Enhancement of biologically active compounds in germinated brown rice and the effect of sun drying

Accepted Manuscript
Enhancement of biologically active compounds in germinated brown rice and the
effect of sun-drying

Patricio J. Cáceres, Elena Peñas, Cristina Martinez-Villaluenga, Lourdes Amigo,
Juana Frias
PII:

S0733-5210(16)30409-X

DOI:

10.1016/j.jcs.2016.11.001

Reference:

YJCRS 2238

To appear in:

Journal of Cereal Science

Received Date:

15 June 2016

Revised Date:

29 September 2016

Accepted Date:

06 November 2016

Please cite this article as: Patricio J. Cáceres, Elena Peñas, Cristina Martinez-Villaluenga, Lourdes
Amigo, Juana Frias, Enhancement of biologically active compounds in germinated brown rice and
the effect of sun-drying, Journal of Cereal Science (2016), doi: 10.1016/j.jcs.2016.11.001

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form.
Please note that during the production process errors may be discovered which could affect the
content, and all legal disclaimers that apply to the journal pertain.


ACCEPTED MANUSCRIPT
HIGHLIGHTS

 Brown rice (BR) is a good source of biologically active compounds

NU
SC
RI
PT

 The content of GABA, TPC and antioxidant activity enhanced during germination of
BR
 Sun-drying maximizes the content of bioactive compounds in GBR

AC

CE

PT

ED

MA

 Sun-dried GBR is highly recommended for its health-promoting properties


ACCEPTED MANUSCRIPT
Enhancement of biologically active compounds in germinated brown
rice and the effect of sun-drying
Patricio J. Cáceresa, Elena Peñasb, Cristina Martinez-Villaluengab, Lourdes Amigoc and

aEscuela

NU
SC
RI
PT

Juana Friasb*
Superior Politécnica del Litoral, ESPOL, Facultad de Ingeniería Mecánica y

Ciencias de la Producción, Campus Gustavo Galindo Km 30.5 Vía Perimetral, P.O. Box
09-01-5863, Guayaquil, Ecuador
bInstitute

of Food Science, Technology and Nutrition

Cierva 3, 28006 Madrid, Spain.
cInstitute

(ICTAN-CSIC), Juan de la

of Food Science Research (CIAL) (CSIC-UAM), Nicolás Cabrera 9, Campus

de Cantoblanco, 28049 Madrid, Spain

MA

*Corresponding author:
Juana Frias

Institute of Food Science, Technology and Nutrition (ICTAN-CSIC), Juan de la Cierva
Tel.: + 34 912587510;
Fax: +34 915644853

ED

3, 28006 Madrid, Spain.

AC

CE

PT

E-mail address:

1


ACCEPTED MANUSCRIPT
Abstract

2

Germinated brown rice (GBR) has been suggested as an alternative approach to mitigate

3

highly prevalent diseases providing nutrients and biologically active compounds. In this

4

study, the content of γ-oryzanol, γ-aminobutyric acid (GABA), total phenolic

5

compounds (TPC) and antioxidant activity of soaked (for 24 h at 28°C) and GBR (for

6

48 and 96 h at 28°C and 34°C) were determined and the effect of sun-drying as an

7

economically affordable process was assessed. Germination improved the content of

8

GABA, TPC and antioxidant activity in a time-dependent manner. Sun-drying increased

9

γ-oryzanol, TPC and antioxidant activity, whereas GABA content fluctuated depending

10

on the previous germination conditions. This study indicates that sun-drying is an

11

effective sustainable process promoting the accumulation of bioactive compounds in

12

GBR. Sun-dried GBR can be consumed as ready-to-eat food after rehydration or

13

included in bakery products to fight non-communicable diseases.

MA

NU
SC

RI
PT

1

14

Keywords: Brown rice; germination; sun-drying; bioactive compounds.

16

ED

15

List of abbreviations:

18

BR: Brown rice

19

GAE: Gallic acid equivalents

20

GABA: Gamma-aminobutyric acid

21

GBR: Germinated brown rice

22

ORAC: Oxygen radical antioxidant capacity

23

TE: Trolox equivalents

24

TPC: Total phenolic compounds

CE

AC

25

PT

17

26
27
28
29

2


ACCEPTED MANUSCRIPT
30

1. Introduction
Rice (Oryza sativa L.) is one of the main cereals produced in the world and the

32

major staple food for almost half of the population worldwide. It has been postulated a

33

positive association between white rice intake and risk factors of cardiovascular

34

diseases, including metabolic syndrome and type 2 diabetes in low and middle-income

35

countries (Izadi and Azadbakht, 2015). In recent years, much attention has been paid on

36

the health benefits of brown rice (BR). BR contains health promoting compounds,

37

including dietary fibre, γ-aminobutyric acid (GABA), vitamins, phenolic compounds

38

and γ-oryzanol that are mainly located in the germ and bran layers, which are removed

39

during rice polishing and milling (Wu et al., 2013).

NU
SC
RI
PT

31

Despite its nutritional value and beneficial physiological effects, BR is not widely

41

consumed because it has poor cooking properties, low organoleptic quality and harsh

42

texture (Wu et al., 2013). Numerous studies have demonstrated that germination

43

improves texture and acceptability of BR and also enhances nutrient and phytochemical

44

bioavailability (Tian et al., 2004). During germination, significant changes in

45

biochemical, nutritional and sensory characteristics occur resulting in the degradation of

46

storage proteins and carbohydrates and promoting the synthesis and accumulation of

47

biofunctional compounds. Germination process generally results in improved levels of

48

vitamins, minerals, fibres and phytochemicals such as ferulic acid, GABA, γ-oryzanol

49

and antioxidant activity (Cho and Lim, 2016).

ED

PT

CE

AC

50

MA

40

Consumption of GBR is receiving increasing attention supported by scientific

51

evidence on its beneficial health effects reducing the risk of diseases such as obesity,

52

cardiovascular diseases, type 2 diabetes, neurodegenerative diseases and osteoporosis

53

and GBR has been identified as a natural and inexpensive substitute of conventional

3


ACCEPTED MANUSCRIPT
54

white rice to improve nutritive and health status of a large population that currently eat

55

rice as staple food (Wu et al., 2013).
Several studies have been carried out to optimize the germination conditions and

57

maximize the beneficial attributes of GBR since the chemical composition of the grains

58

change dramatically during germination (Cáceres et al., 2014a, 2014b; Cho and Lim,

59

2016). Lesser efforts, however, have been dedicated to evaluate the effect of drying

60

processes on the quality of the obtained GBR grains. Most of the research studies

61

focused on the production and characterization of GBR preserve the product by freeze-

62

drying. This technique maintains the color, shape, aroma and nutritional quality of the

63

product and its relevance to preserve nutraceutical compounds has been highlighted,

64

however, the process is slow and requires expensive equipment and, thus, it is rarely

65

used for the preservation of foods on the industrial scale (Karam et al., 2016). Drying

66

techniques as convective drying, hot-air oven, vacuum, osmotic, fluidized bed and

67

superheated steam dehydration are used to achieve water evaporation in shorter times.

68

In GBR, drying procedure affect starch digestibility and GABA content depending on

69

operation conditions (Chungcharoen et al., 2014). These drying methods are still

70

expensive and not always affordable in low and middle-income countries where rice

71

production and transformation is performed with few economic resources.

CE

PT

ED

MA

NU
SC
RI
PT

56

Solar drying is the oldest preservation procedure for agri-food products and

73

widely used to dehydrate rice grains in rice producers´ countries located in tropical

74

areas of the world. Our group has recently optimized germination conditions to

75

maximize the phytochemical content, antioxidant activity and nutritional features

76

(Cáceres et al., 2014a, 2014b) of three certified BR varieties and one experimental

77

cultivar BR grown in Ecuador. This country experiences little variation in daylight

78

hours during the course of the year and temperatures oscillate between 30 and 37 ºC

AC

72

4


ACCEPTED MANUSCRIPT
( />
80

climate conditions that favourably could stabilize GBR towards a cost-effective and

81

sustainable production. Therefore, the aim of the present work was to assess the effect

82

of different germination conditions on γ-oryzanol, GABA, total phenolic compounds

83

and antioxidant activity in a highly produced Ecuadorian rice variety, SLF09. GBR was

84

sun-dried and changes in the content of these biologically active compounds were

85

studied. The consumption of sundried GBR might contribute to the intake of health-

86

promoting compounds in populations where rice is the main food as ready-to-eat meals

87

or soups after rehydration or to supplement functional foods as strategies for combating

88

highly prevalent chronic diseases.

NU
SC
RI
PT

79

90

2. Material and methods

91

2.1. Rice samples

MA

89

Commercial certified brown rice (BR) variety indica SLF09 was supplied by the

93

company INDIA-PRONACA Co, Ecuador. This variety was selected based on its high

94

harvest yield (6 Tm/Ha) and the consumer acceptability characterized by its translucent

95

white center and extra-long shape grain.

96

2.2.

PT

ED

92

CE

Germination process

Germination process was performed as described in Cáceres et al. (2014b). Fifty

98

grams of BR were washed with distilled water and soaked in sodium hypochloride (1:5;

99

w/v) at 28 ºC for 30 min. After draining, BR grains were rinsed with distilled water to

100

neutral pH. BR grains were then soaked in distilled water (1:5; w/v) at 28 ºC for 24 h.

101

Afterwards, soaking solution was removed and the soaked BR grains were obtained.

AC

97

102

Soaked BR were extended on drilled grilles over a moist laboratory paper and

103

they were then covered with the same paper. The grille was placed in plastic

5


ACCEPTED MANUSCRIPT
germination trays containing distilled water in order to maintain the paper always wet

105

by capillarity. Germination trays containing the soaked grains were introduced in a

106

germination cabinet (model EC00-065, Snijders Scientific, Netherlands) provided with

107

a circulating water system to keep the humidity > 90%. GBR were produced at 28 and

108

34 ºC in darkness for 48 and 96 h. Soaked and GBR grains were dehydrated in a freeze-

109

drier (Freeze Mobile G, Virtis Company, INC Gardiner, NY, USA). Freeze-dried grains

110

were finely ground in a ball mill (Glen Creston Ltd., Stanmore, UK), passed through a

111

sieve of 0.5 mm and the obtained flour was stored under vacuum conditions in sealed

112

plastic bags in darkness at 4 ºC until further analysis. Each germination process was

113

carried out in triplicate.

114

2.3.

NU
SC
RI
PT

104

Sun-drying proccess

Fresh soaked and GBR samples produced above were lied out plastic cloths on a

116

single layer 3 mm thick, under sunlight for ~10 h (whole daylight) in Guayaquil

117

(Ecuador), at a latitude of 2º 12’ 21’’ S and a longitude of 79º 54’ 28’’ W, an elevation

118

of 6 m above the sea level, and an average temperature 33.5 ± 3.5 ºC. Sun-dried soaked

119

and GBR were finely ground in a ball mill (Glen Creston Ltd., Stanmore, UK), passed

120

through a sieve of 0.5 mm and the flour obtained was stored under vacuum conditions

121

in sealed plastic bags in darkness at 4 ºC until further analysis. Each drying process was

122

conducted in triplicate.

123

2.4.

ED

PT

CE

Determination of moisture content

AC

124

MA

115

The content of moisture in dried soaked and GBR was determined by keeking the

125

samples at 105 ºC to a constant weight according to AOAC 925.09 (AOAC, 2000).

126

2.5.

Determination of γ-oryzanol.

127

The analysis of γ-oryzanol in rice samples was performed as previously reported

128

(Cho et al., 2012) with some modifications. Briefly, 1 g of sample was mixed with 10

6


ACCEPTED MANUSCRIPT
mL of methanol and further sonicated for 10 min. The mixture was centrifuged at

130

15,000 rpm for 10 min at room temperature (25 ± 2 ºC) and then concentrated to

131

dryness. Samples were then diluted in 1 mL of 100% methanol, filtered through a

132

0.45µm membrane and then analysed by HPLC. The HPLC system consisted of an

133

Alliance Separation Module 2695 (Waters, Milford, USA), a photodiode array detector

134

2996 (Waters) setted at 325 nm wavelengh and Empower II software (Waters). Twenty

135

microliters were injected onto a C18 column (150 x 3.9 mm i.d., 5 μm size, Waters). A

136

gradient mobile phase was pumped at a flow of 1.0 mL/min to separate the -oryzanol

137

components consisting in solvent A (acetonitrile), solvent B (methanol) and solvent C

138

(bi-distilled water) for 50 min as follows: initial isocratic flow 60% solvent A, 35%

139

solvent B and 5% solvent C for 5 min, gradient flow 60% solvent A and 40% solvent B

140

for 3 min keeping it at isocratic flow for 2 min, then gradient flow 22% solvent A and

141

78% solvent B for 10 min, to be maintained isocratically for 15 min, and changing to

142

initial conditions for 5 min and, finaly, isocratic conditions to equilibrate column for 10

143

min. γ-Oryzanol derivatives in rice samples were identified by retention time and

144

spiking the sample with a commercial γ-oryzanol standard solution (Cymit, Spain). The

145

purity of peaks was confirmed by spectra comparison and by mass espectrometry

146

analysis (Cho et al., 2012). Steryl ferulates components of γ-oryzanol were quantified

147

by external calibration curve using γ-oryzanol standard solutions. Replicates samples

148

were independently analyzed and results were expressed in mg γ-oryzanol/100 g of dry

149

matter (DM).

150

2.6.

AC

CE

PT

ED

MA

NU
SC
RI
PT

129

Determination of γ-aminobutyric acid (GABA)

151

γ-Aminobutyric acid (GABA) content was determined by HPLC (Cáceres et al.,

152

2014b). Briefly, 50 L aliquot of concentrated water-soluble extract and 10µL allyl-L-

153

glycine solution (Sigma-Aldrich) used as internal standard were derivatized with 30 µL

7


ACCEPTED MANUSCRIPT

phenyl isothiocyanate (PITC 99%, Sigma-Aldrich) and dissolved in mobile phase A for

155

GABA analysis. An Alliance Separation Module 2695 (Waters, Milford, USA), a

156

photodiode array detector 2996 (Waters) setted at 242nm wavelength and an Empower

157

II chromatographic software (Waters) were used as chromatographic system. A volume

158

of 20µL of sample were injected onto a C18 Alltima 250 x 4.6 mm i.d., 5 μm size

159

(Alltech) column thermostatted at 30 ºC. The chromatogram was developed at a flow

160

rate of 1.0 mL/min by eluting the sample with mobile phase A (0.1 M ammonium

161

acetate pH 6.5) and mobile phase B (0.1 M ammonium-acetate, acetonitrile, methanol,

162

44/46/10, v/v/v, pH 6.5). Replicates samples were independently analyzed and results

163

were expressed as mg GABA/100 g DM.

164

2.7.

NU
SC
RI
PT

154

Determination of total phenolic compounds

The Folin-Ciocalteu’s method was used for the quantification of total phenolic

166

compounds (TPC), as previously reported. The absorbance was measured at 739 nm

167

using a microplate reader (Synergy HT, BioTek Instruments) and TPC were quantified

168

by external calibration using gallic acid (Sigma-Aldrich) as standard. Sample replicates

169

were independently analyzed and results were expressed as mg of gallic acid

170

equivalents (GAE)/100 g DM.

171

2.8.

PT

ED

MA

165

CE

Determination of antioxidant activity
Antioxidant activity was determined by the method of oxygen radical absorbance

173

capacity (ORAC) by fluorescence detection (λexc 485 nm and λem 520 nm) using an

174

automatic multiplate reader (BioTek Instruments), previously described (Cáceres et al.,

175

2014b). Sample replicates were independently analyzed and results were expressed as

176

mg of Trolox equivalents (TE)/100g DM.

AC

172

8


ACCEPTED MANUSCRIPT
177

2.9.

Statistical analysis
Each germination experiment and subsequent drying process were conducted in

179

triplicate. Two extractions were performed for each replicate and the analytical

180

determinations were carried out in triplicate. Data were expressed as mean ± standard

181

deviation. The data obtained from each experimental condition were subjected to one-

182

way analysis of variance (ANOVA) using Duncan test to determine the significant

183

differences at P  0.05 level using Statgraphics Centurion XVI Program, version

184

16.1.17 (Statistical Graphics Corporation, Rockville, Md) for Windows. This

185

programme was also applied for correlation analysis between quantitative variables (γ-

186

oryzanol and TPC) versus ORAC at the experimental processing conditions.

NU
SC
RI
PT

178

187

3. Results

MA

188

In order to study the effect of germination on the relevant biologically active

190

compounds, soaked BR and GBR were freeze-dried, as this drying process minimize its

191

degradation and deterioration. In parallel, fresh soaked and GBR were sun-dried and

192

sample moisture content ranged between 9.5-12.5 %.

193

3.1. Effect of germination on -oryzanol content in brown rice variety SLF09

PT

BR

variety

SLF09

exhibited

four

main

chromatographic

peaks

that

CE

194

ED

189

unambiguously were identified as cycloartenyl ferulate (peak 1), 24-methylene

196

cycloartanyl ferulate (peak 2), campestryl ferulate (peak 3) and sitosteryl ferulate (peak

197

4) (Figure 1), confirmed by spicking with commercial standard γ-oryzanol by HPLC

198

and mass espectrometry analysis. The quantitative results revealed that 24-methylene

199

cycloartanyl ferulate (peak 2) was present in the larger amount,followed by cycloartenyl

200

ferulate (peak 1) and campestryl ferulate (peak 3) and, finally, sitosteryl ferulate (peak

201

4) (Table 1). Total content of γ-oryzanol underwent a significantly decrease (P≤0.05)

AC

195

9


ACCEPTED MANUSCRIPT
during the initial soaking treatment and a 17% reduction was observed. This effect was

203

due to drops exhibited by the individual derivatives: Campestryl ferulate suffered the

204

largest decrease (25%), followed by sitosteryl ferulate (20%) and, in less amount,

205

cycloartenyl and 24-methylene cycloartanyl ferulates (15%, Table 1). Germination

206

process did not bring about further γ-oryzanol losses, since most of the steryl derivative

207

concentrations kept almost unchanged (P≥0.05), and concentrations ranged from 9.2 to

208

9.64 mg/100g DM in GBR grains (Table 1).

NU
SC
RI
PT

202

In an attempt to stablish the proportion of each individual derivative within the

210

total γ-oryzanol content before and after germination, the contribution of each steryl

211

ferulate to the total γ-oryzanol content was calculated (Figure 2). In crude BR, 24-

212

methylene cycloartanyl ferulate was the predominant one (45%), followed by

213

cycloartenyl ferulate (23%), then campestryl ferulate (20%) and, finaly, sitosteryl

214

ferulate (12%). These proportions were mainteined almost invaried after soaking and

215

slight modifications were appreciated in GBR samples. While the contributions of

216

cycloartenyl and sitosteryl ferulates did not change during germination, those for 24-

217

methylene cycloartanyl and campestryl ferulates were modified to aproximately 48 and

218

17%, respectively (Figure 2).

219

3.2. Effect of germination on GABA content in brown rice variety SLF09

CE

PT

ED

MA

209

Table 2 reports the GABA content in ungerminated, soaked and germinated BR.

221

Variety SLF09 showed a concentration of 1.07 mg GABA/100g DM. that increased 7-

222

fold after soaking process carried out at 28 ºC for 24 h. During germination, a gradual

223

and time-dependent accumulation of GABA was achieved and 28 ºC produced larger

224

amounts of this compound than 34 ºC.

225

3.3. Effect of germination on the content of total phenolic compounds in brown rice

226

variety SLF09

AC

220

10


ACCEPTED MANUSCRIPT
Changes in total phenolic compounds (TPC) of BR at different germination

228

conditions are presented in Table 2. The TPC in crude samples corresponded to 132.53

229

mg GAE/100g DM and this content underwent a significantly (P  0.05) decrease after

230

steeping process. Germination, however, led to a sharp increment in the concentration

231

of these compounds with time.

232

3.4. Effect of germination on the antioxidant activity in brown rice variety SLF09

NU

SC
RI
PT

227

The total antioxidant activity of crude, soaked and GBR grains determined by the

234

ORAC-FL method is also collected in Table 2. The antioxidant activity of non-

235

germinated SLF09 grains was 494.81 mg TE/100g DM and soaking did not cause

236

significant (P≥0.05) changes. During germination process, the antioxidant activity

237

increased gradually following a time-dependent pattern and higher temperature led to

238

higher levels. However, there was not found a significant positive correlation between

239

antioxidant activity and γ-oryzanol content of GBR (freeze-dried) samples (Figure 4A).

240

3.5. Effect of sun-drying on the content of -oryzanol, GABA, TPC and antioxitant

241

activity of germinated brown rice variety SLF09

ED

MA

233

Tables 1 and 2 include the content of -oryzanol, GABA, TPC and antioxidant

243

activity in sundried soaked and GBR. This drying process increased the content of -

244

oryzanol a 34 and 48 % in 28 ºC/48h-GBR and 28 ºC/96h-GBR samples, respectively.

245

Sundried 34 ºC/48h-GBR and 34 ºC/96h-GBR increased -oryzanol concentrations a

246

42% (Figure 3) following the accumulation of the individual steryl ferulates during sun-

247

drying (Table 1). Figure 2 illustrates the contributions of individual steryl ferulates to

248

the total -oryzanol content. Sun-drying increased the proportion of campestryl ferulate

249

to approximately 25-26%, whilst cycloartenyl ferulate and 24-methylene cycloartanyl

250

ferulate decreased to 18-19% and 42-43%, respectively, and sitosteryl ferulate was not

251

modified.

AC

CE

PT

242

11


ACCEPTED MANUSCRIPT
The content of GABA in sundried GBR grains is found in Table 2. The largest

253

GABA accumulation was achieved in those samples previously germinated for 96 h,

254

while temperature did not modify GABA content in GBR for 48 h, and soaked BR

255

provided the lowest GABA content. Sun-drying only increased GABA content in

256

soaked and 34 ºC/48h GBR counterparts (41 and 33%, respectively), it did not cause

257

significant GABA modification in 28 ºC/48h GBR, while for those BR grains

258

germinated for 96h, sun-drying led to unexpected GABA losses (99 and 24% at 28 and

259

34ºC, respectively) (Figure 3).

NU
SC
RI
PT

252

Sun-drying brought about slight changes in TPC content of GBR and only in those

261

germinated for 96 h sun-drying led to a significant (P0.05) TPC enhancement (Table 2,

262

Figure 3). However, the antioxidant activity underwent a gradual and significant

263

(P0.05) increase in sundried GBR that was higher for those GBR produced at 28 ºC,

264

althought those germinated at 34 ºC also provided a large ORAC value. In all the

265

samples, sun-drying caused a sharp increment in antioxidant activity compared with the

266

GBR counterparts (Figure 3).

ED

MA

260

In an attempt to elucidate the potential compounds responsible for antioxidant

268

activity, Figure 4 shows the correlation between ORAC values and TPC and γ-oryzanol

269

content in GBR and sundried GBR. A significant positive correlation (P0.05) was

270

found between ORAC and TPC content of GBR (Figure 4B) (r=0.96), and between

271

ORAC and γ-oryzanol (Figure 4C) (r=0.82) and TPC (Figure 4D) (r=0.86) of sundried

272

GBR.

274

CE

AC

273

PT

267

4. Discussion

275

BR variety SLF09 is largely produced in Eduador by INDIA-PRONACA and

276

exported to other Latin American countries. It is one of the long grain rice indica

12

ACCEPTED MANUSCRIPT
varieties highly consumed due to this variety of rice remains loose after cooking. In

278

Ecuador, rice is produced at local farmlands that currently reach overproduction

279

(Cáceres et al., 2014a). The remaing amount after covering human consumption is

280

mainly used for animal feeding and, hence, undervaluaded. Therefore, germination of

281

BR emerges as a simple cost-effective strategy for enhancing the content of bioactive

282

compounds. In addition, economic, effective and sustainable sun-drying provided by

283

Ecuadorian climatology can contribute to the preservation of GBR for further storage,

284

comercialization and consumption as ready-to-eat staple food or incorporated in most

285

atractive functional foods with added-value (Cornejo et al., 2015). In this context, GBR

286

can contribute to reduce the risk of cardiometabolic diseases in those populations where

287

rice constitute the staple food without altering the existing consumption habits (Ochoa-

288

Avilés et al., 2014).

MA

NU
SC
RI
PT

277

The composition of GBR depends on many factors such as genotype diversity,

290

soaking conditions, germination time and temperature, as well as drying process.

291

Germination generally improves the nutritional quality, by augmenting the protein

292

digestibility, vitamins, minerals and health promoting phytochemicals of seeds (Cho

293

and Lim, 2016).

PT

ED

289

BR variery SLF09 provides γ-oryzanol in the form of four main derivatives. A

295

wide range of variation for total γ-oryzanol has been reported in varieties of BR from

296

different geographical origin, from 1.2 mg/100g in BR varieties from the Camargue

297

region of France (Pereira-Caro et al., 2013) to 313 mg/100g in a BR cultivar grown in

298

Taiwan (Huang and Ng, 2012). The amounts of γ-oryzanol found in BR variety SLF09

299

is comparable to those previously reported in three indica cultivars grown in Brazil

300

(Pascual et al., 2013), and in eight cultivars from South Sarawak, Malaysia (Kiing et al.,

301

2009). The contribution of each steryl ferulate to total γ-oryzanol content lies within the

AC

CE

294

13

ACCEPTED MANUSCRIPT
range previously reported in different French rice varieties (Pereira-Caro et al., 2013)

303

and differ to those observed in long BR grain cultivars (Miller and Engel, 2006), in

304

which the largest proportion was accounted by cycloartenyl ferulate (43-48%), followed

305

by 24-methylene cycloartanyl ferulate (26-29%) and, in minor proportions, campestryl

306

ferulate (17-21%) and sitosteryl ferulate (7-8%). The different proportions of individual

307

γ-oryzanol constituents have been attributed to the variability among genotypes.

NU
SC
RI
PT

302

During germination process, γ-oryzanol underwent a significant decrease (15%)

309

that occurred mainly during the initial hydration process, since not further changes

310

during germination were found. Results reported in the literature about the effect of

311

germination on the content of total γ-oryzanol in BR are not coincident possibly due to

312

different germination conditions used. Results presented here are in accordance with

313

those previously reported in several BR cultivars from Malaysia (Kiing et al., 2009)

314

where a decrease of γ-oryzanol after germination at 25 ºC for 24 h was observed, and

315

differ to Thai cultivar RD-6 that underwent an increase after 12 h soaking and further 24

316

h-germination at 28-30 ºC (Moongngarm and Saetung, 2010). During the germination

317

process hydrolytic enzymes are activated and the decrease observed on γ-oryzanol could

318

be due to the induction of feruloyl esterases activity during the initial soaking process

319

(Sancho et al., 1999). In addition, dynamic ferulic acid metabolism during BR hydration

320

may occur (Tian et al., 2004). Nevertheless, results indicate that individual steryl

321

ferulate contribution remained almost constant throughtout germination process, effect

322

that has not been reported previously.

ED

PT

CE

AC

323

MA

308

GBR were sundried and -oryzanol increased between 34 and 48%. These

324

outcomes evidence the accumulation of γ-oryzanol derivatives during drying under solar

325

exposition. It has been reported that sunlight has a profound effect on the biosynthesis

326

of ferulic acid esters by affecting the metabolic activation of enzymes involved in the

14

ACCEPTED MANUSCRIPT
327

defence mechanism to radiation and in the development of new plant structural tissues

328

(Wang et al., 2014). This is the first report describing the effect of sun-drying on γ-

329

oryzanol content and composition evidencing GBR as a rich source of γ-oryzanol.
It is widely recognized that γ-oryzanol is a natural antioxidant. Among its

331

individual components, 24-methylene cycloartenyl ferulate exhibited the greatest

332

antioxidant potential and, together with cycloartenyl ferulate, showed anti-inflammatory

333

properties (Akihisa et al., 2000). γ-Oryzanol is administrated to the treatment of

334

diabetes, menopause, allergies and gastrointestinal inflammatory diseases and one of the

335

most demanding compounds for nutraceutical, pharmaceutical and cosmeceutical

336

preparations (Lemus et al., 2014). Our results show that sun-drying enhance γ-oryzanol

337

content in GBR, and can be considered as a sustainable bio-efficient process to develop

338

γ-oryzanol enriched GBR.

MA

NU
SC
RI
PT

330

GABA is usually present as a minor compound in crude grains, however,

340

germination boosts its accumulation in rice sprouts (Cáceres et al., 2014b). GABA

341

synthesis is usually initiated as consequence of the activation of glutamate

342

decarboxylase (GAD) enzyme during soaking process, activity that increases with

343

germination time (Roohinejad et al., 2011). GAD catalyses the decarboxylation of

344

glutamic acid to GABA and CO2 and it has been established a range between 20 and 40

345

ºC as optimal temperature for enzyme activity (Yang et al., 2013).

CE

PT

ED

339

The drying process of GBR under sunlight had a different effect on GABA

347

depending on the previous germination conditions and higher amounts were only found

348

in soaked BR and 34 ºC/48h GBR. These results can be partly attributable to some

349

remaining GAD activity after germination due to the activity of this enzyme at

350

temperatures below 40 °C (Yang et al., 2013). GABA diminution was observed in those

351

dried samples previously germinated for 96 h, results that could be attributed to

AC

346

15


ACCEPTED MANUSCRIPT
activation of GABA shunt pathway sunlight exposue. These metabolic pathway uses

353

GABA as precursor for the synthesis of succinic acid required in the Krebs cycle (Fait

354

et al., 2008). Nevertheless, the content of GABA in sundried GBR has been described in

355

the present work for the first time, ranging from 12 mg/100g DM in soaked grains to 67

356

mg/100g DM in 34 ºC/96h GBR. GABA has a well-known antihypertensive effect and

357

it has been reported that a daily GABA intake of 20 mg causes a reduction of blood

358

pressure in individuals with pre-hypertension (Inoue et al., 2003). Taking into account

359

that 100 g of sun-dried GBR provide between 1.5 to 3-fold these required amounts, its

360

consumption would contribute to control blood pressure.

NU
SC
RI
PT

352

BR is considered a good source of phenolic compounds and the content in the

362

variety SLF09 is within the range previously reported (Ti et al., 2014). In BR, TPC

363

content increased sharply as consequence of germination time while temperature had a

364

minor influence (Cáceres et al., 2014b). This increment has been partially explained by

365

the production of enzymes that hydrolyse fiber components during GBR germination

366

(Tian et al., 2004). In addition, the action of endogenous esterases can release free

367

phenolics needed for synthesis of more complex compounds providing, at the same

368

time, defence against environmental agents (Lemus et al., 2014). GBR obtained at 34ºC

369

for 96 h in the present work exhibited greater TPC content than those reported

370

previously (Cáceres et al., 2014b; Moongngarm and Saetung, 2010; Ti et al., 2014). Ti

371

et al., (2014) identified protocatechuic, chorogenic, caffeic and ferulic acids as the main

372

phenolic acids in GBR and the later was the most abundant (357 µg/g d.m. after 5 day-

373

germination).

AC

CE

PT

ED

MA

361

374

Sun-drying preserved or, even, increased the content of TPC (Figure 3). Although it

375

might be expected a drop due to their susceptibility to oxidation during light exposure,

376

the TPC rise found could be possibly due to the activation of the phenylpropanoid

16


ACCEPTED MANUSCRIPT
pathway in response to environmental factors and UV-B exposure (Du et al., 2014).

378

Phenolic compounds are considered bioactive compounds with health implications.

379

Particularly, soluble phenolic acids inhibit the oxidation of LDL cholesterol and the cell

380

membrane liposomes attenuating inflammation and enhancing mental health, immunity

381

and protecting against diabetes deterioration (Chandrasekara and Shahidi, 2011).

382

Therefore, sundried GBR can be considered an important source of phenolic

383

compounds with beneficial attributes.

NU
SC
RI
PT

377

The antioxidant activity found in BR was higher than those observed in different

385

Ecuadorian BR by Cáceres et al. (2014b), and differ to those reported by Ti et al. (2014)

386

in BR variety Tianyou 998. This variability on antioxidant activity in crude grains could

387

be attributed to the phenolic composition in different BR genotypes as well as to the

388

contribution of other antioxidant compounds such as γ-oryzanol and vitamin E isomers

389

(Cáceres et al., 2014b; Moongngarm and Saetung, 2010). Germination enhanced the

390

antioxidant potential of BR variety SLF09, in agreement with previous studies (Cáceres

391

et al., 2014b; Ti et al., 2014; Tian et al., 2004). During germination of BR, antioxidant

392

activity was time and temperature dependent, as recently reported (Cáceres et al.,

393

2014b), most likely caused by the accumulation of compounds with peroxyl-scavenging

394

activity such as phenolic compounds, as it was confirmed by the positive correlation

395

obtained between antioxidant activity and TPC (Figure 4B). However, other antioxidant

396

compounds such as tocopherols, tocotrienols, phytates and vitamin C could also

397

contribute to this activity (Frias et al., 2005), and this contribution may explain the lack

398

of significant correlation between the γ-oryzanol content and the activity antioxidant in

399

GBR (Figure 4A). In sundried GBR samples, antioxidant activity was always

400

significantly (P0.05) higher than their germinated counterparts, phenomenon that can

401

be attributed to the increase observed in bioactive compounds such as γ-oryzanol and

AC

CE

PT

ED

MA

384

17


ACCEPTED MANUSCRIPT
polyphenols. This hypothesis was confirmed by the significant positive correlation

403

found between them (Figure 4C and 4D, respectively). Lemus et al. (2014) have

404

recently shown that antioxidant activity of GBR is associated with the prevention of

405

oxidative stress-related diseases. The present work exhibits, by the first time, the

406

antioxidant activity of sun-dried GBR and its consumption could contribute to

407

ameliorate highly societal prevalent degenerative diseases.

NU
SC
RI
PT

402

408
409

4.

Conclusions

Germination conditions affected the content of biologically active compounds of

411

BR variety SLF09. γ-Oryzanol decreased slightly during germination and sun-drying

412

led to an important accumulation. GABA was synthetized during germination in a time-

413

dependent manner and underwent significant rises after sun-drying only in those

414

germinated for 48 h. TPC and antioxidant activity increased during germination and

415

were preserved or even enhanced under solar dehydration. These outcomes show

416

germination as a simple and sustainable process to enhance BR bioactive compounds

417

and reveal, for the first time, the effectiveness of sun-drying for maximizing their

418

accumulation. The obtained sun-dried GBR can be consumed directly after rehydatation

419

as staple food or, after a milling process, can be incorporated in bakery or pasta

420

products. In this context, consumption of sundried GBR can take place as parbolished

421

rice to feed a large world population and contribute to the control of metabolic related

422

disorders.

424

ED

PT

CE

AC

423

MA

410

Acknowledgments

425

This work has received financial support from the project AGL2013-43247R

426

from Ministerio de Economia y Competitividad (Spain) and European Union through

18


ACCEPTED MANUSCRIPT
FEDER Programme. P. J. Cáceres is indebted to the Ministry of High Education,

428

Science, Technology and Innovation (SENESCYT, Ecuador) for the foreign Ph.D. grant

429

and E. Peñas to Ramon y Cajal Programme for financial support. We also acknowledge

430

to INDIA-PRONACA enterprise for providing the BR cultivars.

NU
SC
RI
PT

427

431
432

References

433

Akihisa, T., Yasukawa, K., Yamaura, M., Ukiya, M., Kimura, Y., Shimizu, N., Arai,

434

K., 2000. Triterpene alcohol and sterol ferulates from rice bran and their anti-

435

inflammatory effects. Journal of Agricultural and Food Chemistry 48, 2313-2319.

436
437

AOAC, 2000 Official Methods of Analysis of the Association of Official Analytical
Chemists. 17th Edition.

Cáceres, P.J., Martínez-Villaluenga, C., Amigo, L., Frias, J., 2014a. Assessment on

439

Proximate Composition, Dietary Fiber, Phytic Acid and Protein Hydrolysis of

440

Germinated Ecuatorian Brown Rice. Plant Foods for Human Nutrition 69, 261-267.

441

Cáceres, P.J., Martínez-Villaluenga, C., Amigo, L., Frias, J., 2014b. Maximising the

442

phytochemical content and antioxidant activity of Ecuadorian brown rice sprouts

443

through optimal germination conditions. Food Chemistry 152, 407-414.

PT

ED

MA

438

Cornejo, F., Caceres, P.J., Martínez-Villaluenga, C., Rosell, C.M., Frias, J., 2015.

445

Effects of germination on the nutritive value and bioactive compounds of brown

446

rice breads. Food Chemistry 173, 298-304.

448
449
450

AC

447

CE

444

Chandrasekara, A., Shahidi, F., 2011. Bioactivities and antiradical properties of millet

grains and hulls. Journal of Agricultural and Food Chemistry 59, 9563-9571.

Cho, D.H., Lim, S.T., 2016. Germinated brown rice and its bio-functional compounds.
Food Chemistry 196, 259-271.

19


ACCEPTED MANUSCRIPT
Cho, J.Y., Lee, H.J., Kim, G.A., Kim, G.D., Lee, Y.S., Shin, S.C., Park, K.H., Moon,

452

J.H., 2012. Quantitative analyses of individual γ-Oryzanol (Steryl Ferulates) in

453

conventional and organic brown rice (Oryza sativa L.). Journal of Cereal Science

454

55, 337-343.

NU
SC
RI
PT

451

455

Chungcharoen, T., Prachayawarakorn, S., Tungtrakul, P., Soponronnarit, S., 2014.

456

Effects of Germination Process and Drying Temperature on Gamma-Aminobutyric

457

Acid (GABA) and Starch Digestibility of Germinated Brown Rice. Drying

458

Technology 32, 742-753.

459

Du, W.X., Avena-Bustillos, R.J., Breksa, A.P., McHugh, T.H., 2014. UV-B light as a

460

factor affecting total soluble phenolic contents of various whole and fresh-cut

461

specialty crops. Postharvest Biology and Technology 93, 72-82.

Fait, A., Fromm, H., Walter, D., Galili, G., Fernie, A.R., 2008. Highway or byway: the

463

metabolic role of the GABA shunt in plants. Trends in Plant Science 13, 14-19.

464

Frias, J., Miranda, M.L., Doblado, R., Vidal-Valverde, C., 2005. Effect of germination

465

and fermentation on the antioxidant vitamin content and antioxidant capacity of

466

Lupinus albus L. var. Multolupa. Food Chemistry 92, 211-220.

ED

MA

462

Huang, S.H., Ng, L.T., 2012. Quantification of polyphenolic content and bioactive

468

constituents of some commercial rice varieties in Taiwan. Journal of Food

469

Composition and Analysis 26, 122-127.

471
472
473

CE

Inoue, K., Shirai, T., Ochiai, H., Kasao, M., Hayakawa, K., Kimura, M., Sansawa, H.,

AC

470

PT

467

2003. Blood-pressure-lowering effect of a novel fermented milk containing γ-

aminobutyric acid (GABA) in mild hypertensives. European Journal of Clinical
Nutrition 57, 490-495.

20


ACCEPTED MANUSCRIPT
474

Izadi, V., Azadbakht, L., 2015. Is there any association between rice consumption and

475

some of the cardiovascular diseases risk factors? A systematic review. Advanced

476

Research Yields in Atherosclerosis, 11, 109-115.
Karam, M.C., Petit, J., Zimmer, D., Baudelaire Djantou, E., Scher, J., 2016. Effects of

478

drying and grinding in production of fruit and vegetable powders: A review.

479

Journal of Food Engineering 188, 32-49.

NU
SC
RI
PT

477

480

Kiing, S.C., Yiu, P.H., Rajan, A., Wong, S.C., 2009. Effect of germination on γ-

481

oryzanol content of selected sarawak rice cultivars. American Journal of Applied

482

Sciences 6, 1658-1661.

Lemus, C., Angelis, A., Halabalaki, M., Skaltsounis, A.L., 2014. γ-Oryzanol. An

484

Attractive Bioactive Component from Rice Bran, Wheat and Rice in Disease

485

Prevention and Health, pp. 409-430.

MA

483

Miller, A., Engel, K.H., 2006. Content of γ-oryzanol and composition of steryl ferulates

487

in brown rice (Oryza sativa L.) of European origin. Journal of Agricultural and

488

Food Chemistry 54, 8127-8133.

ED

486

Moongngarm, A., Saetung, N., 2010. Comparison of chemical compositions and

490

bioactive compounds of germinated rough rice and brown rice. Food Chemistry

491

122, 782-788.

CE

PT

489

Ochoa-Avilés, A., Verstraeten, R., Lachat, C., Andrade, S., Van Camp, J., Donoso, S.,

493

Kolsteren, P., 2014. Dietary intake practices associated with cardiovascular risk in

494
495

AC

492

urban and rural Ecuadorian adolescents: A cross-sectional study. BMC Public

Health 14.

496

Pascual, C.S.C.I., Massaretto, I.L., Kawassaki, F., Barros, R.M.C., Noldin, J.A.,

497

Marquez, U.M.L., 2013. Effects of parboiling, storage and cooking on the levels of

21


ACCEPTED MANUSCRIPT
498

tocopherols, tocotrienols and γ-oryzanol in brown rice (Oryza sativa L.). Food

499

Research International 50, 676-681.
Pereira-Caro, G., Cros, G., Yokota, T., Crozier, A., 2013. Phytochemical profiles of

501

black, red, brown, and white rice from the camargue region of France. Journal of

502

Agricultural and Food Chemistry 61, 7976-7986.

NU
SC
RI
PT

500

503

Roohinejad, S., Omidizadeh, A., Mirhosseini, H., Saari, N., Mustafa, S., Meor Hussin,

504

A.S., Hamid, A., Abd Manap, M.Y., 2011. Effect of pre-germination time on amino

505

acid profile and gamma amino butyric acid (GABA) contents in different varieties

506

of Malaysian brown rice. International Journal of Food Properties 14, 1386-1399.

Sancho, A.I., Faulds, C.B., Bartolomé, B., Williamson, G., 1999. Characterisation of

508

feruloyl esterase activity in barley. Journal of the Science of Food and Agriculture

509

79, 447-449.

MA

507

Ti, H., Zhang, R., Zhang, M., Li, Q., Wei, Z., Zhang, Y., Tang, X., Deng, Y., Liu, L.,

511

Ma, Y., 2014. Dynamic changes in the free and bound phenolic compounds and

512

antioxidant activity of brown rice at different germination stages. Food Chemistry

513

161, 337-344.

ED

510

Tian, S., Nakamura, K., Kayahara, H., 2004. Analysis of phenolic compounds in white

515

rice, brown rice, and germinated brown rice. Journal of Agricultural and Food

516

Chemistry 52, 4808-4813.

518
519

CE

Wang, W., Guo, J., Zhang, J., Peng, J., Liu, T., Xin, Z., 2014. Isolation, identification

AC

517

PT

514

and antioxidant activity of bound phenolic compounds present in rice bran. Food

Chemistry 171, 40-49.

520

Wu, F., Yang, N., Touré, A., Jin, Z., Xu, X., 2013. Germinated Brown Rice and Its

521

Role in Human Health. Critical Reviews in Food Science and Nutrition 53, 451-

522

463.

22


ACCEPTED MANUSCRIPT
523

Yang, R., Yin, Y., Guo, Q., Gu, Z., 2013. Purification, properties and cDNA cloning of

524

glutamate decarboxylase in germinated faba bean (Vicia faba L.). Food Chemistry

525

138, 1945-1951.

AC

CE

PT

ED

MA

NU
SC
RI
PT

526

23