Fernando Sesma

Bacteria as vitamin suppliers to their host: a gut microbiota perspective

Food-related lactic acid bacteria (LAB) as well as human gut commensals such as bifidobacteria can de novo synthesize and supply vitamins. This is important since humans lack the biosynthetic capacity for most vitamins and these must thus be provided exogenously. Although vitamins are present in a variety of foods, deficiencies still occur, mainly due to malnutrition as a result of insufficient food intake and because of poor eating habits. Fermented milks with high levels of B-group vitamins (such as folate and riboflavin) can be produced by LAB-promoted and possibly bifidobacteria-promoted biosynthesis. Moreover, certain strains of LAB produce the complex vitamin cobalamin (or vitamin B12). In this review, fermented foods with elevated levels of B-group vitamins produced by LAB used as starter cultures will be covered. In addition, genetic abilities for vitamin biosynthesis by selected human gut commensals will be discussed.

B-group vitamin production by lactic acid bacteria--current knowledge and potential applications.

Although most vitamins are present in a variety of foods, human vitamin deficiencies still occur in many countries, mainly because of malnutrition not only as a result of insufficient food intake but also because of unbalanced diets. Even though most lactic acid bacteria (LAB) are auxotrophic for several vitamins, it is now known that certain strains have the capability to synthesize water‐soluble vitamins such as those included in the B‐group (folates, riboflavin and vitamin B12 amongst others). This review article will show the current knowledge of vitamin biosynthesis by LAB and show how the proper selection of starter cultures and probiotic strains could be useful in preventing clinical and subclinical vitamin deficiencies. Here, several examples will be presented where vitamin‐producing LAB led to the elaboration of novel fermented foods with increased and bioavailable vitamins. In addition, the use of genetic engineering strategies to increase vitamin production or to create novel vitamin‐producing strains will also be discussed. This review will show that the use of vitamin‐producing LAB could be a cost‐effective alternative to current vitamin fortification programmes and be useful in the elaboration of novel vitamin‐enriched products.

Lactobacillus reuteri CRL1098 Produces Cobalamin

We found that Lactobacillus reuteri CRL1098, a lactic acid bacterium isolated from sourdough, is able to produce cobalamin. The sugar-glycerol cofermentation in vitamin B(12)-free medium showed that this strain was able to reduce glycerol through a well-known cobalamin-dependent reaction with the formation of 1,3-propanediol as a final product. The cell extract of L. reuteri corrected the coenzyme B12 requirement of Lactobacillus delbrueckii subsp. lactis ATCC 7830 and allowed the growth of Salmonella enterica serovar Typhimurium (metE cbiB) and Escherichia coli (metE) in minimal medium. Preliminary genetic studies of cobalamin biosynthesis genes from L. reuteri allowed the identification of cob genes which encode the CobA, CbiJ, and CbiK enzymes involved in the cobalamin pathway. The cobamide produced by L. reuteri, isolated in its cyanide form by using reverse-phase high-pressure liquid chromatography, showed a UV-visible spectrum identical to that of standard cyanocobalamin (vitamin B12).

Metabolic engineering of lactic acid bacteria for the production of nutraceuticals

Lactic acid bacteria display a relatively simple and well-described metabolism where the sugar source is converted mainly to lactic acid. Here we will shortly describe metabolic engineering strategies on the level of sugar metabolism, that lead to either the efficient re-routing of the lactococcal sugar metabolism to nutritional end-products other than lactic acid such as L-alanine, several low-calorie sugars and oligosaccharides or to enhancement of sugar metabolism for complete removal of (undesirable) sugars from food materials. Moreover, we will review current metabolic engineering approaches that aim at increasing the flux through complex biosynthetic pathways, leading to the production of the B-vitamins folate and riboflavin. An overview of these metabolic engineering activities can be found on the website of the Nutra Cells 5th Framework EU-project (www.nutracells.com). Finally, the impact of the developments in the area of genomics and corresponding high-throughput technologies on nutraceutical production will be discussed.

Heterofermentative pattern and exopolysaccharide production by Lactobacillus helveticus ATCC 15807 in response to environmental pH

Aims: The objective of this work was to evaluate the fermentation pattern of and the exopolysaccharide (EPS) production by Lactobacillus helveticus ATCC 15807 in milk batch cultures under controlled pH (4·5, 5·0 and 6·2).

Combined Effect of Bacteriocins on the Survival of Various Listeria Species in Broth and Meat System

The antilisterial efficiency of three bacteriocins from lactic acid bacteria, lactocin 705 (produced by L. casei CRL705, 17000 AU/ml), enterocin CRL35 (produced by E. faecium CRL35, 17000 AU/ml), and nisin (2000 IU/ml), was tested in broth, individually and in combination against Listeria monocytogenes and Listeria innocua. Both Listeria species showed an initial decrease in viable counts followed by the regrowth of the survivors after 1 h in the presence of each bacteriocin. A greater antilisterial effect was observed when the bacteriocins were combined in pairs, maximal inhibition being reached when nisin was involved. When a mix of the three bacteriocins was used, no survivors were observed after 24 h of incubation. Similar results were obtained when the bacteriocin combinations were tested in a meat system, indicating that the use of more than one LAB bacteriocin in combination may be effective in preventing the spontaneous emergence of a bacteriocin-resistant Listeria population.

Use of superoxide dismutase and catalase producing lactic acid bacteria in TNBS induced Crohn's disease in mice.

Reactive oxygen species are involved in various aspects of intestinal inflammation and tumor development. Decreasing their levels using antioxidant enzymes, such as catalase (CAT) or superoxide dismutase (SOD) could therefore be useful in the prevention of certain diseases. Lactic acid bacteria (LAB) are ideal candidates to deliver these enzymes in the gut. In this study, the anti-inflammatory effects of CAT or SOD producing LAB were evaluated using a trinitrobenzenesulfonic acid (TNBS) induced Crohns disease murine model. Engineered Lactobacillus casei BL23 strains producing either CAT or SOD, or the native strain were given to mice before and after intrarectal administration of TNBS. Animal survival, live weight, intestinal morphology and histology, enzymatic activities, microbial translocation to the liver and cytokines released in the intestinal fluid were evaluated. The mice that received CAT or SOD-producing LAB showed a faster recovery of initial weight loss, increased enzymatic activities in the gut and lesser extent of intestinal inflammation compared to animals that received the wild-type strain or those that did not receive bacterial supplementation. Our findings suggest that genetically engineered LAB that produce antioxidant enzymes could be used to prevent or decrease the severity of certain intestinal pathologies.

Enterocin CRL35 inhibits late stages of HSV-1 and HSV-2 replication in vitro

The replication of herpes simplex virus (HSV) type 1 and 2 in Vero cells is inhibited in the presence of enterocin CRL35 (ECRL), a bacteriocin produced by Enterococcus faecium CRL35. Attempts to resolve the mode of action of ECRL indicate that virus adsorption and penetration are not affected. Instead, a late step of virus multiplication is hindered since the addition of 100 microg/ml of ECRL at 8h post infection still causes a 90% inhibition of virus release. The effect of ECRL on HSV antigen expression was studied by immunofluorescence using a polyclonal serum and a monoclonal antibody against glycoprotein D (gamma protein). These studies indicated that ECRL impeded the second round of infection, apparently as a consequence of the inhibition of glycoprotein D expression. The replication of syncytial mutants of HSV-1 was significantly inhibited at a ECRL concentration of 25 microg/ml. Both the percentage of fused cells and the polykaryocyte size were affected. Studies on the effect of ECRL on viral protein synthesis showed that in the presence of ECRL, HSV late gamma proteins were not synthesized. From these findings, it is concluded that inhibition of HSV spreading by ECRL is due to the prevention of mainly late glycoprotein synthesis.

The complete coenzyme B12 biosynthesis gene cluster of Lactobacillus reuteri CRL1098

The coenzyme B(12) production pathway in Lactobacillus reuteri has been deduced using a combination of genetic, biochemical and bioinformatics approaches. The coenzyme B(12) gene cluster of Lb. reuteri CRL1098 has the unique feature of clustering together the cbi, cob and hem genes. It consists of 29 ORFs encoding the complete enzymic machinery necessary for de novo biosynthesis. Transcriptional analysis showed it to be expressed as two tandem transcripts of approximately 22 and 4 kb, carrying cobD, cbiABCDETFGHJ, cobA/hemD, cbiKLMNQOP, sirA, hemACBL, and cobUSC, hemD, cobT, respectively. Both transcripts appear to be similarly regulated, and under the conditions assayed are induced in the late-exponential growth phase. Evidence for a regulatory mechanism of negative feedback inhibition by vitamin B(12) itself was observed. Comparative genomics analysis of the coding sequences showed them to be most similar to those coding for the anaerobic coenzyme B(12) pathways previously characterized in a few representatives of the genera Listeria and Salmonella. This contrasts with the trusted species phylogeny and suggests horizontal gene transfer of the B(12) biosynthesis genes. G+C content and codon adaptation index analysis is suggestive that the postulated transfer of these genes was not a recent event. Additional comparative genomics and transcriptional analysis of the sequences acquired during this study suggests a functional link between coenzyme B(12) biosynthesis and reuterin production, which might be implicated in Lb. reuteris success in colonizing the gastrointestinal tract. This information on gene organization, gene transcription and gene acquisition is relevant for the development of (fermented) foods and probiotics enriched in B(12).

Characterization of the melA Locus for α-Galactosidase in Lactobacillus plantarum

ABSTRACT Alpha-galactosides are abundant sugars in legumes such as soy. Because of the lack of α-galactosidase (α-Gal) in the digestive tract, humans are unable to digest these sugars, which consequently induce flatulence. To develop the consumption of the otherwise highly nutritional soy products, the use of exogenous α-Gal is promising. In this framework, we characterized the melA gene for α-Gal in Lactobacillus plantarum. The melA gene encodes a cytoplasmic 84-kDa protein whose enzymatically active form occurs as oligomers. The melA gene was cloned and expressed in Escherichia coli, yielding an active α-Gal. We show that melA is transcribed from its own promoter, yielding a monocistronic mRNA, and that it is regulated at the transcriptional level, i.e., it is induced by melibiose but is not totally repressed by glucose. Posttranscriptional regulation by the carbon source could also occur. Upstream of melA, a putative galactoside transporter, designated RafP, was identified that shows high homology to LacS, the unique transporter for both α- and β-galactosides in Streptococcus thermophilus. rafP is also expressed as a monocistronic mRNA. Downstream of melA, the lacL and lacM genes were identified that encode a heterodimeric β-galactosidase. A putative galM gene identified in the same cluster suggests the presence of a galactose operon. These results indicate that the genes involved in galactoside catabolism are clustered in L. plantarum ATCC 8014. This first genetic characterization of melA and of its putative associated transporter, rafP, in a lactobacillus opens doors to various applications both in the manufacture of soy-derived products and in probiotic and nutraceutical issues.