is rich in polyphenols, flavanoid analysiswas carried out only in Jyothi, accordingly native contained 278 mgcatechin Equivalent/100 g. Steaming resulted in only 7% reductionwhereas in germination, 26% and parboiling, 41% loss wereobserved.

Fig. 2. Effect of processing of Jyothi bran o

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It may be concluded that the content of polyphenol is higher inbran of pigmented variety. Byungrok et al. (2011) also observeddifferences in polyphenol content in bran varieties with highercontent in pigmented varieties. Extent of loss of polyphenols wasminimal in steam stabilized bran. Parboiling of paddy resulted insevere loss of polyphenols in pigmented bran. This may be due toleaching and thermal treatment associated with the paddy par-boiling process (Lamberts et al., 2006). Germination and steamstabilization of paddy results in considerable loss of soluble poly-phenols whereas the loss of bound polyphenols is moderate. Thismay be due to increase of bound polyphenols in germinated brownrice as reported by Tian et al. (2004). Further, the heat processingcan reduce the polyphenolic components as reported by Randhiret al. (2008) in sprouted buckwheat by autoclaving.

3.4. Free radical scavenging activity (Table 4)

Free radical scavenging activity was around 10 times more innative bran from Jyothi pigmented variety than in the non-pigmented varieties. High radical scavenging activity is alsocorrelated with the content of polyphenols. Byungrok et al. (2011)also reported higher free radical scavenging activity in colouredrice bran and its correlation with polyphenols.

n Vitamin E components in total fat.

iotechnological treatments on nutraceutical content and antioxidant.1016/j.jcs.2014.01.025

Table 4Antioxidant properties of differently processed bran.

Parameter Sample IR64 SM Jyothi

Free radicalscavengingactivity (mgCatechin Eq:/100 g)

Raw 211 � 8.0a 175 � 8.0a 2319 � 280.0a

Steamed 164 � 19.0b 148 � 3.0b 2094 � 177.0b

Parboiled 68 � 12.0b 73 � 3.0b 143 � 9.0b

Germinated& steamed

68 � 2.0b 75 � 5.0b 1086 � 60.0b

Total antioxidantactivity (mM a-tocopherolequivalent/g).

Raw 82.2 � 5a 46.2 � 2a 176.1 � 10a

Steamed 63.2 � 6b 42.0 � 1a 133.3 � 9b

Parboiled 19.0 � 2b 23.5 � 3b 16.1 � 0.6b

Germinated& steamed

20.4 � 3b 17.5 � 2b 39.0 � 0.3b

Values are mean � SD of three independent observations. a, b values with differentsuperscripts significantly different at p < 0.05 when raw of each variety comparedwith respective process.

P.M. Pradeep et al. / Journal of Cereal Science xxx (2014) 1e6 5

Physical processing of bran by parboiling resulted in a signifi-cant reduction (58e94%), in activity, in all varieties of bran. How-ever, steam stabilization of bran resulted in only 15e22% loss inactivity in non-pigmented bran, and less significant (10%) loss inpigmented bran. In the case of bran obtained by biotechnologicalprocessing, paddy germination followed by steaming, resulted in53e67% loss of activity in all varieties. These variations in activitydue to different processing methods may be related to the phyto-components present in it. Aguilar-Garcia et al. (2007) on principalcomponent analysis and multiple regression in rice bran indicatedthat FRAP (ferric reducing antioxidant power) was sensitive topolyphenols and total tocotrienols while ORAC (oxygen radicalabsorbance capacity) was sensitive to polyphenols and totaltocopherols.

3.5. Total antioxidant activity (Table 4)

Total antioxidant activity of native bran of Jyothi wasw2.5 timesmore than that of IR64 and Sona masuri. It is related to the contentof all the antioxidant components like tocols, oryzanol, phytos-terols, polyphenols, etc.

Thermal processing such as steaming of IR64 leads to 27% loss inactivity, in the case of parboiled and germinated sample the extentof loss was high (76%). Similarly in Sona masuri, steaming resultedin 9% loss in activity whereas in parboiled, the loss was 49% andgerminated, 62%. In Jyothi, steaming induced 24% loss in activitywhereas loss in parboiled was 91% and germinated 78%. As envis-aged, steaming of bran resulted in mild loss in total antioxidantactivity compared to bran obtained from parboiling or germination.Zhang et al. (2010) also observed a decrease in free radical scav-enging on pressure steaming of buckwheat.

4. Conclusion

Most of the bioactive components and antioxidant propertieswere significantly higher in pigmented Jyothi bran compared to theother two non-pigmented brans. Technological interventionsresulted in changes in nutraceutical properties of the bran fromdifferent rice varieties. In steam stabilized bran, the contents oforyzanol, tocotrienol and tocopherol were higher than in native;retention of soluble polyphenols, bound polyphenols and totalpolyphenols are 80e90%; and free radical scavenging activity andtotal antioxidant activity 75e90%, whereas in the parboiled andbiotechnologically processed bran, the extent of loss of nutraceut-icals was severe except for oryzanol and tocotrienol content. Studyindicates that, through appropriate processing technology, it ispossible to obtain stabilized bran with higher nutraceuticals

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especially from polyphenol rich pigmented rice bran which canform a healthy food ingredient in functional foods.

Acknowledgments

Authors wish to acknowledge the project granted fromMinistryof Food Processing, Government of India and The Director, CSIR-CFTRI for extending support.

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