Effects of cyclic cellulase conditioning and germination treatment on the γ-aminobutyric acid content and the cooking and taste qualities of germinated brown rice

Food Chemistry 289 (2019) 232–239

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Effects of cyclic cellulase conditioning and germination treatment on the γaminobutyric acid content and the cooking and taste qualities of germinated brown rice Qiang Zhanga,b, Nian Liua,b, Shuangshuang Wanga,b, Yang Liuc, Haipeng Lanc,

T

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a

College of Engineering, Huazhong Agricultural University, Wuhan 430070, China Key Laboratory of Agricultural Equipment in Mid-lower Yangtze River, Ministry of Agriculture, Wuhan 430070, China c College of Mechanic and Electrical Engineering, Tarim University, Alar 843300, China b

A R T I C LE I N FO

A B S T R A C T

Keywords: Cyclic cellulase conditioning treatment Germinated brown rice γ-Aminobutyric acid (GABA) Cooking quality Taste quality

A novel method for producing germinated brown rice (GBR) was developed by cyclic cellulase conditioning and germination treatment. Changes in the γ-aminobutyric acid (GABA) content and the cooking and taste qualities of GBR were analysed. The results indicated GABA accumulation reached its highest level under 50 mg/ml cellulase solution treatment for 90 min followed by 32 h germination at 30 °C. The treatment conditions had no significant influence on the cooking or taste qualities of GBR. Correlation analysis showed the taste value of cooked GBR significantly correlated with hardness, springiness, chewiness, and stickiness. The cyclic cellulase conditioning treatment could contribute to improved cooking and taste qualities of GBR and higher GABA content of GBR compared to the soaking treatment. The cooking and taste qualities of GBR were improved by the degradation of the crude fibre of the cortex and the increase in GBR water absorption rate under cyclic cellulase conditioning.

1. Introduction Rice as a health food has become increasingly popular worldwide (Zhang, Jia, Zuo, Fu, & Wang, 2015). Brown rice is the product obtained after rice hulling, which has been reported to contain more bioactive substances than polished white rice, including ferulic acid, oryzanol, tocopherol, and especially γ-aminobutyric acid (GABA) (Moongngarm & Saetung, 2010). However, brown rice is not considered to be a suitable food source due to its poor cooking quality, undesirable flavour and hard texture (Das, Gupta, Kapoor, Banerjee, & Bal, 2008a). Compared with brown rice, the sensory quality of germinated brown rice (GBR) has a certain degree of improvement (Shen et al., 2015). More importantly, a large number of endogenous enzymes are activated and released during brown rice germination (Chen et al., 2016; Cho & Lim, 2018; Li, Liu, & Chen, 2016). The germination process may cause changes in the nutrients in brown rice (Patil & Khan, 2011). The species and contents of physiologically active components in GBR are more abundant than those in brown rice (Cáceres, Peñas, MartinezVillaluenga, Amigo, & Frias, 2017; Zhang, Xia, Li, & Hung, 2018). Among the components, GABA in particular has attracted the most attention due to its unique bioactivity (Cornejo, Caceres, Martínez-

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Villaluenga, Rosell, & Frias, 2015), which includes the physiological activities of lowering blood pressure, improving brain function, activating liver and kidney function, and promoting ethanol metabolism (Imam, Azmi, Bhanger, Ismail, & Ismail, 2012). As an important inhibitory neurotransmitter in the central nervous systems of mammals, GABA is widely distributed in animals and plants (Bown & Shelp, 1997). Studies have shown that the content of GABA in GBR is approximately ten times higher than that in polished white rice and twice that in brown rice (Kim, 2012; Moongngarm & Saetung, 2010). The cooking and taste qualities of GBR refer to various physicochemical and sensory characteristics of GBR during cooking and eating, such as gelatinization, expansibility, rice softness, rice springiness, colour, smell, and palatability (Zhang et al., 2015). The important properties of GBR for consumers are its cooking and taste qualities. GBR, similarly to brown rice, is difficult to cook, and the texture of cooked GBR is hard. When GBR is eaten directly as a staple food, its taste quality is obviously poorer than that of the polished white rice (Sirisoontaralak, Nakornpanom, Koakietdumrongkul, & Panumaswiwath, 2015). Therefore, it is necessary to process the GBR with higher nutrients and better cooking and taste qualities. Germinated grains rich in GABA including GBR have been

Corresponding author at: College of Mechanical and Electronic Engineering, Tarim University, Alar 843300, China. E-mail address: [email protected] (H. Lan).

https://doi.org/10.1016/j.foodchem.2019.03.034 Received 21 September 2018; Received in revised form 7 March 2019; Accepted 9 March 2019 Available online 13 March 2019 0308-8146/ © 2019 Elsevier Ltd. All rights reserved.

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The conditioning treatment was conducted according to the setting time. Following the conditioning treatment, the 105 °C constant weight method was used to verify whether the samples reached the target moisture content under each moisture adding amount with the error controlled at ± 0.5%. The moisture content of brown rice samples reached 30% (w.b.) by cyclic conditioning treatment. The brown rice was then spread in culture dishes covered with gauze. Deionised water was used to moisten the brown rice and gauze. The culture dishes were placed in a constant temperature foster box (CTHI-150(A)B, temperature fluctuation ± 0.2 °C; Shi Dukai Equipment Co., Ltd., Shanghai, China) and germinated for a certain time under the temperature condition set in the experiment. After the germination of the brown rice, GBR was placed into an oven and dried to 15.5% (w.b.) at 50 °C. Finally, the GBR samples were cooled and then sealed using doublelayer self-sealing bags.

continuously investigated (Saikusa, Horino, & Mori, 1994a, 1994b; Ohtsubo, Suzuki, Yasui, & Kasumi, 2005; Kihara, Okada, Iimure, & Ito, 2007; Imam et al., 2012; Cho & Lim, 2016). A large number of studies have reported that water absorption and germination treatment are beneficial for the accumulation of GABA in grains. For example, the content of GABA can be significantly increased when brown rice is soaked in water and germinated (Saikusa, Horino, & Mori, 1994a, 1994b). Further studies have shown that germination treatment of brown rice after segmentation moisture conditioning leads to the accumulation of GABA (Cao, Jia, Han, Liu, & Zhang, 2015). Gradual water absorption and germination treatment greatly promote the accumulation of GABA. It has been reported that cellulase solution treatment is beneficial for GABA generation (Das et al., 2008a; Zhang et al., 2015). Studies have shown that gaseous treatment contributes to GABA accumulation (Komatsuzaki et al., 2007) and have suggested that aeration treatment is favourable to GABA enrichment (Guo, Chen, Song, & Gu, 2011). Although many studies have been done on the optimization of soaking and germination conditions to increase the GABA content of GBR (Thitinunsomboon, Keeratipibul, & Boonsiriwit, 2013; Zhang et al., 2014), the enrichment of GABA in GBR by cyclically spraying the cellulase solution has not yet been reported. GABA may be partially lost at the soaking stage of the brown rice germination process, and GBR is difficult to cook. Therefore, the aims of the present study are to a) investigate the effects of cyclic cellulase conditioning and germination treatment conditions on the GABA content and the cooking and taste qualities of GBR and b) study the microstructures of GBR obtained by different treatments and the changes of moisture adsorption during soaking.

2.4. Experimental design 2.4.1. Determination of the effects of cyclic cellulase conditioning treatment conditions on GABA content 2.4.1.1. Cellulase concentration. The cellulase conditioning treatment was carried out according to the method introduced in Section 2.3. Cellulase concentration levels were controlled at 0 mg/mL, 10 mg/mL, 50 mg/mL and 100 mg/mL. The pH of the cellulase solution was set at 5.0. The treatment was conducted for 90 min at 35 °C. The brown rice samples treated with cellulase were respectively placed in a constant temperature and humidity incubator (CTHI-150(A)B, temperature fluctuation ± 0.2 °C; Shi Dukai Equipment Co., Ltd., Shanghai, China) for 32 h germination at 30 °C. The GBR samples were then collected to determine the content of GABA.

2. Materials and methods 2.1. Brown rice samples

2.4.1.2. Cellulase treatment time. The cellulase conditioning treatment for the brown rice samples was carried out according to the test method in Section 2.3. The cellulase treatment time was set to 0 min, 60 min, 90 min and 120 min. The pH of cellulase was set at 5.0. The cellulase concentration was set at 50 mg/mL. The cellulase treatment temperature was set at 35 °C. The brown rice samples treated with cellulase were placed in a constant temperature and humidity incubator (CTHI-150(A)B, temperature fluctuation ± 0.2 °C; Shi Dukai Equipment Co., Ltd., Shanghai, China) for 32 h germination at 30 °C. After germination, the content of GABA was determined.

The selected rough rice variety in the experiment was Japonica Dongnong 429, which was purchased from the rice experiment station of Northeast Agricultural University (Harbin, China). The rough rice was stored at the temperature of 20 ± 2 °C (Zhang, Liu, Tu, & Wang, 2018). The initial moisture content of rough rice was 12.5% (w.b.). The brown rice was obtained by hulling (THU-35B, Satake, Tokyo, Japan) before the experiment. After sieving, impurities such as mildew grains, embryo free grains, discoloured grains, immature grain and stones were removed artificially. Thereafter, full, plump brown rice was selected at random. It was rinsed three times with tap water, soaked and disinfected for 5 min with 0.5% sodium hypochlorite solution. Then, it was rinsed several times with deionised water. After the surface moisture was drained, it was maintained in reserve.

2.4.2. Determination of the effects of germination treatment conditions on GABA content 2.4.2.1. Germination temperature. The cellulase conditioning treatment was carried out according to the method in Section 2.3. The pH of cellulase was set at 5.0. The treatment temperature of 50 mg/mL cellulase was 35 °C. After cellulase treatment for 90 min, the brown rice samples were placed in a constant temperature and humidity incubator (CTHI-150(A)B, temperature fluctuation ± 0.2 °C; Shi Dukai Equipment Co., Ltd., Shanghai, China) for germination at 15 °C, 20 °C, 25 °C, 30 °C, 35 °C and 40 °C. Following 32 h germination, the GBR samples were collected to measure the content of GABA.

2.2. Enzyme and reagents Commercially available solid cellulases (activity: filter Filter Paper Activity ≥ 40,000 U/g) were purchased from Imperial Jade Bio-technology Co., Ltd., Yinchuan, China. All the chemicals were analytically pure. All the chemicals and reagents were obtained from Hengxing Chemical Reagent Co., Ltd., Tianjin, China and Bodi Chemical Co., Ltd., Tianjin, China. 2.3. Cyclic cellulase conditioning treatment and germination

2.4.2.2. Germination time. The cellulase conditioning treatment was carried out according to the method in Section 2.3. The pH of cellulase was 5.0. The treatment temperature of the 50 mg/mL cellulase was set at 35 °C. After cellulase treatment for 90 min, the brown rice samples were placed in a constant temperature and humidity incubator (CTHI150(A)B, temperature fluctuation ± 0.2 °C; Shi Dukai Equipment Co., Ltd., Shanghai, China) for 0 h, 8 h, 16 h, 24 h, 32 h and 40 h germination at 30 °C. The content of GABA was determined after germination.

One kilogram of pretreated brown rice was used as the test samples. The pH 5.0 cellulase solution at a determined concentration was uniformly sprayed onto the surface of the brown rice samples by spray humidification. The once moisture adding amount (percentage increase in moisture content of brown rice) was set at 1.5%. Following each humidification, the brown rice were sealed in double-layer self-sealing bags and placed under the temperature condition set in the experiment. 233

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2.7. Evaluation of the cooking qualities of GBR

2.4.3. Determination of the effects of the cyclic cellulase conditioning and germination treatment on the texture attributes and cooking and taste qualities of GBR 2.4.3.1. Cellulase concentration. The cellulase conditioning treatment was carried out according to the method in Section 2.3. The pH of cellulase was set at 5.0. The cellulase concentrations were 10 mg/mL, 50 mg/mL and 100 mg/mL. The cellulase treatment was conducted for 90 min at 35 °C. The brown rice samples treated with cellulase were placed in a constant temperature and humidity incubator (CTHI-150(A) B, temperature fluctuation ± 0.2 °C; Shi Dukai Equipment Co., Ltd., Shanghai, China) for 32 h germination at 30 °C. After germination, the texture attributes and the cooking and taste qualities of GBR were measured.

2.7.1. Cooking time The cooking time was determined by the method of Bhattacharya and Sowbhagy (1971) with a slight modification. Five grams of the GBR samples obtained after each treatment were weighed and added to 100 mL of 100 °C boiling water to be cooked for 10 min. Following cooking, the samples were collected every minute. Ten rice grains were randomly selected and squeezed with a flat plate against a black background until the cooked rice had no white core. The duration of this process was the cooking time. 2.7.2. Volume expansion rate The volume expansion rate was determined by the method of Sidhu, Gill, and Bains (1975) with a slight modification. Five grams of each GBR sample were weighed and placed into a measuring cylinder filled with 15 mL water. The total volume Y was measured. Thereafter, the GBR samples were placed in the boiling water bath to be cooked for 20 min. The cooked GBR samples were taken out and transferred into the measuring cylinder filled with 50 mL water. The total volume X was measured. The volume expansion rate is the ratio of X-50 toY-15.

2.4.3.2. Cellulase treatment time. The cellulase conditioning treatment was carried out according to the method in Section 2.3. The pH of cellulase was set at 5.0. The cellulase treatment temperature was set at 35 °C. After the brown rice samples were treated with 50 mg/mL cellulase for 60 min and 120 min, respectively, they were placed in a constant temperature and humidity incubator (CTHI-150(A)B, temperature fluctuation ± 0.2 °C; Shi Dukai Equipment Co., Ltd., Shanghai, China) for 32 h germination at 30 °C. Thereafter, the texture attributes, and the cooking and taste qualities of GBR were determined.

2.7.3. Meal rate The meal rate was determined by the method of Singh, Kaur, Sodhi, and Sekhon (2005) with a slight modification. Five grams of each GBR sample were weighed and put in the boiling water bath to be cooked. The cooking time was set according to the optimum cooking time determined. The cooked GBR samples were taken out and the surface moisture was removed. The mass of the cooked GBR samples was accurately weighed. The meal rate is the ratio of the GBR sample mass after cooking to the GBR sample mass before cooking.

2.4.3.3. Germination temperature. The test method of cellulase conditioning treatment was the same as that described in Section 2.3. The pH of cellulase was adjusted to 5.0. After treatment with 50 mg/mL cellulase for 90 min at 35 °C, the brown rice samples were placed in a constant temperature and humidity incubator (CTHI-150(A)B, temperature fluctuation ± 0.2 °C; Shi Dukai Equipment Co., Ltd., Shanghai, China) for the germination at 15 °C, 20 °C, 25 °C, 35 °C and 40 °C. Following 32 h germination, the GBR samples were collected to determine their texture attributes, and cooking and taste qualities.

2.8. Cooking of GBR The preparation of the cooked GBR samples referred to the method of Zhang et al. (2015). Twenty grams of each GBR sample were added to 30 mL deionised water and soaked for 60 min before cooking. The cooking method of GBR referred to GB/T15682-2008 (rice cooking standard from the Department of Agriculture, P. R. China).

2.5. Preparation of GBR by soaking treatment, cyclic moisture conditioning treatment and cyclic cellulase conditioning treatment The soaking treatment conditions consisted of soaking for 12 h at the soaking temperature of 30 °C and germinating for 32 h at 30 °C. The conditions of cyclic moisture conditioning treatment were as follows. The conditioning treatment of the water solution was conducted at 30 °C for 90 min according to the method in Section 2.3. Germination was then performed for 32 h at the germination temperature of 30 °C. The conditions of cyclic cellulase conditioning treatment were as follows. The brown rice was treated with 50 mg/mL cellulase solution at pH 5.0 for 90 min at 30 °C according to the method described in Section 2.3. After cellulase treatment, the germination was carried out for 32 h at 30 °C. The GABA content, and cooking and taste qualities of GBR obtained by these three treatments were analysed.

2.9. Instrumental texture analysis Texture profile analysis of GBR samples was carried out using a Stable Micro System TA-XT2 texture analyser (Texture Technologies Co., Surrey, UK) with a 2.5 mm cylindrical probe. The speeds of pretest, test and post-test were 1.0, 0.5 and 0.5 mm s−1, respectively, and the deformation ratio was 90%. The force-time curve was recorded and analysed with the TA-XT2 texture analyser software, which recorded the texture characteristics of the cooked GBR including hardness (H1), adhesiveness (A3), cohesiveness (A2/A1), springiness (D2/D1), stickiness (H2), gumminess and chewiness. Gumminess was calculated by Hardness × Cohesiveness and chewiness was obtained by Gumminess × Springiness based on the standard calculations of curve attributes of the texture profile analysis. The measurements were performed in six replicates, and the averages are reported for each sample in Table 2.

2.6. Analysis of the GABA content of GBR Ten grams of each GBR sample were ground and then screened through a 0.25 mm hole sizer. One gram of GBR powder was weighed and stored in an Erlenmeyer flask. Thereafter, it was dissolved in 0.02 mol/L hydrochloric acid solution to which 6% sulfosalicylic acid solution was added. Heating reflux was operated for 5 min in the boiling water bath. After oscillation for 30 min, the solution was moved into a 50 mL volumetric flask and diluted to the calibration with pH 2.2 citrate buffer solution. After standing for 1 h at room temperature, it was centrifuged at 1000 r/min for 15 min. The preparation method of GABA standard solution, and chromatographic conditions was previously described by Zhang et al. (2015). A 0.45 μm filtration membrane was used for filtration. Finally, the GABA content was measured by using an amino acid analyser (L-8800, Hitachi, Hitachinaka, Japan).

2.10. Taste evaluation Ten professionally trained panellists employed at the National Rice Quality Test Centre (Harbin, China) developed a taste profile for cooked GBR samples according to the GB/T15682-1995 method (rice taste standard from the Ministry of Agriculture, P. R. China). During the panel tests, the cooked GBR taste (100 points) was evaluated in terms of smell (25 points), appearance (10 points), colour (10 points), palatability (30 points) and flavour (25 points). Taste evaluation of cooked 234

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GBR referred to Zheng and Lan (2007). The taste qualities of cooked GBR subjected to different cyclic cellulase conditioning and germination treatments are shown in Table 3. 2.11. Moisture adsorption curves of GBR obtained by cyclic moisture and cyclic cellulase conditioning treatments After cyclic moisture conditioning treatment and cyclic cellulase conditioning treatment, the obtained GBR was randomly selected as the test material. The initial moisture content of GBR was 15.5% (w.b.). One hundred millilitres of deionised water was measured, poured into a 250 mL beaker and equilibrated for 1 h at 30 °C. An approximately 10 g sample was weighed and placed into the beaker. The sample was removed every 10 min within the time range of the test (0–120 min). The GBR grains were blotted with a filter paper to remove the moisture on the surface and weighed quickly. The moisture content was calculated on a wet basis. 2.12. Scanning electron microscopy analysis of GBR surface structures After cyclic moisture conditioning treatment and cyclic cellulase conditioning treatment, the obtained GBR samples were placed in a drying dish for drying pretreatment. A layer of conductive metal with a thickness of 5–10 nm was sprayed on the surface of the GBR samples before observation. The coated GBR samples were then placed under a scanning electron microscope (S-3000N, Hitachi Ltd., Japan) and magnified 2000 times to observe the microstructure of GBR cortex. 2.13. Statistical analysis All the experiments were performed in triplicate and the results are reported as the mean ± standard deviation. Both experimental data were analysed using the SPSS program version 16.0 (SPSS Inc., Chicago, IL, USA). Duncan’s multiple range tests were applied to estimate significant differences at a level of p < 0.05. 3. Results and discussion

Fig. 1. Effect of cyclic cellulase conditioning treatment on GABA content in GBR: a cellulase concentration (cellulase treatment time of 90 min); b cellulase treatment time (cellulase concentration at 50 mg/mL). Cellulase solution at pH 5.0; Cellulase treatment temperature was 35 °C. Germination time was 32 h at 30 °C.

3.1. Effect of cyclic cellulase conditioning treatment on the GABA content of GBR 3.1.1. Cellulase concentration Fig. 1a shows the effect of cellulase concentration on the GABA content of GBR. As shown in Fig. 1a, significant differences were found in GABA content at different cellulase concentrations (p < 0.05). The content of GABA increased rapidly with the germination time. When the cellulase concentration was 50 mg/mL, the highest content of GABA was achieved after 32 h germination. The highest content of GABA was 1.32 times higher than that obtained by cyclic moisture conditioning treatment at the optimum germination time. When the cellulase concentration was 50 mg/mL, the content of GABA at the germination duration of 8 h was higher than that obtained by cyclic moisture conditioning treatment at the optimum germination time. These results indicated that the cyclic cellulase conditioning treatment improved the accumulation of GABA, which was consistent with the previous studies of cellulase solution soaking treatment (Das et al., 2008a; Nogata & Nagamine, 2009). Glutamic acid is converted by glutamic acid decarboxylase (GAD) to generate GABA in GBR (Komatsuzaki et al., 2007). The activity of GAD is closely related to the enrichment of GABA (Liu, Zhai, & Wan, 2005). An acidic pH is needed for GAD activity (Saikusa, Horino, & Mori, 1994a, 1994b). An acidic environment during cellulase conditioning treatment increases the activity of GAD, which is beneficial for the generation of GABA. Therefore, the GABA content in the GBR obtained by cellulase conditioning treatment increased. Fig. 1a shows that the GABA content was unchanged when the germination time exceeded 28 h under different concentrations of cellulase. The

GABA content at the cellulase concentration of 100 mg/mL was higher than that at the cellulase concentration of 10 mg/mL, which indicated that excessively low cellulase concentration was not conducive to the accumulation of GABA.

3.1.2. Cellulase treatment time The effect of the cellulase treatment time on GABA content is shown in Fig. 1b. The results showed that the cellulase treatment time had a significant effect on GABA content (p < 0.05). With the germination time, the GABA content increased significantly (p < 0.05). At the cellulase treatment duration of 90 min and the germination duration of 36 h, the accumulation of GABA in GBR reached its highest level, which was 68.15% higher than that produced by cyclic moisture conditioning treatment at the optimum germination time. Moreover, the content of GABA produced by germination after cellulase treatment for 90 min was significantly higher than that by germination after cellulase treatment for 60 min or 120 min (p < 0.05). The result might be attributed to the degradation of the brown rice cortical cellulose by cellulase at the appropriate treatment time. The structural damage to the brown rice cortex helped accelerate the exchange of nutrients inside and outside cells and promote the germination of brown rice. There was a high positive correlation between GAD activity and brown rice germination (Das et al., 2008a). Therefore, the appropriate cellulase treatment can 235

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Table 1 Cooking qualities of GBR subjected to various cyclic cellulase conditioning and germination treatmentsC,D. TreatmentB

CTA

CC10 CC50 CC100 ITCT60 ITCT120 GT15 GT20 GT25 GT35 GT40

17.7 16.5 16.9 17.5 17.1 16.6 16.9 16.8 16.5 17.1

A

VER ± ± ± ± ± ± ± ± ± ±

0.86a 0.73a 1.45a 0.92a 1.86a 1.01a 1.25a 0.82a 1.17a 2.13a

325 386 369 368 378 383 381 386 389 376

MR ± ± ± ± ± ± ± ± ± ±

13b 9a 10a 8a 12a 14a 15a 6a 13a 3a

276 300 290 283 289 303 306 288 296 298

± ± ± ± ± ± ± ± ± ±

6a 11a 10a 11a 8a 9a 15a 12a 16a 11a

CT cooking time (min), VER volume expansion rate (%), and MR meal rate

(%). B CC10/CC50/CC100: brown rice was treated by cyclic cellulase conditioning at the cellulase concentrations of 10, 50, and 100 mg/mL, respectively; ITCT60/ITCT120: brown rice was treated by cyclic cellulase conditioning for 60 and 120 min, respectively; GT15/GT20/GT25/GT35/GT40: brown rice was germinated at 15, 20, 25, 35, and 40 °C, respectively. Cellulase solution at pH 5.0; Cellulase treatment temperature was 35 °C. Germination time was 32 h at 30 °C. C Data was expressed as mean ± standard deviation of triplicate determinations. D Different characters in the same column represent significant difference at p < 0.05.

content significantly increased (p < 0.05). Especially in the first 8 h, the GABA generation rate was faster and the curve was steeper. When the germination time exceeded 16 h, the GABA generation rate decreased slightly. After germination for 24 h, it was reduced to a very low level. At the germination duration of 32 h, the GABA content reached its highest level, which was 7.50 times that of raw brown rice. This may be because during germination, the activated GAD in brown rice continuously acted upon glutamic acid (Crawford, Brown, Breitkreuz, & Guinel, 1994) to produce GABA. Fig. 2. Effect of germination treatment on GABA content in GBR: a germination temperature (germination time of 32 h); b germination time (germination temperature at 30 °C). 50 mg/mL cellulase solution at pH 5.0; Cellulase treatment time was 90 min at 35 °C.

3.3. Effect of cyclic cellulase conditioning and germination treatment on the cooking qualities of GBR The effects of cellulase conditioning and germination treatment on the cooking qualities of GBR are shown in Table 1. The effects of the treatment conditions of cellulase conditioning on GBR cooking time were not significant (p > 0.05). The optimum cooking time was obtained at the cellulase concentration of 50 mg/mL. The cooking time was the longest at the cellulase concentration of 10 mg/mL. Table 1 indicates that a suitable cellulase concentration treatment could reduce the cooking time. It was also found that the germination temperature had no significant effect on the cooking time (p > 0.05). No significant differences were found in the volume expansion rate or meal rate under different treatment conditions of cellulase conditioning (p > 0.05). When the cellulase concentration was 10 mg/mL, the volume expansion rate and the meal rate were at the lowest level. This indicated that the cellulase concentration was too low, which was not conducive to the improvement of the GBR cooking qualities. When the germination temperature was 35 °C and the cellulase concentration was 50 mg/mL, the volume expansion rate was the highest. When the germination temperature was 20 °C and the cellulase concentration was 50 mg/mL, the meal rate reached its highest level. All these results indicated that both the highest volume expansion rate and the highest meal rate of GBR were achieved at the cellulase concentration of 50 mg/mL. Table 1 also shows that the germination temperature had no significant effect on the GBR volume expansion rate or meal rate (p > 0.05).

improve GAD activity to promote the accumulation of GABA. 3.2. Effect of germination treatment on the GABA content of GBR 3.2.1. Germination temperature As shown in Fig. 2a, the highest GABA content was obtained at the germination temperature of 30 °C. The results also showed that the germination temperature had a significant effect on the GABA content (p < 0.05). The GABA content obtained at the optimum treatment temperature was 91.47% higher than that at the germination temperature of 15 °C. The GABA content at the germination temperature of 40 °C was also significantly higher than that at the germination temperature of 15 °C and 20 °C. These results indicated that the relatively low germination temperature was not conducive to the increase of GABA content. When the germination temperature was relatively higher (30–35 °C), the GABA content was also higher. Similar research results have also been reported (Zhang et al., 2014). The catalytic activity of GAD is closely related to the germination temperature (Saikusa, Horino, & Mori, 1994a, 1994b). The most suitable temperature range for the catalytic reaction of GAD in brown rice may be 30–35 °C. 3.2.2. Germination time Fig. 2b illustrates that during the brown rice germination, the GABA 236

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Table 2 Texture attributes of GBR subjected to various cyclic cellulase conditioning and germination treatmentsB,C. TreatmentA CC10 CC50 CC100 ITCT60 ITCT120 GT15 GT20 GT25 GT35 GT40

Hardne-ss (g)

Adhesivene-ss –654 –705 –689 –564 –581 –632 –677 –629 –591 –631

a

3568 ± 102 3180 ± 117b 3346 ± 124b 3621 ± 131a 3537 ± 173a 3124 ± 139b 3235 ± 79b 3269 ± 115b 3312 ± 132b 3167 ± 165b

± ± ± ± ± ± ± ± ± ±

ab

47 36a 76ab 43b 59b 37ab 52ab 38b 40b 53b

Springin-ess 0.43 0.78 0.59 0.61 0.56 0.72 0.65 0.59 0.75 0.68

± ± ± ± ± ± ± ± ± ±

Cohesivene-ss (g.s) b

0.12 0.16a 0.06ab 0.23ab 0.16ab 0.07ab 0.09ab 0.11ab 0.18ab 0.26ab

0.465 0.432 0.475 0.466 0.458 0.445 0.451 0.457 0.463 0.463

± ± ± ± ± ± ± ± ± ±

a

0.023 0.031a 0.024a 0.029a 0.051a 0.027a 0.029a 0.033a 0.036a 0.051a

Gummin-ess 1632 1571 1523 1703 1623 1611 1433 1558 1749 1571

± ± ± ± ± ± ± ± ± ±

Chewin-ess ab

Stickin-ess c

111 86b 53b 71ab 89ab 105b 116b 123b 46a 136b

889 ± 156 1247 ± 48b 1211 ± 101bc 893 ± 98c 1206 ± 147bc 1117 ± 121bc 1236 ± 116bc 996 ± 135c 1589 ± 144a 1714 ± 79a

0.234 0.287 0.211 0.225 0.232 0.278 0.291 0.281 0.278 0.286

± ± ± ± ± ± ± ± ± ±

0.021b 0.014a 0.016b 0.025b 0.014b 0.015a 0.028a 0.029a 0.036a 0.051a

A CC10/CC50/CC100: brown rice was treated by cyclic cellulase conditioning at the cellulase concentrations of 10, 50, and 100 mg/mL, respectively; ITCT60/ ITCT120: brown rice was treated by cyclic cellulase conditioning for 60 and 120 min, respectively; GT15/GT20/GT25/GT35/GT40: brown rice was germinated at 15, 20, 25, 35, and 40 °C, respectively. Cellulase solution at pH 5.0; Cellulase treatment temperature was 35 °C. Germination time was 32 h at 30 °C. B Data was expressed as mean ± standard deviation of triplicate determinations. C Different characters in the same column represent significant difference at p < 0.05.

10 mg/mL. The results showed that an excessively low cellulase concentration was not conducive to an improvement in the GBR smell value. The smell value of cooked GBR is the panellists’ evaluation of the unique odours of cooked GBR by olfactory perception. When the cellulase concentration was 50 mg/mL, the colour value was the highest. The colour value of the cooked GBR refers to the panellists’ evaluation by visual perception (Guerrero, O'Sullivan, Kerry, & de la Caba, 2015). The optimum value of appearance was found at the cellulase concentration of 50 mg/mL. The appearance of cooked GBR is the overall observation in terms of shape, integrity and transparency. The highest palatability value appeared at the cellulase concentration of 50 mg/mL. Palatability refers to the acceptable or agreeable degree to the palate or taste of cooked GBR. The flavour value of cooked GBR was the highest at the cellulase concentration of 50 mg/mL. Flavour refers to the taste impression of cooked GBR and is mainly determined by the senses of taste and smell. The results showed that there was no significant correlation between germination temperature and GBR taste quality (p > 0.05). Table 4 presents the correlations between the texture attributes and the taste qualities of cooked GBR. The smell of cooked GBR was significantly correlated (p < 0.05) with hardness (r = −0.94), springiness (r = 0.79) and stickiness (r = 0.68). There was a significant correlation (p < 0.05) between colour and hardness (r = −0.83), as well as stickiness (r = 0.72). The correlation analysis results also revealed that palatability was significantly correlated (p < 0.05) with hardness (r = −0.68), springiness (r = 0.90), chewiness (r = 0.76), and stickiness (r = 0.67). Appearance showed no significant correlation with overall texture attributes. The flavour of cooked GBR was positively

3.4. Effect of cyclic cellulase conditioning and germination treatment on the texture attributes of cooked GBR Table 2 summarises the texture attributes of the cooked GBR after different cyclic cellulase conditioning and germination treatments. No significant effect of the cyclic cellulase conditioning treatment conditions on the hardness, adhesiveness, springiness, cohesiveness, gumminess, chewiness, and stickiness of cooked GBR was found (p > 0.05). The hardness of cooked GBR is the force required to bite through the sample using the molars. Adhesiveness is the degree to which the kernels adhere to contacting substances. The springiness of cooked GBR is the degree of recovery to its original shape after partial compression. The cohesiveness index is defined as the degree to which the grains deform rather than crumble, crack, or break when biting with the molars. The gumminess of cooked GBR is the degree to which the kernels adhere to each other. Chewiness refers to the amount of work required to chew the samples. Stickiness is the degree of binding between kernels from the stretched state to the original state. In this study, it was also found that the germination temperature had no significant influence on all the texture attributes of cooked GBR (p > 0.05). 3.5. Effect of cyclic cellulase conditioning and germination treatment on the taste qualities of GBR The results in Table 3 show the effect of the cellulase conditioning and germination treatment conditions on the taste qualities of cooked GBR. The lowest smell value was found at the cellulase concentration of

Table 3 Taste qualities of GBR subjected to various cyclic cellulase conditioning and germination treatmentsB,C. TreatmentA

Smell (points)

CC10 CC50 CC100 ITCT60 ITCT120 GT15 GT20 GT25 GT35 GT40

16.2 19.3 18.9 17.3 16.8 20.5 19.5 18.8 18.9 19.3

± ± ± ± ± ± ± ± ± ±

Color (points) 1.2b 0.6ab 1.5ab 1.7ab 1.3ab 2.6a 1.8ab 0.7ab 1.5ab 0.8ab

6.6 7.7 7.1 6.3 6.6 7.5 7.2 8.2 7.2 7.4

± ± ± ± ± ± ± ± ± ±

Appearance (points)

1.3ab 0.5ab 0.6ab 0.7b 0.8ab 0.9ab 1.1ab 0.7a 0.6ab 1.5ab

6.2 8.1 7.8 7.7 7.8 6.9 8.3 7.2 7.6 7.1

A

± ± ± ± ± ± ± ± ± ±

0.4b 1.3a 0.6a 0.7a 0.9a 0.8ab 0.8a 1.6a 1.5a 0.4a

Palatability (points) 16.5 21.1 18.3 18.9 19.3 20.1 19.6 18.7 20.5 21.5

± ± ± ± ± ± ± ± ± ±

0.9b 1.3a 1.2ab 0.9a 0.6a 1.0a 2.7a 1.6a 2.3a 1.8a

Flavour (points) 18.8 21.3 19.5 19.3 20.9 20.6 20.4 21.2 22.7 20.1

± ± ± ± ± ± ± ± ± ±

3.6b 0.9ab 1.3b 2.2b 1.4ab 1.1ab 1.6ab 2.7ab 0.8a 1.2ab

CC10/CC50/CC100: brown rice was treated by cyclic cellulase conditioning at the cellulase concentrations of 10, 50, and 100 mg/mL, respectively; ITCT60/ ITCT120: brown rice was treated by cyclic cellulase conditioning for 60 and 120 min, respectively; GT15/GT20/GT25/GT35/GT40: brown rice was germinated at 15, 20, 25, 35, and 40 °C, respectively. Cellulase solution at pH 5.0; Cellulase treatment temperature was 35 °C. Germination time was 32 h at 30 °C. B Data was expressed as mean ± standard deviation of triplicate determinations. C Different characters in the same column represent significant difference at p < 0.05. 237

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Table 4 Correlation coefficients (r) between the texture attributes and the taste qualities of cooked GBR. r

Smell

Color

Appearance

Palatability

Flavor

Taste value

Hardness Adhesiveness Springiness Cohesiveness Gumminess Chewiness Stickiness

−0.94** 0.40 0.79** −0.46 −0.38 0.45 0.68*

−0.83** 0.44 0.49 −0.48 −0.39 0.26 0.72*

−0.14 0.14 0.48 −0.25 −0.31 0.23 0.11

−0.68* −0.002 0.90** −0.50 −0.003 0.76* 0.67*

−0.46 −0.15 0.68* −0.41 0.23 0.52 0.60

−0.83** 0.18 0.93** −0.56 −0.17 0.65* 0.76**

Note: *Significant at p < 0.05, **extremely significant at p < 0.01.

obtained after cyclic cellulase conditioning treatment absorbed water sufficiently, resulting in a high gelatinization degree of starch (Saikusa, Horino, & Mori, 1994a, 1994b). Therefore, cyclic cellulase conditioning treatment was an effective method to improve the cooking and taste qualities of GBR.

correlated (p < 0.05) with springiness (r = 0.68). Consequently, the taste value of cooked GBR was significantly correlated (p < 0.05) with hardness (r = −0.83), springiness (r = 0.93), chewiness (r = 0.65), and stickiness (r = 0.76). The results illustrate the influences of the texture attributes on the taste qualities of cooked GBR. The relationships between the texture attributes and the taste qualities explain the reasons for which cellulase conditioning and germination treatment can improve the taste quality of cooked GBR. These results agree with those reported by other researchers (Kunze, 2008; Zheng, Liu, Chen, Ding, & Jin, 2011).

3.8. Effect of cellulase treatment on GBR microstructure The microstructures of the GBR samples obtained by different treatments under the scanning electron microscope are shown in Fig. S2. Fig. S2a illustrates that the cortical structure of GBR obtained by cyclic moisture conditioning treatment was still compact and intact. Fig. S2b shows that an obvious porous and loose structure appeared in the cortex of GBR obtained by cyclic cellulase conditioning treatment. The reason was that cellulase selectively degraded cortical non-starch polysaccharides and destroyed the GBR cortical structure, which made the moisture more easily penetrate the cortex during GBR cooking (Das et al., 2008a). At the same time, the crude fibre content of GBR decreased. All these changes contributed to the improvement of the cooking and taste qualities of GBR (Das, Banerjee, & Bal, 2008b). Das et al. (2008a) confirmed that the bran layer of brown rice was degraded and became thinner due to the action of cellulase on the cortical nonstarch polysaccharides of brown rice. Water was absorbed quickly through the bran layer, leading to faster gelatinization of the rice during cooking. Zhang et al. (2015) observed that the cellulase treatment damaged the crude fibre structure of the GBR cortex to reduce the hardness of GBR, which improved the GBR cooking quality.

3.6. Comparison of the GABA content and cooking and taste qualities of GBR obtained by different treatments The GABA content, cooking time, volume expansion rate, meal rate and taste quality of GBR obtained by different treatments are shown in Table S1. The GABA contents of GBR obtained by different treatments were significantly different (p < 0.05). The GABA content of GBR obtained by the soaking treatment was the lowest. The GABA content of GBR obtained by the cyclic cellulase conditioning treatment was the highest, which was dependent on GAD activity (Saikusa, Horino, & Mori, 1994a, 1994b). The acid environment formed during the cellulase treatment may increase GAD activity, which contributes to the accumulation of GABA. According to the research of Hill-Venning et al. (1996), GABA in brown rice is easily soluble in water. Part of GABA may be dissolved in the soaking solution and lost during soaking. Therefore, cyclic cellulase conditioning treatment is conducive to the accumulation of GABA in GBR. Based on Table S1, the cooking time of GBR obtained after cyclic cellulase conditioning treatment was the shortest, and the volume expansion rate, meal rate and taste value were the highest. The softness of cooked rice is related to the cooking time. The cooking time even determines the stickiness of the cooked rice to a large extent (Patil & Khan, 2012). A shorter cooking time helps to lower the loss of nutrients during the rice cooking. Through the comprehensive analysis of the various indicators of cooking and taste characteristics, it is revealed that cyclic cellulase conditioning treatment can improve the cooking and taste qualities of GBR.

4. Conclusions A feasible method applied to producing GBR by cyclic cellulase conditioning and germination treatment was evaluated in this study. The cellulase concentration, cellulase treatment time, germination temperature and germination time had significant effects on the GABA content of GBR. Suitable treatment conditions were helpful in increasing GABA content. The effects of cyclic cellulase conditioning treatment conditions and germination temperature on the texture and taste attributes of cooked GBR were not significant. When the cellulase concentration was 50 mg/mL, the cooking time of GBR was the shortest and its volume expansion rate, meal rate and overall taste qualities were the highest. Additionally, the correlation analysis showed a significant influence of texture attributes on the taste qualities of cooked GBR. The relationships between the texture attributes and the taste characteristics explained the mechanism by which cyclic cellulase conditioning and germination treatment can improve the taste quality of GBR. Moreover, the results of the GBR soaking test showed that the water absorption rate of GBR obtained after cyclic cellulase conditioning treatment significantly accelerated. The microstructure observation indicated that the cortex of GBR obtained after cellulase treatment was degraded. Therefore, the present study indicates that cyclic cellulase conditioning and germination treatment is an effective method to enhance GABA in GBR and improve its cooking and taste qualities.

3.7. Moisture adsorption change during GBR soaking Fig. S1 describes the moisture adsorption curves of the GBR obtained after cyclic moisture conditioning treatment and cyclic cellulase conditioning treatment at the soaking temperature of 30 °C. The moisture content of GBR samples increased gradually with the soaking time. At different time intervals, different water absorption rates were observed. In the initial stage of soaking, the moisture content increased rapidly. In the final stage of soaking, the moisture content increased more slowly and gradually stabilized. It can also be seen that under the condition of cyclic cellulase conditioning treatment, the water absorption rate of GBR accelerated significantly. This may be because the degradation of the GBR cortex weakened the barrier of the moisture into the GBR interior. At the same soaking time, the starch of GBR 238

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Conflict of interest

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