Glutamate and pH affect GABA production and acid stress tolerance in Levilactobacillus brevis LB12

Introduction

Levilactobacillus brevis is recognized as the most efficient lactic acid bacteria (LAB) species for the production of gamma‑aminobutyric acid acid (GABA) because it possesses a complete gad operon (gadR, gadB, gadC, and gadA). We investigated the effects of glutamate availability and pH conditions on GABA production, gad gene regulation, and acid stress tolerance in Lvb. brevis LB12, which are critical factors for developing robust and competitive starter cultures.

Methods

Lvb. brevis LB12 batch cultivations were carried out in bioreactors under either controlled pH (6.5, to maximize biomass) or uncontrolled pH (to activate the GABA system), with or without glutamate (Glu) supplementation. Acidification adaptation phases (down to pH 4.5) were applied to evaluate the role of low pH on the GABA pathway. Transcriptional regulation of gad operon was analysed via RT-qPCR, and cell survival under acid stress was evaluated.

Results and Discussion

Glu supplementation and GAD activation significantly improved survival under acid stress, with high GABA-producing cells showing enhanced resistance. This confirms that GABA biosynthesis acts as a robust acid-response mechanism where glutamate decarboxylation consumes intracellular protons to ensure pH homeostasis and acid protection. Gene expression analysis confirmed that the gad operon genes were strongly upregulated by glutamate and low pH. Conversely, gadA expression was less affected, indicating distinct regulatory patterns between the two decarboxylase isoforms and identifying gadR as the key driver of operon activation.

Conclusions

GABA biosynthesis is a central component of the acid-adaptation strategy of Lvb. brevis LB12. Optimizing bioreactor parameters to trigger this pathway offers a scalable strategy to improve starter culture robustness during industrial food processing.