Ph.D. Dissertation:

Screening Newly GABA-Producing Lactic Acid Bacteria Isolated from Honey bee and Its Optimization and Application in soy Milk and Honey Juice

Gamma-aminobutyric acid (GABA), a four-carbon non-protein amino acid, is an inhibitory neurotransmitter in the mammalian central nervous system. It can be abundantly found in nature and is synthesized by glutamic acid decarboxylase. It serves several important physiological functions, including reduction in blood pressure and treatment of insomnia and depression. A number of bacteria and algae can produce significant amounts of GABA, among which lactic acid bacteria are widely used in food industries considering their nontoxicity. In the present study, a number of lactic acid bacteria, isolated from honeybees, were screened and optimized to produce GABA; then, they were examined in soy milk and honey syrup. The purpose of the first phase of the study was to investigate the probiotic characteristics and antibiotic resistance of 5 lactic acid bacteria isolated from honey in the honeybee gut. According to the results, all the isolated bacteria had desirable probiotic characteristics, including acid resistance, resistance to bile salts, resistance to gastric juice (pepsin & trypsin), inhibition of hemolytic activity, and hydrolysis of L-arginine. Furthermore, their non-resistance to antibiotics was confirmed. In the second section of the study, the potential of GABA production by bacteria was evaluated in the MRS agar at pH of 5 with 50 mM glucose for 72 hours. The results showed that the bacteria had the greatest ability to produce GABA (0.6290 mM). In the third phase of the study, synthetic growth and GABA production, as well as pH changes in the MRS agar containing 50 mM glutamic acid (pH, 5; temperature, 37°), were performed every 3 hours for 72 hours. The growth rates were measured at OD= 600 nm by a spectrophotometer, GABA production was evaluated via high-performance liquid chromatography (HPLC), and pH changes were determined by a pH meter. The highest growth rate, GABA production, and pH were 1.86 after 18 hours, 0.453 mM after 51 hours, and 4.86 after 42 hours, respectively. In the fourth phase, as the optimal conditions for fermentation of microorganisms were different, the key factors affecting the amount of GABA production, including the level of glutamic acid, ambient temperature, initial pH, and duration of incubation, were investigated independently to determine the changing range of each factor and its effect on GABA production during fermentation. The 400 mM glutamic acid at pH of 5 and temperature of 37°C for 60 hours was optimal. Moreover, the maximum amount of the produced GABA was 1.1, 0.576, 0.563, and 0.574 mM, respectively in each analysis. In the fifth phase, the conditions for bacteria-producing GABA were optimized using the independent factors of time, temperature, glutamic acid and pH using a central composite design in order to produce the highest amount of GABA. A total of 30 treatments were designed, and the results showed that the amount of produced GABA varied from 0.87 to 4.3 mM, depending on the conditions during fermentation. The results of variance analysis showed that the effects of all 4 parameters on the amount of produced GABA were significant. For 4.3 mM production of GABA, the optimal conditions are as follows: temperature, 37.71?; pH, 5.26, glutamic acid level, 506.99; duration, 53 hours. In the sixth phase, the optimal conditions for the production of functional probiotic beverages in soy milk and honey syrup were investigated. The best treatment included soy milk producing 6% GABA, which was statistically significant compared to other treatments with soy milk. The lowest production rate was reported with the honey syrup with 12% concentration (3.090 mM), which was significantly different from other treatments. The results of this study showed that by increasing the concentration of honey syrup and decreasing the concentration of soy milk, the overall acceptance of similar samples increased, as the highest total acceptance scores on the first and seventh days of production were attributed to the 12% honey syrup sample and the lowest to 12% soy milk, which were statistically significant. In general, the functional probiotic beverage of honey syrup 12% with 3.095 mM GABA and microbial population of 8.437 was the optimal sensory and mental treatment.

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