Lactococcus lactis is one of the most extensively used lactic acid bacteria for the manufacture of dairy products. Exploring the biodiversity of L. lactis is extremely promising both to acquire new knowledge and for food and health-driven applications. L. lactis is divided into four subspecies: lactis, cremoris, hordniae and tructae, but only subsp. lactis and subsp. cremoris are of industrial interest. Due to its various biotopes, Lactococcus subsp. lactis is considered the most diverse. The diversity of L. lactis subsp. lactis has been assessed at genetic, genomic and phenotypic levels. Multi-Locus Sequence Type (MLST) analysis of strains from different origins revealed that the subsp. lactis can be classified in two groups: “domesticated” strains with low genetic diversity, and “environmental” strains that are the main contributors of the genetic diversity of the subsp. lactis. As expected, the phenotype investigation of L. lactis strains reported here revealed highly diverse carbohydrate metabolism, especially in plant- and gut-derived carbohydrates, diacetyl production and stress survival. The integration of genotypic and phenotypic studies could improve the relevance of screening culture collections for the selection of strains dedicated to specific functions and applications.

Gut disorders associated to irritable bowel syndrome (IBS) are combined with anxiety and depression. Evidence suggests that microbially produced neuroactive molecules, like γ-aminobutyric acid (GABA), can modulate the gut-brain axis. Two natural strains of Lactococcus lactis and one mutant were characterized in vitro for their GABA production and tested in vivo in rat by oral gavage for their antinociceptive properties. L. lactis NCDO2118 significantly reduced visceral hypersensitivity induced by stress due to its glutamate decarboxylase (GAD) activity. L. lactis NCDO2727 with similar genes for GABA metabolism but no detectable GAD activity had no in vivo effect, as well as the NCDO2118 ΔgadB mutant. The antinociceptive effect observed for the NCDO2118 strain was mediated by the production of GABA in the gastro-intestinal tract and blocked by GABA_B receptor antagonist. Only minor changes in the faecal microbiota composition were observed after the L. lactis NCDO2118 treatment. These findings reveal the crucial role of the microbial GAD activity of L. lactis NCDO2118 to deliver GABA into the gastro-intestinal tract for exerting antinociceptive properties in vivo and open avenues for this GRAS (Generally Recognized As safe) bacterium in the management of visceral pain and anxious profile of IBS patients.

Background: γ-aminobutyric acid (GABA) is a bioactive compound produced by lactic acid bacteria (LAB). The diversity of GABA production in the Lactococcus genus is poorly understood. Genotypic and phenotypic approaches were therefore combined in this study to shed light on this diversity. A comparative genomic study was performed on the GAD-system genes (gadR, gadC and gadB) involved in GABA production in 36 lactococci including L. lactis and L. cremoris species. In addition, 132 Lactococcus strains were screened for GABA production in culture medium supplemented with 34 mM L-glutamic acid with or without NaCl (0.3 M). Results: Comparative analysis of the nucleotide sequence alignments revealed the same genetic organization of the GAD system in all strains except one, which has an insertion sequence element (IS981) into the P_gadCB promoter. This analysis also highlighted several deletions including a 3-bp deletion specific to the cremoris species located in the P_gadR promoter, and a second 39-bp deletion specific to L. cremoris strains with a cremoris phenotype. Phenotypic analysis revealed that GABA production varied widely, but it was higher in L. lactis species than in L. cremoris, with an exceptional GABA production of up to 14 and 24 mM in two L. lactis strains. Moreover, adding chloride increased GABA production in some L. cremoris and L. lactis strains by a factor of up to 16 and GAD activity correlated well with GABA production. Conclusions: This genomic analysis unambiguously characterized the cremoris phenotype of L. cremoris species and modified GadB and GadR proteins explain why the corresponding strains do not produce GABA. Finally, we found that glutamate decarboxylase activity revealing GadB protein amount, varied widely between the strains and correlated well with GABA production both with and without chloride. As this protein level is associated to gene expression, the regulation of GAD gene expression was identified as a major contributor to this diversity.

Irritable bowel syndrome (IBS) is a functional gastrointestinal disorder characterized by its main symptom, visceral hypersensitivity (VH), which is aggravated by stress. Gut–brain interactions and gut bacteria may alleviate IBS symptoms, including VH. γ‐amino butyric acid (GABA), produced notably by lactic acid bacteria (LAB), shows promising result in IBS symptoms treatment. In bacteria, GABA is generated through glutamate decarboxylase (GAD) metabolism of L‐glutamic acid, maintaining intracellular pH. In mammals, GABA acts as an inhibitory neurotransmitter, modulating pain, stress, and anxiety. Therefore, utilizing GABA‐producing LAB as a therapeutic approach might be beneficial. Our previous work showed that a GABA‐producing Lactococcus lactis strain, NCDO2118, reduced VH induced by acute stress in rats after a 10‐day oral treatment. Here, we identified the strain CNCM I‐5388, with a four‐fold higher GABA production rate under the same conditions as NCDO2118. Both strains shared 99.1% identical GAD amino acid sequences and in vitro analyses revealed the same optimal pH for GAD activity; however, CNCM I‐5388 exhibited 17 times higher intracellular GAD activity and increased resistance to acidic pH. Additionally, in vivo experiments have demonstrated that CNCM I‐5388 has faster anti‐VH properties in rats compared with NCDO2118, starting from the fifth day of treatment. Finally, CNCM I‐5388 anti‐VH effects partially persisted after 5‐day treatment interruption and after a single oral treatment. These findings highlight CNCM I‐5388 as a potential therapeutic agent for managing VH in IBS patients.

Irritable bowel syndrome (IBS) is a multifactorial disorder of the gut‐brain axis, characterized by visceral hypersensitivity (VH). Psychobiotics, through GABA synthesis, are good candidates to alleviate gastrointestinal discomfort. Here, we analyzed the GABA‐producer Lactococcus lactis CNCM I‐5388 as an active‐enzyme postbiotic to relieve VH mediated by psychological stress. L. lactis CNCM I‐5388 was inactivated by ethanol while maintaining its glutamate decarboxylase (GAD) activity. This EtOH‐treated nonviable form was given daily orally for 1, 5, or 10 days to female Wistar rats in comparison with viable L. lactis CNCM I‐5388 or vehicle. Visceral sensitivity was measured by electromyography before and after partial restraint stress (PRS). GABA was quantified in the stomach collected from rats and in the gastric compartment of TIM‐1 human gut model in fed state. A daily treatment for 5 and 10 days by L. lactis CNCM I‐5388 both in its viable and nonviable forms counteracted VH promoted by PRS. However, only viable L. lactis CNCM I‐5388 tended to reduce VH after a single administration. After 5‐day treatment, only under PRS conditions, the production of GABA within the stomach was enhanced in rats treated with viable or nonviable L. lactis CNCM I‐5388. This increase was confirmed by using the TIM‐1 human gut model. We found that a postbiotic with an active‐GAD enzyme of L. lactis CNCM I‐5388, similarly to its viable psychobiotic form, exerts anti‐VH properties in an IBS‐like rat model. These effects are associated with GABA production in the stomach where the low pH promotes GAD activity.