Effect of Pre-Germination Time on Amino Acid Profile and Gamma Amino Butyric Acid (GABA) Contents in Different Varieties of Malaysian Brown Rice

Abstract

Eighteen varieties of Malaysian brown rice were evaluated for their crude protein, total glutamic acid, and gamma amino butyric acid contents after pre-germination at different times. The crude protein and total glutamic acid content increased significantly in all the varieties after pre-germination. Gamma amino butyric acid content increased dramatically with time during the pre-germination process. A significant (p < 0.05) positive correlation was observed between protein, glutamic acid, and gamma amino butyric acid contents before and after pre-germination. The brown rice varieties containing higher glutamic acid and/or protein content before the pre-germination process provided more gamma amino butyric acid content during pre-germination.

Keywords:

• Brown rice
• Unpolished rice
• Pre-Germination
• Functional compounds
• γ-Oryzanol

INTRODUCTION

Rice (Oryza sativa L.) is one of the most important crops for more than a third of the world's population in addition to wheat and corn. Rice is planted on about 148 million hectares annually, or on 11% of the world's cultivated land. World production of rice has risen steadily from about 200 million tons of paddy rice in 1960 to 645 million tons in 2007. Rice accounts for over 22% of global energy intake. More than 90% of the world's rice is grown and consumed in Asia where 60% of the earth's people live.

Brown rice (BR), or “hulled rice,” is un-milled or partly milled rice produced by removing its external hard husk. BR grains contain more nutritional components, such as dietary fibers, phytic acids, gamma oryzanol, and vitamins B and E, than the ordinary milled rice grains.[1] Furthermore, BR contains an efficient functional compound called gamma amino butyric acid (GABA). These bio-functional components are commonly found in the germ and bran layers mainly removed by the refining process.[2]

GABA is a well-known non-protein-based amino acid that is widely distributed in both animals and plants namely with a number of important biological properties. It is one of the major inhibitory neurotransmitters in the central nervous system found in several peripheral tissues. GABA is primarily synthesized by the decarboxylation of L-glutamic acid in the presence of glutamate decarboxylase (GAD). The alteration of GABA and GAD levels in the brain was found to result in many neurological disorders, such as seizures, Parkinson's disease, stiff-man syndrome, and schizophrenia.[3] In addition, medical studies have proven that GABA has a remarkable effect in curing various diseases. It was reported to reduce blood pressure by intravenous administration in experimental animals[4,5] and in human subjects.[6] GABA has also been proven to have potential as a therapeutic agent against the renal damage involved in acute renal failure,[7] have an anti-ulcer effect due to inhibit the pepsin secretion,[8] and affect the cardiovascular diseases and diabetes by controlling the postprandial blood glucose concentration without increasing the insulin secretion.[9] Recently, the interest in the utilization of GABA as a nutritional compound has enormously increased. With all the health-giving benefits of GABA, various processes, such as fermentation, enzymatic treatment, gaseous treatment, and (pre) germination, have been suggested by previous researchers to enhance the GABA concentration.10–14

The term “pre-germination” is often used to describe the soaking, or steeping process, of cereal in water. Pre-germination is one the most common methods to enhance GABA and other nutrients, such as dietary fiber and gamma oryzanol, through endogenous enzyme activation in BR.[13] The pre-germination treatment of rice results in chemical alteration of molecules stored in both the embryo and endosperm into novel substances. The number of PGBR-containing products is increasing in the Asian food market because they contain higher amounts of GABA and other nutritional compounds than the ordinary polished rice-containing products.[15]

Although GABA is not found in protein structure, glutamic acid as a main precursor of GABA is one of 20 proteinogenic amino acids. Plant-based proteins are very rich in terms of glutamic acid, thus explaining why the protein concentration level of plants could be attributed to high glutamic acid content and probably high GABA concentration. According to the importance of GABA as a bio-functional component and difficulty of measuring GABA and glutamic acid levels in BR and PGBR by using HPLC, finding an alternative indicator method for GABA analysis seems to be essential. Therefore, main hypotheses of the present study were to prove that (i) brown rice varieties containing higher protein and/or glutamic acid concentration levels have higher GABA content in non-germinated brown rice, and consequently, (ii) they produce higher GABA content after pre-germination compared to those varieties containing medium and low concentration levels of protein and/or glutamic acid.

MATERIALS AND METHODS

Reagents and Standards

Acetonitrile, methanol (HPLC grade), sulphuric acid (nitrogen-free), sodium hypochlorite, sodium hydroxide, hydrochloric acid (37%), and ammonium acetate were provided by Merck (Darmstadt, Germany). L-α-amino-n-butyric acid, triethylamine, phenylisothiocyanate (PITC), amino acid standard AAS18, and GABA standard were purchased from Sigma-Aldrich (St. Louis, MO, USA). Glacial acetic acid (≥99.7%) was supplied by Fisher (Leics, UK).

Samples

Thirty-five Malaysian BR varieties cultivated by the Malaysian Agriculture Research and Development Institute were used as our samples in this study. Samples were harvested in 2005 and stored in a refrigerator at 4°C for six months. Cleaned paddy rice was de-husked by using a Satake rice machine (Satake Engineering Co. LTD, Tokyo, Japan), then the husked BR was ground into rice flour and subsequently sieved through a 355-μm screen for analytical use.

Pre-Germination

Pre-germination of BR was carried out as described in the previous study.[12] Selected varieties were sterilized in 0.1% sodium hypochlorite solution for 30 min, and then washed with distilled water. Subsequently, 1 kg of each variety was soaked in 30 L of double-distilled water at 30°C. Soaking water was replaced with fresh double-distilled water every 12 h to avoid any possible fermentation. Sampling was performed after 24, 48, 72, and 96 h of the soaking process. PGBR seeds were air dried, ground into flour by a mortar and a pestle, and subsequently sieved through a 355-μm screen for further analysis.

Crude Protein Analysis

The crude protein content of 18 BR varieties were measured according to the AOAC (1955) official method, and the protein content was calculated by multiplying a conversion factor of 5.95.[13]

Quantitative Analysis of GABA and Total Amino Acids

The total amino acid and GABA contents were determined by using a reversed-phase high performance liquid chromatography.[16] Non-germinated and air-dried pre-germinated BR seeds were weighed (the weight was equivalent to 4% of protein) and hydrolyzed by adding 15 ml 6N HCl to the sample and mixing well in a stoppered test tube for 24 h at 110°C. Hydrolysates were analyzed by the HPLC gradient system with the precolumn PITC derivatization. Subsequently, 20 μL of the sample was injected into the HPLC system equipped with a HPLC photodiode array detector (model MD-2010; JASCO, Tokyo, Japan). The linear gradient system was used with buffer A (0.1 M ammonium acetate, pH 6.5) and buffer B (0.1 M ammonium acetate containing acetonitrile and methanol, 44:46:10, v/v, pH 6.5) at a flow-rate of 1 ml/min, by using a C18 reversed phase column (Thermal C18 5U, 250 × 4.6 mm) in an oven at 43°C. The UV absorption detection at a wavelength of 254 nm was employed to measure the total content of amino acids. The results were analyzed by using the Borwin chromatography software (Version 1.5, Jasco Co. Ltd., Japan). Protein, glutamic acid, and GABA contents of all 18 varieties were analyzed before pre-germination and also after 24, 48, 72, and 96 h of the pre-germination procedure. Besides the glutamic acid contents, the amino acid profile of all selected varieties was measured.

Statistical Analysis

Data were expressed as the mean ± SD in triplicate. The data obtained from the measurements were subjected to univariate analysis of variance and least significant difference tests (LSD) to determine the significant differences among the samples in terms of GABA, crude protein, and total glutamic acid contents. Significant differences among mean values were determined by Tukey's test significance defined at p < 0.05. Pearson's correlation coefficient (r ²) was applied to investigate the possible correlations between GABA, glutamic acid, and protein contents. The data analysis was performed using the Minitab vs. 14 statistical package (Minitab Inc., PA, USA).

RESULTS AND DISCUSSION

After measuring the GABA contents, 35 varieties were blocked based on their GABA concentrations into three groups (high, medium, and low). Six varieties from each group were randomly selected for pre-germination namely high GABA rice (HGR, 0.08–0.11 mg/g), medium GABA rice (MGR, 0.04–0.07 mg/g), and low GABA rice (LGR, 0–0.03 mg/g) varieties.

Protein, Amino Acid Profile, and GABA Contents in Non-Germinated Seeds

The results showed a wide variation for most of the amino acids among the selected non-germinated varieties. As shown in Table 1, the concentration ranges for different amino acids widely differ depending on the BR variety. The present study was in agreement with Reddy and Sekhar, ^[17] who pointed out an extensive variation range for most of amino acids in rice varieties; while other researchers ^[18] relatively found low variation of the amino acid content in milled rice. In this study, the variation range was more highlighted with leucine and phenylalanine contents. As compared to the USDA Standard Reference, ^[19] the inferior, comparable, and superior concentration values of total amino acids were observed depending on the BR variety. This study revealed that there was a significant (p < 0.05) variation in GABA content of various non-germinated BR varieties (0.01–0.1 mg/g). MRQ74 had the highest GABA content (0.10 mg/g) among all the varieties, while MR232 showed the least GABA concentration (0.01 mg/g), thus, indicating a ten times difference between the highest and the least GABA contents. Kihara et al.[20] also found relatively large differences among GABA contents of 43 barley varieties. This finding was in agreement with that reported by Saikusa et al.^[14] They reported considerable differences in GABA concentrations of the BR germs. This could be explained by the genetic variations of different brown rice varieties.

Table 1 Amino acid profile of non-germinated brown rice varieties (mg/g of dry wt ± S.D.).^a

Variety	Asp	Ser	Gly	His	Arg	Thr	Ala	Pro
MRQ 74	9.0 ± 0.2	3.3 ± 0.2	4.2 ± 0.3	0.8 ± 0.0	4.6 ± 0.2	2.5 ± 0.4	5.8 ± 0.3	3.8 ± 0.2
Sekencang	8.1 ± 0.2	3.6 ± 0.4	3.9 ± 0.1	1.3 ± 0.1	4.4 ± 0.1	2.3 ± 0.1	4.9 ± 0.5	3.6 ± 0.2
Kadaria	8.3 ± 0.3	4.7 ± 0.1	4.3 ± 0.1	1.6 ± 0.1	4.6 ± 0.2	2.1 ± 0.1	4.6 ± 0.3	3.8 ± 0.3
MR 220	7.9 ± 0.5	4.4 ± 0.2	4.1 ± 0.1	1.0 ± 0.1	4.6 ± 0.1	2.0 ± 0.1	4.8 ± 0.1	3.6 ± 0.3
MR 123	7.7 ± 0.4	4.7 ± 0.2	4.4 ± 0.3	1.6 ± 0.1	5.2 ± 0.1	2.8 ± 0.3	4.8 ± 0.1	3.9 ± 0.3
MR 219	7.6 ± 0.2	3.6 ± 0.4	3.7 ± 0.2	1.5 ± 0.2	4.7 ± 0.3	2.1 ± 0.1	4.8 ± 0.4	3.5 ± 0.3
Pulut Siding	6.6 ± 0.2	3.9 ± 0.2	3.5 ± 0.2	1.4 ± 0.2	4.2 ± 0.1	2.3 ± 0.2	4.2 ± 0.3	3.2 ± 0.1
Pulut Hitam 9	6.8 ± 0.2	4.4 ± 0.2	3.8 ± 0.5	1.6 ± 0.1	4.8 ± 0.6	2.5 ± 0.3	4.4 ± 0.2	3.5 ± 0.2
MRQ50	7.0 ± 0.2	4.1 ± 0.1	3.8 ± 0.1	1.3 ± 0.2	4.3 ± 0.0	2.2 ± 0.2	4.4 ± 0.0	3.3 ± 0.1
Manik	6.6 ± 0.5	3.1 ± 0.1	3.2 ± 0.1	1.1 ± 0.1	3.8 ± 0.1	2.1 ± 0.1	3.8 ± 0.2	3.1 ± 0.3
MR 84	7.2 ± 0.1	3.7 ± 0.1	4.1 ± 0.1	0.9 ± 0.0	4.4 ± 0.3	2.8 ± 0.2	4.7 ± 0.2	3.6 ± 0.1
MR 159	5.8 ± 0.6	3.5 ± 0.2	3.4 ± 0.2	1.2 ± 0.1	3.9 ± 0.4	2.4 ± 0.2	3.8 ± 0.1	3.2 ± 0.1
Sekembang	6.0 ± 0.4	2.7 ± 0.2	2.8 ± 0.6	0.8 ± 0.0	3.4 ± 0.3	1.4 ± 0.0	3.5 ± 0.1	2.8 ± 0.2
Makmur	5.6 ± 0.2	3.0 ± 0.1	3.0 ± 0.1	0.9 ± 0.1	3.5 ± 0.1	1.7 ± 0.2	3.5 ± 0.1	2.7 ± 0.3
MR 103	6.5 ± 0.2	3.1 ± 0.1	3.1 ± 0.2	1.1 ± 0.1	3.7 ± 0.2	1.8 ± 0.1	3.8 ± 0.4	3.0 ± 0.1
MR 232	8.0 ± 0.3	3.4 ± 0.3	3.2 ± 0.1	1.6 ± 0.1	3.9 ± 0.2	2.1 ± 0.1	5.1 ± 0.1	3.3 ± 0.1
Bahagia	7.3 ± 0.2	3.5 ± 0.2	3.4 ± 0.2	1.4 ± 0.4	4.1 ± 0.1	2.1 ± 0.1	4.3 ± 0.2	3.4 ± 0.1
Malinja	6.9 ± 0.2	5.8 ± 0.4	3.2 ± 0.2	1.2 ± 0.3	3.9 ± 0.3	1.7 ± 0.1	4.2 ± 0.4	3.2 ± 0.3
MRQ 74	1.7 ± 0.1	2.5 ± 0.2	1.7 ± 0.3	3.7 ± 0.1	11.4 ± 0.1	1.3 ± 0.1	2.6 ± 0.1
Sekencang	1.3 ± 0.1	2.3 ± 0.1	1.5 ± 0.3	3.8 ± 0.1	7.4 ± 0.2	1.4 ± 0.1	2.5 ± 0.1
Kadaria	1.5 ± 0.1	2.2 ± 0.2	1.1 ± 0.1	3.9 ± 0.1	6.5 ± 0.1	1.4 ± 0.1	2.4 ± 0.2
MR 220	2.1 ± 0.1	2.6 ± 0.1	1.8 ± 0.4	3.5 ± 0.1	10.0 ± 0.2	1.9 ± 0.1	2.6 ± 0.1
MR 123	2.2 ± 0.0	2.3 ± 0.2	1.7 ± 0.3	3.6 ± 0.1	12.3 ± 0.1	4.0 ± 0.1	2.6 ± 0.1
MR 219	2.3 ± 0.1	2.4 ± 0.2	1.4 ± 0.1	3.3 ± 0.1	8.0 ± 0.2	4.5 ± 0.1	2.5 ± 0.1
Pulut Siding	1.4 ± 0.2	2.3 ± 0.2	1.3 ± 0.2	3.6 ± 0.1	4.7 ± 0.1	1.4 ± 0.1	2.0 ± 0.1
Pulut Hitam 9	2.0 ± 0.1	2.3 ± 0.1	1.4 ± 0.2	3.5 ± 0.1	12.2 ± 0.1	4.0 ± 0.2	2.4 ± 0.1
MRQ50	2.1 ± 0.1	2.0 ± 0.1	1.4 ± 0.3	3.5 ± 0.1	11.2 ± 0.1	3.4 ± 0.1	2.1 ± 0.1
Manik	1.0 ± 0.1	1.9 ± 0.1	1.1 ± 0.2	2.9 ± 0.1	5.8 ± 0.2	1.0 ± 0.1	2.0 ± 0.2
MR 84	1.4 ± 0.1	2.1 ± 0.1	1.1 ± 0.1	3.7 ± 0.1	8.1 ± 0.1	1.8 ± 0.1	2.4 ± 0.1
MR 159	1.8 ± 0.1	1.9 ± 0.2	1.2 ± 0.1	3.0 ± 0.2	7.8 ± 0.1	3.2 ± 0.1	1.9 ± 0.1
Sekembang	1.1 ± 0.1	1.9 ± 0.1	1.2 ± 0.0	3.1 ± 0.1	7.6 ± 0.1	1.1 ± 0.2	1.8 ± 0.2
Makmur	1.1 ± 0.2	1.6 ± 0.1	1.0 ± 0.1	2.7 ± 0.1	4.5 ± 0.1	1.4 ± 0.1	1.6 ± 0.1
MR 103	1.1 ± 0.1	1.7 ± 0.1	1.1 ± 0.2	2.9 ± 0.1	4.9 ± 0.1	2.1 ± 0.1	2.0 ± 0.1
MR 232	0.8 ± 0.1	2.3 ± 0.1	1.2 ± 0.2	3.4 ± 0.1	4.9 ± 0.1	1.2 ± 0.2	2.3 ± 0.1
Bahagia	1.0 ± 0.1	2.2 ± 0.1	1.3 ± 0.1	2.9 ± 0.1	3.2 ± 0.2	0.8 ± 0.1	2.3 ± 0.2
Malinja	1.4 ± 0.4	2.0 ± 0.5	1.3 ± 0.2	3.0 ± 0.5	3.1 ± 0.3	1.2 ± 0.2	2.1 ± 0.1
^aS.D.: standard deviation (three replicates).
Note: Protein (g/100 g).

The results also showed that there was a considerable variation (10.10–15.20 mg/g) in the total glutamic acid content among all non-germinated BR varieties. The highest and least glutamic acid contents were observed in MRQ74 and Makmur, respectively (Table 2). Protein content of different BR varieties varied from 6.99 to 10.17%. MRQ74 had the highest protein content (10.17%), whereas Makmur showed the least protein concentration level (6.99%) among all the BR varieties (Table 3).

Table 2 Glutamic acid content in brown rice varieties at different pre-germination times

		Pre-germination times (mg/g of dry wt ± S.D.)
Group	Variety	0	24	48	72	96
HGR	MRQ 74	15.2 ± 0.6^a	16.4 ± 0.5^a	19.4 ± 0.5^b	18.8 ± 0.5^b	21.3 ± 0.3^c
	Sekencang	14.1 ± 0.4^a	19.5 ± 0.3^b	15.9 ± 0.4^c	15.2 ± 0.5^ac	20.2 ± 0.5^b
	Kadaria	14.3 ± 0.2^a	19.6 ± 0.1^b	14.6 ± 0.4^a	17.2 ± 0.5^c	19.3 ± 0.3^b
	MR 220	14.8 ± 0.7^a	18.6 ± 0.6^b	16.9 ± 0.5^c	17.5 ± 0.2^bc	17.5 ± 0.3^bc
	MR 123	14.7 ± 0.3^a	20.4 ± 0.1^b	19.7 ± 0.5^b	20.1 ± 0.3 ^b	20.1 ± 0.6^b
	MR 219	13.0 ± 0.9^a	17.6 ± 0.1^b	14.6 ± 0.5^c	14.2 ± 0.5^ac	16.6 ± 0.4^b
MGR	Pulut Siding	13.2 ± 0.1^a	18.0 ± 0.1^b	16.5 ± 0.4^c	16.7 ± 0.4^c	21.0 ± 0.2^d
	Pulut Hitam 9	13.1 ± 0.1^a	17.5 ± 0.3^b	15.2 ±0.2^c	16.8 ± 0.5^b	17.6 ± 0.4^b
	MRQ50	13.9 ± 0.6^a	18.6 ± 0.2^b	13.5 ± 0.3^a	18.3 ± 0.2^b	18.3 ± 0.8^b
	Manik	11.9 ± 0.6^a	14.3 ± 0.1^b	15.8 ± 0.5^c	12.7 ± 0.5^a	15.6 ± 0.6^bc
	MR 84	13.6 ± 0.5^a	13.9 ± 0.1^a	12.0 ± 0.2^b	13.4 ± 0.4^a	17.1 ± 0.6^d
	MR 159	11.8 ± 0.4^a	15.0 ± 0.5^b	16.3 ± 0.4^c	15.3 ± 0.4^bc	16.1 ± 0.4^c
LGR	Sekembang	11.1 ± 0.6^a	16.3 ± 0.1^b	13.6 ± 0.4^c	15.8 ± 0.1^bd	14.7 ± 0.4^d
	Makmur	10.1 ± 0.1^a	15.5 ± 0.1^b	11.1 ± 0.1^c	14.8 ± 0.5^bd	16.1 ± 0.4^be
	MR 103	11.1 ± 0.0^a	15.9 ± 0.3^b	16.2 ± 0.4^b	13.3 ± 0.4^c	16.3 ± 0.6^b
	MR 232	13.6 ± 0.5^a	13.6 ± 0.1^a	12.8 ± 0.5^ab	12.0 ± 0.2^b	12.7 ± 0.4^ab
	Bahagia	13.3 ± 0.2^a	17.4 ± 0.4^b	13.4 ± 0.3^a	13.2 ± 0.4^a	17.9 ± 0.5^b
	Malinja	12.4 ± 0.4^a	17.0 ± 0.4^b	14.0 ± 0.5^c	12.4 ± 0.3^a	15.0 ± 0.4^c
^a–eLetters indicate significant difference at p < 0.05.
Note: Standard deviation (three replicates).
HGR: High GABA rice; MGR: Medium GABA rice; LGR: Low GABA rice.

Table 3 Protein content in brown rice varieties at different pre-germination times

		Pre-germination times (g/100g ± S.D.)
Group	Variety	0	24	48	72	96
HGR	MRQ 74	10.17 ± 0.2^a	11.93 ± 0.4^b	11.80 ± 0.5^b	11.03 ± 0.3^abc	12.27 ± 0.4^bd
	Sekencang	9.89 ± 0.1^a	11.58 ± 0.2^b	12.13 ± 0.5^b	11.66 ± 0.5^b	11.96 ± 0.05^b
	Kadaria	9.82 ± 0.2^a	11.71 ± 0.3^b	11.58 ± 0.3^b	12.22 ± 0.2^b	12.21 ± 0.4^b
	MR 220	8.72 ± 0.2^a	10.89 ± 0.2^b	10.41 ± 0.1^b	10.48 ± 0.0^b	10.69 ± 0.5^b
	MR 123	9.80 ± 0.3^a	12.00 ± 0.3^b	11.78 ± 0.2^b	11.96 ± 0.4^b	12.30 ± 0.5^b
	MR 219	8.55 ± 0.3^a	10.40 ± 0.2^b	10.55 ± 0.3^b	10.52 ± 0.5^b	10.13 ± 0.1^b
MGR	Pulut Siding	8.06 ± 0.2^a	9.99 ± 0.4^b	10.61 ± 0.1^b	10.21 ± 0.1^b	10.60 ± 0.35^b
	Pulut Hitam 9	8.67 ± 0.3^a	10.32 ± 0.1^b	10.82 ± 0.3^b	10.82 ± 0.5^b	11.06 ± 0.2^b
	MRQ50	8.90 ± 0.3^a	11.10 ± 0.5^b	11.17 ± 0.5^b	11.24 ± 0.2^b	10.82 ± 0.3^b
	Manik	7.65 ± 0.0^a	9.34 ± 0.4^b	9.31 ± 0.4^b	9.44 ± 0.2^b	9.04 ± 0.2^b
	MR 84	8.56 ± 0.0^a	10.27 ± 0.3^b	10.20 ± 0.5^b	10.28 ± 0.2^b	10.30 ± 0.3^b
	MR 159	7.63 ± 0.1^a	9.44 ± 0.5^b	9.12 ± 0.5^b	9.53 ± 0.3^b	9.02 ± 0.1^b
LGR	Sekembang	7.38 ± 0.0^a	9.46 ± 0.1^b	9.65 ± 0.5^b	9.99 ± 0.5^b	9.92 ± 0.4^b
	Makmur	7.00 ± 0.0^a	9.01 ± 0.4^b	9.57 ± 0.2^cb	8.60 ± 0.4^db	8.96 ± 0.2^cdb
	MR 103	7.85 ± 0.1^a	9.44 ± 0.4^b	9.57 ± 0.4^b	9.85 ± 0.2^b	9.92 ± 0.3^b
	MR 232	7.62 ± 0.2^a	8.41 ± 0.1^b	8.60 ± 0.1^b	8.33 ± 0.1^b	8.55 ± 0.3^b
	Bahagia	7.87 ± 0.3^a	9.71 ± 0.3^b	9.99 ± 0.3^b	9.88 ± 0.2^b	10.02 ± 0.3^b
	Malinja	8.02 ± 0.4^a	10.21 ± 0.4^b	9.38 ± 0.5^b	9.72 ± 0.5^b	9.98 ± 0.2^b
^a–dLetters indicate significant difference at p < 0.05.
Note: Standard deviation (three replicates).
HGR: High GABA rice; MGR: Medium GABA rice; LGR: Low GABA rice.

The results indicated that GABA had a significant (p < 0.05) positive correlation with the glutamic acid (r ² = 0.578) and protein (r ² = 0.704) contents. As illustrated by Oh and Choi,[21] glutamic acid as a precursor of GABA is one of the most abundant amino acids in BR. The results of this study also demonstrated a significant (p < 0.05) positive correlation between glutamic acid and protein (r ² = 0.837). Besides the non-germinated BR, the results also showed a wide variation for most of the amino acids of BR varieties at different pre-germination times (24, 48, 72, and 96 h).

Protein, Amino Acid Profile, and GABA Contents after 24, 48, 72, and 96 Hour of Pre-Germination

In most cases, the content of most of the amino acids increased after 24 h of pre-germination. After 24 h of pre-germination, the concentration range of different amino acids were found to be as follows: aspartic acid (7.1 to 10 mg/g), serine (3.1 to 5.1 mg/g), glycine (3 to 4.7 mg/g), histidine (1 to 1.9 mg/g), arginine (4.1 to 6.3 mg/g), threonine (3.6 to 6 mg/g), alanine (4.2 to 6.9 mg/g), proline (3.2 to 4.8 mg/g), tyrosine (1 to 3.1 mg/g), valine (2.2 to 6.3 mg/g), methionine (1.2 to 2.2 mg/g), isoleucine (2.3 to 3.8 mg/g), leucine (7.5 to 15.6 mg/g), phenylalanine (2.1 to 3.7 mg/g), and lysine (2.1 to 3.4 mg/g).

This study revealed that there was a significant (p < 0.05) variation in GABA content of various 24 h PGBR varieties (0.02 to 0.2 mg/g). MRQ74 had the highest GABA content (0.2 mg/g) among all the varieties, while MR232 and Bahagia showed the least GABA concentration (0.02 mg/g) (Table 4). The results also showed that there was a considerable variation (13.6 to 20.4 mg/g) in the total glutamic acid content among all 24-h PGBR varieties. The least and highest glutamic acid contents were observed in MR232 and MR123, respectively (Table 3). Protein content of different BR varieties ranged from 8.41 to 12%. MR123 and MR232 had the highest (12%) and least (8.41%) protein content among all the varieties after 24 h of pre-germination (Table 3). The results also showed that GABA contents had a significant (p < 0.05) positive correlation with total glutamic acid (r ² = 0.426) and protein contents (r ² = 0.746) in the 24-h pre-germinated samples. Results also indicated that glutamic acid and protein contents had a significant (p < 0.05) positive correlation (r ² = 0.770).

Table 4 GABA content in brown rice varieties at different pre-germination times

		Pre-germination times (mg/g of dry wt ± S.D.)
Group	Variety	0	24	48	72	96
HGR	MRQ 74	0.10 ± 0.04^a	0.20 ± 0.02^b	0.44 ± 0.03^c	0.94 ± 0.04^d	1.81 ± 0.06^e
	Sekencang	0.08 ± 0.01^a	0.13 ± 0.02^a	0.24 ± 0.04^b	0.57 ± 0.05^c	1.00 ± 0.04^d
	Kadaria	0.05 ± 0.01^a	0.07 ± 0.03^a	0.10 ± 0.04^a	0.28 ± 0.07^b	0.60 ± 0.05^c
	MR 220	0.10 ± 0.03^a	0.15 ± 0.03^a	0.29 ± 0.05^b	0.79 ± 0.04^c	1.18 ± 0.04^d
	MR 123	0.09 ± 0.01^a	0.18 ± 0.04^b	0.30 ± 0.04^c	0.64 ± 0.03^d	1.14 ± 0.02^e
	MR 219	0.08 ± 0.01^a	0.12 ± 0.02^a	0.22 ± 0.03^b	0.62 ± 0.05^c	1.20 ± 0.04^d
MGR	Pulut Siding	0.03 ± 0.01^a	0.05 ± 0.03^a	0.11 ± 0.03^a	0.20 ± 0.05^b	0.35 ± 0.04^c
	Pulut Hitam 9	0.06 ± 0.02^a	0.12 ± 0.03^ab	0.20 ± 0.04^b	0.55 ± 0.04^c	0.84 ± 0.04^d
	MRQ50	0.09 ± 0.01^a	0.16 ± 0.04^ab	0.27 ± 0.05^b	0.64 ± 0.05^c	0.97 ± 0.06^d
	Manik	0.05 ± 0.02^a	0.09 ± 0.04^a	0.24 ± 0.05^b	0.50 ± 0.04^c	0.75 ± 0.02^d
	MR 84	0.05 ± 0.01^a	0.10 ± 0.03^a	0.21 ± 0.06^b	0.42 ± 0.05^c	0.80 ± 0.04^d
	MR 159	0.04 ± 0.02^a	0.07 ± 0.02^ab	0.15 ± 0.04^b	0.36 ± 0.04^c	0.66 ± 0.06^d
LGR	Sekembang	0.06 ± 0.02^a	0.11 ± 0.03^a	0.21 ± 0.05^b	0.54 ± 0.04^c	0.85 ± 0.04^d
	Makmur	0.03 ± 0.01^a	0.05 ± 0.02^a	0.09 ± 0.04^a	0.22 ± 0.04^b	0.43 ± 0.04^c
	MR 103	0.02 ± 0.01^a	0.03 ± 0.02^a	0.07 ± 0.02^a	0.20 ± 0.06^b	0.36 ± 0.03^c
	MR 232	0.01 ± 0.00^a	0.02 ± 0.02^a	0.03 ± 0.02^a	0.08 ± 0.03^a	0.17 ± 0.04^b
	Bahagia	0.02 ± 0.01^a	0.02 ± 0.01^a	0.04 ± 0.01^a	0.09 ± 0.02^b	0.14 ± 0.01^c
	Malinja	0.02 ± 0.01^a	0.03 ± 0.02^a	0.06 ± 0.01^a	0.11 ± 0.01^b	0.18 ± 0.02^c
Note: Standard deviation (three replicates).
^a–eLetters indicate significant difference at p < 0.05.
HGR: High GABA rice; MGR: Medium GABA rice; LGR: Low GABA rice.

The concentration ranges of target amino acids, namely aspartic acid (5.6 to 10.3 mg/g), serine (2.5 to 4.8 mg/g), glycine (2.6 to 4.5 mg/g), histidine (0.6 to 1.9 mg/g), arginine (4 to 5.7 mg/g), threonine (2.2 to 6.1 mg/g), alanine (3.8 to 6.1 mg/g), proline (2.9 to 4.7 mg/g), tyrosine (0.9 to 3.3 mg/g), valine (1.7 to 6.5 mg/g), methionine (1 to 2 mg/g), isoleucine (1.8 to 4.1 mg/g), leucine (6.2 to 15.7 mg/g), phenylalanine (0.7 to 4 mg/g), and lysine (1.13 to 3.3 mg/g) were observed after 48 h of pre-germination.

This study revealed that there was a significant (p < 0.05) variation in GABA content of various BR varieties (0.03 to 0.44 mg/g). MRQ74 had the highest GABA content (0.44 mg/g) among all the varieties; while MR232 showed the least GABA concentration (0.03 mg/g) (Table 4). The results also showed that there was a considerable variation (11.13 to 19.7 mg/g) in the total glutamic acid content among all BR varieties. The least and highest glutamic acid contents were observed in Makmur and MR123, respectively (Table 3). The protein concentration level of different varieties varied from 8.6 to 12.13%. Sekencang had the highest protein content (12.13%), whereas MR232 showed the least protein concentration level (8.6%) among all the varieties pre-germinated for 48 h (Table 4). A significant (p < 0.05) positive correlation (r ² = 0.595) was also found between GABA and total glutamic acid. Furthermore, the significant (p < 0.05) positive correlation (r ² = 0.609) was observed between GABA and protein contents of the 48-h pre-germinated varieties. Results also indicated that the glutamic acid and protein contents had a significant (p < 0.05) positive correlation (r ² = 0.498).

The concentration ranges of target amino acids, including aspartic acid (6.3 to 10.4 mg/g), serine (2.6 to 5.3 mg/g), glycine (2.8 to 4.7 mg/g), histidine (0.7 to 2 mg/g), arginine (4.3 to 6.4 mg/g), threonine (2.7 to 6.5 mg/g), alanine (3.8 to 6.6 mg/g), proline (3 to 4.9 mg/g), tyrosine (0.3 to 3.3 mg/g), valine (1.7 to 6.2 mg/g), methionine (1 to 1.9 mg/g), isoleucine (0.8 to 3.9 mg/g), leucine (8.7 to 16.3 mg/g), phenylalanine (0.5 to 4.3 mg/g), and lysine (1.5 to 3.5 mg/g), were observed after 72 h of pre-germination.

The result showed that there was a significant (p < 0.05) variation in GABA content of various BR varieties (0.08 to 0.94 mg/g). MRQ74 had the highest GABA content (0.94 mg/g) among all the varieties, while MR232 showed the least GABA concentration (0.08 mg/g) (Table 4). The results also showed that there was a considerable variation (12 to 20.1 mg/g) in the total glutamic acid content among all BR varieties. The least and highest glutamic acid contents were observed in MR232 and MR123, respectively (Table 2). Protein content of different BR varieties ranged from 8.33 to 12.22%. Kadaria had the highest protein content (12.22%), whereas MR232 had the least protein level (8.33%) among all the varieties after 48 h of pre-germination (Table 3). The results also showed that GABA contents had a significant (p < 0.05) positive correlation with total glutamic acid (r ² = 0.667) and protein contents (r ² = 0.540) after 72 h of pre-germination. The glutamic acid and protein contents were also significantly (p < 0.05) correlated (r ² = 0.714).

In the 96-h pre-germinated BR seeds, the increase in most of the amino acids observed compared to 72-h pre-germinated seeds. The subsequent changes in the concentration level of target amino acids were found to be as follows: aspartic acid (6.3 to 13 mg/g), serine (3.3 to 6.3 mg/g), glycine (3.2 to 6.2 mg/g), histidine (1.3 to 2.6 mg/g), arginine (5 to 6.3 mg/g), threonine (4.3 to 6.6 mg/g), alanine (4.1 to 8.8 mg/g), proline (3.2 to 6.4 mg/g), tyrosine (1.5 to 4.3 mg/g), valine (4.3 to 8.8 mg/g), methionine (1.1 to 2.7 mg/g), isoleucine (2.5 to 5.4 mg/g), leucine (9.8 to 18 mg/g), phenylalanine (1.3 to 5.5 mg/g), and lysine (2.2 to 4.5 mg/g).

This study showed that there was a significant (p < 0.05) variation in GABA content of various 96-h PGBR varieties (0.14 to 1.81 mg/g). MRQ74 had the highest GABA content (1.81 mg/g) among all the varieties, while Bahagia showed the least GABA concentration (0.14 mg/g) (Table 4). The results also showed that there was a considerable variation (12.7 to 21.3 mg/g) in the total glutamic acid content among all BR varieties. The least and highest glutamic acid contents were observed in MR232 and MRQ74, respectively (Table 2). Protein content of different varieties varied from 8.55 to 12.3%. MR123 had the highest protein content (12.3%); conversely, MR232 showed the least protein concentration (8.55%) among all 96-h pre-germinated varieties (Table 3). In 96-h pre-germinated BR varieties, GABA content was significantly (p < 0.05) correlated with the total glutamic acid (r ² = 0.493) and protein (r ² = 0.589) contents. A significant (p < 0.05) positive correlation (r ² = 0.842) was observed between glutamic acid and protein contents.

Protein, Glutamic Acid, and GABA Changes in High, Medium, and Low GABA Rice Groups (HGR, MGR, and LGR) at Different Pre-Germination Times

The GABA changes in the HGR group ranged between 0.12 to 0.2 mg/g after 24 h pre-germination, while the higher variations of GABA content were observed after 48 h (0.22–0.44 mg/g), 72 h (0.57–0.94 mg/g), and 96 h (0.97–1.81 mg/g) pre-germination, thus, ensuring the significant (p < 0.05) positive effect of pre-germination time on GABA accumulation. After 72 h of pre-germination, GABA contents were 7 to 9.5 times higher compared to non-germinated varieties classified in the HGR group (Table 4). The range of GABA changes in the MGR group was 0.07 to 0.12 mg/g after 24 h, 0.10 to 0.24 mg/g after 48 h, 0.28 to 0.55 mg/g after 72 h, and 0.60 to 0.85 mg/g after 96 h of pre-germination. After 72 h of pre-germination, GABA contents were 5.5 to 9 times higher compared to non-germinated brown rice seeds in the MGR group.

The GABA contents of LGR brown rice varieties ranged from 0.02 to 0.05 mg/g after 24 h. The subsequent ranges of GABA contents after 48, 72, and 96 h pre-germination processes were found to be 0.03 to 0.11 mg/g, 0.08 to 0.22 mg/g, and 0.14 to 0.43 mg/g, respectively. As clearly shown in Table 4, GABA contents of 72-h pre-germination samples were 7- to 8-fold higher compared to non-germinated BR seeds in LGR group. The quantity of GABA accumulation in BR varieties was observed in order of the initial GABA content (i.e., HGR > MGR > LGR). In fact, BR varieties containing higher initial higher GABA content provided higher GABA concentration after pre-germination and vice versa.

The present study showed that GABA content significantly (p < 0.05) increased with prolonged pre-germination time in most of the varieties, especially in the HGR and MGR groups. This finding was in agreement with the Ohtsubo et al.^[13] and Saikusa et al.^[14] studies, and it was found that GABA increased dramatically after water soaking of BR varieties. They found in their study that the GABA content in BR germs progressively increased as the incubation proceeded in most of the cultivars. Compared with other data (13.6 and 16.6 mg/100 g for Japanese rice) reported by Ito and Ishikawa,^[15] GABA content of Malaysian germinated BR increased 5–6 times because of the longer soaking time. Previous researchers indicated that the GABA content of germinated BR was almost ten times higher than the GABA concentration of milled rice,^[13,22] and found that GABA content after 72 h was 11.5 times higher than GABA concentration of non-germinated BR. The results of the present study also revealed that GABA concentration of HGR, MGR, and LGR varieties after 72 h of pre-germination followed a wide range between 4.5 to 10 times higher than GABA concentration of non-germinated BR seeds. The amount of GABA accumulated in BR varieties during water soaking varied enormously depending on the cultivar. For instance, the GABA production of MRQ74 was 0.20 mg/g after 24 h, 0.44 mg/g after 48 h, 0.94 mg/g after 72 h, and 1.81 mg/g after 96 h of pre-germination. Ohtsubo et al.^[13] also reported that the GABA content increased during germination, showing 0.11 mg/g after 24 h, 0.27 mg/g after 48 h, 0.69 mg/g after 72 h, and 1.49 mg/g after 96 h.

Protein content significantly (p < 0.05) increased in all the varieties after 24 h of pre-germination (Table 3). In all the varieties, protein content did not change after 48, 72, and 96 h of pre-germination except for two varieties (MRQ74 and Makmur). In MRQ74, the protein level decreased after 72 h; while it significantly (p < 0.05) increased after 96 h. In Makmur, protein content increased after 48 h of pre-germination. Conversely, it significantly (p < 0.05) decreased after 72 h of pre-germination, thus indicating the significant (p < 0.05) effect of BR variety and pre-germination time on protein content. These data clearly showed the protein biosynthesis during water soaking. Mostafa et al.[23] reported a noticeable increase in total protein with prolonging the germination time. Previous researchers also observed an increase in the total and protein nitrogen contents during germination.[24] It is presumed that the cytoplasmic components essential for protein synthesis, such as tRNAs, amino acids, and enzymes (aminoacyl-tRNA synthetases), exist in the dry seed in sufficient quantities, thereby inducing the resumption of protein synthesis in the seed upon imbibitions.[25] The results also showed that GABA contents were higher in varieties with high protein content compared to those with medium and low ones. In other words, high GABA and protein contents were observed in HGR > MGR > LGR groups.

The glutamic acid contents of the non-germinated and pre-germinated samples are shown in Table 2. The results revealed that total glutamic acid significantly (p < 0.05) increased after 24 h of pre-germination in most cases except for MRQ74, MR84, and MR232. However, the results did not show a typical change in the pattern of glutamic acid with prolonging the pre-germination time. In most of the varieties (e.g., Sekencang, Kadaria, MR220, MR219, Pulut Siding, Pulut Hitam 9, Sekembang, Makmur, Bahagia, and Malinja), total glutamic acid level significantly (p < 0.05) decreased after 48 h of pre-germination, while the increase in glutamic acid content was observed after 72 h and 96 h of pre-germination compared to non-germinated samples. In Table 2, after 24 h of pre-germination, the variety MR232 was found to have the lowest concentration of glutamic acid. However, when the timing is increased, the glutamic acid concentration did not fall much, while in the case of another variety, Makmur, the fall in glutamic acid content is high. By this way, it found the timings required for the different varieties for the higher expression of the required acids. The results also showed that GABA contents of BR varieties containing high glutamic acid content were found to be higher compared to those varieties containing medium and low glutamic acid concentration. In MRQ74, the total concentration of glutamic acid enhanced with prolonging of the pre-germination time, thereby increasing GABA content.

The content of glutamic acid in rice germ is relatively high among the different grain fractions, in addition to its high GAD activity, making rice germ a good material for GABA accumulation. Komatsuzaki et al.^[12] reported that during water absorption the GAD enzyme was activated and glutamic acid was converted to GABA. Therefore, GABA content variation might be explained by the different GAD activity or glutamic acid and protein contents, which could vary with the breed and purity of the rice germ. Zhang et al.[26] selected trypsin as a protease to hydrolyze the germ protein and produce glutamic acid for GABA accumulation. They found that after the photolytic hydrolyzes of germ protein, the amount of GABA yield significantly increased. The results of this study revealed the correlation between protein and GABA content in different pre-germination times. This phenomenon might be attributed to the high activities of endogenous peptidase, which cause protein hydrolysis and subsequently increase glutamic acid concentration, the main precursor of GABA. The progressive increase in GABA content observed during water soaking indicated that the amount of glutamic acid in the BR varieties was sufficient enough for the GAD activity to exceed that of the GABA-decomposing enzymes.^[14]

CONCLUSIONS

The effect of pre-germination on GABA content clearly depended on BR variety and pre-germination times, thus reflecting the longer pre-germination time, which provided the higher GABA concentration level. Moreover, it was revealed that the GABA contents in pre-germinated samples were dependent on the initial GABA content of its source. Non-germinated varieties containing higher GABA contents produced more GABA during different pre-germination times compared to those varieties with medium or low GABA contents. The present study could be useful when BR varieties are selected based on their GABA contents for functional food consumption. The results also showed a variation between protein and glutamic acid contents in different Malaysian BR varieties. Since GABA contents of BR varieties containing high glutamic acid and protein contents were found to be higher compared to those varieties containing medium and low glutamic acid and protein concentrations, the protein and glutamic acid contents could be considered as the indicators for selection of the BR varieties with high GABA content. Considering the considerable GABA accumulation induced by simple pre-germination treatment using water soaking, the proposed treatment is recommended to provide an appropriate functional food.

ACKNOWLEDGMENTS

The authors wish to express their sincere gratitude to Dr. Boo Huey Chern for her kind cooperation. This work was fully supported by the GABA biosynthesis project, the Ministry of Education, Malaysia.