Germination of Six Indonesian Brown Rice: Evaluation of Antioxidant, Bioactive Compounds, Fatty Acids and Pasting Properties

Germination can improve the palatability and alter physicochemical, nutritional, and nutraceutical value of brown rice. This study aimed to evaluate the antioxidant, bioactive compounds, fatty acids, and pasting proles from six Indonesian brown rice varieties during germination. The germination was carried out through a complete soaking method for up to 120 h, and the samples were taken every 24 h germination. Results The results showed that germination increased GABA content in brown rice. The highest level of GABA, up to 126.55 mg/100g, obtained in rice var. Inpari 43, after 120 h. Germination also affected the phenolic content, antioxidant capacity, and γ-oryzanol content, but no consistent trends were observed among the varieties. Fatty acid compositions of germinated brown rice showed no changes during germination. The pasting properties of samples changed signicantly after germination, especially in peak viscosity, nal viscosity, breakdown, and setback value.


Introduction
Rice is becoming one of the grain crops cultivated and consumed by the majority of people worldwide, especially in Asia and Africa region. It approximately contributes to half of the world population as a source of carbohydrate (Pengkumsri et al. 2015; Gong et al. 2017). Rice is considered a strategic commodity in Indonesia, not only as of the most primary food crop that cultivated by farmers but also as a staple food for about 240 million Indonesian people (Munarko et al. 2020).
Nowadays, increasing awareness to consume whole food as a healthy food has increased. Consuming whole food in regular diets has been associated with the reduced risk of chronic diseases, including cardiovascular disease, obesity, type II diabetes, and cancer (Gong et al. 2017). Brown rice or unpolished rice is considered as whole food because only the inedible outer layer part of the paddy is removed, while the bran layer still intact to the endosperm. A number of studies have shown that brown rice contains a high amount of antioxidant activity and bioactive compounds, including phenolic compounds, γ-oryzanol, and unsaturated fatty acid ( The germination process is able to improve the texture and taste of brown rice (Cho and Lim 2016). During germination, many hydrolytic enzymes such as α-amylase, β-amylase, and protease are activated. They hydrolyzed starch and protein resulted in an increase of oligosaccharides, sugars, and amino acids (Mohan et al. 2010). These changes also affected the physical characteristics, including its pasting properties of germinated brown rice (GBR) (Xu et al. 2012). Besides, germination also enhanced bioactive compounds such as γ-aminobutyric acid (GABA) (Ohtsubo et al. 2005; Zhang et al. 2014; Cáceres et al. 2017). GABA is a non-protein amino acid that has some essential physiological functions such as antihypertensive and anti-stress effects on human health (Iimure et al. 2009).
Commonly, the germination process may apply two procedures, i.e., a simple soaking method by steeping of the seeds in water, and temporary immersion followed by atmospheric germination (Cho and Lim 2016). The simple soaking method is the easiest procedure to produce GBR compared to the atmospheric germination. This method is suitably applied at the low production scale (individual or household scale), and in the areas where people easily access brown rice to produce GBR. In Indonesia, rice is commonly cultivated at local farmlands to cover their daily consumption. Therefore, it is easy to produce GBR at the household scale by applying the simple soaking method.
There are many rice varieties cultivated in Indonesia. In the last two decades, Indonesian Center for Rice Research has released many varieties that can be cultivated by farmers. Several varieties, such as Inpari 42, Inpari 43, Situ Bagendit, Inpari 17, and Inpara 3, are potentially high-yield varieties that have been developed. Inpari 42 and Inpari 43 are lowland irrigated rice cultivars that have low amylose content. Situ Bagendit is an upland rice variety with intermediate amylose content and is commonly cultivated by farmers in both dryland and paddy elds. Inpari 17 and Inpara 3 are categorized as high amylose rice and cultivated in lowland and swampland, respectively. Moreover, IPB University has released a new variety, namely IPB 3S. This variety includes lowland elds with intermediate amylose content (Munarko et al. 2020). Although many studies have reported the effect of germination on chemicals and bioactive compounds in many varieties over the world (Ohtsubo et

Brown rice preparation
Brown rice samples were prepared by the hulling process (Yanmar, Japan). The whole kernels were separated from broken rice using rice grader (Ogawa Seiki.Co.Ltd, Japan) and followed by manual sortation. Brown rice samples were vacuum sealed and stored at 4 °C for the further germination process.
Germination procedure Germination procedure of brown rice followed the method of Ohtsubo et al. (2005) with a modi cation. Brown rice was sanitized using 0.1% sodium hypochlorite solution for 30 minutes and followed by rinsing in tap water. The sample was taken as 0 h treatment (ungerminated). The production of GBR was performed by soaking the sanitized brown rice in water (1:10, w/v) and incubated it in hot air sterilizer to maintain the water temperature at 33 ± 2 °C. The soaking water was removed and changed every four hours. The samples were removed after 24, 48, 72, 96, and 120 h and washed by using tap water before dried into a freeze dryer (Labconco, US). The dried GBR was ground using a blender (Miyako, Indonesia), sieved (40 mesh), vacuum sealed, and stored at − 20 °C before analysis.
Evaluation of GABA (γ-aminobutyric acid) content GABA content was analyzed duplicate according to the method of Zhang et al. (2014) with a slight modi cation in sample preparation. Sample (1.0 g) was placed in a plastic tube, and 5 mL deionized water was added. The mixture was then extracted for an hour, followed by a centrifugation at 3000 rpm for 30 minutes and ltered. A half milliliter of supernatant was added with 0.2 mL borate buffer (pH 9), 1.0 mL phenol reagent 6% (w/v), and 0.4 mL sodium hypochlorite 9% and then mixed vigorously. The tube was then boiled in the water bath for 10 minutes and cooled immediately in ice water for 20 minutes to develop a blue color. The absorbance was measured by a UV-Vis spectrophotometer (Thermo Scienti c 150, US) at 645 nm. Standard curve calibration of GABA was prepared to determine GABA concentration in the samples and expressed as mg GABA/100 g dried samples.
Determination of total phenolic content and antioxidant capacity Phenolic compounds were extracted using ethanol extraction (Munarko et al. 2020). The rice our (approximately 1.5 g) was extracted with 20 mL of ethanol 80% (v/v) using a shaker (Innova2300, new Brunswick scienti c) for 30 minutes and centrifuged at 6000 rpm (Hermle Z 383 K, Wehingen, Germany) at 4 °C for 30 minutes. The supernatant was collected into a dark bottle and stored at 4 °C.
Total phenolic content (TPC) was analyzed duplicate by the modi ed Folin-Ciocalteau method (Qiu et al. 2010). Extract (0.2 mL) was mixed with 1.8 mL of 10 × diluted Folin-Ciocalteau reagent (freshly prepared) and 1.8 mL of Na 2 CO 3 60 g/L. After reacting for 90 minutes, the mixture was analyzed by a UV-Vis spectrophotometer (Thermo Scienti c 150, US) at 725 nm. The absorbance was compared to the standard curve of gallic acid and expressed as mg gallic acid equivalent per 100 g dry samples (mg GAE/100 g).
Determination of DPPH scavenging activity was measured duplicate by the method originally developed by Brand-Williams et al. (1995) with a modi cation. The phenolic extract of rice (0.3 mL) and 0.7 mL of distilled water were mixed with 3.0 mL of freshly DPPH solution 140 µM and incubated in the darkroom for 60 minutes. The mixture was then analyzed using a UV-Vis spectrophotometer (Thermo Scienti c 150, US) at 515 nm. The antioxidant activity was expressed by mg ascorbic acid equivalent per 100 g of dried sample (mg AAE/100 g).

Analysis of γ-oryzanol
Analysis of γ-oryzanol applied a partial extraction method (Lilitchan et al. 2008). Two identical rice our samples (1.0 g) were extracted with isopropanol using different volumes (4 mL and 8 mL) and centrifuged for 10 minutes at 2500 rpm (Eppendorf 5810R). The absorbance of the supernatant was measured using a UV-Vis spectrophotometer (Thermo Scienti c 150, US) at 326 nm. Total γ-oryzanol of the sample was compared to the γ-oryzanol standard curve and then calculated as follows (Eq. 1): Where y is the concentration of γ-oryzanol in the rice samples (expressed as mg/100g dry sample), is the concentration of γ-oryzanol in 4 mL extract, and is the concentration of γ-oryzanol in 8 mL extract.

Analysis of fatty acid composition
Lipid extraction applied the method of Bligh and Dyer (1959) with a modi cation (Munarko et al. 2020). Sample (5.0 g) was mixed with distilled water, chloroform, and methanol to reach chloroform:methanol:water became 1:2: followed by centrifugation at 4000 rpm (Hermle Z 383 K, Wehingen, Germany) for 10 minutes. The sample was added chloroform and water containing 0.85% of KCl to reach the nal ratio of 2:2:1.8 of chloroform:methanol:water (v/v/v). The mixture was centrifuged at 4000 rpm (Hermle Z 383 K, Wehingen, Germany) for 10 minutes and then ltered to remove the solid. The supernatant was allowed to separate into two phases. The lower chloroform phase was collected and evaporated using nitrogen gas at 50 °C.
Fatty acid derivatization was prepared by using the BF 3

Analysis of pastingpro le
Pasting pro les of brown rice our were carried out duplicate by using a rapid visco analyzer (RVA) (Tec-Master, Newport Scienti c, Australia). Sample (3.0 g, moisture content 14%) was diluted in 25 g of aquadest, followed by heating and cooling cycle with constant stirring. The sample was heated at 50 °C for 1 min in advance of heating to 95°C at 6 °C/min and maintained for 5 min. The temperature reduced to 50 °C at 6 °C/min and then held at 50 °C for 5 min.

Statistical analysis
Analysis of the variance and signi cance of the differences among the samples were conducted by analysis of variance (ANOVA) procedure and Duncan's multiple range test of SPSS software version 22.

Results And Discussion
The effect of germination on GABA content The changes in GABA content during germination are presented in Fig. 1. GABA contents in brown rice relatively low, ranging from 19-27 mg/100 g. The accumulation of GABA increased signi cantly during germination. GABA content in GBR var. Inpari 42, Inpari 43, and IPB 3S increased after soaked for 24 h. However, decreasing in GABA content was The increase of GABA during germination was closely related to the activation of some enzymes that converts glutamate to succinate via GABA, called GABA shunt. The rst step applies the direct and irreversible α-decarboxylation of glutamate by glutamate decarboxylase (GAD enzyme). The second enzyme is GABA transaminase (GABA-T) which catalyzes the reversible conversion of GABA to succinic semialdehyde using either pyruvate or α-ketoglutarate as amino receptors. The last step of the GABA shunt is catalyzed by succinic semialdehyde dehydrogenase (SSADH), which is irreversible oxidizing succinic semialdehyde to succinate (Shelp et al. 1999). Increasing GABA content was related to the increasing activity of the GAD enzyme. According to Zhang et al. (2014), GAD enzyme activity increased during germination in brown rice var. Guichao 2 and Jing 305. At the longer germination time, GAD activity decreased and followed by decreasing GABA content. In the case of GABA content in var. Inpara 3 from our research, the decrease of GABA content after 96 h and 120 h germination might be related to the decrease of GAD activity; thus the rate of GABA production was lower than the conversion of GABA to succinate.
The effect of germination on total phenolic content and antioxidant capacity The TPC of red non-waxy rice (red Jasmine) variety decreased signi cantly, although the DPPH scavenging activity did not change after germination. In contrast, black waxy rice and white non-waxy rice (KDML105) increased both of total phenolic content and the antioxidant activity. From this study, it is important to pay attention that the technological properties applied in the germination process might affect the accumulation of TPC and antioxidant activity in GBR.
The effect of germination on γ-oryzanol content The γ-oryzanol is a bioactive compound in the lipophilic fraction that be the major substance found in brown rice. It consists of ten or more compounds with ester bonds between ferulic acid and triterpenes (Cho and Lim 2016). γoryzanol content in brown rice during germination is shown in Table 1. The highest γ-oryzanol content of ungerminated brown rice was found in var. Situ Bagendit (57.62 ± 2.72 mg/100 g) and var. Inpara 3 (57.40 ± 2.12 mg/100 g). The γ-oryzanol content from var. Inpari 42 and var. Inpari 17 did not change after soaking for 24 h, but they decreased after the longer soaking time. In var. IPB 3S, the γ-oryzanol content did not change notably during 48 h of germination. It decreased slightly at 72 and 96 h and then increased up to the highest level after germination for 120 h. Moreover, the γ-oryzanol content in Inpari 43 slightly increased after soaking for 24 h; even it decreased afterward. Fatty acid pro les of germinated brown rice The fatty acid composition was analyzed for ungerminated brown rice and selected GBR at 120 h germination ( Table 2). The germination process did not affect the composition of fatty acids as indicated by the identical fatty acids compositions in ungerminated brown rice and GBR. Both in ungerminated brown rice and GBR 120 h, the composition of fatty acids was dominated by palmitic acid (C16:0), oleic acid (C18:1 cis), and linoleic acid (C18:2), which contributed to 17-19%, 33-36%, and 37-41% of the fatty acid composition, respectively. The remaining minor compounds of fatty acids consisted of myristic (C14:0), palmitoleic (C16:1), stearic (C18:0), linolenic (C18:3), arachidic acid (C20:0), cis-11 eicosenoic (C20:1), docosanoic acid (C22:0). Based on its saturation, both ungerminated brown rice and GBR were dominated by unsaturated fatty acids that contributed to 77-79% of total fatty acid content, whereas saturated fatty acids only contributed approximately 21-23%. These results were comparable to the study of Jayadeep and Malleshi (2011) in brown rice var. IR 64 and BPT that reported no obvious changes in fatty acid compositions after germination. Note: BR = Brown rice; GBR = Germinated brown rice (at 120 h); SFA = Saturated fatty acids; USFA = Unsaturated fatty acids; TFA = Total fatty acids; All numbers are presented in % (mg/100 mg total fatty acid)

List of Figures
The effect of germination on pasting pro les of germinated brown rice The pasting pro les of six Indonesian brown rice varieties during germination was presented in Fig. 3. The germination process signi cantly modi ed the pasting pro les of brown rice for all rice varieties. In this study, germination of brown rice by the soaking method considerably impact the reduction of peak viscosity, trough viscosity, breakdown, setback, and nal viscosity. The lowered peak viscosity occurred especially after soaking for 48 h or longer.
The pasting behavior of food materials during the heating and cooling process in uences the quality of nal the products (Xu et al. 2012). The GBR in all varieties experienced the decline of peak viscosity, trough viscosity, breakdown, setback, and nal viscosity. The decrease of peak viscosity was attributed to the presence of endogenous hydrolytic enzymes activity such as amylase enzyme, which hydrolyzed starch to smaller molecules (Wichamanee and Teerarat 2012; Wu et al. 2013). It is well documented in the previous study that α-amylase, as well as β-amylase enzymes, increased as germination progressing, thus, leading to a decrease in peak viscosity (Mohan et al. 2010;Pinkaew et al. 2016). Germination also reduced trough and breakdown viscosity which related to the stability of the our during the heating process (Mohan et al. 2010). After heating at a particular time, the pasting our was then cooled down to obtain the nal viscosity and setback value. The setback and nal viscosity usually represent the retrogradation tendency. In this study, the reduction of setback value indicates that GBR is more stable against retrogradation (Yuliana and Akhbar 2020).

Conclusion
The germination process from six Indonesian rice varieties by a simple soaking method, was found to signi cantly increase the GABA content of all Indonesian varieties used in this study. The highest GABA content was obtained in GBR var. Inpari 43 which increased consistently up to 4.7-fold after soaking 120 h. The lowest trend of GABA content found in var. In part 3 that increased 1.6-fold after 72 h soaking. TPC, antioxidant activity, and γ-oryzanol contents did not increase after germination, as well as fatty acid compositions. Germination in uenced the pasting pro les of GBR. The longer period of soaking time caused the lower peak viscosity, breakdown, and setback value. Based on this, GBR was less viscous and more stable during heating and cooling. Availability of data and materials All data generated or analysed during this study are included in this published article.