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

 Brown rice BR is a good source of biologically active compounds  The content of GABA, TPC and antioxidant activity enhanced during germination of BR  Sun-drying maximizes the content

Enhancement of biologically active compounds in germinated brown rice and the

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

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

 The content of GABA, TPC and antioxidant activity enhanced during germination of BR

 Sun-drying maximizes the content of bioactive compounds in GBR

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

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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 Juana Friasb*

aEscuela 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 (ICTAN-CSIC), Juan de la Cierva 3, 28006 Madrid, Spain

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

de Cantoblanco, 28049 Madrid, Spain

1 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

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

30 1 Introduction

31 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)

40 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)

50 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

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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)

56 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

72 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

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79 (http://www.serviciometeorologico.gob.ec/meteorologia/boletines/bol_anu2015),

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

89

90 2 Material and methods

91 2.1 Rice samples

92 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 Germination process

97 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

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

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104 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 Sun-drying proccess

115 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 Determination of moisture content

124 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

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129 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 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

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154 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 Determination of total phenolic compounds

165 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 Determination of antioxidant activity

172 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.ACCEPTED MANUSCRIPT

177 2.9 Statistical analysis

178 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

187

188 3 Results

189 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

194 BR variety SLF09 exhibited four main chromatographic peaks that

195 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)

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202 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)

209 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

220 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

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227 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

233 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

242 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

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252 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)

260 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)

267 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

273

274 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

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277 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)

289 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)

294 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

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302 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

308 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

323 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

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