Food Chemistry 161 (2014) 337–344

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Dynamic changes in the free and bound phenolic compounds and antioxidant activity of brown rice at different germination stages Huihui Ti 1, Ruifen Zhang 1, Mingwei Zhang ⇑, Qing Li, Zhencheng Wei, Yan Zhang, Xiaojun Tang, Yuanyuan Deng, Lei Liu, Yongxuan Ma Sericultural and Agri-food Research Institute, Guangdong Academy of Agricultural Sciences, Guangzhou 510610, PR China

a r t i c l e

i n f o

Article history: Received 28 January 2014 Received in revised form 2 April 2014 Accepted 4 April 2014 Available online 13 April 2014 Keywords: Brown rice Germination stage Phenolics Flavonoids Antioxidant activity

a b s t r a c t Germinated brown rice is a good source of the phenolics associated with antioxidant effects. Germination significantly increased by 63.2% and 23.6% the total phenolic and flavonoid contents, respectively. The percentage contribution of bound phenolics to total was 42.3% before and decreased slightly to 37.6% after germination. The percentage contribution of bound flavonoids to total, 51.1%, was the same before and after germination. The change in the amounts of free and bound forms indicated that transformations could occur during the germination process. Six individual phenolics were detected by HPLC. The levels of ferulic, coumaric, syringic, and caffeic acid significantly increased. The ratio of bound ferric reducing antioxidant power to total was basically constant, while germination increased the ratio of bound oxygen radical absorbance capacity to total. This indicated that the increase of bound phenolics exerts beneficial health effects throughout the digestive tract after absorption and may reduce mutations. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Rice (Oryza sativa L.) is the major staple food for nearly half of the world’s population. Brown rice is a rice seed from which only the husk is dehulled, and it consists of the embryo, endosperm, and bran. Compared with polished rice, brown rice contains more abundant health-promoting substances, including dietary fibres, vitamins, gamma-amino butyric acid (GABA), and c-oryzanol. Despite its nutritional value and physiological functions, brown rice is not widely consumed because it has poor cooking properties and a harsh texture mainly because of the high fibre content in the bran. Numerous studies have shown that the nutritional and chemical profiles of cereal grains are altered because of germination. Presently, germinated brown rice (GBR) is one of the most popular germinated cereal products, and it has gained a great deal of attention as an alternative to brown rice because of its improved texture. GBR is simply produced by soaking brown rice grains in water until they begin to bud (Komatsuzaki et al., 2007). During germination, some molecules are degraded for the respiration and synthesis of new cell constituents, which causes significant changes in the biochemical, nutritional and sensory characteristics ⇑ Corresponding author. Tel.: +86 2087237865; fax: +86 2087236354. 1

E-mail address: [email protected] (M. Zhang). These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.foodchem.2014.04.024 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.

of cereals. On one hand, enzymes are activated to hydrolyse carbohydrates and proteins, which makes these nutrients more easily digestible by the human body (Komatsuzaki et al., 2007; Palmiano & Juliano, 1973). Additionally, germination improves the organoleptic quality of brown rice because the enzymatic hydrolysis of the polymeric materials softens the rice bran and often improves its flavour (Hunt, Johnson, & Juliano, 2002). On the other hand, the germination process generally results in improved nutritional values of cereal not only by decreasing the anti-nutritional components, such as phytic acid, but also by increasing the levels of vitamins, minerals, fibres and phytochemicals, such as ferulic acid, GABA, and c-oryzanol. Thus, GBR is believed to have a greater nutritional and physiological value compared with non-germinated brown rice. Phenolic compounds, as one of the most abundant groups of active phytochemicals in brown rice, exist in free and insolublebound forms (Adom & Liu, 2002). Several studies have analysed the total phenolic levels and their antioxidant activities of germinated brown rice. However, these studies actually reported only the content of solvent-extractable free phenolics after 24 h or after a five day germination treatment (Donkor, Stojanovska, Ginn, Ashton, & Vasiljevic, 2012; Moongngarm & Saetung, 2010). An extraction procedure mainly for free phenolics was used in those studies for determining the antioxidant activities. Such an extraction procedure may underestimate the total phenolic contents and the antioxidant activity if the bound fraction has not been

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included (Donkor et al., 2012; Moongngarm & Saetung, 2010). Moreover, complex biochemical reactions occurred during the germination process, and thus, it is not clear how the transformation of free and bound phenolics, and the antioxidant capacity, vary throughout brown rice’s germination process. Therefore, the objectives of the present study were (1) to investigate dynamic changes in the free and bound phytochemical (phenolics and flavonoids) contents and their antioxidant activities; and (2) to characterise the dynamic changes in the composition and content of individual phenolics in free and bound forms at different germination stages. We expect to provide information which can contribute to appropriately concentrate these antioxidant nutritions of brown rice according to the needs of the foods production in relation to germinated brown rice.

further analysis. The morphological illustrations of GBR at different stages are shown in Fig. 1. 2.3. Extraction of free phenolics

2. Materials and methods

The free phenolic compounds of the GBR samples were extracted using the method previously reported (Sun, Chu, Wu, & Liu, 2002). Briefly, 0.5 g of GBR was mixed with 50 mL of chilled acidified methanol (95% methanol and 1 M HCl 85:15, v/v). The mixture was homogenised using a XHF-D homogenizer at 10,000 rpm for 5 min in an ice-bath. The supernatants were obtained by centrifugation at 2500g for 10 min and concentrated under a vacuum at 45 °C until the filtrate had been evaporated. The concentrated filtrate was then reconstituted to a final volume of 10 mL with chilled acidified methanol. The extracts were stored at 20 °C prior to further analysis.

2.1. Chemicals and reagents

2.4. Extraction of bound phenolics

6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), 2, 20 -Azobis-(2-amidinopropane) dihydrochloride (ABAP) and 30 , 60 -Dihydroxyspiro[isobenzofuran-1(3H), 90 -(9H)-xanthene]-3-one disodium salt (FL) were purchased from Sigma Chemical Co. Gallic acid, chlorogenic acid, protocatechuic acid, catechin, caffeic acid, syringic acid, p-coumaric acid, ferulic acid and syringic acid were purchased from Aladdin (Shanghai, China). HPLC-grade acetic acid and acetonitrile were obtained from Fisher (Suwanee, GA, USA). All other chemicals used were of an analytical grade or above.

Bound phenolics were extracted according to the previous literature (Naczk & Shahidi, 1989; Sun et al., 2002). Briefly, the residue from the above free phenolic extraction was hydrolysed with 40 mL of 2 M NaOH at room temperature for 1 h with continuous shaking under nitrogen gas. The mixture was defatted with hexanes and then neutralised with concentrated HCl. The remaining mixture was then extracted five times with EtOAc. The supernatants were combined and evaporated at 45 °C to dry. The bound phenolics were reconstituted with distilled water to a final volume of 10 mL and then stored at 20 °C prior to further use.

2.2. GBR preparation

2.5. Determination of total phenolic content

Fresh brown rice, Tianyou 998, was obtained from the Experimental Station of the Rice Research Institute of the Guangdong Academy of Agricultural Sciences in 2012. Rice grains were airdried and stored at room temperature. Then, they were dehusked to obtain brown rice. Five grams of brown rice were treated for 30 min with 25 mL of 0.07% sodium hypochlorite. Then, the seeds were watered to a neutral pH and soaked in distiled water for 5.5 h and shaken every 30 min. The hydrated seeds were placed on a germination tray on wet laboratory paper and then they were covered. The tray was introduced into a germination machine (G-120 Snijders International S. L., Holland) and the wet paper was in contact with the germinator’s circulating water, keeping the seeds always wet by capillary action (Frías, Miranda-Zárate, & Vidal-Valverde, 2005). The germination process was performed in the darkness at 20 °C with 99% relatively humidity. Five stages, G1–G5, of germination at 17, 24, 30, 35 and 48 h, respectively, were investigated. Three samplings were taken randomly at each stage. The sprouts were freeze-dried, ground and passed through a 0.5 mm pore-sized sieve. The flour obtained was stored in darkness, under vacuum conditions in desiccators at 4 °C prior to

The total phenolic content was analysed using the Folin Ciocalteu (FC) colorimetric method described previously by Dewanto, Wu, Adom, and Liu (2002). Briefly, 0.125 mL of the extracts was mixed with 0.5 mL of distilled water and subsequently with 0.125 mL of FC reagent. After 6 min, 1.25 mL of a 7% aqueous Na2CO3 solution was added to the mixture. Then, water was added to bring the total volume to 3 mL. The mixtures were incubated for 90 min, and the absorbance of the resulting blue colour was measured at 760 nm using a Shimadzu UV-1800 spectrometer (Shimadzu Inc., Kyoto, Japan). Gallic acid was used as the standard, and the total phenolic content was expressed as milligrams of gallic acid equivalents (GAE) per 100 g of sample dry weight (DW). 2.6. Determination of total flavonoid content The total flavonoid content was determined using a modified colorimetric method described previously (Dewanto et al., 2002). A 0.3 mL aliquot of the extracts was added to a tube containing 1.5 mL of distilled water. Then, 0.09 mL of 5% NaNO2 solution was added to the mixture. Five min later, 0.18 mL of 10% AlCl36H2

Fig. 1. Brown rice at different germination stages. (A) Before germination, 0 h; (B) germinated for 17 h; (C) germinated for 24 h; (D) germinated for 30 h; (E) germinated for 35 h; and (F) germinated for 48 h.

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O solution was added. At 11 min, 0.6 mL of 1 M NaOH solution was added. Distilled water was added to bring the total volume to 3 mL, and the absorbance was determined immediately at 510 nm using a Shimadzu UV-1800 spectrometer. The total flavonoid content was expressed as milligrams of (+)-catechin equivalents (CE) per 100 g of sample DW. Additional dilutions were necessary if the absorbance measurement was above the linear range of the (+)catechin standard curve. 2.7. Determination of the phenolic composition The samples were analysed on an Agilent 1200HPLC system (Waldbronn, Germany) equipped with an Agilent 1200 series VWD detector and an autosampler using a 250 mm  4.6 mm, 5 lm Agilent Zorbox SB-C18 column (Palo Alto, CA, USA). The mobile phase was a 0.4% aqueous solution of acetic acid (solution A) and acetonitrile (solution B). The gradient used was as follows: 0–40 min, 5–25% of solution B; 40–45 min, 25–35% of solution B; and 45–50 min, 35–50% of solution B. The mobile phase was pumped at a constant flow rate of 1.0 mL/min. The injection volume was 20 lL, and the run time was 50 min. The column temperature was kept at 30 °C. The phenolics were detected at 280 nm. Prior to analysis, the samples were filtered through a 0.25 lm membrane filter (Millipore, Billerica, MA, USA). The identification of each peak was primarily based on a comparison of their retention time with the known standards. Over 94% of these phenolics was recovered. 2.8. Antioxidant activity determined by ferric reducing antioxidant power (FRAP) assay The FRAP assay was performed by modifying a previous method (Benzie & Strain, 1996). The FRAP reagent was prepared by mixing 25 mL of 300 mM acetate buffer (5.1 g of CH3COONa3H2O and 20 mL of CH3COOH pH 3.6), 2.5 mL TPTZ solution (10 mM TPTZ in 40 mM HCl), and 2.5 mL of 20 mM FeCl36H2O solution, and then incubating at 37 °C before use. A 0.03 mL aliquot of the above extract was added to a tube containing 0.09 mL of distilled water. The mixture was incubated with 0.9 mL of the FRAP reagent for 30 min in the darkness at room temperature. The absorbance was measured at 593 nm using a Shimadzu UV-1800 spectrometer. Trolox was used as the standard to establish a standard curve. The FRAP value was expressed as milligrams of Trolox equivalents (TE) per 100 g of sample DW. 2.9. Antioxidant activity determined by oxygen radical absorbance capacity (ORAC) assay The ORAC assay was performed according to a modified method from our laboratory (Zhang, Zhang, Zhang, & Liu, 2010). Briefly, GBR extract dilutions were prepared daily in 75 mM phosphate buffer (pH 7.4). The assay was carried out in black-walled 96-well

plates (Corning Scientific, Corning, NY, USA). The outside wells of the plate were not used because there was much more variation. The final reaction mixture of each well contained 20 lL of extracts or 20 lL of Trolox standard (range = 6.25–50 lM) and 200 lL of fluorescein (final concentration = 0.96 lM). The reaction mixture was pre-incubated at 37 °C for 20 min before rapidly adding the 2,20 -azobis(2-amidinopropane) dihydrochloride (ABAP) solution (20 lL, 119 mM) using a multichannel pipette. Fluorescence intensity was read from the top using a Fluoroskan Ascent FL platereader (Thermo Labsystems, Franklin, MA, USA) with an excitation wavelength of 485 nm and an emission wavelength of 538 nm for 35 cycles every 4.5 min. ORAC was expressed as micromoles of Trolox equivalents per gram of DW. 2.10. Statistical analysis Data were reported as mean ± SD for the three samplings at each stage. Different samples were analysed using an ANOVA followed by SNK-q test. A value of p < 0.05 was considered to be statistically significant. Pearson correlation tests were conducted to determine the correlation between variables. All of the statistical analyses were performed using the SPSS statistical package version 13.0 (SPSS Inc. Chicago, IL, USA). 3. Results 3.1. Effect of germination treatment on the total phenolic content and ratio of free and bound fractions to the total phenolics Changes of the free, bound, and total phenolic contents at different germination stages are presented in Table 1. The free, bound, and total phenolic contents increased during the germination process. The free phenolic contents ranged from 158.6 to 187.5 mg GAE/ 100 g DW during the germination process (G1–G5), which increased by 58.1–87.0% compared with those before germination. The free phenolic content increased significantly, by approximately 64.6%, at the first stage (G1). Then, a gradual increase in the free phenolic content from stage G1 to stage G3 was observed, and the content increased by 87.0% at G3. The free phenolic contents decreased at the final time point measured (G5), but had still increased by 76.7% compared with the content before germination. The free phenolic content was the highest at G3, followed by G4 and G5. The free phenolic contents of the last two time points were not significantly different (p > 0.05). The percentage contribution of free phenolics to the total increased to 66.0% at stage G3, and then decreased to 62.4% at the end of germination. The bound phenolic contents ranged from 90.3 to 106.6 mg GAE/100 g DW during the germination process, which gradually increased from 22.5% to 44.6% compared with that before germination. The bound phenolic content was the highest at G5 followed by G4 and G2. There was an insignificant increase between stages

Table 1 Total phenolic content during the germination of brown rice and the percentage contributions of free and bound fractions to the total phenolics. Germination stage

G0 G1 G2 G3 G4 G5 * **

Phenolics (mg of gallic acid equiv/100 g of DW) Free form

Bound form

Total phenolics

100.3 ± 9.8a* (57.7)** 164.6 ± 13.5c (64.6) 158.6 ± 11.0b (61.5) 187.5 ± 7.1e (66.0) 173.8 ± 3.0d (63.3) 177.2 ± 9.0d (62.4)

73.7 ± 5.1a (42.3) 90.3 ± 7.6b (35.4) 99.1 ± 8.0 cd (38.5) 96.6 ± 5.1c (34.0) 100.6 ± 9.7d (36.7) 106.6 ± 6.7e (37.6)

174.0 ± 10.8a 254.9 ± 13.8b 257.6 ± 16.8b 284.1 ± 6.9d 274.4 ± 13.4c 283.9 ± 11.3d

Values with no letters in common in each column are significantly different (p < 0.05). Values in parentheses indicate the percentage contribution to the total phenolics.

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G4 and G2 (p > 0.05). The bound phenolic content was the lowest at G3. The ratio of bound phenolics to the total was slightly lower during the germination process than before germination. The total phenolic contents varied from 254.9 to 284.1 GAE/ 100 g DW. The trend of changes in the content was consistent with that of the free phenolics. Free phenolics contributed significantly to the increase of the total phenolic content in G1. The total phenolic content significantly increased, by 46.5%, during the first stage. Then, a steady increase in the total phenolic content between stages G1 and G3 was observed, and its content reached the maximum value of 284.1 GAE/100 g DW, up 63.3%, at G3. There was an insignificant increase during the remainder of the germination process (G3–G5). The total phenolic content of GBR substantially increased, by 63.2%, compared with that of brown rice at G5. The highest total phenolic content was detected at G3 and G5 followed by G4, with somewhat similar total phenolic contents at the first two time points (p > 0.05). The lowest total phenolic contents were found at G1 and G2 during the germination treatment process (p < 0.05). 3.2. Effect of germination treatment on the total flavonoid content and ratio of free and bound fractions to the total flavonoids Changes of the free, bound, and total flavonoid contents at different germination stages are presented in Table 2. The free flavonoid contents ranged from 29.5 to 75.4 mg CE/ 100 g DW during the germination process (G1–G5). The free flavonoid content significantly increased from 11.3% in the G1 stage to 51.7% in the G2 stage (p < 0.05), and then a significant increase of 23.4% at stage G5 (p < 0.05) was observed when compared with the level before germination. The relative contributions of free flavonoid to the total flavonoid levels increased during the G1 and G2 stages and then decreased during the remaining stages. The percentage contribution of the free fraction to the total reached its lowest value of 24.0% at G2 and its highest value of 48.9% at G5, which was the same percentage as before germination. The bound flavonoid contents ranged from 74.1 to 97.4 mg GAE/ 100 g DW. The bound flavonoid content significantly increased to approximately 52.4% during the G1 and G2 stages (p < 0.05). It then significantly decreased during the remainder of the germination process, but still increased by 23.6% at G5 compared with the content before germination. The bound flavonoid content was the highest at G2, followed by G1. The bound flavonoid contents were not significantly different at G3, G4 and G5 (p > 0.05). The percentage contribution of bound flavonoids to the total reached the highest level, 76%, at G2 and the lowest level of 51.1% at G5, which was the same as before germination. The total flavonoid content increased from 114.5 to 154.4 mg CE/100 DW (G1–G5). The level was 124.9 mg CE/100 DW before germination. The total flavonoid content of GBR first increased by 11.3% at G1, then slowly decreased between stages G2 and G4, and finally significantly increased by 23.6% at G5 compared with

the level before germination. The total flavonoid contents were the highest at G1 and G5 (p < 0.05), which showed no significant difference between them (p > 0.05). The total flavonoid contents were not significantly different among G2, G3 and G4 (p > 0.05). 3.3. Effect of germination treatment on phenolic composition Six phenolic compounds, protocatechuic acid, chlorogenic acid, caffeic acid, syringic acid, ferulic acid and coumaric acid, were identified in the free and bound fractions of GBR. The contents of these six compounds in GBR and the ratio of their free and bound fractions to the total are presented in Table 3. HPLC analyses showed that the compositions of the free and bound phenolics of brown rice during germination did not change, but the content levels of the phenolics were significantly different (p < 0.05). Protocatechuic acid, which only existed in free form, first decreased and then increased by 72.4% at G5 (61.9 lg/g DW). Chlorogenic acid was also detected in free form, with its content ranging from trace amounts in G1, G3 and G4 to 20.8 lg/g DW at G5, which was a similar content level compared with that before germination (22.0 lg/g DW). Caffeic acid, which was also detected in free form, showed a significant increase during germination (p < 0.05) and reached 280.5 lg/g DW (an increase of 2231.7%) at G5. Syringic acid existed in both free and bound forms, with the contents ranging over the germination process from a trace amount to 2.9 lg/g DW and from 1.7 to 6.4 lg/g DW, respectively. Coumaric acid was only detected in bound forms and significantly increased by from 106.1% to 244.7% during germination (G1–G5). Ferulic acid mainly existed in the bound form, and the percentage contribution to the total content increased from 95.5% to 99.1% during germination (G1–G5). Bound and total ferulic acid contents were 121.6 and 123.1 lg/g DW, respectively before germination. The bound and total ferulic acid contents gradually increased from 89.5% to 182.7% and from 91.8% to 190.1%, respectively, during germination (G1–G5). Interestingly, the levels of coumaric acid, at 357.1 lg/g DW, and ferulic acid, at 117.9 lg/g DW, were substantially increased after germination, which were 2.8 and 3.4 times, respectively, as much as the levels in brown rice. 3.4. Effect of germination treatment on antioxidant capacity by FRAP assay The free, bound, and total antioxidant activities of GBR as determined by the FRAP assay and the percentage contribution of each fraction to the total at different germination times are presented in Table 4. The FRAP values of free fractions ranged from 111.6 to 139.0 mg TE/100 g DW during germination (G1–G5). The FRAP value of free fraction increased by 19.0% during the first 17 h (G1) (p < 0.05) and then decreased at G2 to the same level as before germination (p > 0.05). During the remainder of the germination process, the

Table 2 Total flavonoid content during the germination of brown rice and the percentage contributions of free and bound fractions to the total flavonoids. Germination stage

G0 G1 G2 G3 G4 G5 * **

Flavonoids (mg of catechin equiv/100 g of DW) Free form

Bound form

Total phenolics

61.1 ± 2.8d* (48.9)** 54.2 ± 3.6c (36.4) 29.5 ± 0.1a (24.0) 38.8 ± 3.3b (33.6) 40.4 ± 1.2b (35.7) 75.4 ± 6.7e (48.9)

63.9 ± 3.6a (51.1) 84.9 ± 3.6bc (63.6) 97.4 ± 5.9c (76.0) 78.2 ± 5.3ab (66.4) 74.1 ± 4ab (64.3) 79.0 ± 2.5ab (51.1)

124.9 ± 8.4a 139.1 ± 11.3b 126.9 ± 6a 117.0 ± 5a 114.5 ± 10.2a 154.4 ± 9.2b

Values with no letters in common in each column are significantly different (p < 0.05). Values in parentheses indicate the percentage contribution to the total flavonoids.

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H. Ti et al. / Food Chemistry 161 (2014) 337–344 Table 3 Changes in the main phenolic acids during brown rice germination. Phenolics

Germ. stage

Protocatechuic acid

Chlorogenic acid

Caffeic acid

Syringic acid

Coumaric acid

Ferulic acid

* **

Free form

Bound form *

G0 G1 G2 G3 G4 G5 G0 G1 G2 G3 G4 G5 G0 G1 G2 G3 G4 G5 G0 G1 G2 G3 G4 G5 G0 G1 G2 G3 G4 G5 G0 G1 G2 G3 G4 G5

**

35.9 ± 2.5d (100) 11.3 ± 1.9a (100) 24.3 ± 2.1c (100) 11.9 ± 1.9a (100) 18.4 ± 1.7b (100) 61.9 ± 2.6e (100) 22.0 ± 2.1b (100) TrB 9.7 ± 0.7a (100) Tr Tr 20.8 ± 1.9b (100) 12.03 ± 1.18a (100) 17.8 ± 0.1a (100) 43.5 ± 2.2b (100) 42.0 ± 2.1b (100) 57.7 ± 1.9c (100) 280.5 ± 19.1d (100) Tr 0.4 ± 0.1a (8.7) 0.7 ± 0.1b (14.0) 0.9 ± 0.1c (15.7) 1.3 ± 0.3d (18.7) 2.9 ± 1.9e (31.7) nd nd nd nd nd nd 1. 6 ± 0.2a (1.3) 5. 6 ± 0.3c (2.4) 5.8 ± 0.1c (2.3) 1.3 ± 0.2a (0.5) 2.3 ± 0.6b (0.9) 13.2 ± 1.1e (3.7)

A

nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd 1.7 ± 0. 1a (100) 3.9 ± 0.1b (91.3) 4.5 ± 0.3c (86.0) 4.8 ± 0.1 cd (84.3) 5. 7 ± 0.2e (81.3) 6.4 ± 0.3f (68.3) 34.2 ± 1.1a (100) 70.5 ± 2.7b (100) 77.0 ± 1.8c (100) 77.7 ± 1.1c (100) 82.9 ± 1.9d (100) 117.9 ± 10.3e (100) 121. 6 ± 10.1a (98.7) 230.5 ± 12.8b (97.6) 242.8 ± 18.2bc (97.7) 246.8 ± 10.2c (95.5) 263.6 ± 13.8d (99.1) 343.8 ± 21.8e (96.3)

Total 35.9 ± 2.5d 11.3 ± 1.9a 24.3 ± 2.1c 11.9 ± 1.9a 18.3 ± 1.7b 61.9 ± 2.6e 22.0 ± 2.1b Tr 9. 7 ± 0.7a Tr Tr 20.8 ± 1.9b 12.03 ± 1.18a (100) 17.8 ± 0.1a 43.5 ± 2.2b 41.9 ± 2.1b 57.7 ± 1.9c 280.5 ± 19.1d 1.7 ± 0.1a 4.3 ± 0.1b 5.2 ± 0.2c 5.8 ± 0.2c 6.9 ± 0.5d 9.4 ± 0.6e 34.2 ± 1.1a 70.5 ± 2.7b 77.0 ± 1.8c 77.7 ± 1.1c 82.9 ± 1.9d 117.9 ± 10.3e 123.1 ± 9.7a 236.1 ± 11.7b 248. 6 ± 13.4c 248.0 ± 9.3c 265.9 ± 10.3d 357.1 ± 19.3e

Values with no letters in common in each column are significantly different (p < 0.05). Values in parentheses indicate the percentage contribution to the total. A nd – Not detected. B Tr – Trace.

Table 4 Total antioxidant activity during the germination of brown rice and the percentage contributions of each fraction to the total as determined by the FRAP assay. Germination stage

FRAP(mg Trolox equiv/100 g DW) Free form

Bound form

G0 G1 G2 G3 G4 G5

107.9 ± 7.0a* (56.7)** 128.4 ± 1.7b (59.2) 111.6 ± 6.9a (52.3) 139.0 ± 0.7c (60.2) 123.1 ± 5.8b (54.1) 128.4 ± 2.5b (54.9)

82.3 ± 5.9a (43.3) 88.3 ± 3.3ab (40.8) 101.7 ± 1.1c (47.7) 91.8 ± 7.3b (39.8) 104.6 ± 0.8c (45.9) 105.5 ± 2.1c (45.1)

Total

190.2 ± 12.9a 216.7 ± 1.7bc 213.3 ± 7.7b 230.9 ± 7.3d 227.7 ± 6.7 cd 233.8 ± 0.5d

* Values with no letters in common in each column are significantly different (p < 0.05). ** Values in parentheses indicate the percentage contribution to the total.

FRAP value of free fraction increased by 28.8% at G3 (p < 0.05). It then decreased, but was still 19.0% (p < 0.05) higher than before germination. Nevertheless, the FRAP value of free fraction only increased by 4.1% (p > 0.05) between stages G4 and G5. The FRAP value of free fraction was the highest at G3, and then decreased to some extent at G5. The FRAP values of free fractions at G1, G4 and G5 were much higher than that at G2. The percentage contribution of the free fraction to the total antioxidant activity was 56.7% before germination, and ranged during germination (G1– G5) from 52.3% to 60.2%. The FRAP values of the bound fractions ranged from 88.3 to 105.5 mg TE/100 g DW during germination (G1–G5). During the

G1–G3 stages, the FRAP value of the bound fraction increased by 23.6% (reaching 101.7 mg TE/100 g DW) at G2, then decreased at G3, and significantly increased by 28.2% at the final measurement. The FRAP value of the bound fraction was the highest at G5, followed by G4, G2, and G3. The FRAP values of the bound fractions at G2, G4, and G5 were not significantly different (p > 0.05). The FRAP value of the bound fraction was the lowest at G1. The percentage contribution of the bound fractions to the total antioxidant activity increased from 39.8% to 47.7% during germination, with an average of 43.8%, indicating that the percentage contribution of the bound fraction to the total was stable before and after germination. The total FRAP value gradually increased, ranging from 213.3 to 233.8 mg TE/100 g DW during germination (G1–G5). The total FRAP values at G4 and G5, with increases of 19.7% and 22.9%, respectively, were much higher than those at the other germination stages. Moreover, the FRAP values of the first two stages were not significantly different (p > 0.05). 3.5. Effect of germination treatment on antioxidant capacity by ORAC assay The free, bound, and total antioxidant activities of GBR and the percentage contribution of each fraction to the total at different germination times are expressed as ORAC values in Table 5. The ORAC values of the free fractions ranged from 32.1 to 48.5 lmol TE/g DW during germination (G1–G5) with increases from 9.2% to 65.0%. The ORAC value of the free fraction was

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significantly increased by 51.4%, reaching 44.5 lmol TE/g DW at G2. The ORAC values of the free fractions increased between stages G1 and G2 (p < 0.05), then slightly decreased at G3 (p > 0.05), and insignificantly increased during the remainder of the germination process (G3–G5) (p > 0.05). The ORAC value of free fraction reached the maximum value of 48.5 lmol TE/g DW at G5, followed by G4, G2, and G3. The ORAC values of free fractions at G2, G3, G4 and G5 were significantly much higher than that at G1 (p < 0.05). The difference among the first four stages was not significant (p > 0.05). The percentage contribution of the ORAC value of free fraction to the total ranged from 66.7% to 71.6% during germination (G1– G5), with an average of 70.1%, indicating that the percentage contribution of the ORAC value of free fraction to the total significantly decreased during the germination process compared with the percentage contribution (76.2%) before germination. The ORAC values of the bound fraction ranged from 13.4 to 24.2 lmol TE/g DW during germination (G1–G5). The ORAC value of bound fraction gradually increased from 45.7% to 163.0% compared with that before germination (9.2 lmol TE/g DW). It reached its highest level (24.2 lmol TE/g DW) at G5, followed by G2, G4, and G3. The difference among the last three time points was not significant (p > 0.05). The ORAC value of bound fraction was the lowest at G1. The percentage contribution of ORAC value of bound fraction to the total ranged from 28.4% to 33.3%, with an average of 28.9%, indicating that the contribution of the bound fraction significantly increased during the germination process. The total ORAC value ranged from 45.5 to 72.7 lmol TE/g DW, increasing from 17.6% to 87.9% compared with that before germination (38.7 lmol TE/g DW). The trend of the total ORAC values was consistent with that of the free ORAC values. The total ORAC value increased by 63.3% at G2 (p < 0.05), then slightly decreased at G3 (p > 0.05), and insignificantly increased (3.8%) between stages G2 and G4 (p > 0.05). The total ORAC value significantly increased by 87.9% (p < 0.05). 4. Discussion It is well known that the germination process generally improves the nutritional quality of cereal grains, not only by reducing the anti-nutritive compounds, but by augmenting the levels of nutrients and physiologically active substances. During the germination process, enzyme synthesis and kernel modification take place, which can result in the enhancement of intrinsic phenolic compounds and antioxidant activity (Kaukovirta-Norja, Wilhelmsson, & Poutanen, 2004). In our study, the phenolic content in brown rice was significantly increased (p < 0.05) after germination treatment. In fact, these phenomena have been reported in other cereals, such as wheat, rice, barley, oats, and rye (Donkor et al., 2012). Our study confirmed this conclusion and was also in agreement with previous reports (Donkor et al.,

Table 5 Total antioxidant activity during the germination of brown rice and the percentage contribution of each fraction to the total as determined by the ORAC assay.

*

Germination stage

ORAC (lmol of Trolox equiv/g of DW) Free form

Bound form

Total

G0 G1 G2 G3 G4 G5

29.4 ± 3.2a* (76.2)** 32.1 ± 3.3a (70.5) 44.5 ± 0.5b (70.3) 40.5 ± 3.7b (71.2) 46.9 ± 4.5b (71.6) 48.5 ± 5.7b (66.7)

9.2 ± 1.0a (23.8) 13.4 ± 1.7b (29.5) 18.8 ± 1.6c (29.7) 16.4 ± 2.1bc (28.8) 18.6 ± 2.0c (28.4) 24.2 ± 0.9d (33.3)

38.7 ± 3.3a 45.5 ± 4.1b 63.2 ± 1.1 cd 56.8 ± 1.2c 65.6 ± 3.9d 72.7 ± 6.0e

Values with no letters in common in each column are significantly different (p < 0.05). ** Values in parentheses indicate the percentage contribution to the total.

2012; Moongngarm & Saetung, 2010) that GBR displayed a higher total phenolic concentration than untreated brown rice grains. Donkor et al. (2012) reported that seven selected important grains, wheat, oats, barley, rye, sorghum, buckwheat and brown rice, contained substantial amounts of total phenolics, with brown rice having about 50 lg/mL (ferulic acid equivalent) after a 5-day germination treatment. It is difficult to compare their results with ours because of the different standard and germination time. Moongngarm and Saetung (2010) observed that the phenolic content of brown rice increased from 70.3 to 84.3 mg GAE/100 g after 24 h of germination treatment. The total phenolic contents after germination treatment in the present study are significantly higher than that reported in this previous study. The previously reported phenolic content in GBR was even lower than that in brown rice before germination in the present study (174.0 mg GAE/100 g). Cáceres, Martínez-Villaluenga, Amigo, and Frias (2014) reported that the total phenolic content increased from 57.6 to 306.7 mg GAE/100 g. The increase was much higher than that seen in our study. Possible explanations for this discrepancy include the different extraction methods used (the contribution of the bound fraction was not included in the previous study), the different varieties tested, and growing conditions. In addition, the different germination time or the procedural differences may also lead to the significant differences in phenolic content, such as the temperatures and soaking times used. During the 24 h soaking period before germination in a previous study by Bishnoi , Khetarpaul and Yadav (1994), increases of total phenolic compounds were observed in brown rice, whereas inversely, all identified compounds underwent a drastic decrease in beans, peas, and lentils. Additionally, we have documented the changes in the phenolic content of brown rice at five germination stages, which had not been reported previously. In this study, the total phenolics reached the highest level (284.1 GAE/100 g DW) with an increase of 63.3% after 30 h of germination, and showed no significant increase (p > 0.05) during the subsequent germination process. An increase was observed after nine days of germination in lupin seeds, with a rise of 63% (Duenas, Hernandez, Estrella, & Fernandez, 2009). A report by Fernandez-Orozco et al. (2006) also found an increase of 53% of total phenols (expressed as mg of catechin) in lupin sprouts after nine days. A rise in the total phenols of chickpeas was also reported after five days of germination (Khattak, Zeb, Bibi, Khalil, & Khattak, 2007). From the above information, we can deduce that the total phenolic content after 30 h of germination increased by the same proportion when compared with the content after several days of germination. A germination treatment can enhance the respiration of brown rice and the probability of microbial infection increases with the extension of germination time, leading to the excessive deposition of nutritional properties. Therefore, a prolonged germination treatment was not carried out after 48 h of germination time in our study. Large amounts of bound phenolics in cell walls are covalently conjugated to cell wall components, such as cellulose, pectin and polysaccharides, through ester bonds (Naczk & Shahidi, 1989). They cannot be extracted through common techniques, which results in an underestimation of the total phenolic content. In this study, the data showed that most of the brown rice phenolics were distributed in free forms. The bound forms contributed 42.3% of the total phenolics in brown rice. This result was different than in a previous study that found the percentage contributions of free phenolics to the total were 15%, 25%, 25% and 38% in corn, wheat, oats and rice, respectively (Adom & Liu, 2002). During the germination process, the percentage contribution of the bound phenolics fluctuated and finally decreased to 37.6%, whereas the bound flavonoids contribution changed in a ‘‘V’’ shape (51.0% before and after germination), which indicated that there are transformations between the bound and free forms. The free and bound phenolic

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contents were significantly increased at the first germination stage, which may be partly explained by the production of enzymes to break down cell walls surrounding compounds during germination (Kaukovirta-Norja et al., 2004). Additionally, we found that the free phenolic content significantly increased, whereas the bound phenolic contents decreased, in stages G2 and G3. The rise of the free phenolic content coincided with a decline in the bound phenolic content, although not to the same extent, which may indicate that the increased free phenolics come from (1) the liberation of the bound phenolics (some enzymes that are synthesised to degrade storage macromolecules can liberate bound phenolics); and (2) the synthesis of phenolics in response to the germination treatment. During the remainder of the germination process, a direct opposite trend was detected in both free and bound forms. The increase of the bound phenolics may be explained by the polymerisation and oxidation of phenolics, and by certain changes in enzymes involved in the synthesis and degradation of free or bound phenolics. These observations were not consistent with those found in oats, in which the free phenolics increased and the bound phenolics decreased (Xu et al., 2009). The data in our study explained that the increase of free phenolics was not entirely from the degradation of bound phenolics. The bound phenolics cannot be directly digested by human enzymes, thus we hypothesise that the bound phenolics may be absorbed when slowly and continuously released into the lower gastrointestinal tract by the simultaneous action of b-glucosidases and esterases of the microflora in the colon (Vitaglione, Napolitano, & Fogliano, 2008). Some literature indicated that phenolics from the cell wall had antimutagenic properties and that this physiological activity could have significance in the preventative activity of the diet against cancer. Therefore, the increase of bound phenolics during germination may exert beneficial health effects throughout the digestive tract after absorption and may reduce mutations. The bound flavonoids showed an opposite trend during the germination process, and interestingly, the distribution of flavonoids in free and bound forms before and after germination did not change, which indicated a stable transformation between free and bound flavonoids. Flavonoids are reactive compounds with no steric inhibition due to additional side chains; therefore, many flavonoids more easily form polymers (bound forms). The synthesised flavonoid pathway may be activated during germination by the phenylpropanoid metabolic pathway, during which the intermediates may further generate acetyl coenzyme A esters (CoA) that are converted to flavonoids. The changes in the flavonoid content during germination could be explained by various types of key enzymes or cofactors being synthesised, which leads to the production of the flavonoids. Phenolic compounds, particularly hydroxycinnamates in their bound forms, are found in significant quantities in rice grains. The most abundant phenolic compound in brown rice was ferulic acid, followed by coumaric acid, and caffeic acid. Most of the ferulic and coumaric acids were present in bound forms, whereas less, or trace amounts, of other compounds were detected in bound forms, which is similarly reported by Zhou, Robards, Helliwell, and Blanchard (2004). After germination, changes in their concentrations were observed and, while ferulic acid was still the most abundant, there was now more caffeic acid than coumaric acid. Our study showed that these hydroxycinnamic acids were significantly increased during germination and a sharp increase in caffeic acid occurred during the last stage of the process. The changes observed in the germination process could be partly explained by the action of the endogenous esterases. Or rather, all these simple phenylpropanoids are produced from cinnamate, which is synthesised from phenylalanine by the action of phenylalanine ammonia-lyase via a series of hydroxylation, methylation, and dehydration reactions (Dixon & Paiva, 1995). The increased activity of phenylal-

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anine ammonia-lyase during germination (Walton, 1968) could catalyse the synthesis of phenolics. This may partly explain the increase of free phenolic forms during the germination process. In fact, Tian, Nakamura, and Kayahara (2004) found that 6-Oferuloylsucrose and 6-O-sinapoylsucrose were the major soluble phenolic compounds in brown rice and that they significantly decreased during germination, while the levels of free ferulic and sinapinic acids increased significantly. The result may provide evidence for the increase of ferulic acid in our study (transformation between ester-linked ferulic acid and free ferulic acid). The increases of bound ferulic and coumaric acids may be because of the conversion (polymerisation) from free phenolics. However, the protocatechuic acid first decreased and then increased, and chlorogenic acid initially disappeared and then increased to the same level after germination when compared with their raw grains. The disappearance of chlorogenic acid may be attributed to the production of complex phenolic acids. These facts mean that not all phenolic acids had higher raw grains. In our study, the antioxidant activities of brown rice phenolics during germination were evaluated by the methods of FRAP and ORAC. The FRAP assay is based on electron transfer (ET), and the ORAC assay is based on hydrogen atom transfer (HAT). It is concluded that the ORAC assay is chemically more relevant to chain-breaking antioxidant activity, while the FRAP assay is a test to determine the total antioxidant power using antioxidants as reductants in a redox-linked colorimetric method. Therefore, a single antioxidant activity assay cannot adequately determine the activity of all antioxidants in a complex system. The antioxidant activities of brown rice during the germination process as determined by the FRAP and ORAC assays were consistent. The results of this work showed that the total FRAP value fluctuated and finally increased by the combined contribution of free and bound fractions. The total ORAC value of brown rice significantly increased during germination, which was consistent with other publications (Bolivar, Cevallos, & Luis, 2010). Donkor et al. (2012) reported that the antioxidant activity of GBR using different analysis and quantification methods. Although it is difficult to compare the antioxidant activity of GBR from our study with that from other investigators, it was reported to have a much higher value than that of brown rice. In our study, the change in the ORAC value of brown rice was consistent with that of the phenolic profiles, indicating that phenolics play an important role in the ORAC antioxidant activity, which was positively correlated with the phenolic contents. The phytochemical constituents appear to work by blocking the free radical chain reaction process.

5. Conclusion Recently, GBR has become one of the most popular germinated cereal products, and it gained considerable attention because of its improved texture and nutritional properties compared with brown rice. Here, we present data showing that GBR has a high content of phenolics and flavonoids, and has potent antioxidant activities. A proposed germination treatment (30 h) shows much higher levels of total phenolics and antioxidant activity, while a prolonged germination treatment has no significant effects on them. The physiological and biochemical reactions resulting from germination are extremely complex processes. Therefore, along with the development of low cost processes for increasing and diversifying the use of common grain varieties, the data in our study indicated that an appropriate germination time would be necessary for the enrichment of phenolic contents in GBR. The free and bound phenolic and flavonoid contents and their antioxidant activity were increased after germination compared with before germination in brown rice. However, they show signif-

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icant differences at different stages of germination. The total phenolic contents and antioxidant activity mainly existed in free form during the germination process, while the total flavonoids were mostly present in the bound form. The free and bound phytochemicals (phenolics and flavonoids) in GBR found in this study provide an explanation for the better health benefits associated with GBR than brown rice. GBR can be used as a natural resource in the food and pharmaceutical industries. Acknowledgements We appreciate the financial support from the National ‘‘948’’ Project (2014-Z52), Special Fund for Public Welfare Industry (agriculture) Research Project (201303071), Project of National Key Technology Research and Development Program for the 12th Five-year Plan (2012BAD33B10 & 2012BAD33B08). References Adom, K. K., & Liu, R. H. (2002). Antioxidant activity of grains. Journal of Agricultural and Food Chemistry, 50, 6182–6187. Benzie, I. F. F., & Strain, J. J. (1996). The ferric reducing ability of plasma (FRAP) as a measure of ‘‘antioxidant power’’: The FRAP assay. Analytical Biochemistry, 239, 70–76. Bishnoi, S., Khetarpaul, N., & Yadav, R. K. (1994). Effect of domestic processing and cooking methods on phytic acid and polyphenol content of peas. Plant Foods for Human Nutrition, 45, 381–388. Bolivar, A., Cevallos, C., & Luis, C. Z. (2010). Impact of germination on phenolic content and antioxidant activity of 13 edible seed species. Food Chemistry, 119, 1485–1490. Cáceres, P. J., Martínez-Villaluenga, C., Amigo, L., & Frias, J. (2014). Maximising the phytochemical content and antioxidant activity of Ecuadorian brown rice sprouts through optimal germination conditions. Food Chemistry, 152, 407–414. Dewanto, V., Wu, X., Adom, K. K., & Liu, R. H. (2002). Thermal processing enhances the nutritional value of tomatoes by increasing total antioxidant activity. Journal of Agricultural and Food Chemistry, 50, 3010–3014. Dixon, R. A., & Paiva, N. L. (1995). Stress induced phenylpropanoid metabolism. Plant Cell, 7, 1085–1097. Donkor, O. N., Stojanovska, L., Ginn, P., Ashton, J., & Vasiljevic, T. (2012). Germinated grains – sources of bioactive compounds. Food Chemistry, 135, 950–959. Duenas, M., Hernandez, T., Estrella, I., & Fernandez, D. (2009). Germination as a process to increase the polyphenol content and antioxidant activity of lupin seeds (Lupinus angustifolius L.). Food Chemistry, 117, 599–607.

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