Lactic acid bacteria (LAB) potential in the food industry and in the biotechnological sector is a well-established interest. LAB potential in counteracting especially food-borne infections has received growing attention, but despite being a road full of promises is yet poorly explored. Furthermore, the ability of LAB to produce antimicrobial compounds, both by ribosomal synthesis and by decrypting them from proteins, is of high value when considering the growing impact of multidrug resistant strains. The antimicrobial potential of 14 food-derived lactic acid bacteria strains has been investigated in this study. Among them, four strains were able to counteract Listeria monocytogenes growth: Lactococcus lactis SN12 and L. lactis SN17 by high lactic acid production, whereas L. lactis 41FLL3 and Lactobacillus sakei I151 by Nisin Z and Sakacin P production, respectively. Strains Lactococcus lactis MG1363, Lactobacillus rhamnosus 17D10 and Lactobacillus helveticus 4D5 were tested and selected for their potential attitude to hydrolyze caseins. All the strains were able to release bioactive peptides with already known antimicrobial, antihypertensive and opioid activities. These features render these strains or their bioactive molecules suitable for use in food as biocontrol agents, or as nutraceutical supplements to treat mild disorders such as moderate hypertension and children insomnia. These results highlight once again that LAB potential in ensuring food safety, food nutraceutical value and ultimately in favoring human health is still underexplored and underexploited.

GABA (γ-aminobutyric acid) production has been widely described as an adaptive response to abiotic stress, allowing bacteria to survive in harsh environments. This work aimed to clarify and understand the relationship between GABA production and bacterial growth conditions, with particular reference to osmolarity. For this purpose, Lactococcus lactis NCDO 2118, a GABA-producing strain, was grown in glucose-supplemented chemically defined medium containing 34 mM L-glutamic acid, and different concentrations of salts (chloride, sulfate or phosphate ions) or polyols (sorbitol, glycerol). Unexpectedly, our data demonstrated that GABA production was not directly related to osmolarity. Chloride ions were the most significant factor influencing GABA yield in response to acidic stress while sulfate ions did not enhance GABA production. We demonstrated that the addition of chloride ions increased the glutamic acid decarboxylase (GAD) synthesis and the expression of the gadBC genes. Finally, under fed-batch conditions in a complex medium supplemented with 0.3 M NaCl and after a pH shift to 4.6, L. lactis NCDO 2118 was able to produce up to 413 mM GABA from 441 mM L-glutamic acid after only 56 h of culture, revealing the potential of L. lactis strains for intensive production of this bioactive molecule.

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.

Backslopping, which involves using the whey from a previous batch as a starter for a new production run, is one of the oldest traditional cheese-making methods and is still widely used today. However, this technique can sometimes lead to acidification failures, necessitating the use of industrial starters to quickly restart production and minimise economic loss. This study investigated the effect of using a well-defined indigenous starter culture on raw milk during backslopping. The microbial composition and technological characteristics of successive subcultures were analyzed during one month of backslopping with and without the starter added. The results showed that the indigenous starter remained highly stable in skim milk, predominantly drove homolactic fermentation. The raw milk alone displayed greater initial microbial diversity and metabolic variability but then stabilised and Lactococcus lactis became dominant from day four onwards of backslopping. The combination of the indigenous starter with the raw milk, rapidly established L. lactis dominance, directing the ecosystem towards homolactic fermentation from the start of backslopping. The addition of the indigenous starter impacted particularly subdominant populations and technological properties, resulting in an acidification that met the requirements of the Rocamadour PDO from the start of the backslopping. From day fifteen onwards, comparison of bacterial communities with and without the starter added became differentiated, demonstrating that adding the indigenous starter reduced variability in bacterial composition. Notably, coexistence between the indigenous starter and the raw milk flora was observed, suggesting that this could be a promising option for farmer producers to strengthen the link between the product and its microbial terroir.