Introduction

Rice is the staple food for about half of the world’s population (FAO, 2007). Simply, rice refers to white rice, also known as polished rice, which is produced by further milling brown rice and removing the bran and most of the germ layers from it. A significant proportion of nutrients of white rice are removed during the milling because most of them remain in the outer bran layer of rice (Saleh et al., 2019; Zhou et al., 2002). Compared with white rice, brown rice is richer in nutrient components, such as fibers, vitamins, iron, calcium, and γ-aminobutyric acid (GABA) (Patil and Khan, 2011). Although brown rice is healthier than white rice (Dinesh Babu et al., 2009), brown rice is not widely accepted as an appropriate staple food because of its coarse texture and difficulty in cooking (Komatsuzaki et al., 2007). Therefore, a technique that helps overcome these limitations could be of great significance.

The development of germinated brown rice (GBR) technique appeared to overcome some of the limitations of brown rice. GBR is prepared after soaking the brown rice in water and thereby keeping it moist for an extended period of time. The soaking and germination of brown rice not only soften the texture and make it easily cookable but also enhance the nutritional value. Germination modifies the contents of existing nutrients as well as releases new nutrient components (Kayahara et al., 2001), making GBR more nutritious. GBR is considered healthier than white rice and unterminated brown rice, especially in the prevention of diet-related diseases, such as obesity, type 2 diabetes, and colorectal cancers (Imam et al., 2014). Consumption of GBR is better for controlling postprandial blood glucose levels without increasing insulin secretion in subjects with hyperglycemia (Ito et al., 2005) than white rice. Similarly, intake of GBR is regarded as a protectant of cell proliferation and apoptosis as well as of heart failure owing to myocardial ischemia (Petchdee et al., 2020).

Lately, elicitation approaches have been emphasized with the aim of enhancing the phytochemical and biological activity of germinated seeds (Liu et al., 2019). The treatment of brown rice with sugars, calcium chloride, and glutamic acid increased the concentration of GABA, polyphenols, and flavonoids (Jeong et al., 2018; Oh, 2003; Sim et al., 2020). In a separate study, calcium chloride in GBR enhanced the accumulation of bioactive compounds and antioxidant capacities (Choe et al., 2021). Lactobacillus acidophilus-fermented GBR activated apoptotic pathway, which may inhibit preneoplastic lesions of the colon (Li et al., 2019). Cold plasma treatment increased the concentration of gamma oryzanols in the GBR (Yodpitak et al., 2019) which is considered to increase muscle strength (Eslami et al., 2014). The treatment of brown rice with cellulase solution greatly increased GABA content in GBR (Zhang et al., 2019). Similarly, red onion solution enhanced the antioxidant capacity and GABA content as well as made the rice slightly softer and stickier than that germinated in water (Nakamura et al., 2020).

Turmeric (Curcuma longa L.) contains a number of nutrients and bioactive constituents, including curcumin that has antioxidant, anti-inflammatory, antibacterial, antidepressant, antidiabetic, antitumor properties (Farhood et al., 2019; Naeini et al., 2019; Soleimani et al., 2018). Different extracts obtained from plant sources have been used as elicitors to treat the germinating grains. Considering the health benefits of GBR and turmeric, this study aimed to investigate the effect of turmeric extracts on the nutritional and antioxidant properties of GBR.

Materials and Methods

Materials

Brown rice of a Korean rice cultivar Ilpum Byeo was used in this study. Turmeric powder (Jindo Turmeric Agricultural Cooperative Corporation, Jeollanam-do, Korea) was purchased from a local market in Daegu, Korea. Three concentrations 1, 3, and 5% (w/v) of turmeric solutions were prepared by mixing the turmeric powder with tap water.

Cultivation of germinated brown rice and preparation of sample powder

Brown rice (1 ㎏) was washed with tap water and soaked in the three different concentrations (1, 3, and 5%, w/v) of turmeric solutions or tap water alone (0%) for 1 h. Each treatment consisted of three replicates. After 1 h of soaking, the moistened brown rice samples were put into netted plastic bags and incubated at 35℃ for 36 h to allow germination. During the 36-h of incubation, the germinating brown rice was moistened every 1 h by brief dipping into the respective solutions (0, 1, 3, and 5%) used for soaking. The germinated brown rice (GBR) samples were named based on the concentration of turmeric used for soaking the brown rice i.e., TE-0: GBR cultivated with 0% turmeric powder, TE-1: GBR cultivated with 1% turmeric powder, TE-3:GBR cultivated with 3% turmeric powder, and TE-5: GBR cultivated with 5% turmeric powder, respectively.

The 36-h old GBR samples were harvested and kept in a deep freezer (-70℃) for 24 h before lyophilization. The lyophilized GBR samples were ground into powder using a commercial grinder (HIL-G-501, Hanil Co., Seoul, Korea).

Color measurement

The Hunter’s color values of the powdered GBR samples were measured following a method described earlier (Kim et al., 2014). The L (lightness), a (redness), and b (yellowness) values were determined using a Chroma Meter (CR-300, Minolta Corp, Tokyo, Japan). A calibration plate (Minolta Corp.; YCIE = 94.5, XCIE = 0.3160, YCIE = 0.330) and a standard plate (Hunter Associates Laboratory Inc., Reston, VA, USA; L = 97.51, a = -0.18, b = 1.67) were used to standardize the instrument with D65 illuminant.

Free amino acid analysis

The free amino acid profile was determined following the methods described earlier (Je et al., 2005; Kim et al., 2016). In brief, 1.5 g sample powder was homogenized (12,000 rpm, 2 min) with 10 mL of ice-cold 6% (v/v) perchloric acid in an ice bath using an ACE homogenizer (Nissei AM-7, Nihonseikei Kaisha Ltd, Tokyo, Japan), followed by an ice-incubation for 30 min and centrifugation (4,600 × g, 15 min). The supernatant was filtered through a filter paper (Whatman No. 41). The filtrate pH was adjusted to 7 using a 33% (w/v) KOH solution, and centrifuged (4,600 × g, 10 min). The precipitate of potassium perchlorate was separated and the pH of the mixture was adjusted to 2.2 using 10 M HCl and then distilled water was added to make the final volume 50 mL. The mixture and lithium citrate buffer (pH 2.2) were mixed at a 2:1 ratio to determine the amino acid profile using an automatic amino acid analyzer (Biochrom-20, Pharmacia Biotech Co., Uppsala, Sweden).

Determination of mineral content

A previously described method (Skujins, 1998) was followed to measure the amount of mineral elements using an inductively coupled plasma atomic emission spectrometer (ICP AES, Varian Vista, Victoria, Australia). Sample powder (500 ㎎) was digested in a mixture of 65% HNO₃ (15 mL) and 35% H₂O₂ (2 mL). The mixture was diluted with an equal volume of distilled water. The amount of mineral elements was determined using ICP AES after calibrating the instrument with known standards.

Preparation of sample extracts for antioxidant assays

The extracts for antioxidant assays were prepared as in a previous report (Park et al., 2020). One gram of sample powder was extracted with 10 mL of absolute methanol using a shaking incubator (250 rpm, 25℃) for 6 h. The mixture was centrifuged (1660 × g, 10 min), followed by filtration of the supernatant using a syringe filter (0.2 ㎛). The filtrate was used for antioxidant analyses.

Determination of 1,1-diphenly-2-picrylhydrazyl (DPPH) radical scavenging activity

The DPPH free radical scavenging potential was determined following the methods described earlier (Blois, 1958; Dhungana et al., 2015). In short, 100 mL of the extracts and 100 mL of freshly prepared 0.05% (w/v) methanolic solution of DPPH were mixed in a 96-well microplate, followed by 30 min of incubation at room temperature at dark conditions. Immediately, the absorbance of the reaction mixtures was examined at 517 nm using a spectrophotometer (Multiskan GO, Thermo Fisher Scientific Oy, Vantaa, Finland).

Determination of peroxidase (POD) activities

The POD activities were measured using the guaiacol method as described earlier (Zhang and Kirkham, 1994) with some modifications. One hundred milliliters of supernatant was added to the reaction mixture containing 1.0 mL of 2% H₂O₂, 2.9 mL of 50 mM phosphate buffer (pH 5.5), and 1.0 mL of 50 mM guaiacol. For the control, phosphate buffer was used instead of the enzyme. The absorbance values were measured at 470 nm for 3 min, and the POD activity was determined as a unit change per minute.

Determination of total polyphenol content

The total polyphenol content (TPC) was measured according to the Folin-Ciocalteau method (Singleton et al., 1999) as described by Dhungana et al. (2016). The sample extract (50 mL) and 2% (w/v) aqueous Na₂CO₃ (1000 mL) were mixed in microtubes and allowed to react at room temperature for 3 min. Then, 50 mL of 1 N Folin-Ciocalteau reagent was added to the mixture and incubated for 30 min at room temperature under dark conditions. After the incubation of 30 min, the absorbance was measured at 750 nm using a microplate spectrophotometer (Multiskan GO; Thermo Fisher Scientific). The TPC of the samples was determined using the calibration curve plotted using gallic acid (GA) as standard.

Total flavonoid content analysis

The total flavonoid content (TFC) was determined following the methods described earlier (Dhungana et al., 2016; Zhishen et al., 1999). The sample extract (100 mL), absolute methanol (500 μL), 10% AlCl₃ (50 μL), 1 M HCl (50 μL), and distilled water (300 μL) were mixed in microtubes and incubated for 30 min at room temperature under dark condition. Then, the absorbance values of the reaction mixtures were measured at 510 nm using a microplate spectrophotometer (Multiskan GO; Thermo Fischer Scientific). The TFC was calculated using the calibration curve drawn using quercetin as a standard.

Determination of superoxide dismutase (SOD)-like activity

The methods described by Adhikari et al. (2019) and Dubey et al. (2015) were adopted to determine the SOD-like activities on the basis of 50% reduction of nitro blue tetrazolium. Sample powder (0.5 g) was homogenized in a phosphate extraction buffer (5 mL). The homogenized mixture was centrifuged (15,000 × g) for 20 min. One hundred microliters of supernatant were added to the reaction mixture consisting of nitro blue tetrazolium (2.25 mM), 100 mM phosphate buffer (pH 7.8), methionine (200 mM), sodium carbonate (1.5 M), and EDTA (3 mM). The reaction was initiated by adding 0.4 mL (2 µM) of riboflavin and placing it under fluorescent light (15 W) for 15 min. A reaction mixture without the sample extract was considered as a control. The reaction was stopped by turning the light off and keeping the reaction mixtures in dark. The absorbance of reaction mixtures was measured at 560 nm using a microplate spectrophotometer (Multiskan GO, Thermo Fischer Scientific).

Statistical analysis

Data were analyzed using analysis of variance in SAS 9.4 (SAS Institute, Cary, NC, USA). The significant differences between treatment means were determined using the Tukey test (p < 0.05). The average values of three replicates were reported unless otherwise mentioned.

Results

Hunter’s color value

The treatment of turmeric extracts did not significantly affect the color value of GBR except for the yellowness value (Table 1). In the case of the yellowness value, the turmeric-treated GBR samples had significantly higher values. However, the difference in concentration of turmeric did not show any significant variation among TE-1, TE-3, and TE-5.

Mineral content

Turmeric treatment significantly influenced the mineral content of GBR (Table 2) although the effect was not consistent for an individual mineral element with the concentration of the extract. The amount of Fe and Mn were reduced in the turmeric-treated samples in a concentration-dependent manner. Four mineral elements Ca, K, Mg, and Na were significantly lower in TE-3 followed by TE-5 as compared to the control and TE-1. The Cu and Zn contents were significantly highest in TE-1. Treatment of lower concentration (1%) slightly increased (33.95.5 ㎎/㎏) but higher (3 and 5%) concentrations of turmeric extract reduced (1735.8 - 2393.7 ㎎/㎏) the total mineral content in germinated brown rice.

Amino acid content

Unlike mineral content, a positive influence of turmeric extract was observed on the availability of amino acids in GBR. The amount of many amino acids was significantly increased with the concentration of turmeric extracts (Table 3). All three components (essential, non-essential, and other free amino acids) were increased in a concentration-dependent manner. A total of 25 amino acids were detected, whereas 8 amino acids were not detectable in four treatments. The amount of essential amino acids was increased by 58.3, 71.5, and 88.3% with the application of 1, 3, and 5% concentrations of turmeric, respectively. The increment for non-essential amino acids was lower i.e., 38.7, 47.8, and 62.3% in TE-1, TE-3, and TE-5, respectively.

Antioxidant potential

As found in the free amino acid profile, the overall antioxidant potential of GBR was significantly improved although DPPH free radical scavenging potential was decreased by turmeric treatment (Table 4). The DPPH free radical scavenging potential of GBR was reduced with the increasing concentrations of turmeric extracts. The peroxidase and SOD-like activities and total polyphenol content were significantly highest in TE-5, indicating a concentration-dependent incremental effect of turmeric.

Discussion

The effect of three (1, 3, and 5% w/v) concentrations of turmeric extracts on the color, nutrient, and antioxidant properties of germinated brown rice was investigated considering Hunter’s color; amino acid, mineral, total polyphenol, and total flavonoid contents; and DPPH, POD, and SOD-like activities. Although measurement of specific plant growth regulars in turmeric extracts was not carried out in the present study, roles of some growth regulators can be expected to alter the nutrient and antioxidant activities of GBR as in the previous studies with calcium chloride (Choe et al., 2021), selenium (Liu and Ning, 2021), the extracts of persimmon fruit powder (Kim et al., 2017), Pu-erh tea (Kim et al., 2020), and lacquer stem (Kwak et al., 2017). The alterations in the nutrient and antioxidant activities of GBR might be due to the absorption of various phytochemicals present in turmeric (Zhang and Kitts, 2021) during soaking and subsequent germination (Lintschinger et al., 2000).

The higher yellowness value of the turmeric-treated GBR samples might be owing to the color of turmeric although such reports are not available. The color of a food product is one of the major visible traits that could determine the willingness of consumers to buy the product (Udomkun et al., 2018).

The soaking and subsequent dipping of brown rice in the mineral-containing turmeric (Kotha and Luthria, 2019) extracts might have increased the mineral contents of GBR at low concentration (TE-1), however, inhibitory (Akter et al., 2018) and herbicidal (Ibáñez and Blázquez, 2019) effects of turmeric might have adversely influenced to reduce the mineral content at high concentrations (TE-3 and TE-5). In the previous studies, the mineral content of germinating seeds was increased with zinc sulfate application in soybean sprouts (Xu et al., 2012; Zou et al., 2014) and selenium treatment in cereal sprouts (Lintschinger et al., 2000). Elements like Zn, Cu, Ca, and Mg commonly lack in human diets (White and Broadley, 2009). Mg, K, and Ca are beneficial against hypertension (Houston and Harper, 2008) and Zn contributes to growth, development, differentiation, DNA synthesis, RNA transcription, and cellular apoptosis (MacDiarmid, 2000). Thus, the treatment of brown rice with low concentrations of turmeric could be useful to increase mineral content.

The essential amino acids must be supplied through diets because they cannot be synthesized de novo by an organism at the required rate. In this perspective, turmeric treatment could be an effective way to increase the availability of essential amino acids in GBR. The content of glutamic acid, a precursor for γ-amino-n-butyric acid (GABA) synthesis (Nikmaram et al., 2017), was increased in GBR with turmeric treatments. The mineral elements present in turmeric might have played an influential role in the activation of diamine oxidase activity resulting in elevated GABA content in the turmeric-applied GBR (Wang et al., 2016). In previous studies, similar results with an increased amino acid content, including GABA, were found with lacquer (Kwak et al., 2017) and Pu-erh tea (Kim et al., 2020) treatment in soybean sprouts. Glycine and GABA, two of the many other non-essential amino acids increased in the turmeric-treated GBR, have beneficial roles related to brain and memory enhancement, neurological diseases; anxiety relief, sedation, anticonvulsant, and muscle relaxation functions (Krogsgaard-Larsen, 1989; Mody et al., 1994; Oh and Oh, 2004).

Several enzymes, including peroxidases and superoxide dismutases (SODs), are reactive oxygen scavengers that protect living organisms against harmful oxidative damages (Dvořák et al., 2021). Peroxidase may increase the accumulation of phenolic compounds in germinated brown rice as in olive fruits (Cirilli et al., 2017). Similarly, the substantial increment in the antioxidant potentials in the turmeric-treated GBR might be owing to the elements such as calcium (Kotha and Luthria, 2019) and/or phenolic compounds (Mughal, 2019; Li et al., 2011) in turmeric. Similar results of high phenolic contents in GBR were found after treatment of high phenolic- containing onions (Gennaro et al., 2002; Griffiths et al., 2002; Nakamura et al., 2020) and soybean sprouts produced with lacquer treatment (Kwak et al., 2017). Various enzymatic and non-enzymatic antioxidants, including SOD, catalase, glutathione transferase, carotenoids, glutathione peroxidase, vitamin C, vitamin E, and polyphenols contribute to antioxidant activities (Kurutas, 2015). Additionally, several factors like the oxidation conditions, partitioning characteristics, and condition of oxidizable substrate collectively define the antioxidant potential of foods (Frankel and Meyer, 2000). Hence, a visible difference in the content of an antioxidant polyphenol, for instance, not necessarily contributes to higher antioxidant activity. In the present study, the turmeric- treated GBR had higher total polyphenol content than the control but the latter showed a higher DPPH free radical scavenging than the turmeric-treated ones.

In conclusion, the effect of turmeric extracts on the nutrient content and antioxidant potential of GBR was investigated. The yellowness of turmeric-treated GBR was significantly higher than that of the untreated control. Treatment of 1% turmeric extract slightly increased but 3 and 5% concentrations reduced the total mineral content in GBR. The amount of essential, non-essential, and total amino acids, including, GABA were increased with the concentration of turmeric extract. Similarly, the overall antioxidant potential of GBR was higher with the higher concentration of turmeric treatment. The results indicated that turmeric treatment could enhance the nutritional and functional value of GBR.