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Thiamine or vitamin B1, an essential micronutrient mainly involved in energy production, has a beneficial impact on the nervous system, and its deficiency can be associated with the development and progression of neurodegenerative diseases. The aim of this work was to select thiamine-producing lactic acid bacteria (LAB) and study their physiological effects using neuron cell cultures. In this study, 23 LAB able to produce thiamine were identified by growth in thiamine-free synthetic medium. Intra- and extracellular thiamine concentrations were determined using a microbiological method and results confirmed by HPLC techniques. A wide variation in vitamin production was found showing that this property was not only species specific but also a strain-dependent trait. Five of these strains were pre-selected for their capacity to produce higher concentrations of thiamine. Only the pre-treatment with the intracellular extract of Lactiplantibacillus (L.) plantarum CRL 1905 increased significantly neuronal survival in N2a cells' model of neurotoxicity (MPP + ) with thiamine deficiency conditions (amprolium). Furthermore, amprolium-resistant variants of CRL 1905 were isolated by exposition of the strain to increasing concentrations of this toxic thiamine analogue. The variant A9 was able to increase more than 2 times the intracellular thiamine production of the original strain. A9 bacterial extract significantly prevented neuronal cell death and the increase of IL-6. The amprolium-resistant strain A9 showed a modulating and neuroprotective effect in an in vitro model of neurotoxicity constituting a potential bio-strategy to counteract thiamine deficiencies and thus prevent or treat neurodegenerative diseases. KEY POINTS: • LAB can produce thiamine in a species- and strain-dependant manner. • L. plantarum CRL 1905 significantly reduce MPP + -induced neurotoxicity in N2a cells. • Amprolium-resistant strain A9 has neuroprotective effect and prevents IL-6 increase.

Recent studies have shown that inflammatory diseases are becoming more frequent throughout the world. The causes of these disorders are multifactorial and include genetic, immunological, and environmental factors, and intestinal microbiota dysbiosis. The use of beneficial microorganisms has shown to be useful in the prevention and treatment of disorders such as colitis, mucositis, and even colon cancer by their immune-stimulating properties. It has also been shown that certain vitamins, especially riboflavin and folate derivatives, have proven to be helpful in the treatment of these diseases. The application of vitamin-producing lactic acid bacteria, especially strains that produce folate and riboflavin together with immune-stimulating strains, could be used as adjunct treatments in patients suffering from a wide range of inflammatory diseases since they could improve treatment efficiency and prevent undesirable side effects in addition to their nutrition values. In this review, the most up to date information on the current knowledge and uses of vitamin-producing lactic acid bacteria is discussed in order to stimulate further studies in this field.

Inflammatory bowel diseases are chronic and relapsing-remitting disorders that affect the gastrointestinal tract. Previously, the administration of folate and riboflavin-producing lactic acid bacteria (LAB) or an immune-modulating strain showed beneficial effects as they were able to reduce the acute inflammation in mouse models. The aim of this work was to evaluate a mixture of vitamin-producing and immune-modulating LAB administering together with an anti-inflammatory drug during the remission period of a mouse model of recurrent colitis. BALB/c mice were intrarectally instilled with trinitrobenzene sulfonic acid (TNBS) and those who recovered from this acute challenge were given the LAB mixture, mesalazine, or the combination of both (mesalazine + LAB) during 21 days, followed by a second challenge with TNBS. Control mice instilled with ethanol (vehicle of TNBS) and receiving the different treatments were also evaluated in order to study the effect of chronic anti-inflammatory therapy. The combination of mesalazine and LAB mixture was the most effective to decrease the intestinal damage at macroscopic and histological levels and to reduce pro-inflammatory cytokines (IL-6 and TNF-α) in intestinal fluids. In animals instilled with ethanol, mesalazine produced a loss of body weight and intestinal damages with increased IL-6. These side effects were prevented by the co-administration of mesalazine and the LAB mixture. The LAB blend did not affect the primary anti-inflammatory treatment, was able to improve it, and also prevented the side effects of this therapy.

The cell envelope-associated proteinase (CEP) of Lactobacillus delbrueckii subsp. lactis CRL 581 (PrtL) has an essential role in bacterial growth, contributes to the flavor and texture development of fermented products, and can release bioactive health-beneficial peptides during milk fermentation. The genome of L. delbrueckii subsp. lactis CRL 581 possesses only one gene that encodes PrtL, which consists of 1924 amino acids and is a multidomain protein anchored to the cell via its W domain. PrtL was extracted from the cell under high ionic strength conditions using NaCl, suggesting an electrostatic interaction between the proteinase and the cell envelope. The released PrtL was purified and biochemically characterized; its activity was maximal at temperatures between 37 and 40 °C and at pH between 7 and 8. Under optimal conditions, PrtL exhibited higher affinity for succinyl-alanyl-alanyl-prolyl-phenylalanine-p-nitroanilide than for succinyl-alanyl-glutamyl-prolyl-phenylalanine-p-nitroanilide, while methoxy-succinyl-arginyl-prolyl-tyrosyl-p-nitroanilide was not degraded. A similar α- and β-casein degradation pattern was observed with the purified and the cell envelope-bound proteinase. Finally, on the basis of its specificity towards caseins and the unique combination of amino acids at residues thought to be involved in substrate specificity, PrtL can be classified as a representative of a new group of CEP.

Lactobacillus fermentum CECT 5716, isolated from human milk, has immunomodulatory, anti-inflammatory, and anti-infectious properties, as revealed by several in vitro and in vivo assays, which suggests a strong potential as a probiotic strain. In this work, some phenotypic properties of L. fermentum CECT 5716 were evaluated, and the genetic basis for the obtained results was searched for in the strain genome. L. fermentum CECT 5716 does not contain plasmids and showed neither bacteriocin nor biogenic amine biosynthesis ability but was able to produce organic acids, glutathione, riboflavin, and folates and to moderately stimulate the maturation of mouse dendritic cells. No prophages could be induced, and the strain was sensitive to all antibiotics proposed by European Food Safety Authority (EFSA) standards, while no transmissible genes potentially involved in antibiotic resistance were detected in its genome. Globally, there was an agreement between the phenotype properties of L. fermentum CECT 5716 and the genetic information contained in its genome.

Ability of industrially relevant species of thermophilic lactobacilli strains to hydrolyze proteins from animal (caseins and β-lactoglobulin) and vegetable (soybean and wheat) sources, as well as influence of peptide content of growth medium on cell envelope-associated proteinase (CEP) activity, was evaluated. Lactobacillus delbrueckii subsp. lactis (CRL 581 and 654), L. delbrueckii subsp. bulgaricus (CRL 454 and 656), Lactobacillus acidophilus (CRL 636 and 1063), and Lactobacillus helveticus (CRL 1062 and 1177) were grown in a chemically defined medium supplemented or not with 1 % Casitone. All strains hydrolyzed mainly β-casein, while degradation of αs-caseins was strain dependent. Contrariwise, κ-Casein was poorly degraded by the studied lactobacilli. β-Lactoglobulin was mainly hydrolyzed by CRL 656, CRL 636, and CRL 1062 strains. The L. delbrueckii subsp. lactis strains, L. delbrueckii subsp. bulgaricus CRL 656, and L. helveticus CRL 1177 degraded gliadins in high extent, while the L. acidophilus and L. helveticus strains highly hydrolyzed soy proteins. Proteinase production was inhibited by Casitone, the most affected being the L. delbrueckii subsp. lactis species. This study highlights the importance of proteolytic diversity of lactobacilli for rational strain selection when formulating hydrolyzed dairy or vegetable food products.

Alpha-galactosidase (alpha-Gal) enzyme, which is encoded by the melA gene hydrolyzes alpha-1,6 galactoside linkages found in sugars, such as raffinose and stachyose. These alpha-galacto-oligosaccharides (alpha-GOS), which are found in large quantities in vegetables, such as soy, can cause gastrointestinal disorders in sensitive individuals because monogastric animals (including humans) do not posses alpha-Gal in the gut. The use of microbial alpha-Gal is a promising alternative to eliminate alpha-GOS in soy-derived products. Using degenerate primers, the melA gene from Lactobacillus (L.) fermentum CRL722 was identified. The complete genomic sequence of melA (2223 bp), and of the genes flanking melA, were obtained using a combination of polymerase chain reaction-based techniques, and showed strong similarities with the alpha-Gal gene of thermophilic microorganisms. The alpha-Gal gene from L. fermentum CRL722 was cloned and the protein purified from cell-free extracts of the native and recombinant strains using various techniques (ion exchange chromatography, salt precipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and ultra-filtration); Its main biochemical properties were determined. The enzyme was active at moderately high temperatures (55 degrees C) and stable at wide ranges of temperatures and pH. The thermostable alpha-Gal from L. fermentum CRL722 could thus be used for technological applications, such as the removal of alpha-GOS found in soy products. The complete melA gene could also be inserted in other micro-organisms, that can survive in the harsh conditions of the gut to degrade alpha-GOS in situ. Both strategies would improve the overall acceptability of soy-derived products by improving their nutritional value.

The influence of environmental pH on biological activity of Bifidobacterium longum CRL 849 grown in MRS-raffinose was evaluated. At pH 6.0, 5.5 and 5.0, raffinose was completely consumed by this microorganism, showing different consumption rates at each pH value (between 3.03 and 0.76 mmol l(-1) h(-1)). At pH 4.5, the growth was lowest. The removal of raffinose was due to the alpha-galactosidase (alpha-gal) activity of this bifidobacteria, which was highest at pH 6.0-5.5 (1,280-1,223 mU ml(-1)). The production of beta-glucosidase (beta-glu) showed a similar pattern to alpha-gal activity with major values. The yield of organic acids produced during raffinose consumption was also highest at pH 6.0-5.5. The results of this study will allow the selection of the optimum growth conditions of B. longum CRL 849, with elevated levels of alpha-gal to be used in the reduction of nondigestible alpha-oligosaccharide in soy products and beta-glu activities involved in isoflavone conversion to bioactive forms when used as starter culture.

In this study, the behaviour of Lactobacillus fermentum CRL 722 and CRL 251 were evaluated under different pH conditions (pH 6.0, 5.5, 5.0, 4.5) and without pH control. Growth was similar under all conditions assayed except at pH 4.5. These microorganisms were able to eliminate raffinose, a nondigestible alpha-oligosaccharide (NDO) found in soy products, showing a consumption rate of 0.25 g l(-1) h(-1) (pH 6.0-5.0). The removal of raffinose was due to the high alpha-galactosidase (alpha-gal) activities of these lactic acid bacteria, which was highest at pH 5.5 (5.0 U/ml). The yield of organic acids produced during raffinose consumption was also highest at this pH. The results of this study will allow selection of the optimum growth conditions of L. fermentum with elevated levels of alpha-gal to be used in the reduction of NDO in soy products when used as starter cultures.