BIOSYNTHESIS OF GAMMA AMINO BUTYRIC ACID IN FERMENTED RICE BY RHIZOPUS OLIGOSPORUS.
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 .
Gamma-amino butyric acid also has physiological function to depress the elevation of systolic blood pressure  and improve discrimination learning (Ishkawa and Saito, 1978). 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".
Gamma amino butyric acid is found in 3 phases i.e., gases, liquid and solid (Majumdar and Guha, 1988). It is found mostly as a zwitter ion, in which carboxyl group deprotonated and the amino group protonated 
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].
MATERIALS AND METHODS
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.
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, .
RESULTS AND DISCUSSION
(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,  worked on different varieties of brown rice and observed that GABA accumulation increased with time.
Komatsuzaki, et al,  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;  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  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,  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,  reported that at 30oC the mycelial growth was slow. These results are comparable with our results.
DETECTION OF GABA IN FERMENTED RICE
(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,  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,  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, . 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,  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.
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Department of Zoology, Govt. College of Science Wahdat Road, LAHORE. Email:email@example.com
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|Author:||Qureshi; Anwar, A.M.|
|Date:||Jun 30, 2011|
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