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: frias@ictan.csic.es ACCEPTED MANUSCRIPT Abstract Germinated brown rice (GBR) has been suggested as an alternative approach to mitigate highly prevalent diseases providing nutrients and biologically active compounds In this study, the content of γ-oryzanol, γ-aminobutyric acid (GABA), total phenolic compounds (TPC) and antioxidant activity of soaked (for 24 h at 28°C) and GBR (for 48 and 96 h at 28°C and 34°C) were determined and the effect of sun-drying as an economically affordable process was assessed Germination improved the content of GABA, TPC and antioxidant activity in a time-dependent manner Sun-drying increased 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 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 ACCEPTED MANUSCRIPT 30 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 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 diabetes, neurodegenerative diseases and osteoporosis 53 and GBR has been identified as a natural and inexpensive substitute of conventional 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 ACCEPTED MANUSCRIPT (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 NU SC RI PT 79 90 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 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 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 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 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 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 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, g of sample was mixed with 10 ACCEPTED MANUSCRIPT mL of methanol and further sonicated for 10 The mixture was centrifuged at 130 15,000 rpm for 10 at room temperature (25 ± ºC) and then concentrated to 131 dryness Samples were then diluted in 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., 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 as follows: initial isocratic flow 60% solvent A, 35% 139 solvent B and 5% solvent C for min, gradient flow 60% solvent A and 40% solvent B 140 for keeping it at isocratic flow for 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 and, finaly, isocratic conditions to equilibrate column for 10 143 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 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., 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 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 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 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 ACCEPTED MANUSCRIPT 527 Figure captions 528 Figure Chromatogram of γ-oryzanol standard Peak 1, cycloartenyl ferulate; peak 530 2, 24-methylenecycloartanyl ferulate; peak 3, campesteryl ferulate; peak 4, sitosteryl 531 ferulate NU SC RI PT 529 532 533 Figure Contribution of the individual steryl ferulates to total content of γ- 534 oryzanol in crude, soaked, and germinated brown rice and effect of sun-drying 535 1, cycloartenyl ferulate; 2, 24-methylenecycloartanyl ferulate; 3, campesteryl 536 ferulate; 4, sitosteryl ferulate 537 Figure Effect of sun-drying on bioactive compounds and antioxidant activity of 539 soaked and germinated brown rice, indicating increase percentages (positive y-axe) or 540 decrease percentages (negative y-axe) MA 538 ED 541 Figure Correlation analysis between antioxidant activity with the content of γ- 543 oryzanol (A) and TPC (B) for GBR, and between antioxidant activity with the content 544 of γ-oryzanol (C) and TPC (D) for sundried GBR R2 indicates the percentage of 545 variation explained by the model and r indicates the correlation 548 549 CE 547 AC 546 PT 542 24 Table Content of γ-oryzanol components (mg/100g d.m.) in crude, soaked and germinated brown rice variety SFL09 and 551 the effect of sun-drying RI PT 550 552 24-Methylene Sitosteryl Campestryl ferulate cycloartanyl ferulate ferulate (Peak 3) (Peak 4) (Peak 2) 2.60±0.05b 4.98±0.07d 2.24±0.03b Freeze-dried Soaked 28ºC, 24h Germinated 2.21±0.04aA 4.27±0.06aA 1.67±0.05aA 28ºC, 48h 2.22±0.06aA 4.32±0.12abA 28ºC, 96h 2.32±0.07aA 4.52±0.10bcA 34ºC, 48h 2.33±0.13aA 4.56±0.20cA 34ºC, 96h 2.36±0.11aA 4.58±0.19cA Sun-dried Soaked 28ºC, 24h Germinated 2.63±0.11bB 6.07±0.18eB 28ºC, 48h 2.60±0.05bB 28ºC, 96h 3.56±0.05cB 34ºC, 48h 3.24±0.06cB Total γoryzanol 1.34±0.02b 11.17±0.10b 1.08±0.02aA 9.23±0.08aA 1.61±0.05aA 1.05±0.03aA 9.20±0.20aA 1.58±0.03aA 1.09±0.02aA 9.52±0.17aA 1.59±0.15aA 1.11±0.08aA 9.59±0.56aA 1.60±0.09aA 1.10±0.05aA 9.64±0.42aA 3.56±0.13cB 1.82±0.03bB 14.08±0.19bB 6.08±0.17eB 3.65±0.09cB 1.78±0.03bB 14.09±0.21bB 7.70±0.09fB 4.64±0.09eB 2.30±0.03cB 18.18±0.17dB 7.23±0.04fB 4.15±0.04dB 2.11±0.01cB 16.75±0.09cB CE PT ED MA NU Crude SC BR samples Cycloartenyl ferulate (Peak 1) 554 differences among germination conditions (P≤0.05 according to Duncan’s test) Uppercase letters indicate statistical 555 differences among drying process for a same germination conditions (P≤0.05 according to Duncan’s test) AC 553 34ºC, 96h 3.09±0.12cB 7.21±0.11fB 4.36±0.15eB 2.07±0.02cB 16.73±0.07cB Data are the mean values ± standard deviation of three independent experiments (n=3) Lowercase letters indicate statistical 25 ACCEPTED MANUSCRIPT 556 Table Content of γ-aminobutyric acid (GABA), total phenolic compounds (TPC) and 557 antioxidant activity (ORAC) of crude, soaked and germinated brown rice and the effect of sun- 558 drying 559 BR samples GABA (mg/100g d.m.) TPC (mg GAE/100g dm) Crude 1.07±0.09a 132.53±2.78b 494.81±19.71a 7.46±0.12bA 113.23±7.77aA 508.41±12.49abA Germinated 28ºC, 48h 34.84±2.78dA 187.17±3.19dA 554.85±17.59bA 28ºC, 96h 99.03±4.83fB 298.23±13.48eA 977.47±62.49dA 34ºC, 48h 24.33±0.44cA 176.48±3.02cA 622.80±18.60cA 34ºC, 96h 83.60±2.67eB 382.99±10.44gA 1079.35±69.70dA NU SC RI PT Freeze-dried Soaked 28ºC, 24h MA Sun-dried 12.75±0.50gB 118.14±5.30fA 547.66±25.22eA Germinated 28ºC, 48h 36.41±2.67hA 190.29±8.55gA 978.63±30.33fB 28ºC, 96h 49.85±4.62iA 359.22±12.35hB 1283.25±74.04iB 34ºC, 48h 36.50±1.36hB 195.13±18.26gA 826.82±54.82gB 429.34±17.54iB 1174.88±45.48hA PT 34ºC, 96h ED Soaked 28ºC, 24h 560 ORAC (mg TE/100g d.m.) 66.94±1.21jA Data are the mean values ± standard deviation of three independent experiments (n=3) Lowercase 562 letters indicate statistical differences among germination conditions (P≤0.05 according to Duncan’s 563 test) Uppercase letters indicate statistical differences among drying process for a same germination 564 conditions (P≤0.05 according to Duncan’s test) AC 565 CE 561 26 Peak MANUSCRIPT ACCEPTED 566 567 568 569 570 NU SC RI PT 571 572 573 574 Peak 575 576 577 578 579 580 MA 581 582 583 ED 584 Peak 585 586 PT 587 588 CE 589 590 593 594 AC 591 592 Peak Figure 27 ACCEPTED MANUSCRIPT 595 596 597 598 Germinated brown rice NU SC RI PT 599 CE PT ED Germinated brown rice AC 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 MA 600 Figure 28 ACCEPTED MANUSCRIPT 636 637 638 60 NU SC RI PT 40 20 Soaked 28 °C - 48 h 28 °C - 96 h 34 °C - 48 h 34 °C - 96 h -20 -40 -60 -80 -100 γ-Oryzanol 641 642 Figure ED 643 644 645 AC CE PT 646 647 ORAC MA 639 640 TPC GABA -120 29 ACCEPTED MANUSCRIPT C A 648 649 650 652 653 y=353.8x – 2589.36 r=0.47 654 R =0.22 656 r= 0.82 R =0.68 D B 657 y=127.759x – 1073.91   655 NU SC RI PT 651 658 Figure Correlation analysis between antioxidant activity with the content of γ-oryzanol (A) 660 and TPC (B) for GBR, and between antioxidant activity with the content of γ-oryzanol (C) 661 and TPC (D) for sundried GBR R2 indicates the percentage of variation explained by the 662 model ED MA 659 y=2.494x + 160.61 r=0.96 665 666 669 R =0.73 Figure AC 668 r=0.86 CE 664 667 y=2.0952x – 396.82 PT 663 R =0.92 30 [...]... Contribution of the individual steryl ferulates to total content of γ- 534 oryzanol in crude, soaked, and germinated brown rice and effect of sun- drying 535 1, cycloartenyl ferulate; 2, 24-methylenecycloartanyl ferulate; 3, campesteryl 536 ferulate; 4, sitosteryl ferulate 537 Figure 3 Effect of sun- drying on bioactive compounds and antioxidant activity of 539 soaked and germinated brown rice, indicating increase... 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... 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. .. 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... 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... 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. .. 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 with the content of γ- 543 oryzanol (A) and TPC (B) for GBR, and between antioxidant activity with the content 544 of γ-oryzanol (C) and TPC (D) for sundried GBR R2 indicates the percentage of 545 variation explained by the model and r indicates the correlation 548 549 CE 547 AC 546 PT 542 24 Table 1 Content of γ-oryzanol components (mg/100g d.m.) in crude, soaked and germinated brown rice variety... 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... 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,