Production of recombinant glycosidases fused with Usp45 and SpaX to avoid the purification and immobilization stages.

The elucidation of the physicochemical properties of glycosidases is essential for their subsequent technological application, which may include saccharide hydrolysis processes and oligosaccharide synthesis. As the application of cloning, purification and enzymatic immobilization methods can be time consuming and require a heavy financial investment, this study has validated the recombinant production of the set of Lacticaseibacillus rhamnosus fucosidases fused with Usp45 and SpaX anchored to the cell wall of Lacticaseibacillus cremoris subsp cremoris MG1363, with the aim of avoiding the purification and stabilization steps. The cell debris harboring the anchored AlfA, AlfB and AlfC fucosidases showed activity against p-nitrophenyl α-L-fucopyranoside of 6.11 ±â€¯0.36, 5.81 ±â€¯0.29 and 9.90 ±â€¯0.58 U/mL, respectively, and exhibited better thermal stability at 50 °C than the same enzymes in their soluble state. Furthermore, the anchored AlfC fucosidase transfucosylated different acceptor sugars, achieving fucose equivalent concentrations of 0.94 ±â€¯0.09 mg/mL, 4.11 ±â€¯0.21 mg/mL, and 4.08 ±â€¯0.15 mg/mL of fucosylgalatose, fucosylglucose and fucosylsucrose, respectively.

Enterocins Produced by Enterococci Isolated from Breast-Fed Infants: Antilisterial Potential.

Enterocins are bacteriocins synthesized by Enterococcus strains that show an interesting antimicrobial effectiveness against foodborne pathogens such as Listeria monocytogenes. The objectives of this study were to identify and analyze the expression of enterocin genes of Enterococcus isolated from breast-fed infants and evaluate their ability to inhibit three human isolates of virulent L. monocytogenes, as well as some probiotic bacteria. The susceptibility of the strains of L. monocytogenes to fifteen antibiotics was tested, detecting their resistance to cefoxitin (constitutively resistant), oxacillin, and clindamycin. The production of enterocins A, B, and P was observed in Enterococcus faecium isolates, while enterocin AS-48 was detected in an Enterococcus faecalis isolate. AS-48 showed antilisterial activity by itself, while the joint action of enterocins A and B or B and P was necessary for inhibiting L. monocytogenes, demonstrating the synergistic effect of those combinations. The presence of multiple enterocin genes does not assure the inhibition of L. monocytogenes strains. However, the expression of multiple enterocin genes showed a good correlation with the inhibition capacity of these strains. Furthermore, the potential beneficial strains of lactobacilli and bifidobacteria examined were not inhibited by any of the enterocins produced individually or in combination, with the exception of Bifidobacterium longum BB536, which was inhibited by enterocin AS-48 and the joint production of enterocins A and B or B and P. The enterocins studied here could be candidates for developing alternative treatments against antibiotic-resistant bacterial infections. Moreover, these selected enterocin-producing E. faecium strains isolated from breast-fed infants could be used as probiotic strains due to their antilisterial effect, as well as the absence of virulence factors.

Food phenolics and Lactiplantibacillus plantarum.

Phenolic compounds are important constituents of plant food products. These compounds play a key role in food characteristics such as flavor, astringency and color. Lactic acid bacteria are naturally found in raw vegetables, being Lactiplantibacillus plantarum the most commonly used commercial starter for the fermentation of plant foods. Hence, the metabolism of phenolic compounds of L. plantarum has been a subject of study in recent decades. Such studies confirm that L. plantarum, in addition to presenting catalytic capacity to transform aromatic alcohols and phenolic glycosides, exhibits two main differentiated metabolic routes that allow the biotransformation of dietary hydroxybenzoic and hydroxycinnamic acid-derived compounds. These metabolic pathways lead to the production of new compounds with new biological and organoleptic properties. The described metabolic pathways involve the action of specialized esterases, decarboxylases and reductases that have been identified through genetic analysis and biochemically characterized. The purpose of this review is to provide a comprehensive and up-to-date summary of the current knowledge of the metabolism of food phenolics in L. plantarum.

Characterization and stabilization of GluLm and its application to deglycosylate dietary flavonoids and lignans.

This study describes the characterization of the recombinant GH3 aryl-ß-glucosidase "GluLm" from Limosilactobacillus mucosae INIA P508, followed by its immobilization on an agarose support with the aim of developing an efficient application to increase the availability and concentration of flavonoid and lignan aglycones in a vegetal beverage. In previous studies, heterologous GluLm-producing strains demonstrated a great capacity to deglycosylate flavonoids. Nevertheless, the physicochemical properties and substrate spectrum of the enzyme remained unknown up to now. A high production of purified GluLm was achieved (14 mg L-1). GluLm exhibited optimal activity at broad ranges of pH (5.0-8.0) and temperature (25-60°C), as well as high affinity (Km of 0.10 mmol L-1) and specific constant (86554.0 mmol L-1 s-1) against p-nitrophenyl-ß-D-glucopyranoside. Similar to other GH3 ß-glucosidases described in lactic acid bacteria, GluLm exhibited ß-xylosidase, ß-galactosidase, and ß-fucosidase activities. However, this study has revealed for the first time that a GH3 ß-glucosidase is capable to hydrolyze different families of glycosylated phenolics such as flavonoids and secoiridoids. Although it exhibited low thermal stability, immobilization of GluLm improved its thermostability and allowed the development of a beverage based on soybeans and flaxseed extract with high concentration of bioactive isoflavone (daidzein, genistein), lignan (secoisolariciresinol, pinoresinol, and matairesinol), and other flavonoid aglycones. KEY POINTS: • Limosilactobacillus mucosae INIA P508 GluLm was purified and biochemically characterized • Immobilized GluLm efficiently deglycosylated flavonoids and lignans from a vegetal beverage • A viable application to produce vegetal beverages with a high content of aglycones is described.

Riboflavin bio-enrichment of soy beverage by selected roseoflavin-resistant and engineered lactic acid bacteria.

Some lactic acid bacteria (LAB) have the ability to synthesize riboflavin, a trait linked to the presence of ribG, ribB, ribA and ribH genes located in the rib operon. Previous screening of riboflavin producers identified several LAB strains belonging to different species with this ability, but none of them surpassed 0.25 mg/L production of the vitamin. In this study, we explored two strategies to obtain riboflavin-overproducing strains: by roseoflavin selection of mutants, and by the transformation of selected strains with plasmids pNZ:TuR.rib or pNZ:TuB.rib containing the genes ribG, ribB, ribA and ribH from Lactococcus cremoris MG1363. The resulting riboflavin-overproducing strains were able to produce yields between 0.5 and 6 mg/L in culture media and several of them were selected for the fermentation of soy beverages. Riboflavin in bio-enriched soy beverages was evaluated by direct fluorescence measurement and high-performance liquid chromatography-fluorescence analysis. Soy beverages fermented with the recombinant strains Lactococcus cremoris ESI 277 pNZ:TuB.rib and Lactococcus lactis INIA 12 pNZ:TuR.rib showed the highest riboflavin yields (>5 mg/L) after 24 h fermentation. On the other hand, roseoflavin-resistant mutant Limosilactobacillus fermentum INIA P143R2 was able to enrich fermented soy beverages with 1.5 mg/L riboflavin. Riboflavin-overproducing LAB strains constitute a good option for riboflavin enrichment of soy beverages by fermentation and the commercialization of such beverages could be very useful to prevent riboflavin deficiency.

Effect of fermented soy beverage in aged female mice model.

Soy beverage is a rich source of phytoestrogens isoflavones, with potential benefits on health. The effect of those compounds depends greatly on their bacterial metabolization into their aglycone forms. This study evaluated the health effects of two soy beverages, non-fermented (SB) and fermented with Bifidobacterium pseudocatenulatum INIA P815 (FSB), in acyclic and cyclic C57BL/6J aged female mice as a model of menopause and premenopause, respectively. SB and FSB treatments were administrated for 36 days and, subsequently, body weight, lipid and inflammatory profile and fertility were analyzed and compared. In addition, hepatic gene expression and faecal microbiota composition were also assessed. After fermentation, FSB presented a high content in the aglycones daidzein and genistein and a higher antioxidant activity. FSB treated cyclic mice showed a significant increase in the number of retrieved oocytes and zigotes. Differences in serum lipids were observed in triglycerides, which were lower in FSB than in SB groups. None of the treatments influenced the inflammatory profile or caused a dramatic change in the intestinal microbiota profile or hepatic gene expression in any of the groups. Our data showed that FSB provided greater health benefits than SB in lipid profile and fertility in cyclic mice. These beneficial effects could be attributed to the fermentation process, which produces more bioavailable and bioactive compounds, achieving a greater impact on health.

Fermented soy beverages as vehicle of probiotic lactobacilli strains and source of bioactive isoflavones: A potential double functional effect.

Soy beverages can be a source of bioactive isoflavones, with potential human health benefits. In this work, the suitability of three Lacticaseibacillus and three Bifidobacterium probiotic strains as functional starters for soy beverage fermentation were evaluated, alongside with the effect of refrigerated storage on the viability of the strains and the isoflavone composition of the fermented beverages. The three bifidobacteria strains suffered a decrease in their viability during refrigeration and only Bifidobacterium breve INIA P734 produced high concentrations of bioactive isoflavones. Meanwhile, L. rhamnosus GG and L. rhamnosus INIA P344 produced high levels of aglycones and, with L. paracasei INIA P272, maintained their viability during the refrigeration period, constituting promising starters to obtain functional soy beverages that could gather the benefits of the bioactive isoflavone aglycones and the probiotic strains. Moreover, the three lactobacilli caused an increase in the antioxidant capacity of the fermented beverages, which was maintained over the refrigerated storage.

Isoflavone Metabolism by Lactic Acid Bacteria and Its Application in the Development of Fermented Soy Food with Beneficial Effects on Human Health.

Isoflavones are phenolic compounds (considered as phytoestrogens) with estrogenic and antioxidant function, which are highly beneficial for human health, especially in the aged population. However, isoflavones in foods are not bioavailable and, therefore, have low biological activity. Additionally, their transformation into bioactive compounds by microorganisms is necessary to obtain bioavailable isoflavones with beneficial effects on human health. Many lactic acid bacteria (LAB) can transform the methylated and glycosylated forms of isoflavones naturally present in foods into more bioavailable aglycones, such as daidzein, genistein and glycitein. In addition, certain LAB strains are capable of transforming isoflavone aglycones into compounds with a greater biological activity, such as dihydrodaidzein (DHD), O-desmethylangolensin (O-DMA), dihydrogenistein (DHG) and 6-hydroxy-O-desmethylangolensin (6-OH-O-DMA). Moreover, Lactococcus garviae 20-92 is able to produce equol. Another strategy in the bioconversion of isoflavones is the heterologous expression of genes from Slackia isoflavoniconvertens DSM22006, which have allowed the production of DHD, DHG, equol and 5-hydroxy-equol in high concentrations by engineered LAB strains. Accordingly, the consequences of isoflavone metabolism by LAB and its application in the development of foods enriched in bioactive isoflavones, as well as health benefits attributed to their consumption, will be addressed in this work.

Polymerase chain reaction for molecular detection of the genes involved in the production of riboflavin in lactic acid bacteria.

Some lactic acid bacteria (LAB) strains have the ability to synthesize riboflavin, a trait linked to the presence of ribG, ribB, ribA and ribH genes in the rib operon. Multiple sequence alignments of these genes showed that these sequences are not identical in different LAB species, so primers designed to detect these genes in one species do not always work with others. Therefore, we designed degenerate primers based on sequences from Lactococcus lactis MG1363, Levilactobacillus brevis ATCC 367 and Limosilactobacillus fermentum IFO3956, and established optimal PCR conditions for the detection of rib genes in different LAB species. Simultaneously, we selected riboflavin-producing LAB strains from our bacterial collection belonging to the species L. brevis, L. fermentum, L. lactis, Leuconostoc mesenteroides and Lactiplantibacillus plantarum, and we were able to detect ribG, ribB, ribA and ribH genes in these strains by PCR using the designed primers. Thus, the development of degenerate primers and optimal PCR conditions for the detection of ribG, ribB, ribA and ribH genes in LAB allowed the detection and the selection of potential riboflavin-producing strains of different species, which could be good candidates for the development of riboflavin-enriched functional foods.

Characterization and stabilization of the α-L-fucosidase set from Lacticaseibacillus rhamnosus INIA P603.

This study describes the molecular identification, biochemical characterization, and stabilization of three recombinant AlfA, AlfB, and AlfC fucosidases from Lacticaseibacillus rhamnosus INIA P603. Even though previous studies revealed the presence of fucosidase activity in L. rhamnosus extracts, the identification of the fucosidases, their physicochemical properties, and the substrate spectrum remained unknown. Although the presence of alfB is not common in strains of L. rhamnosus, fucosidases from L. rhamnosus INIA P603 were selected because this strain exhibited higher fucosidase activity in culture and the complete set of fucosidases. A high yield of purified recombinant AlfA, AlfB, and AlfC fucosidases was obtained (8, 12, and 18 mg, respectively). AlfA, AlfB, and AlfC showed their optimal activities at pH 5.0 and 4.0 at 60 °C, 40 °C, and 50 °C, respectively. Unlike 3-fucosyllactose, all three recombinant fucosidases were able to hydrolyze 2'-fucosyllactose (2'-FL), and their activities were improved through their immobilization on agarose supports. Nevertheless, immobilized AlfB exhibited the highest hydrolysis, releasing 39.6 µmol of fucose mg enzyme-1 min-1. Only the immobilized AlfB was able to synthetize 2'-FL. In conclusion, the enzymatic properties elucidated in this study support the potential ability of fucosidases from L. rhamnosus INIA P603 to hydrolyze fucosylated substrates as well as justifying interest for further research into AlfB for its application to catalyze the synthesis of fucosylated prebiotics. KEY POINTS: • Few strains of L. rhamnosus exhibited alfB on their chromosomes. • Fucosidases from L. rhamnosus INIA P603 were characterized and stabilized. • Although all the fucosidases hydrolyzed 2'-FL, only AlfB transfucosylated lactose.

Promoters for the expression of food-grade selectable markers in lactic acid bacteria and bifidobacteria.

The genetic engineering of bacteria for food applications has biosafety requirements, including the use of non-antibiotic selectable markers. These can be gene-encoding bacteriocin immunity proteins, such as nisI and pedB, which require the use of promoters to ensure optimal expression. Our aim was to search for promoters for the expression of pediocin (pedB) and nisin (nisI) immunity genes, which could allow the selection of a wide variety of transformed lactic acid bacteria (LAB) and bifidobacteria strains. Eight promoters from LAB or bifidobacteria were initially studied using evoglow-Pp1 as the reporter gene in Lactococcus lactis NZ9000, resulting in the selection of P32, P3N, PTuR and PEF-P, which exhibited a strong constitutive expression. These promoters were further tested for the expression of the food-grade selectable markers pedB and nisI in agar diffusion assays with pediocin and nisin, respectively. The results obtained demonstrated that both the PTuR and PEF-P promoters allowed a good level of expression of nisI and pedB in the LAB and bifidobacteria strains tested. A suitable concentration of nisin or pediocin could be established for the selection of the strains transformed with vectors harbouring the combination of the selected promoters and markers nisI and pedB, and this was successfully applied to different strains of LAB and bifidobacteria. Therefore, PTuR and PEF-P promoters are excellent candidates for the expression of nisI and/or pedB as selectable markers in LAB and bifidobacteria, and they are suitable for use in food grade vectors to allow the selection of genetically engineered strains. KEY POINTS: • Food-grade vectors require non-antibiotic selectable markers such as pedB and nisI. • Eight promoters from LAB or bifidobacteria were initially tested in L. lactis NZ9000. • PTuR and PEF-P efficiently drove the expression of pedB and nisI in LAB and bifidobacteria.

Evoglow-Pp1 and mCherry proteins: a dual fluorescent labeling system for lactic acid bacteria.

Fluorescent proteins are widely used for cell and protein tracking. Most of these proteins show a high signal and need the presence of oxygen to emit fluorescence. Among them, the fluorescent protein mCherry stands up because of its bright signal and fast maturation. Furthermore, the anaerobic cyan-green fluorescent protein Evoglow-Pp1 allows fluorescent detection under anaerobic conditions. In this work, we modified the pNZ:TuR.aFP plasmid, which harbors the gene encoding Evoglow-Pp1 and the promoter of elongation factor Tu from Limosilactobacillus reuteri CECT925, to obtain a plasmid containing the mrfp gene encoding the monomeric mCherry (pNZ:TuR.mCherry). Moreover, both genes were cloned together (pNZ:TuR.aFP.mCherry) developing a chimeric protein; and with a stop codon between them (pNZ:TuR.aFP.STOP.mCherry) resulting in the expression of both Evoglow-Pp1 and mCherry proteins separately under the influence of the same promoter. Lactococcus lactis, Lacticaseibacillus casei, Lactiplantibacillus plantarum, Limosilactobacillus fermentum, Lacticaseibacillus rhamnosus, and L. reuteri strains were transformed with the previously mentioned plasmids, showing an excellent red (pNZ:TuR.mCherry), green (pNZ:TuR.aFP), and red combined with green (pNZ:TuR.aFP.mCherry and pNZ:TuR.aFP.STOP.mCherry) fluorescence signal. Both fluorescence emissions were stable in strains transformed with pNZ:TuR.aFP.STOP.mCherry, while differences in the red or green fluorescence emission were observed in some of the strains harboring pNZ:TuR.aFP.mCherry. Moreover, these plasmids allowed strains differentiation in a complex environment, such as fecal microbiota. Hence, we present the plasmid pNZ:TuR.aFP.STOP.mCherry as a useful tool for the labeling of lactobacilli strains, which would be functional under anoxic conditions, thanks to Evoglow-Pp1, while having the high brightness and good photostability of mCherry. KEY POINTS: • LAB transformed with pNZ:TuR.mCherry expressed the red fluorescent protein mCherry. • LAB transformed with pNZ:TuR.aFP.mCherry developed a fusion of both proteins Evoglow-Pp1 and mCherry. • LAB with pNZ:TuR.aFP.STOP.mCherry expressed both fluorescent proteins separately.

Architecture Insight of Bifidobacterial α-L-Fucosidases.

Fucosylated carbohydrates and glycoproteins from human breast milk are essential for the development of the gut microbiota in early life because they are selectively metabolized by bifidobacteria. In this regard, α-L-fucosidases play a key role in this successful bifidobacterial colonization allowing the utilization of these substrates. Although a considerable number of α-L-fucosidases from bifidobacteria have been identified by computational analysis, only a few of them have been characterized. Hitherto, α-L-fucosidases are classified into three families: GH29, GH95, and GH151, based on their catalytic structure. However, bifidobacterial α-L-fucosidases belonging to a particular family show significant differences in their sequence. Because this fact could underlie distinct phylogenetic evolution, here extensive similarity searches and comparative analyses of the bifidobacterial α-L-fucosidases identified were carried out with the assistance of previous physicochemical studies available. This work reveals four and two paralogue bifidobacterial fucosidase groups within GH29 and GH95 families, respectively. Moreover, Bifidobacterium longum subsp. infantis species exhibited the greatest number of phylogenetic lineages in their fucosidases clustered in every family: GH29, GH95, and GH151. Since α-L-fucosidases phylogenetically descended from other glycosyl hydrolase families, we hypothesized that they could exhibit additional glycosidase activities other than fucosidase, raising the possibility of their application to transfucosylate substrates other than lactose in order to synthesis novel prebiotics.

The commensal bacterium Lactiplantibacillus plantarum imprints innate memory-like responses in mononuclear phagocytes.

Gut microbiota is a constant source of antigens and stimuli to which the resident immune system has developed tolerance. However, the mechanisms by which mononuclear phagocytes, specifically monocytes/macrophages, cope with these usually pro-inflammatory signals are poorly understood. Here, we show that innate immune memory promotes anti-inflammatory homeostasis, using as model strains of the commensal bacterium Lactiplantibacillus plantarum. Priming of monocytes/macrophages with bacteria, especially in its live form, enhances bacterial intracellular survival and decreases the release of pro-inflammatory signals to the environment, with lower production of TNF and higher levels of IL-10. Analysis of the transcriptomic landscape of these cells shows downregulation of pathways associated with the production of reactive oxygen species (ROS) and the release of cytokines, chemokines and antimicrobial peptides. Indeed, the induction of ROS prevents memory-induced bacterial survival. In addition, there is a dysregulation in gene expression of several metabolic pathways leading to decreased glycolytic and respiratory rates in memory cells. These data support commensal microbe-specific metabolic changes in innate immune memory cells that might contribute to homeostasis in the gut.

Heterologous production of equol by lactic acid bacteria strains in culture medium and food.

The isoflavones daidzin and genistin, present in soybeans, can be transformed by the intestinal microbiota into equol and 5-hydroxy-equol, compounds with enhanced availability and bioactivity, although these are only produced by a fraction of the population. Hence, there is an interest in the production of these compounds, although, to date, few bacteria with biotechnological interest and applicability in food have been found able to produce equol. In order to obtain lactic acid bacteria able to produce equol, the daidzein reductase (dzr), dihydrodaidzein reductase (ddr), tetrahydrodaidzein reductase (tdr) and dihydrodaidzein racemase (ifcA) genes, from Slackia isoflavoniconvertens DSM22006, were cloned into the vector pNZ:TuR, under a strong constitutive promoter (TuR). Lactococcus lactis MG1363, Lacticaseibacillus casei BL23, Lactiplantibacillus plantarum WCFS1, Limosilactobacillus fermentum INIA 584L and L. fermentum INIA 832L, harbouring pNZ:TuR.tdr.ddr, were able to produce equol from dihydrodaidzein, while L. fermentum strains showed also production of 5-hydroxy-equol from dihydrogenistein. The metabolization of daidzein and genistein by the combination of strains harbouring pNZ:TuR.dzr and pNZ:TuR.tdr.ddr showed similar results, and the addition of the correspondent strain harbouring pNZ:TuR.ifcA resulted in an increase of equol production, but only in the L. fermentum strains. This pattern of equol and 5-hydroxy-equol production by L. fermentum strains was also confirmed in cow's milk supplemented with daidzein and genistein and incubated with the different combination of strains harbouring the constructed plasmids. Bacteria generally recognized as safe (GRAS), such as the lactic acid bacteria species used in this work, harbouring these plasmids, would be of value for the development of fermented vegetal foods enriched in equol and 5-hydroxy-equol.

The use of Lactobacillus plantarum esterase genes: a biotechnological strategy to increase the bioavailability of dietary phenolic compounds in lactic acid bacteria.

In Lactobacillus plantarum the metabolism of hydroxybenzoic and hydroxycinnamic acid derivatives follows a similar two-step pathway, an esterase action followed by a decarboxylation. The L. plantarum esterase genes involved in these reactions have been cloned into pNZ8048 or pT1NX plasmids and transformed into technologically relevant lactic acid bacteria. None of the strains assayed can hydrolyse methyl gallate, a hydroxybenzoic ester. The presence of the L. plantarum tannase encoding genes (tanALp or tanBLp) on these bacteria conferred their detectable esterase (tannase) activity. Similarly, on hydroxycinnamic compounds, esterase activity for the hydrolysis of ferulic acid was acquired by lactic acid bacteria when L. plantarum esterase (JDM1_1092) was present. This study showed that the heterologous expression of L. plantarum esterase genes involved in the metabolism of phenolic acids allowed the production of healthy compounds which increase the bioavailability of these dietary compounds in food relevant lactic acid bacteria.

Catabolite responsive elements as a strategy for the control of heterologous gene expression in lactobacilli.

Genes involved in the transport and catabolism of carbohydrates are usually controlled through the binding of the catabolite control protein A (CcpA) to the catabolite-responsive elements (cre) of target genes in Gram-positive bacteria. In this work, we show how the elimination of the cre sites in Lactobacillus casei BL23 promoters induced by sorbitol (PgutF), maltose (PmalL), and myo-inositol (PiolT) allowed the induction of gene expression in media supplemented with sorbitol, maltose, and myo-inositol, respectively, even in the presence of glucose. This was studied using plasmids encoding the anaerobic fluorescent protein evoglow-Pp1 as a reporter. In addition, gutF cre site was introduced into a bile inducible promoter (P16090) and into the constitutive promoter of the elongation factor P (PEf-P) of L. casei BL23. The existence of the cre site blocked gene expression in the P16090 inducible promoter in the presence of glucose, while it had no influence on the expression of the PEf-P constitutive one. These results demonstrated that the introduction or elimination of cre sites in inducible promoters allows the control and modification of their heterologous genetic expression, showing how the cre site, the transcriptional regulator, and CcpA interact to control gene expression in inducible genes. KEY POINTS: • Cre sequences regulate gene expression in inducible promoters in L. casei BL23. • Cre sites do not affect gene expression in constitutive promoters in L. casei BL23. • Cre sequences could control heterologous genic expression in lactobacilli.

Bile-induced promoters for gene expression in Lactobacillus strains.

Bioengineering of probiotics allows the improvement of their beneficial characteristics. In this work, we develop a molecular tool that would allow the activation of desirable traits in probiotics once they reach the intestine. The activity of upstream regions of bile-inducible genes of Lactobacillus casei BL23 and Lactobacillus plantarum WCFS1 was analyzed using plasmids encoding an anaerobic fluorescent protein as reporter. The promoter P16090 from Lb. casei BL23 was selected and its bile induction confirmed in Lb. casei BL23, Lb. plantarum WCFS1, and in Lactobacillus rhamnosus and Lactobacillus reuteri strains. However, the induction did not occur in Lactococcus lactis MG1363 or Bifidobacterium strains. Studies with different bile compounds revealed the importance of cholic acid in the bile induction process. Induction of fluorescence was also confirmed for transformed Lb. casei BL23 under simulated colonic conditions and in the presence of intestinal microbiota. The developed vector, pNZ:16090-aFP, constitutes a promising tool suitable for the expression of genes of interest under intestinal conditions in probiotic strains of the species Lb. casei, Lb. plantarum, Lb. rhamnosus, and Lb. reuteri.

Incomplete metabolism of phytoestrogens by gut microbiota from children under the age of three.

Phytoestrogens are plant-derived polyphenols with structural and functional similarities to mammalian oestrogens. The aim of this work was to study the metabolism of phytoestrogens by children's intestinal microbiota and to compare it with previous results in adults. Faecal samples of 24 healthy children were subjected to phytoestrogen fermentation assay. Only one child produced equol, while O-desmethylangolensin was found in all. Urolithin production was detected in 14 children and enterolactone in 10. Further comparison with the metabolism of phytoestrogens by adult intestinal microbiota reflected that glycitein, dihydrogenistein, urolithins D and E, enterolactone, secoisolariciresinol and arctigenin were the most important metabolites differentiating between adult and child microbial gut metabolism. Although the child intestinal microbiota showed the ability to metabolise isoflavones, ellagitannins and lignans to a certain extent, it generally showed a reduced metabolism of phytoestrogens, with a lack of 5-hydroxy equol and enterodiol, and less urolithins and enterolactone producers.

Probiotic Bacteria for Healthier Aging: Immunomodulation and Metabolism of Phytoestrogens.

Age-related degeneration gives rise to a number of pathologies, many of them associated with imbalances of the microbiota and the gut-associated immune system. Thus, the intestine is considered a key target organ to improve the quality of life in senescence. Gut microbiota can have a powerful impact in the deterioration linked to aging by its nutritional and immunomodulatory activity. Reduced numbers of beneficial species and low microbial biodiversity in the elderly have been linked with pathogenesis of many diseases. A healthy lifestyle with an elderly customized diet including probiotics can contribute to reducing the chronic proinflammatory status and other age-related pathologies. Beneficial effects of probiotic lactic acid bacteria and bifidobacteria to alleviate some of these disorders based on their immunomodulatory properties as well as their capacity to produce bioactive metabolites from dietary phytoestrogens are summarized. On one hand, the preservation of gut barrier integrity and an increased ability to fight infections are the main reported immune benefits of probiotics. On the other hand, the intake of a diet rich in phytoestrogens along with the presence of selected probiotic bacteria may lead to the production of equol, enterolignans, and urolithins, which are considered protective against chronic diseases related to aging.