1. Introduction
With an increase in people’s awareness of health, the preference for whole grains over refined food is increasing in popularity, and brown rice (BR) and germinated brown rice (GBR) have gradually replaced white rice (WR) as staple foods [
1]. BR, which has light-brown coloration, has the inedible outer husk removed after harvesting, retaining the bran, aleurone layers, germ, and endosperm. Rice bran is nutrient-rich, containing polyunsaturated fatty acids, a vitamin B complex, vitamin E, γ-oryzanol, dietary fiber, etc. [
2]. As a result, BR has many physiological functions, such as adjusting the intestinal flora to stimulate bowel movements [
3] and the prevention of cancer [
4]. In addition, since the glycemic index of BR (55) is lower than that of refined WR (64), BR delays the rise in blood sugar, which aids blood glucose stabilization in patients with type II diabetes [
5]. However, because BR has a rough texture, is unpalatable, and is not easily masticated, it has not been widely adopted. To combat this, researchers have found that germination treatment can improve these sensory shortcomings of BR. GBR is attained by soaking the whole kernel of BR in water until its embryo begins to bud. During germination, the chemical compositions of rice change drastically. Hydrolytic enzymes are activated to decompose large molecular substances into small molecular compounds [
6]. The content of the significant and functional component, γ-aminobutyric acid (GABA), is substantially increased with increasing germination time. It has been reported that GABA is an inhibitory transmitter in the mature brain [
7].
Bacillus species are widely used by the fermentation industry.
Bacillus subtilis subsp.
natto (
B. natto) is commonly used in the commercial production of Japanese food. During the growth process,
B. natto is capable of producing extracellular enzymes that decompose proteins, carbohydrates, fats, and other macromolecules. Natto, a common fermented soy product with
B. natto, is rich in amino acids, organic acids, oligosaccharides, and other components that are easily absorbed by the body. In particular, natto contains nattokinase (NK), which was first discovered by Sumi et al. [
8]. A large number of studies have confirmed that NK has many beneficial effects on cardiovascular health. NK has potent antithrombotic [
9], antihypertensive [
10], anti-atherosclerotic, lipid-lowering [
11], and neuroprotective [
12] activities. Moreover, it has been reported that the ether extracts of rice bran subjected to fermentation with
B. natto show good antioxidant activity [
13]. Rice grains contain fiber and antioxidants, such as ferulic acid, phytic acid, tocopherol, oryzanol, which are known for their anticancer properties [
14,
15]. Therefore, the fermentation of rice with
B. natto is of great interest.
The rice samples in this experiment are all of the Japonica rice variety. GBR and BR were used as test subjects, WR was used as a control, and the B. natto strain was added for fermentation. The nutrient composition (protein, fat, ash, and moisture), functional ingredients (γ-oryzanol, GABA, and free amino acids), and biological activity (DPPH radical scavenging capability and nattokinase activity) of each of the three rice materials were compared before and after fermentation.
2. Materials and Methods
2.1. Samples
BR (Oryza sativa, Tainan 11) was harvested in 2018, and GBR samples were supplied by Asia Rice Biotech Company (Taipei, Taiwan). According to the Asian Rice Biotech Company, the GBR was produced under the condition that the brown rice was completely germinated in water for 22 h at a temperature of 37 °C. WR was obtained after removing the bran of BR by milling for 10 s using a model MR1000E polishing machine (Hosokawa Company, Ltd., Tokyo, Japan). The rice samples were vacuum-packed and stored at −18 °C. Rice powder was obtained as follows: 50 g of rice grain was pulverized with a blade mill (fixed at 20,000 rpm), grinding for 5 s twice.
2.2. Chemicals and Standards
Analytical-standard γ-oryzanol was purchased from Wako Pure Chemical Industries (Osaka, Japan). Standards of amino acids and GABA were obtained from Sigma-Aldrich (St. Louis, MO, USA). Certified HPLC-grade solvents n-hexane and isopropanol were purchased from Echo Chemical (Miaoli, Taiwan). All other reagents used for the extraction and analysis were analytical grade or purer and were purchased from Mallinckrodt Pharmaceuticals (Dublin, Ireland) and J. T. Baker Chemicals (Center Valley, PA, USA). Nutrient broth (NB) was purchased form Acumedia Manufacturers (Lansing, MI, USA).
2.3. Fermentation
In the present study, the
B. natto (CKU-20) strain, obtained from Dr. Shih’s lab at the Department of Nutrition and Health Sciences, Chinese Culture University (Taipei, Taiwan), was used as the test organism. To prepare the inoculum, two successive transfers of the test organism were performed in NB at 40 °C and 150 rpm for 24 h. The activated culture was then inoculated into NB and incubated at 40 °C for 16 h, when the viable population was ca. 108 CFU/mL. Rice samples were fermented with
B. natto following the procedures described by Hung [
16]. After rinsing the rice grains with tap water for 30 s, the washed rice samples (100 g) were mixed with 100 mL of medium (0.1% (
w/
w) peptone, 0.1% (
w/
w) yeast extract) and then sterilized in an autoclave at 121 °C for 20 min. After cooling, the steamed rice samples were inoculated with the test organism by evenly spraying with a 1 mL spore suspension of
B. natto. After thorough mixing, the inoculated rice substrate was placed into a rotary incubator and then incubated for 60 h at 37 °C, 95% RH, and 150 rpm. The fermented samples were aged in a 4 °C refrigerator for 24 h and then subjected to freeze-drying. The fermentation was conducted in triplicate for each rice sample.
2.4. Chemical Composition Determination
The chemical composition was determined according to the AACC method [
17]. The moisture content was determined using a hot air oven at 130 °C for 1 h. The protein content (N × 5.95) was analyzed by the Kjeldahl method. The crude fat content was determined with a Soxhlet extraction method. The ash content was measured at 590 °C for 8 h. All analyses were performed in triplicate.
2.5. Analysis of Free Amino Acids and GABA
The extraction procedure was modified from Jannoey et al. [
18]. Rice powder (250 mg) was placed in 800 μL of 70% (
v/v) ethanol solution. The mixture was mixed for 1 min and then centrifuged at 13,000×
g at 4 °C for 10 min. The supernatant was collected. The above extraction was repeated. The collected supernatant (3 mL) was filtered and analyzed by liquid chromatography–electrospray ionization tandem mass spectrometry (LC-ESI-MS).
For the identification of free amino acids and GABA, a Finnigan LXQ linear-ion trap mass spectrometer (Thermo Scientific, Waltham, MA, USA) was employed. The operation conditions of the MS detector were fragmentation range: 70, mass range: 50–300 m/z, sheath gas flow rate: 30 arb unit, auxiliary gas flow rate: 12 arb unit, sweep gas flow rate: 1 arb unit, spray voltage: 5.5 kV, capillary voltage: −17 V, and capillary temperature: 290 °C.
Free amino acids and GABA were analyzed with the Survey HPLC system and an Aquasil C18 column (250 × 2.1 mm, 5 μm) (Thermo Fisher Scientific, Waltham, MA, USA). A linear gradient system was used with mobile phase A (0.1% ammonium perfluorovalerate in water) and mobile phase B (0.1% ammonium perfluorovalerate in acetonitrile); A: 95–85%, 85–40%, and 40–95% were used at 0–10 min, 10–20 min, and 20–21 min, respectively, and then 95% A was used for another 9 min. The flow rate was 0.2 mL/min and the sample volume was 5 μL. The data were acquired and processed by using Xcalibur software (Thermo Fisher Scientific, Waltham, MA, USA).
2.6. Analysis of γ-Oryzanol
Extraction of γ-oryzanol was performed according to AACC Method 30-10 [
17] and the method of Heinemann et al. [
19] with a slight modification. The rice sample (4 g) was added with 8 mL of 95% ethyl alcohol and stirred. Next, 20 mL of 4 N HCl was added and heated in a water bath at 70–80 °C for 90 min. After cooling, the mixture was added to 20 mL of 95% ethyl alcohol. Subsequently, petroleum ether (25 mL) was added and shaken vigorously for 1 min. The supernatant was centrifuged at 10,000×
g for 20 min. The above steps were repeated twice. The supernatants were combined and evaporated to dryness. The dried material was dissolved in 2 mL of HPLC-grade hexane. An aliquot of the sample was filtered for HPLC analysis.
γ-Oryzanol was analyzed using a silica-gel column (250 × 4.6 mm, 5 μm, SPS100-5, Chromatorex, Thermo Fisher Scientific, Waltham, MA, USA) at 30 °C and a KNAUER Smartline series 1000 HPLC (Advanced Scientific, Berlin, Germany) equipped with a photodiode array detector (Jasco MD-215 plus, Tokyo, Japan). Detection was accomplished by measuring the absorbance at 330 nm. The mobile phase was ethyl acetate/acetic acid/n-hexane (isocratic at 1.8:1.8:98.4 (v/v/v)), with a flow rate of 1.5 mL/min. The content was quantified by comparison of the peak area with that of a γ-oryzanol standard curve. The standard curve concentration of γ-oryzanol was formulated in five standard solutions with concentrations ranging from 0–800 ppm.
2.7. Determination of DPPH Scavenging Activity
The DPPH scavenging activity of the rice samples was measured as described by Liyana-Pathirana and Shahidi [
20] with some modifications. The crude rice extract was prepared by soaking 0.1 g of the sample powder in 1 mL of 50% methanol solution for 1 h and then centrifuging at 7500×
g for 10 min. The supernatant (0.1 mL) was mixed with 1.9 mL of DPPH (Fluka Chemie, Buchs, Switzerland) in methanol (0.02 mg/mL). The mixtures were left for 30 min and then measured at 517 nm (UV-2250PC, Shimadzu Co., Kyoto, Japan). The scavenging activity was calculated as follows:
2.8. Determination of Nattokinase Activity
The nattokinase activity was determined according to Jorge et al. [
21]. Rice powder (1 g) was suspended in 20 mL of boric acid saline buffer (0.05 M H
3BO
3, 0.05 M KCl, pH 7.8), settled for 20 min, and then filtered. Boric acid saline buffer (1.4 mL) and fibrinogen solution (0.05% (
w/
v), 0.4 mL) were combined in a vial and kept in a water bath (37 °C) for 5 min. Then, 0.1 mL thrombin (20 U/mL) was added and kept in the water bath (37 °C) for a further 10 min. To this clot, 0.1 mL of sample extract was added and boric acid buffer was as a control. After incubation (37 °C, 60 min), trichloroacetic acid (0.2 M, 2 mL) was added. The vials were kept in the water bath for a further 20 min and then centrifuged at 3000×
g for 5 min. One unit of enzyme activity is defined as the amount of enzyme required to produce an increase of 1.0 in the absorbance at 275 nm in 60 min.
2.9. Statistical Analysis
Data were analyzed using the SAS software version 9 (SAS Institute, Cary, NC, USA). Analysis of variance (ANOVA) and Duncan’s tests were performed. In all cases, the significance was established at p ≤ 0.05. All experiments were carried out in triplicate unless otherwise stated.
4. Conclusions
The nutritional and sensorial properties of cereals and pseudocereals could be enhanced by their germination and fermentation. Thus leads to improved product properties by changing increased nutritional value and better digestibility of the grains making them better food material than the raw grains [
39]. In addition, compared with the fermented white rice, the fermented brown rice had higher nutritional components, flavor, and antioxidant activity [
25]. In this study, GBR fermented with
B. natto yields improved nutritional value and better functional properties than fermented BR or WR. Thus, GBR could be used for developing rice-based products with enhanced nutritional value. The
B. natto fermentation conditions should be further optimized to improve the content of functional components, as although the nattokinase activity of fermented GBR is considerably higher than those of fermented BR and WR, it is lower than that of commercial natto products. In future research, the scope of the study can be expanded to explore the optimum growth conditions for
B. natto fermentation of GBR, with the potential of promoting different active and/or functional components. Such improvements may then encourage increased production of processed BR products.