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This research aims to optimize the production process of gamma-aminobutyric acid (GABA) in fermented pineapple juice using probiotics and acetic acid bacteria (AAB), which are microorganisms with the potential to enhance GABA levels. This process has been developed to improve the nutritional value of fermented pineapple juice and to increase the economic value of Thai pineapples, which have long suffered from low market prices. This study focuses on determining the optimal conditions for GABA production by examining factors such as sugar content, pH levels, fermentation duration, and L-glutamate concentration, as well as the co-cultivation of probiotics and acetic acid bacteria. The experiments are conducted using controlled fermentation techniques, and the bioactive components of the fermented juice are analyzed with advanced instruments such as HPLC and GC-MS. The findings of this research are expected to contribute to the development of formulations and production processes for a high-GABA pineapple-based functional beverage. This product could offer health benefits such as stress reduction, cognitive function enhancement, and relaxation while also strengthening the potential of Thailand’s fermented food and beverage industry.