Printer Friendly


Byline: Qureshi and A.M Anwar

ABSTRACT: The purpose of this research work was to enhance the production of gamma amino butyric acid in fermented food through solid state fermentation. The rice was used as substrate. The fermentation was carried out using Rhizopus oligosporus. Steamed rice was incubated aerobically with Rhizopus oligosporus M-90 for 40 h at 35oC and then anaerobically incubated for 6 h by replacement of the atmosphere with hydrogen. It was calculated that the GABA accumulation in the aerobically fermented rice was less than anaerobically fermented rice.

Key Words: Biosynthesis, GABA, Fermented Rice, Rhizopus oligosporus


Gamma amino butyric acid (GABA) is a depressive neurotransmitter in the sympathetic nervous system [1,2]. It acts beyond inhibitory transmission and regulates inhibitory synapse development. In the central nervous system (CNS) it acts on two distinct types of receptor: an iron channel, i.e., an "ionotropic" receptor permeable to Cl and HCO3 (GABAA receptors) and a G-protein coupled "metabotropic" receptor that is linked to various effectors mechanisms (GABAB receptors). GABAA receptors play structural roles in synapse maturation and stabilization [3].

Gamma-amino butyric acid also has physiological function to depress the elevation of systolic blood pressure [4] and improve discrimination learning (Ishkawa and Saito, 1978)[5]. GABA also induces hypotensive effects, diuretic effects, treatment of epilepsy and tranquilizer effects [6,7,8]. Gamma amino butyric acid is present in a variety of daily foods such as yogurt, Koren Kimchi and a type japans pickle, "shibazuke"[9].

Gamma amino butyric acid is found in 3 phases i.e., gases, liquid and solid (Majumdar and Guha, 1988)[10]. It is found mostly as a zwitter ion, in which carboxyl group deprotonated and the amino group protonated [11]

Gamma butyric acid is biosynthesized in plants and microorganisms via dscarboxylation of glutamate which is induced by various stresses [12,13]. The neurotransmitter GABA is synthesized from glutamate by the action of glutamate decarboxylase. Glutamate decarboxylase (GAD) is the unique enzyme to catalyze the conversion of L-glutamate or its salts to GABA through the single-step a-decarboxylation [14,15].



The culture of Rhizopus oligosporous M-90 used for fermentation was procured from, Food and Biotechnology Research Center, PCSIR laboratories complex Lahore, and rice was used as substrate for fermentation, were purchased from local market.

Preparation of Inoculum

Added 2 ml of sterilized distilled water in 4 to 7 days old culture of Rhizopus oligosporous M-90. Mixed the spores properly in test tube near the flame in laminar flow hood.

Fermentation Methodology

100 gm of rice were soaked for 10 minutes. After soaking the rice were boiled in distilled water for 5 minutes. The boiled rice were inoculated with 1.0ml suspension of spore of Rhizopus oligosporous M-90 about 106/ml. The inoculated rice were packed in sterilized plastic bags and incubated at 350C under aerobic conditions for 40 hours. After aerobic cultivation the fermented rice was inocubated at 35oC in anaerobic jar which was charged with hydrogen gas for 6 hrs. The cultivation time and temperature varied.

Estimation of GABA

Estimation of GABA in the fermented rice was done by thin layer chromatography as described by Gokani, et al, [16].


(a) Effect of soaking time:

The soaking time of 10 min was found to be most suitable for the production of cake. The accumulation of GABA increases with increase in soaking time. Varanyanond, et al, [17] worked on different varieties of brown rice and observed that GABA accumulation increased with time.

Komatsuzaki, et al, [18] reported that amino acids stored in the rice as storage protein, decomposed by water absorption changed into transportable amides. Therefore, on water absorption, glutamate decarboxylase (GAD) is activated and glutamic acid is converted into GABA.

(b) Effect of Inoculum Size:

Among the various inoculums sizes used, best growth of Rhizopus oligosporous was observed with 1.0ml inoculums size as evident from better cake formation. Feng, et al; [19] worked on barley fermentation. According to them Rhizopus oligosporous was inoculated at approximately 104spores /g moist substrate, a barley cake with dense mycelia growth was obtained after 20 hours. With inoculation approximately 102 spores/ g moist barley, thus fungus grew more slowly and a barley cake with dense mycelia growth was not obtained until after 28 hous to 32 hours. When Rhizopus oligosporous was inoculated at approximately 106spores/g moist barley, the time for obtaining dense mycelia growth was shortened to 15 hrs to 20 hrs. However, the growth was uneven. Similar results have also been reported by Nout and Kiers [20] for Soybean tempe fermentation.

(c) Effect of Incubation Period

The incubation period of about 35 to 40 hours was preferred for best fungus growth. The glutamate decarboxylase (GAD) activity increases with the increase in time of incubation and then decrease after 40 hrs. Aoki, et al, [21] worked on soybean and report the similar findings.

Table 1: Production of GABA in fermented rice.

Table 1: Production of GABA in fermented rice.

Aerobic Fermentation###Aerobic###Follow up time Anaerobic Fermentation

Temp (degC)###Fermentation Time (hrs)

###Time (hrs)###GABA production (present +/absent-)


###(hrs) (hrs) (hrs) (hrs)



(d) Effect of Incubation Temperature

Among different incubation temperatures 35oC was found to be best for the maximum growth of the fungus. Steinkraus et al, [22] reported that at 30oC the mycelial growth was slow. These results are comparable with our results.


(a) Effect of Aerobic Cultivation Time:

The growth of Rhizopus oligosporous was observed in small amount after 30 hrs of aerobic cultivation. The growth increases after 40 hrs of aerobic cultivation, while the GABA amount of fermented rice remains low in level. Aoki, et al, [21] worked on soybean. The soybean was inoculated with Rhizopus oligosporous. The report showed that maximum yield of GABA was obtainedat 37oC. According to Huang, et al, [3] the temperature ranging between 25oC to 40oC give more yield of GABA and its yield decrease with further increase in temperature.

(b) Effect of Anaerobic Cultivation Time:

The GABA content in fermented rice increases with the progress in anaerobic cultivation. The GABA was detected at 35oC after 6 hrs of anaerobic cultivation of 40 hrs aerobically cultivated fermented rice (Table 1). The results are in accordance with the results reported earlier by Aoki, et al, [21]. They reported that the production of GABA increases during anaerobic cultivation.

(c) Effect of Replacement of Air (hydrogen gas) on

Accumulation of GABA:

The GABA content in fermented rice increase with decrease in oxygen concentrations. The change in the atmosphere increase the GABA content. During anaerobic fermentation the atmosphere of oxygen was replaced with hydrogen which accumulates GABA in greater yield. Aoki, et al, [21] reported that the GABA content in fermented soybean increased with the decrease of initial oxygen concentration. When the atmosphere was replaced by inert gas i.e., hydrogen or nitrogen gas, Rhizopus oligosporous accumulated a greater amount of GABA.


1. Ohkuma, S., Katsura, M., and Hirouchi, M., GABA and GABA receptors. Neurotransmitter, 19: 167-180(1997).

2. Nishikawa, T., GABAsystem. Clinical Neuroscience, 14: 404-407(1996).

3. Huang, J.Z., and Scheifele, P., GABA and neuroligin singling: linking synaptic activity and adhesion in inhibitory synapse development. Current opinion in neurobiology; 1(18): 77-83(2008).

4. Vemulapalli, S., and Barletta, M., The role of sympathetic nervous system in the cardiovascular effects of systemically administered gamma amino butyric acid. Arch. Int. Pharmacodyn., 267: 46-58(1984).

5. Ishikawa, K., and Saito, S., Effect of intraventricular gamma amino butyric acid on discrimination learning in rats. Psychopharmacology, 56: 127-132(1978).

6. Jakobs, C., Jaeken, J., and Gibson, K.M., Inherited disorders of GABA metabolism. J Inherit. Metb. DIS, 16: 704-715(1993).

7. Cohen, I., Navarro, V., and Ciemenceau, S., On the origin of interictal activity in human temporal lobe epilepsy in vitro. Science, 298: 1418-1421(2002).

8. Komatsuzaki, N., Shima, J., kawamoto, S., komose, H., and kimura, T., producion of gamma amino butyric acidby lactobacillus paracsei isolated from traditional fermented foods. Food Microbiology, 22:497-504(2005).

9. Hayakawa, K., Ueno, Y., Kawamura, S., Taniguchi, R., and Oda, K., Production of gamma amino butyric acid by lactic acid bacteria. Seibutsu Kougaku, 75: 139-244(1997).

10. Majumdar, D., and Guha, S., Conformation, electrostatic potential and pharmacophoric pattern of GABA (gamma-amino butyric acid) and several GABA inhibitors. Journal of Molecular Structure: Theochem., 180: 125-140(1988).

11. Sapse, A. M., Molecular Orbital Calculation for Amino Acids and Peptides. Birkhausr, ISBN 0817638938(2000)..

12. Kono, I., and Himeno, k., Accumulation of gamma amino butyric acid in beni-koji after anaerobic incubation. J. Brew. Soc. Japan, 97: 785-790(2000).

13. Shelp, B.J., Bown, A.W., and McLean, M.D., Metabolism and functions of gamma amino butyric acid. Trends Plant Sci., 4: 446-452(1999).

14. Ueno, H., Enzymatic and structural aspects on glutamate decarboxylase. J Mol Catal B: Enzym., 10: 67-79(2000)..

15. Battaglioli, G., Liu, H., and Martin, D.L., Kinetic differences between the isoforms of glutamate decarboxylase: implication for the regulation of GABA synthesis. J Neurochem, 86: 879-887(2003).

16. Gokani, V.N., thakker, M.U., Patel, J.G., Ghosh, S.K., and Chatterjee, S.K., Thin layer chromatographic method for estimation of gamma amino butyric acid from brain. Indian J Physiol Pharmacol, 23:101-104(1979).

17. Varanyanond, W., Tungtrakul, P., Surojanametakul, V., Watanasiritham, L., and Luxiang, W., Effect of water soaking on gamma amino butyric acid in germs of different Thai rice varieties. Kasetsart J. (Nat. Sci), 39: 411-415(2005).

18. Komatsuzaki, N., Tsukahara, K., Toyoshima, H., Suzuki, T., Shimizu, N., and Kimura, T., (2007). Effect of soaking and gaseous treatment on GABA content in germinated brown rice. Journal of Food Engineering, 78 (2): 556-560.

19. Feng, X.M., Olsson, J., Swanberg, M., Schnurer, J.,Ronnow, D., Image analysis for monitoring the barley tempeh fermentation process. J Appl. Microb.;103(9): 1113-1121(2007).

20. Nout, M.J.R., and Kiers, J. L., Tempe fermentation, innovation and functionally: update into the third millennium. J Apl. Microb., 98 (4): 789-805(2005).

21. Aoki, H., Uda, I., Tagami, K., Furuya, Y., and Fujimoto, K., The production of a new tempeh-like fermented soybean containing a high level of gamma amino butyric acid by anaerobic incubation with Rhizopus. Biosci. Biotechnol. Biochem; 67(5):1018-23(2003).

22. Steinkraus, K.H., Hwa, Y.B., Van Buren, J.P., Provident, M.I and Hand, D.B., Studies on Tempe an Indonesian fermented soybean food. Journal of Food Science, 25: 777-788(1960).

Department of Zoology, Govt. College of Science Wahdat Road, LAHORE.
COPYRIGHT 2011 Asianet-Pakistan
No portion of this article can be reproduced without the express written permission from the copyright holder.
Copyright 2011 Gale, Cengage Learning. All rights reserved.

Article Details
Printer friendly Cite/link Email Feedback
Author:Qureshi; Anwar, A.M.
Publication:Science International
Article Type:Report
Geographic Code:9PAKI
Date:Jun 30, 2011

Terms of use | Privacy policy | Copyright © 2019 Farlex, Inc. | Feedback | For webmasters