Gamma-aminobutyric acid (GABA) plays a key role in mammals as the major inhibitory neurotransmitter of the central nervous system. Although GABA may not be able to cross the human blood-brain barrier, it was approved as a food ingredient because of its benefits to the host after oral administration including anti-hypertensive, anti-depressant and anti-inflammatory activities. Considering the current trend toward the development of new functional and natural products and that microbial fermentation is one of the most promising methods to produce this non-protein amino acid, the in situ production of GABA through fermentation of strawberry and blueberry juices by the efficient GABA producer strain, Levilactobacillus brevis (formerly known as Lactobacillus brevis) CRL 2013, was evaluated. A high GABA production (262 mM GABA) was obtained after fermenting strawberry juice supplemented with yeast extract for 168 h, being GABA yield significantly higher in strawberry juices than in the blueberry ones. Thus, GABA-enriched fermented strawberry juice (FSJ) was selected to carry out in vivo and in vitro studies. The in vitro functional analysis of the GABA-enriched FSJ demonstrated its ability to significantly decrease the expression of cox-2 gene in LPS stimulated RAW 264.7 macrophages. In addition, in vivo studies in mice demonstrated that both, L. brevis CRL 2013 and the GABA-enriched FSJ were capable of reducing the levels of peritoneal, intestinal and serum TNF-α, IL-6, and CXCL1, and increasing IL-10 and IFN-γ in mice exposed to an intraperitoneal challenge of LPS. Of note, the GABA-enriched FSJ was more efficient than the CRL 2013 strain to reduce the pro-inflammatory factors and enhance IL-10 production. These results indicated that the CRL 2013 strain exerts anti-inflammatory effects in the context of LPS stimulation and that this effect is potentiated by fermentation. Our results support the potential use of L. brevis CRL 2013 as an immunomodulatory starter culture and strawberry juice as a remarkable vegetable matrix for the manufacture of GABA-enriched fermented functional foods capable of differentially modulating the inflammatory response triggered by TLR4 activation.

The proteolytic system of Lactobacillus plays an essential role in bacterial growth, contributes to the flavor development of fermented products, and can release bioactive health-beneficial peptides during milk fermentation. In this work, a genomic analysis of all genes involved in the proteolytic system of L. delbrueckii subsp. lactis CRL 581 was performed. Genes encoding the cell envelope-associated proteinase, two peptide transport systems, and sixteen peptidases were identified. The influence of the peptide supply on the transcription of 23 genes involved in the proteolytic system of L. delbrueckii subsp. lactis was examined after cell growth in a chemically defined medium (CDM) and CDM supplemented with Casitone. prtL, oppA 1, optS, optA genes as well as oppDFBC and optBCDF operons were the most highly expressed genes in CDM; their expression being repressed 6- to 115-fold by the addition of peptides. The transcriptional analysis was confirmed by proteomics; the up-regulation of the PrtL, PepG, OppD and OptF proteins in the absence of peptides was observed while the DNA-binding protein YebC was up-regulated by peptides. Binding of YebC to the promoter region of prtL, oppA 1, and optS, demonstrated by electrophoretic mobility shift assays, showed that YebC acts as a transcriptional repressor of key proteolytic genes.

Proteolytic starter cultures with intrinsic immunomodulatory activities are desirably features for the development of functional foods, which would significantly reduce the cost of their production (one-strain starter) having an additional beneficial effect on the host. In this work, Lactobacillus delbrueckii strains were selected according to their ability to efficiently hydrolyse β-casein and to modulate the immune system. Among 36 strains evaluated, the highest proteolytic activities were found for L. delbrueckii subsp. lactis CRL581 and L. delbrueckii subsp. bulgaricus CRL656. The immunomodulatory effect of both strains and their β-casein hydrolysates (CRL581 and CRL656 hydrolysates, respectively) were studied in a murine model. Balb/c mice were fed lactobacilli or their hydrolysates for three days. One day after the last lactobacilli or hydrolysate treatments, mice were challenged with the Toll-like receptor 3 (TLR3) agonist poly(I:C) by intraperitoneal injection. Before and after poly(I:C) challenge the phagocytic and microbicidal activity of peritoneal macrophages, intestinal immunoglobulin A (IgA), cytokine profile, and histological analysis of the intestine were analysed. L. delbrueckii subsp. lactis CRL581 significantly increased the activation of peritoneal macrophages as well as the levels of intestinal IgA, interleukin (IL)-10 and interferon (IFN)-γ when compared to untreated controls. In addition, the CRL581 strain was able to significantly reduce the intestinal inflammatory damage triggered by TLR3 activation. L. delbrueckii CRL581 increased the levels of IL-10, IFN-γ and IFN-β, and reduced tumour necrosis factor alpha and IL-6 concentrations in the intestine of poly(I:C)-challenged mice. No immunomodulatory effects were observed for the CRL656 strain or for the CRL581 or CRL656 hydrolysates. The results of this work show that the technologically relevant and high proteolytic strain L. delbrueckii CRL581 is able to beneficially modulate the intestinal innate antiviral immune response. Although further studies with the CRL581 strain are required to corroborate and deepen its immunological effects, this bacterium is an interesting alternative for the development of new functional foods with antiviral capabilities.

Previously, we isolated lactic acid bacteria from the slime of the garden snail Helix aspersa Müller and selected Weissella viridescens UCO-SMC3 because of its ability to inhibit in vitro the growth of the skin-associated pathogen Cutibacterium acnes. The present study aimed to characterize the antimicrobial and immunomodulatory properties of W. viridescens UCO-SMC3 and to demonstrate its beneficial effect in the treatment of acne vulgaris. Our in vitro studies showed that the UCO-SMC3 strain resists adverse gastrointestinal conditions, inhibits the growth of clinical isolates of C. acnes, and reduces the adhesion of the pathogen to keratinocytes. Furthermore, in vivo studies in a mice model of C. acnes infection demonstrated that W. viridescens UCO-SMC3 beneficially modulates the immune response against the skin pathogen. Both the oral and topical administration of the UCO-SCM3 strain was capable of reducing the replication of C. acnes in skin lesions and beneficially modulating the inflammatory response. Of note, orally administered W. viridescens UCO-SMC3 induced more remarkable changes in the immune response to C. acnes than the topical treatment. However, the topical administration of W. viridescens UCO-SMC3 was more efficient than the oral treatment to reduce pathogen bacterial loads in the skin, and effects probably related to its ability to inhibit and antagonize the adhesion of C. acnes. Furthermore, a pilot study in acne volunteers demonstrated the capacity of a facial cream containing the UCO-SMC3 strain to reduce acne lesions. The results presented here encourage further mechanistic and clinical investigations to characterize W. viridescens UCO-SMC3 as a probiotic for acne vulgaris treatment.

Lactobacillus delbrueckii subsp. delbrueckii TUA4408L is able to differentially modulate the innate immune response of porcine intestinal epithelial cells triggered by TLR4 activation. This strain also has a remarkable ability to grow on plant substrates. These two immunological and biotechnological characteristics prompted us to evaluate whether the soymilk by-product okara fermented with the TUA4408L strain can serve as an immunobiotic feed with the ability to beneficially modulate the intestinal immunity of piglets after weaning to improve their productivity. Our in vivo studies demonstrated that the administration of immunobiotic TUA4408L-fermented okara feed significantly increased piglet growth performance and meat quality. These positive effects were associated with the ability of the TUA4408L-fermented okara feed to beneficially modulate both intestinal microbiota and immunity in pigs. The immunobiotic feed improved the abundance of the beneficial bacteria Lactobacillus and Lactococcus in the gut of pigs, reduced blood markers of inflammation, and differentially regulated the expression of inflammatory and regulatory cytokines in the intestinal mucosa. These findings indicate that the immunobiotic TUA4408L-fermented okara feed could be an economical and environmentally friendly option to improve the growth performance and immune health of pigs.

Previously, we demonstrated that nasally administered Corynebacterium pseudodiphteriticum 090104 (Cp) or its bacterium-like particles (BLPs) increase the resistance of mice against bacterial and viral respiratory pathogens by modulating the innate immunity. In this work, we evaluated the ability of Cp and BLPs to stimulate alveolar macrophages, and to enhance the humoral immune response induced by a commercial vaccine against Streptococcus pneumoniae. In the first set of experiments, Cp or the BLPs were incubated with primary cultures of murine alveolar macrophages and the phagocytic activity, and the production of cytokines was evaluated. The results revealed that Cp and BLPs were efficiently phagocyted by respiratory macrophages and that both treatments triggered the production of TNF-α, IFN-γ, IL-6, and IL-1β. In the second set of experiments, 3-week-old Swiss mice were intranasally immunized at days 0, 14, and 28 with the pneumococcal vaccine Prevenar®13 (PCV), Cp + PCV, or BLPs + PCV. On day 33, samples of bronco-alveolar lavages (BAL) and serum were collected for the study of specific antibodies. In addition, immunized mice were challenged with S. pneumoniae serotypes 6B or 19F on day 33 and sacrificed on day 35 (day 2 post-infection) to evaluate the resistance to the infection. Both Cp + PCV and BLPs + PCV groups had higher specific serum IgG and BAL IgA antibodies than the PCV control mice. In addition, the mice that were immunized with Cp + PCV or BLPs + PCV had lower lung and blood pneumococcal cell counts as well as lower levels of BAL albumin and LDH, indicating a reduced lung damage compared to the control mice. Improved levels of anti-pneumococcal antibodies were also detected in the serum and BAL samples after the challenges with the pathogens. The results demonstrated that C. pseudodiphteriticum 090104 and its bacterium-like particles are capable of stimulating the respiratory innate immune system serving as adjuvants to potentiate the adaptive humoral immune response. Our study is a step forward in the positioning of this respiratory commensal bacterium as a promising mucosal adjuvant for vaccine formulations aimed at combating respiratory infectious diseases.

Lactobacillus delbrueckii subsp. lactis CRL 581 beneficially modulates the intestinal antiviral innate immune response triggered by the Toll-like receptor 3 (TLR3) agonist poly(I:C) in vivo. This study aimed to characterize further the immunomodulatory properties of the technologically relevant starter culture L. delbrueckii subsp. lactis CRL 581 by evaluating its interaction with intestinal epithelial cells and macrophages in the context of innate immune responses triggered by TLR3. Our results showed that the CRL 581 strain was able to adhere to porcine intestinal epithelial (PIE) cells and mucins. The CRL 581 strain also augmented the expression of antiviral factors (IFN-α, IFN-β, Mx1, OAS1, and OAS2) and reduced inflammatory cytokines in PIE cells triggered by TLR3 stimulation. In addition, the influence of L. delbrueckii subsp. lactis CRL 581 on the response of murine RAW macrophages to the activation of TLR3 was evaluated. The CRL 581 strain was capable of enhancing the expression of IFN-α, IFN-β, IFN-γ, Mx1, OAS1, TNF-α, and IL-1β. Of note, the CRL 581 strain also augmented the expression of IL-10 in macrophages. The results of this study show that the high proteolytic strain L. delbrueckii spp. lactis CRL 581 was able to beneficially modulate the intestinal innate antiviral immune response by regulating the response of both epithelial cells and macrophages relative to TLR3 activation.

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