Comprehensive characterization of γ-aminobutyric acid (GABA) production by Levilactobacillus brevis CRL 2013: insights from physiology, genomics, and proteomics.

Introduction: Levilactobacillus brevis CRL 2013, a plant-derived lactic acid bacterium (LAB) with immunomodulatory properties, has emerged as an efficient producer of γ-aminobutyric acid (GABA). Notably, not all LAB possess the ability to produce GABA, highlighting the importance of specific genetic and environmental conditions for GABA synthesis. This study aimed to elucidate the intriguing GABA-producing machinery of L. brevis CRL 2013 and support its potential for safe application through comprehensive genome analysis. Methods: A comprehensive genome analysis of L. brevis CRL 2013 was performed to identify the presence of antibiotic resistance genes, virulence markers, and genes associated with the glutamate decarboxylase system, which is essential for GABA biosynthesis. Then, an optimized chemically defined culture medium (CDM) was supplemented with monosodium glutamate (MSG) and yeast extract (YE) to analyze their influence on GABA production. Proteomic and transcriptional analyses were conducted to assess changes in protein and gene expression related to GABA production. Results: The absence of antibiotic resistance genes and virulence markers in the genome of L. brevis CRL 2013 supports its safety for potential probiotic applications. Genes encoding the glutamate decarboxylase system, including two gad genes (gadA and gadB) and the glutamate antiporter gene (gadC), were identified. The gadB gene is located adjacent to gadC, while gadA resides separately on the chromosome. The transcriptional regulator gadR was found upstream of gadC, with transcriptional analyses demonstrating cotranscription of gadR with gadC. Although MSG supplementation alone did not activate GABA synthesis, the addition of YE significantly enhanced GABA production in the optimized CDM containing glutamate. Proteomic analysis revealed minimal differences between MSG-supplemented and non-supplemented CDM cultures, whereas YE supplementation resulted in significant proteomic changes, including upregulation of GadB. Transcriptional analysis confirmed increased expression of gadB and gadR upon YE supplementation, supporting its role in activating GABA production. Conclusion: These findings provide valuable insights into the influence of nutrient composition on GABA production. Furthermore, they unveil the potential of L. brevis CRL 2013 as a safe, nonpathogenic strain with valuable biotechnological traits which can be further leveraged for its probiotic potential in the food industry.

Wastewater monitoring of SARS-CoV-2 gene for COVID-19 epidemiological surveillance in Tucumán, Argentina.

Wastewater-based epidemiology provides temporal and spatial information about the health status of a population. The objective of this study was to analyze and report the epidemiological dynamics of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the province of Tucumán, Argentina during the second and third waves of coronavirus disease 2019 (COVID-19) between April 2021 and March 2022. The study aimed to quantify SARS-CoV-2 RNA in wastewater, correlating it with clinically reported COVID-19 cases. Wastewater samples (n = 72) were collected from 16 sampling points located in three cities of Tucumán (San Miguel de Tucumán, Yerba Buena y Banda del Río Salí). Detection of viral nucleocapsid markers (N1 gene) was carried out using one-step reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Viral loads were determined for each positive sample using a standard curve. A positive correlation (p < 0.05) was observed between viral load (copies/mL) and the clinically confirmed COVID-19 cases reported at specific sampling points in San Miguel de Tucumán (SP4, SP7, and SP8) in both months, May and June. Indeed, the high viral load concurred with the peaks of COVID-19 cases. This method allowed us to follow the behavior of SARS-CoV-2 infection during epidemic outbreaks. Thus, wastewater monitoring is a valuable epidemiological indicator that enables the anticipation of increases in COVID-19 cases and tracking the progress of the pandemic. SARS-CoV-2 genome-based surveillance should be implemented as a routine practice to prepare for any future surge in infections.

Inorganic phosphate modifies stationary phase fitness and metabolic pathways in Lactiplantibacillus paraplantarum CRL 1905.

Inorganic phosphate (Pi) concentration modulates polyphosphate (polyP) levels in diverse bacteria, affecting their physiology and survival. Lactiplantibacillus paraplantarum CRL 1905 is a lactic acid bacterium isolated from quinoa sourdough with biotechnological potential as starter, for initiating fermentation processes in food, and as antimicrobial-producing organism. The aim of this work was to evaluate the influence of the environmental Pi concentration on different physiological and molecular aspects of the CRL 1905 strain. Cells grown in a chemically defined medium containing high Pi (CDM + P) maintained elevated polyP levels up to late stationary phase and showed an enhanced bacterial survival and tolerance to oxidative stress. In Pi sufficiency condition (CDM-P), cells were ~ 25% longer than those grown in CDM + P, presented membrane vesicles and a ~ 3-fold higher capacity to form biofilm. Proteomic analysis indicated that proteins involved in the "carbohydrate transport and metabolism" and "energy production and conversion" categories were up-regulated in high Pi stationary phase cells, implying an active metabolism in this condition. On the other hand, stress-related chaperones and enzymes involved in cell surface modification were up-regulated in the CDM-P medium. Our results provide new insights to understand the CRL 1905 adaptations in response to differential Pi conditions. The adjustment of environmental Pi concentration constitutes a simple strategy to improve the cellular fitness of L. paraplantarum CRL 1905, which would benefit its potential as a microbial cell factory.

Improvement of Key Molecular Events Linked to Alzheimer's Disease Pathology Using Postbiotics.

In the past 50 years, life expectancy has increased by more than 20 years. One consequence of this increase in longevity is the rise of age-related diseases such as dementia. Alzheimer's disease (AD) is the most common form of dementia, accounting for 60-70% of cases. AD pathogenesis is not restricted to the neuronal compartment but includes strong interactions with other brain cells, particularly microglia triggering the release of inflammatory mediators, which contribute to disease progression and severity. There is growing evidence revealing the diverse clinical benefits of postbiotics in many prevalent conditions, including neurodegenerative diseases. Here, we tested the ability of bacterial conditioned media (BCM) derived from selected lactic acid bacteria (LAB) strains to regulate core mechanisms relevant to AD pathophysiology in the microglia cell line BV-2. Levilactobacillus brevis CRL 2013, chosen for its efficient production of the neurotransmitter GABA, and Lactobacillus delbrueckii subsp. lactis CRL 581, known for its anti-inflammatory properties, were selected alongside Enterococcus mundtii CRL 35, a LAB strain that can significantly modulate cytokine production. BCM from all 3 strains displayed antioxidant capabilities, reducing oxidative stress triggered by beta-amyloid oligomers (oAß1-42). Additionally, BCM effectively mitigated the expression of inflammatory cytokines, namely, TNF-α, IL-1ß, and IL-6 triggered by oAß1-42. Furthermore, our study identified that BCM from CRL 581 inhibit the activity of acetylcholinesterase (AChE), a crucial enzyme in AD progression, in both human erythrocytes and mouse brain tissues. Notably, the inhibitory effect was mediated by low-molecular-weight components of the BCM. L. delbrueckii subsp. lactis CRL 581 emerged as a favorable candidate for production of postbiotics with potential benefits for AD therapy since it demonstrated potent antioxidant activity, reduction of cytokine expression, and partial AChE inhibition. On the other hand, E. mundtii CRL 35 showed that the antioxidant activity failed to inhibit AChE and caused induction of iNOS expression, rendering it unsuitable as a potential therapeutic for AD. This study unveils the potential benefits of LAB-derived postbiotics for the development of new avenues for therapeutic interventions for AD.

In Silico Comparative Genomic Analysis Revealed a Highly Conserved Proteolytic System in Lactobacillus delbrueckii.

Lactobacillus delbrueckii, the type species of the genus Lactobacillus, is widely recognized as the primary starter culture in the dairy industry due to its proteolytic activity, which enables it to growth in milk. In this study, a comprehensive genomic analysis of the proteolytic system was conducted on L. delbrueckii strains. The analysis included 27 genomes of L. delbrueckii, with a specific focus on the key enzyme involved in this system, the cell envelope-associated proteinase (CEP). The amino acid sequences, as well as the protein-structure prediction of the CEPs, were compared. Additionally, syntenic analysis of the genomic locus related to the CEPs revealed high conservation in L. delbrueckii subsp. bulgaricus strains, while L. delbrueckii subsp. lactis strains exhibited greater variability, including the presence of insertion sequences, deletions, and rearrangements. Finally, the CEP promoter region and putative regulatory elements responsible for controlling the expression of the proteolytic system in lactobacilli were investigated. Our genomic analysis and in silico characterization of the CEPs contribute to our understanding of proteolytic activity and the potential applications of these lactic acid bacteria in the dairy industry. Further research in this area will expand our knowledge and potential practical uses of these findings.

Green synthesis, characterization and applications of iron and zinc nanoparticles by probiotics.

The synthesis of nanoparticles (NPs) by microorganisms is one of the most promising areas of research in modern nanotechnology since microorganisms can easily act as real nanofactories of industrially relevant compounds. Recent studies suggest that probiotic bacteria have an intrinsic potential to synthesize metal NPs when grown in the presence of metal ions. In such conditions, they can reduce metal ions through different biochemical mechanisms occurring both intra and extracellularly, and leading to the production of NPs. Different approaches have proposed the synthesis of silver, gold, titanium or selenium NPs from probiotics, with promising health related effects. However, their use for the production of iron and zinc NPs has been scarcely reported. Considering the nutritional relevance of iron and zinc, a thorough approach about the synthesis of iron and zinc NPs by probiotics was addressed, including the factors affecting the synthesis processes, the mechanisms of synthesis, and the physical and chemical characterization of NPs. The impact of products containing probiotics and minerals has applications in many different fields going beyond the food industry and representing a powerful strategy as economic engine for very diverse industries and countries.

Lactobacillus delbrueckii CRL 581 Differentially Modulates TLR3-Triggered Antiviral Innate Immune Response in Intestinal Epithelial Cells and Macrophages.

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-ß, 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-ß, IFN-γ, Mx1, OAS1, TNF-α, and IL-1ß. 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.

Insights into 6S RNA in lactic acid bacteria (LAB).

BACKGROUND: 6S RNA is a regulator of cellular transcription that tunes the metabolism of cells. This small non-coding RNA is found in nearly all bacteria and among the most abundant transcripts. Lactic acid bacteria (LAB) constitute a group of microorganisms with strong biotechnological relevance, often exploited as starter cultures for industrial products through fermentation. Some strains are used as probiotics while others represent potential pathogens. Occasional reports of 6S RNA within this group already indicate striking metabolic implications. A conceivable idea is that LAB with 6S RNA defects may metabolize nutrients faster, as inferred from studies of Echerichia coli. This may accelerate fermentation processes with the potential to reduce production costs. Similarly, elevated levels of secondary metabolites might be produced. Evidence for this possibility comes from preliminary findings regarding the production of surfactin in Bacillus subtilis, which has functions similar to those of bacteriocins. The prerequisite for its potential biotechnological utility is a general characterization of 6S RNA in LAB. RESULTS: We provide a genomic annotation of 6S RNA throughout the Lactobacillales order. It laid the foundation for a bioinformatic characterization of common 6S RNA features. This covers secondary structures, synteny, phylogeny, and product RNA start sites. The canonical 6S RNA structure is formed by a central bulge flanked by helical arms and a template site for product RNA synthesis. 6S RNA exhibits strong syntenic conservation. It is usually flanked by the replication-associated recombination protein A and the universal stress protein A. A catabolite responsive element was identified in over a third of all 6S RNA genes. It is known to modulate gene expression based on the available carbon sources. The presence of antisense transcripts could not be verified as a general trait of LAB 6S RNAs. CONCLUSIONS: Despite a large number of species and the heterogeneity of LAB, the stress regulator 6S RNA is well-conserved both from a structural as well as a syntenic perspective. This is the first approach to describe 6S RNAs and short 6S RNA-derived transcripts beyond a single species, spanning a large taxonomic group covering multiple families. It yields universal insights into this regulator and complements the findings derived from other bacterial model organisms.

Transcriptional Responses of Herbaspirillum seropedicae to Environmental Phosphate Concentration.

Herbaspirillum seropedicae is a nitrogen-fixing endophytic bacterium associated with important cereal crops, which promotes plant growth, increasing their productivity. The understanding of the physiological responses of this bacterium to different concentrations of prevailing nutrients as phosphate (Pi) is scarce. In some bacteria, culture media Pi concentration modulates the levels of intracellular polyphosphate (polyP), modifying their cellular fitness. Here, global changes of H. seropedicae SmR1 were evaluated in response to environmental Pi concentrations, based on differential intracellular polyP levels. Cells grown in high-Pi medium (50 mM) maintained high polyP levels in stationary phase, while those grown in sufficient Pi medium (5 mM) degraded it. Through a RNA-seq approach, comparison of transcriptional profiles of H. seropedicae cultures revealed that 670 genes were differentially expressed between both Pi growth conditions, with 57% repressed and 43% induced in the high Pi condition. Molecular and physiological analyses revealed that aspects related to Pi metabolism, biosynthesis of flagella and chemotaxis, energy production, and polyhydroxybutyrate metabolism were induced in the high-Pi condition, while those involved in adhesion and stress response were repressed. The present study demonstrated that variations in environmental Pi concentration affect H. seropedicae traits related to survival and other important physiological characteristics. Since environmental conditions can influence the effectiveness of the plant growth-promoting bacteria, enhancement of bacterial robustness to withstand different stressful situations is an interesting challenge. The obtained data could serve not only to understand the bacterial behavior in respect to changes in rhizospheric Pi gradients but also as a base to design strategies to improve different bacterial features focusing on biotechnological and/or agricultural purposes.

Improvement of Myelopoiesis in Cyclophosphamide-Immunosuppressed Mice by Oral Administration of Viable or Non-Viable Lactobacillus Strains.

Myelosuppression is the major dose-limiting toxicity of cancer chemotherapy. There have been many attempts to find new strategies that reduce myelosuppression. The dietary supplementation with lactic acid bacteria (LAB) improved respiratory innate immune response and the resistance against respiratory pathogens in immunosupressed hosts. Although LAB viability is an important factor in achieving optimal protective effects, non-viable LAB are capable of stimulating immunity. In this work, we studied the ability of oral preventive administration of viable and non-viable Lactobacillus rhamnosus CRL1505 or L. plantarum CRL1506 (Lr05, Lr05NV, Lp06V or Lp06NV, respectively) to minimize myelosuppressive and immunosuppressive effects derived from chemotherapy. Cyclophosphamide (Cy) impaired steady-state myelopoiesis in lactobacilli-treated and untreated control mice. Lr05V, Lr05NV and Lp06V treatments were the most effective to induce the early recovery of bone marrow (BM) tissue architecture, leukocytes, myeloid, pool mitotic and post-mitotic, peroxidase positive, and Gr-1Low/High cells in BM. We selected the CRL1505 strain for being the one capable of maintaining its myelopoiesis-enhancing properties in its non-viable form. Although the CRL1505 treatments do not modify the Cy ability to induce apoptosis, both increased the incorporation of BrdU in BM cells. Consequently, Lr05NV and Lr05V treatments were able to promote early recovery of LSK cells (Lin-Sca-1+c-Kit+ cells), multipotent progenitors (Lin-Sca-1+c-Kit+CD34+ cells), and myeloid cells (Gr-1+Ly6G+Ly6C- cells) with respect to the untreated Cy control. In addition, these treatments were able to increase the frequency of IL17A-producing innate lymphoid cells in the intestinal lamina propria (IL-17A+RORγt+CD4-NKp46+ cells) after Cy injection. These results were correlated with an increase in the IL-17A serum levels, a GM-CSF high expression and a CXCL12 lower expression in BM. Therefore, both Lr05V and Lr05NV treatments are able to activate beneficially the IL-17A/GM-CSF axis and accelerate the recovery of Cy-induced immunosuppression by increasing BM myeloid precursors. We demonstrated for the first time the beneficial effect of CRL1505 strain on myelopoiesis affected by a chemotherapeutic drug. Furthermore, Lr05NV could be a good and safe resource for reducing chemotherapy-induced leukopenia. The results are a starting point for future research and open up broad prospects for future applications of the immunobiotics.

Enhancement of γ-aminobutyric acid (GABA) production by Lactobacillus brevis CRL 2013 based on carbohydrate fermentation.

Gamma aminobutyric acid (GABA) is a non-protein amino acid that is widely distributed in nature and its physiological importance goes beyond its role as an inhibitory neurotransmitter of the central nervous system in mammals. Since microbial fermentation is one of the most promising methods to obtain GABA, the production of this metabolite by several strains of lactic acid bacteria isolated from quinoa and amaranth sourdoughs was investigated. Lactobacillus brevis CRL 2013 produced the highest GABA levels, reaching 265 mM when optimal culture conditions were set up. The fermentative profile showed that CRL 2013 was able to catabolize carbohydrates through the phosphoketolase pathway yielding variable amounts of lactic acid, acetate and ethanol, which depended on the type of carbon source available and the presence of external electron acceptors such as fructose. Enhanced growth parameters and low GABA synthesis were associated to pentose fermentation. This impairment on GABA production machinery was partially overpassed by the addition of ethanol to the culture media. These results support the potential use of L. brevis CRL 2013 as a starter culture for the manufacture of GABA-enriched functional foods and provide further insights to the understanding of the GAD system regulation in lactic acid bacteria.

Exploring the Genome of Fructobacillus tropaeoli CRL 2034, a Fig-Origin Strain that Produces High Levels of Mannitol from Fructose.

We report the draft genome sequence of Fructobacillus tropaeoli CRL 2034, a strain isolated from ripe fig in Tucumán province, Argentina. The interest in studying the genome of this fructophilic lactic acid bacterium strain was motivated by its ability to produce high levels of mannitol from fructose. This polyol has multiple industrial applications; however, it is mainly used as low calorie sugar in the food industry. The assembled genome of this strain consists of a 1.66-Mbp circular chromosome with 1465 coding sequences and a G+C content of 44.6%. The analysis of this genome supports the one step reaction of fructose reduction to mannitol by the mannitol 2-dehydrogenase enzyme, which together with a fructose permease, were identified as involved in mannitol synthesis. In addition, a phylogenetic analysis was performed including other Leuconostocaceae members to which the Fructobacillus genus belongs to; according to the 16S rRNA gene sequences, the strain CRL 2034 was located in the Fructobacillus clade. The present genome sequence could be useful to further elucidate regulatory processes of mannitol and other bioactive metabolites and to highlight the biotechnological potential of this fruit-origin Fructobacillus strain.

Nucleotide sequence and analysis of pRC12 and pRC18, two theta-replicating plasmids harbored by Lactobacillus curvatus CRL 705.

The nucleotide sequences of plasmids pRC12 (12,342 bp; GC 43.99%) and pRC18 (18,664 bp; GC 34.33%), harbored by the bacteriocin-producer Lactobacillus curvatus CRL 705, were determined and analyzed. Plasmids pRC12 and pRC18 share a region with high DNA identity (> 83% identity between RepA, a Type II toxin-antitoxin system and a tyrosine integrase genes) and are stably maintained in their natural host L. curvatus CRL 705. Both plasmids are low copy number and belong to the theta-type replicating group. While pRC12 is a pUCL287-like plasmid that possesses iterons and the repA and repB genes for replication, pRC18 harbors a 168 amino acid replication protein affiliated to RepB, which was named RepB'. Plasmid pRC18 also possesses a pUCL287-like repA gene but it was disrupted by an 11 kb insertion element that contains RepB', several transposases/IS elements, and the lactocin Lac705 operon. An Escherichia coli / Lactobacillus shuttle vector, named plasmid p3B1, carrying the pRC18 replicon (i.e. repB' and replication origin), a chloramphenicol resistance gene and a pBluescript backbone, was constructed and used to define the host range of RepB'. Chloramphenicol-resistant transformants were obtained after electroporation of Lactobacillus plantarum CRL 691, Lactobacillus sakei 23K and a plasmid-cured derivative of L. curvatus CRL 705, but not of L. curvatus DSM 20019 or Lactococcus lactis NZ9000. Depending on the host, transformation efficiency ranged from 102 to 107 per µg of DNA; in the new hosts, the plasmid was relatively stable as 29-53% of recombinants kept it after cell growth for 100 generations in the absence of selective pressure. Plasmid p3B1 could therefore be used for cloning and functional studies in several Lactobacillus species.

Immunomodulatory Properties of a γ-Aminobutyric Acid-Enriched Strawberry Juice Produced by Levilactobacillus brevis CRL 2013.

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.

Metabolic shift in the production of corrinoid compounds by Lactobacillus coryniformis in the absence of purines.

Lactobacillus coryniformis CRL 1001 and L. reuteri CRL 1098 have the complete genes necessary to synthesize pseudo-cobalamin as final product in a vitamin B12 free commercial medium. Unlike vitaminB12 (the most biologically active form), the pseudo-cobalamin contains adenine instead of 5,6-dimethlbenzimidazole (DMB) in the Coα-ligand. Considering the vitamin B12-gene clusters of these bacteria, the aim of this work was to analyze the production of corrinoids with DMB (vitamin B12) instead of adenine (pseudo-B12) as lower ligand base in a vitamin B12 free chemically defined medium (CDM) without purines. Genome-wide screening of genes related to purine metabolism showed that both strains possess all pur genes necessary for the synthesis of inositol monophosphate, the main precursor for purine biosynthesis. Accordingly, both strains were able to grow in B12 free CDM without purines, with the supplementation of different synthetic intermediaries. Isolated compounds with positive vitamin B12 activity were quantified and characterized by LC/MS-MS. Total corrinoids values were higher for both strains in comparison to those obtained in vitaminB12 free commercial medium. Interestingly, CRL 1001 strain synthesized cobalamin, suggesting that this strain is able to activate DMB as nitrogenous base instead adenine when it is in excess in a purine-free medium. The present paper represents the first demonstration of a partial metabolic shift to produce vitamin B12 in a Lactobacillus strain.

Effect of cheese containing Lactobacillus delbrueckii subsp. Lactis CRL 581 on blood pressure in prehypertensive and stage 1 hypertensive subjects / Efecto de un queso con LactobacillusdelbrueckiisubspLactis CRL 581 sobre la hipertensión arterial en individuos con prehipertensión o estadio 1 de hipertensión arterial

Hypertension is one of the main risk factors for cardiovascular disease. Functional foods containing bioactive peptides have been proposed as a strategy to decrease blood pressure (BP) in subjects under no pharmacological treatment. The aim of this study was to compare the effect of low-sodium, low-fat (LSLF) cheese and LSLF cheese containing Lactobacillus delbrueckii subsp. lactis CRL 581 (LSLF581) on BP in pre-hypertensive and stage 1 hypertensive subjects. Sixty-one pre-hypertensive and stage 1 hypertensive subjects assigned to one of twos (LSLF, n= 29 and LSLF581, n= 32) participated in this 12-month prospective, randomized, double-blind, crossover trial. Twenty-four h ambulatory BP monitoring was performed at the beginning and at the end of each four-week study period. Systolic and diastolic BP decreased in both study groups, but differences between groups were not significant (systolic, -1.78 and -0.2 mmHg; diastolic, -1.54 and -0.42 mmHg in LSLF581 and LSLF, respectively). Although our results could not support a BP lowering effect of LSLF581, small BP reductions could favorably prevent cardiovascular disease development.

La hipertensión arterial es uno de los principales factores de riesgo de enfermedad cardiovascular. Los alimentos funcionales que contienen biopéptidos constituyen una estrategia útil para disminuir la presión arterial (PA) en personas que no están bajo tratamiento farmacológico. El objetivo del estudio fue comparar el efecto de un queso bajo en sodio y bajo en grasas (BSBG) y el mismo queso con Lactobacillus delbrueckii subsp. lactis CRL 581 (BSBG581) sobre la PA en personas con prehipertensión y estadio 1 de hipertensión arterial. Realizamos un estudio prospectivo, randomizado, cruzado y doble ciego durante 12 meses en 61 personas con prehipertensión y estadio 1 de hipertensión arterial, asignadas a dos grupos: BSBG (n= 29) y BSBG581 (n= 32). Se realizó monitoreo ambulatorio de la PA (MAPA) durante 24 h al comienzo y al final de cada etapa del estudio (cuatro semanas). La PA sistólica y diastólica disminuyó en ambos grupos, aunque las diferencias entre grupos no fueron significativas (sistólica, -1.78 y -0.2 mmHg; diastólica -1.54 y -0.42 mmHg en BSBG581 y BSBG respectivamente). Aunque nuestros resultados no pueden confirmar el efecto hipotensor del queso BSBG581, las reducciones moderadas de la PA podrían prevenir el desarrollo de enfermedad cardiovascular.

Diversity and Functional Properties of Lactic Acid Bacteria Isolated From Wild Fruits and Flowers Present in Northern Argentina.

Lactic acid bacteria (LAB) are capable of converting carbohydrate substrates into organic acids (mainly lactic acid) and producing a wide range of metabolites. Due to their interesting beneficial properties, LAB are widely used as starter cultures, as probiotics, and as microbial cell factories. Exploring LAB present in unknown niches may lead to the isolation of unique species or strains with relevant technological properties. Autochthonous rather than allochthonous starter cultures are preferred in the current industry of fermented food products, due to better adaptation and performance of autochthonous strains to the matrix they originate from. In this work, the lactic microbiota of eight different wild tropical types of fruits and four types of flowers were studied. The ability of the isolated strains to produce metabolites of interest to the food industry was evaluated. The presence of 21 species belonging to the genera Enterococcus, Fructobacillus, Lactobacillus, Lactococcus, Leuconostoc, and Weissella was evidenced by using culture-dependent techniques. The isolated LAB corresponded to 95 genotypically differentiated strains by applying rep-PCR and sequencing of the 16S rRNA gene; subsequently, representative strains of the different isolated species were studied for technological properties, such as fast growth rate and acidifying capacity; pectinolytic and cinnamoyl esterase activities, and absence of biogenic amine biosynthesis. Additionally, the strains' capacity to produce ethyl esters as well as mannitol was evaluated. The isolated fruit- and flower-origin LAB displayed functional properties that validate their potential use in the manufacture of fermented fruit-based products setting the background for the design of novel functional foods.

PrfA activation in Listeria monocytogenes increases the sensitivity to class IIa bacteriocins despite impaired expression of the bacteriocin receptor.

BACKGROUND: The scope of the present work was to characterize the activity of class IIa bacteriocins in Listeria (L.) monocytogenes cells that constitutively express an activated form of PrfA, the virulence master regulator, since bacteriocin sensitivity was only characterized in saprophytic cells so far. The mannose phosphotransferase system (Man-PTS) has been shown to be the class IIa bacteriocin receptor in Listeria; hence, special attention was paid to its expression in virulent bacteria. METHODS: L. monocytogenes FBprfA* cells were obtained by transconjugation. Bacterial growth was studied in TSB and glucose containing-minimal medium. Sensitivity to antimicrobial peptides was assessed by killing curves. Membranes of L. monocytogenes FBprfA* cells were characterized using proteomic and lipidomic approaches. RESULTS: The mannose phosphotransferase system (Man-PTS) was downregulated upon expression of PrfA*, and these cells turned out to be more sensitive to enterocin CRL35 and pediocin PA-1, while not to nisin. Proteomic and lipidomic analysis showed differences between wild type (WT) and PrfA* strains. For instance, phosphatidic acid was only detected in PrfA* cells, whereas, there was a significant decline of plasmalogen-phosphatidylglycerol in the same strain. CONCLUSIONS: Our results support a model in which Man-PTS acts just as a docking molecule that brings class IIa bacteriocins to the plasma membrane. Furthermore, our results suggest that lipids play a crucial role in the mechanism of action of bacteriocins. GENERAL SIGNIFICANCE: This is the first demonstration of the link between L. monocytogenes virulence and the bacterial sensitivity toward pediocin-like peptides.

Draft Genome Sequence of Enterococcus faecalis Strain CECT7121, a Corn Silage Isolate with Antibacterial Activity against Gram-Positive Pathogens.

Enterococcus faecalis CECT7121 is a corn silage probiotic bacterium that shows antibacterial activity against Gram-positive pathogens from different origins. Its genome sequence is 2.9 Mb long with a G+C content of 37.3%. Genome annotation identified three bacteriocin gene clusters in the genome.