Freeze-dried immobilized Lactiplantibacillus plantarum B13 for gamma aminobutyric acid biosynthesis

Gamma-aminobutyric acid (GABA) is a four carbon non-protein amino acid. GABA intended for use in food and pharmaceutical products must fulfill strict safety requirements and hence cannot be chemically synthesized. Therefore, biotechnological approach for GABA production appears to be promising due to simple procedure, high catalytic efficiency, and environmental compatibility. Although various GABA-producing strains have been isolated from different sources, much work is still needed to exploit and characterize additional GABA- producers that provide a potential basis for developing fermented health- oriented products. This study was aimed to enhance the yield of GABA by locally isolated lactic acid bacteria (LAB), Lactiplantibacillus plantarum B13 by using natural support for biocatalyst immobilization. L. plantarum B13 was classified as LAB (Gram-positive, catalase-negative and oxidase-negative strain) and potential probiotic (high survivality in acidic and bile salt (≥ 50 %); high autoaggregation (73.03 %), high viable cell counts in phenol (7 – 9 log CFU/mL), possessed hydrophilic nature due to adherence to chloroform (72.48 %), exerted antimicrobial activity (against E. coli, S. aureus, S. typhimurium, and B. subtilis) and antioxidant activity (77.27 %)). The optimum fermentation parameters were determined by using one-factor-at-a time (OFAT). The optimum conditions for yielded the highest GABA (based on thin layer chromatography and UV spectroscopy method) were 45 hours of fermentation time, 35C, pH 5.5, 5 % (w/v) MSG, 0.7 mM PLP and 60 g/L glucose based on the highest GABA productivity (0.424 g/L/h). To increase the cell stability and productivity, L. plantarum B13 was then immobilized on selected vegetables (okra, onion, sweet potato, bell pepper and taro). The best natural support was selected based on GABA production and cell retention (by using cell count method). Onion was the found as the best natural support evaluated based on GABA yield (18.854 g/L) and cell retention (83 %). It was then employed in repeated batch fermentations to compare its reusability with the free-cell fermentation. The onion-immobilized cells were able to be reused with a greater number of fermentation batches compared to the free cell. The GABA yield was reduced to below 50 % of the initial yield after the 8th and 6th batch of fermentations for the immobilized cells and free cell, respectively. Additionally, cell viability and productivity may reduce during the storage period. Therefore, freeze-dried cell storage studies were conducted to investigate the ability to retain cell viability and synthesize GABA. The potential probiotic (free-cell and onion-immobilized L. plantarum B13) and its postbiotic (supernatant containing GABA) were subsequently freeze-dried with a combination of 10 % (w/v) skimmed milk and 10 % (w/v) sucrose as the cryoprotectant to obtain three different probiotic powder preparations (FCP: free cell+ postbiotic; FC: free cell+ postbiotic+ cryoprotectant; IC: immobilized cell+ postbiotic+ cryoprotectant). After 8 weeks of storage at 40C, the IC showed the best performances (7.29 log CFU/mL; survival rate, 81.52 %; and GABA yield, 11.31 g/L) than the FC (7.02 log CFU/mL, 72.82 % survival rate, 8.71 g/L GABA) and FCP (6.06 log CFU/mL, 62.00 % survival rate, 4.89 g/L GABA). Overall, the results proved that the immobilized cells are more effective as compared to the free cell for maintaining high cell viability, survivability, and GABA yield. The findings of this study also suggest the potential use of immobilized L. plantarum B13 in the development of GABA-enriched products.

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