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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., This research was funded by the Spanish Ministry of Science and Innovation grants PID2020-119630RB-I00 and RYC2019-026368-I.

5 Pág., 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., This work was supported by the project PID2020-11960RB-I00 from the Spanish Ministry of Science and Innovation .

12 Pág. Departamento de Tecnología de Alimentos​​, 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., Funding This study was funded by the project PID2020-11960RB-I00 from the Spanish Ministry of Science, Innovation and Universities

Lignans and flavonoids are found in plants in their glycosylated forms and need to be hydrolyzed to aglycones to become bioavailable. Putative β-glucosidase genes from Lactobacillus mucosae INIA P508 were inserted into the plasmid pNZ:TuR. The strain Lactococcus lactis MG1363 harboring the plasmid pNZ:TuR.glu913 showed high β-glucosidase activity and was able to transform secoisolariciresinol diglucoside (SDG) into secoisolariciresinol (SECO). Lactic acid bacteria and Bifidobacterium strains harboring pNZ:TuR.glu913 were incubated with a soy beverage supplemented with flax seed extracts. SDG was almost completely consumed by the transformed strains, while concentration of SECO greatly increased. Moreover, these strains showed high deglycosylation of the isoflavone glycosides daidzin and genistin. In addition, other lignan and flavonoid aglycones were produced, i.e. matairesinol, pinoresinol, quercetin, and eriodyctiol. These deglycosylase activities were maintained when this glucosidase gene was cloned in a food grade vector, pLEB590, and transformed into L. lactis MG1363. This is the first report of the use of a food grade plasmid that confers the ability to efficiently catalyze the deglycosylation of lignans, isoflavonoids, flavones, and flavanones. The recombinant bacteria of this study would be of value for the development of fermented vegetal foods enriched in bioavailable forms of lignans and flavonoids.

Among vegetable functional products, soy-based foods stand out for their high content in protein and flavonoids,mainly isoflavones. The fermentation of soy beverages with selected bacterial strains is a way to obtain a product,enriched in isoflavone aglycones, alongside with other isoflavone derivates and other bioactive flavonoids. Two bacterial strains with high activity on the transformation of these compounds, Limosilactobacillus mucosae INIA P508 and Bifidobacterium pseudocatenulatum INIA P815, were tested for the fermentation of soy beverage. Both strains produced high concentration of the aglycones daidzein and genistein, in addition to a significant increase (P < 0.01) of glycitein, O-desmethylangolensin, 6′′ -hydroxy-O-desmethylangolensin, tetrahydrodaidzein and other flavonoid aglycones such as kaempferol, naringenin and luteolin. Two alternatives to conserve the developed functional beverages were explored, cold storage and thermal treatment. Refrigeration did not affect the concentration of isoflavones, even observing an increase in aglycones. Similarly, the thermal treatment and further room temperature storage did not reduce the amounts of daidzein and genistein. Therefore, both strains could be used for the development of soy beverages enriched in bioactive isoflavones and other bioactive flavonoids which would maintain their concentrations in refrigeration or at room temperature after heat treatment.

2 Pág. Departamento de Tecnología de Alimentos​​ (INIA), Limosilactobacillus reuteri is a beneficial bacterium that inhabits the gastrointestinal tract of different mammals. Diverse beneficial effects have been attributed to specific strains, in part mediated by the production of reuterin. Here, we report the draft genome sequence of L. reuteri INIA P572, a reuterin-producing strain isolated from pig feces., This work was supported by project RTA2017-00002-00-00 from the Spanish Ministry of Science and Innovation.

11 Pág., Lacticaseibacillus paracasei INIA P272 and Lacticaseibacillus rhamnosus INIA P344, isolated from breast-fed infants, are two promising bacterial strains for their use in functional foods according to their demonstrated probiotic and technological characteristics. To better understand their probiotic characteristics and evaluate their safety, here we report the draft genome sequences of both strains as well as the analysis of their genetical content. The draft genomes of L. paracasei INIA P272 and L. rhamnosus INIA P344 comprise 3.01 and 3.26 Mb, a total of 2994 and 3166 genes and a GC content of 46.27 % and 46.56 %, respectively. Genomic safety was assessed following the EFSA guidelines: the identification of both strains was confirmed through Average Nucleotide Identity, and the absence of virulence, pathogenic and antibiotic resistance genes was demonstrated. The genome stability analysis revealed the presence of plasmids and phage regions in both genomes, however, CRISPR sequences and other mechanisms to fight against phage infections were encoded. The probiotic abilities of both strains were supported by the presence of genes for the synthesis of SCFA, genes involved in resistance to acid and bile salts or a thiamine production cluster. Moreover, the encoded exopolysaccharide biosynthesis genes could provide additional protection against the deleterious gastrointestinal conditions, besides which, playing a key role in adherence and coaggregation of pathogenic bacteria together with the high number of adhesion proteins and domains encoded by both genomes. Additionally, the bacteriocin cluster genes found in both strains, could provide an advantageous ability to compete against pathogenic bacteria. This genomic study supports the probiotic characteristics described previously for these two strains and satisfies the safety requirements to be used in food products., This work was supported by projects PID2020-11960RB-I00 from the Spanish Ministry of Science and Innovation, and RMP2015-00001-00-00 from the National Institute for Agricultural and Food Research and Technology (INIA) and the Spanish Agency for Research (AEI-MCIU) co-financed by the European Regional Development Fund

8 Pág. Departamento de Tecnología de Alimentos​​, Three strains with technological and probiotic properties (Lactococcus lactis INIA 650, Lacticaseibacillus paracasei INIA P272 and Bifidobacterium breve INIA P734) were used in different combinations as adjunct cultures for the manufacture of pasteurized ewe's milk. The three strains showed good viability during cheese manufacture and ripening. Moreover, they all were able to survive after the addition of the cheese samples to an in vitro colonic model. The nisin-producing strain L. lactis INIA 650 was able to produce the antimicrobial peptide nisin not only during cheese manufacture and ripening, but also in the colonic model. None of the strains combinations added to the cheese as adjunct cultures had a negative influence in their physicochemical or sensory characteristics when compared to control cheese. This work shows the suitability of cheese as vehicle for a multi-strain combination with potential dual effect in food and gut., This work was supported by project RTA2017-00002-00-00 from the Spanish Ministry of Science and Innovation

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.

Centro de Investigación en Sanidad Animal (CISA), 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., This work was supported by RTA2017-00002–00-00 project from the Ministry of Economy and Competitiveness (Spain).

Departamento de Tecnología de los Alimentos, 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., This work was supported by project RTA2017-00002-00-00 from the Spanish Ministry of Science, Innovation and Universities, 7 Pág.

Departamento de Tecnología de Alimentos​​ (INIA), The draft genome sequence of Bifidobacterium breve INIA P734, a strain shared by mother and child, is reported. It consists of 50 contigs, with 2,391,925 bp, 2,099 genes, and a G+C content of 58.8%. The genome analysis revealed the absence of antibiotic resistance and pathogenicity-related genes., This work was supported by project no. RTA2017-00002-00-00 from the Spanish Ministry of Science and Innovation and RMP2015-00001-00-00 from the National Institute for Agricultural and Food Research and Technology (INIA) and the Spanish Agency for Research (AEI-MCIU), cofinanced by the European Regional Development Fund., 2 Pág.

9 Pág. Departamento de Tecnología de Alimentos​​, 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., This work was supported by RTA2017-00002-00-00 project from the Ministry of Economy and Competitiveness (Spain).

8 Pág., Dihydrodaidzein (DHD) and dihydrogenistein (DHG) are intermediate compounds in the production of equol and 5-hydroxy-equol from daidzein and genistein by certain intestinal bacteria. In this work, we explored the heterologous expression of the daidzein reductase gene from Slackia isoflavoniconvertens DSM22006, responsible for the formation of DHD and DHG, in nine lactic acid bacteria and Bifidobacterium strains under a strong constitutive promoter in the plasmid pNZ:TuR.dzr. All the transformed strains showed high production of DHD and DHG both from pure daidzein and genistein and from the isoflavone glycosides present in soy beverage. In addition, Lactococcus lactis MG1363 pNZ:TuR.dzr, expressing the recombinant daidzein reductase, incremented the production of equol by equol-producing intestinal microbiotas in a colonic environment. Nevertheless, other recombinant strains tested showed the opposite effect to L. lactis MG1363 pNZ:TuR.dzr in the production of equol by intestinal microbiota. Here, we describe for the first time the production of 5-hydroxy-equol by human intestinal microbiota, both from genistein and DHG. The use of DHD and DHG as substrates, compared to daidzein and genistein, resulted in an increment of equol and 5-hydroxy-equol production by intestinal microbiota. The recombinant strains developed would be of value for the development of fermented soy beverage enriched in DHD and DHG with the aim of facilitate equol and 5-hydroxy-equol production by intestinal microbiota., This work was supported by project RTA2017-00002-00-00 from the Spanish Ministry of Science, Innovation and University.

Flaxseed and soybean are an important source of phytochemical compounds, mainly lignans and isoflavones, but also of flavones, flavanones and flavonols. These compounds appear mainly glycosylated in plant food and are converted into aglycones by the intestinal microbiota, increasing their bioavailability. In this work, we analyse the ability of Bifidobacterium strains (n = 25) to produce lignan and flavonoids aglycones from flaxseed and soybean extracts. Most of the Bifidobacterium strains increased the concentrations of secoisolariciresinol, daidzein, genistein, naringenin, eriodyctiol, luteolin and apigenin. Moreover, Bifidobacterium pseudocatenulatum and Bifidobacterium breve strains showed high production of herbacetin, increased the kaempferol concentration and produced quercetin and quercetagetin. B. pseudocatenulatum INIA P815 and B. breve INIA P367 produced 32.37 ± 2.81 and 28.64 ± 3.36 mg respectively of herbacetin g−1 of lignan extract. Bifidobacterium strains transformed the glycosides of a wide range of flavonoids into their aglycones, increasing the antioxidant activity and improving their bioavailability., This study was funded by RTA2017‐00002‐00‐00 from the Spanish Ministry of Science, Innovation and Universities.

Urolithins are microbial metabolites derived from ellagic acid by the action of intestinal bacterial. Potential beneficial effects of urolithins A and B on certain types of cancer and on muscular health during aging have been demonstrated. In this work, urolithin production by 48 strains of bifidobacteria and 1070 bacterial isolates from the faeces of a healthy woman whose intestinal microbiota produced uroltihins A and B was screened. Bifidobacterium pseudocatenulatum INIA P815 was the only strain able to produce urolithins A and B from ellagic acid. Influence of different media on the strain metabolism showed that the production of urolithins could be regulated. The identification of a bifidobacterial strain producing urolithins A and B opens the possibility for its application as probiotic in functional foods and in the development of nutraceuticals., This work was supported by project RM2012-00004-00-00 from theSpanish Ministry of Economy and Competitiveness (MINECO, Madrid, Spain).

Phytoestrogens are plant polyphenols similar to human estrogens. Isoflavones, ellagitanins, and lignans are metabolized by intestinal bacteria into equol, urolithins, and enterolignans, respectively, which are more bioavailable and bioactive, having beneficial effects on health. In this paper, we analysed the production of equol, 5-hydroxy-equol, enterodiol and enterolactone by 70 strains of Lactobacillus, Lactococcus and Enterococcus. Enterodiol and enterolactone production was found in Lactobacillus salivarius INIA P183, Lb. salivarius INIA P448 and Lactobacillus gasseri INIA P508, in levels around 46 µM and 6 µM respectively. However, we did not find neither equol nor 5-hydroxy-equol producing strains. Furthermore, analysis of the ellagic acid metabolism was extended to a total of 138 lactic acid bacteria strains, although none of them produced urolithins. This is the first time that simultaneous production of enterolactone and enterodiol is described in bacteria with probiotic potential, open their possible application in functional foods., This work was supported by project RM2012-00004-00-00 from the Spanish Ministry of Economy and Competitiveness (MINECO, Madrid, Spain). J.M. Landete has a postdoctoral contract with the research program “Ramón y Cajal” (MINECO).

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.

Lignans are phytoestrogens found in plant foods and especially in flax seeds. Although lignans present a low bioavailability, their transformation into the enterolignans enterodiol (END) and enterolactone (ENL) is associated with beneficial effects on human health. Lignan metabolism was studied in 90 lactic acid bacteria and bifidobacteria finding a wide distribution of the ability to deglycosylate secoisolariciresinol diglucoside (SDG) into secoisolariciresinol (SECO). Previous results indicate that enterolignans need a consortium of intestinal bacteria to be produced. In this paper we describe the obtaining of between 1.12 μM and 4.18 μM of END from lignan extracts by the action of a sole strain, Bifidobacterium adolescentis INIA P784. However, END production was not obtained from the pure lignans SECO, matairesinol and sesamin. To our knowledge, the first probiotic bacterium capable of producing END from lignan extracts is described in this work, showing potential applications in biotechnology, in functional foods and as probiotic. © 2016 Elsevier Ltd

Many food safety-related studies require the tracking of inoculated food-borne pathogens to monitor their fate in food complex environments. In the current study, we demonstrate the potential of plasmids containing the fluorescence protein gene evoglow-Pp1 (Evocatal, Dusseldorf, Germany) as a real-time reporter system for Listeria strains. This anaerobic fluorescent protein provides an easily detectable phenotype of microorganisms for food safety studies. This work is the first to report a reliable method to identify fluorescently labeled Listeria strains in food ecosystems. © 2017 American Dairy Science Association

Lactobacillus species are attractive hosts for the expression of heterologous proteins, antigens, vaccines, and drugs due to their GRAS (generally recognized as safe) status. The bioengineering techniques open new possibilities of improving Lactobacillus strains. In this regard, the control of the gene expression in Lactobacillus strains through the adequate native or engineered promoters acquires a key role in the development of biotechnological applications and for their function as probiotic bacteria. Depending on the objective sought, the protein produced and the strain used, inducible or constitutive promoters can be chosen. Whereas, when a fine-tuning of gene expression is required, the development of synthetic promoter libraries could be the best approach. In this work, we revise the main constitutive and inducible natural promoters from Lactobacillus strains or from other genus that have been applied in Lactobacillus, as well as the few engineered promoters developed for these bacteria.

Detection of bioactive peptides in complex ecosystems like intestinal environment is a difficult task. In this study, we developed two new bioreporters for nisin based on Lactococcus lactis NZ9000 transformed with the vector pNZ:Nis-aFP or pNZ:Nis-mCherry, that encoded for the anaerobic fluorescent protein evoglow-Pp1 (aFP) or the fluorescent protein mCherry, respectively. The biosensors were used to study nisin A production by L. lactis INIA 650 in milk and in a colonic model. The use of L. lactis NZ9000 pNZ:Nis-aFP as a biosensor allowed the detection of nisin produced by L. lactis INIA 650 in milk, but not in the in vitro colonic model. In milk, this reporter was induced by direct addition of 10 ng/ml nisin while, in the colonic model, nisin concentrations of 50 ng/ml were necessary. However, the reporter system based on pNZ:Nis-mCherry showed a higher sensibility, detecting nisin concentrations of 1 ng/ml produced by L. lactis INIA 650 in colonic media using agar diffusion or cross streak bioassays.

Isoflavones intake is associated with health benefits. The metabolism of isoflavones by bacteria plays a key role in their biotransformation. Therefore, commercial soy drink was fermented by 11 lactic acid bacteria (LAB) and 9 bifidobacteria strains. The majority of the strains showed deglycosylation of the isoflavone glycosides present in soy drink and appearance of the aglycones daidzein, genistein and glycitein. Moreover, we observed the further transformation of daidzein into O-desmethylangolensin (O-DMA) and tetrahydrodaidzein, alongside with dihydrodaidzein (DHD) and a putative isomer of DHD. On the other hand, genistein was transformed by nearly all strains into 6-hydroxy-O-desmethylangolensin (6-hydroxy-O-DMA), but no dihydrogenistein production was registered. A high concentration of 2-(4-hydroxyphenyl)-propionic acid was observed, suggesting the degradation of O-DMA and 6-hydroxy-O-DMA. The potential of LAB and Bifidobacterium strains to produce functional soy drink enriched with bioactive isoflavones is demonstrated in this work., This work was supported by project RTA2017-00002-00-00 from the Spanish Ministry of Science, Innovation and Universities.

17 Pág., Phytoestrogens (PE) are compounds found in plants such as soy (isoflavones), flax seeds and cereals (lignans) and pomegranates (ellagitannins). PE have shown estrogenic/antiestrogenic, antioxidant, anti-inflammatory, antineoplastic and apoptotic activities. The human studies are showing promising although inconsistent results about the beneficial effects of PE on ameliorating the menopausal symptoms or reducing the risk of certain cancers, cardiovascular disease or diabetes. The effects of PE on the organism are mediated by the intestinal microbiota, which transforms them into bioactive PE such as genistein, equol, enterolignans and certain urolithins. In this work, we review the most recent findings about the bacteria able to metabolize PE, together with the latest studies on the effects of PE on health. In addition, we describe the possible factors hindering the demonstration of the beneficial effect of PE on health, evincing the importance of measuring the actual circulating PE in order to encompass the variability of PE metabolism due to the intestinal microbiota. With this in mind, we also explore an approach to ensure the access to bioactive PE., This work was supported by projects RM2012-00004-00-00 and RTA2017-00002-00-00 from the Ministry of Science, Innovation and Universities (Spain)

Bifidobacterium longum INIA P132 and Bifidobacterium infantis INIA P731, isolated from infant-faeces, were investigated in this work. Regarding the probiotic and technological potential of the bifidobacteria, both were resistant to gastrointestinal tract simulated conditions. B. longum showed high survival upon freezing and thawing as well as lyophilisation and was able to grow in milk. B. infantis had higher adhesion capacity to human Caco-2 cells than the commercial probiotic Bifidobacterium animalis BB12 strain. Moreover, both bacteria secrete heteropolysaccharides (HePS) composed of rhamnose, galactose and glucose. In a dextran sodium sulphate-induced enterocolitis model in zebra fish larvae, treatment with each HePS preparation resulted in a decrease of the larval mortality. In addition, the HePS from B. longum immunomodulated in vitro human macrophages treated with the inflammatory Escherichia coli O111:B4 lipopolysaccharide. Thus, both studied bifidobacteria and their HePS have potential beneficial effects on health and thus, to their application in functional foods., This work was supported by a grant from de Department of Environment, Territorial Planification, Agriculture and Fishing and the project no. IT 866-13 from the Basque Government, by the Spanish Ministry of Science and Innovation (AGL 2015-65010-C3-1-R) and by the Spanish Ministry of Economy and Competitiveness (RM12-00004-00-00 and RTA2013-00029-00-00).

Technological processes in the dairy industry and the further passage through the gastrointestinal tract could impair viability and functionality of probiotic bifidobacteria. In the present work, the growth in milk of nine bifidobacterial strains shared by mother and child, their survival to freeze-drying and cold storage, and their behavior in a model cheese were investigated. All the strains exhibited high stability to the technological conditions studied when compared with two commercial strains.Bifidobacterium breveINIA P734 andBifidobacterium bifidumINIA P671 as adjunct cultures maintained high stability during manufacture and ripening of cheese. Both strains showed, at the end of ripening period, resistance to simulated gastrointestinal conditions. Moreover, their presence did not affect negatively the quality of cheese.B. breveINIA P734 andB. bifidumINIA P671 could be considered as potential candidates for their use in cheese as adjunct cultures.

The aim of this study was to test the compatibility of two selected Lactobacillus strains with different prebiotic carbohydrates in order to develop a synbiotic cheese. Initially, the growth of the probiotic lactobacilli with the prebiotic formulations was monitored in optimized growth medium and in milk. Afterwards, semi-hard cheeses were manufactured with the selected combination of Lb. paracasei INIA P272 and fructo-oligosaccharides (FOS). Results showed that the addition of FOS did not affect Lb. paracasei viability in the cheese after 28 days of ripening, but a higher viability of this strain was achieved in the presence of FOS when cheese was tested in a colonic model. Thus, the inclusion of FOS in the cheese could have a beneficial effect by increasing the probiotic strain viability in the host.

The effect of Serrano and Iberian dry-cured ham processing and ripening on Listeria monocytogenes inactivation at the surface of whole hams was investigated. Salted hams were surface inoculated (6.5 log CFU) with a cocktail of 4 L. monocytogenes strains isolated from environment and products of a meat industry. Serrano and Iberian hams were ripened for 16 and 24 months, respectively. A decrease of at least 4.6 log units on the surface of Serrano ham was recorded after 4 months for L. monocytogenes counts, which remained under the detection limit thereafter. L. monocytogenes declined by >5 log units on the surface of Iberian ham during the first 9 months and was not detected afterwards. The higher nitrite content of Serrano ham might have accelerated the decrease of the pathogen. This study validates the inactivation of L. monocytogenes on the surface of whole dry-hams during extended ripening.

The majority of cases of listeriosis are associated with the consumption of contaminated food. Some strains of Listeria monocytogenes can persist over months or years in meat processing plants increasing the risk of product contamination. The presence of L. monocytogenes was examined in 10 dry-cured ham processing facilities. A total of 1801 samples were collected from environment and equipment, during processing (1095) and after cleaning and disinfection (706). These samples were taken from non-food contact (736) and food contact (1065) surfaces. In addition, 204 samples from ham surfaces were also analysed. Prevalence varied from 6% to 34% among facilities, and was higher during processing than after cleaning and disinfection (24.8% vs 11.0%) and from non-food than from food contact surfaces (22.6% vs 17.4%). L. monocytogenes serotype 1/2a was predominant (53.9%), followed by 1/2c (26.0%) and 1/2b (15.3%) and less frequently 4b (4.8%). A total of 142 different pulsotypes were registered. Potential persistent L.monocytogenes strains were isolated in 9 out the 10 facilities, with no more than 6 pulsotypes in a given plant. Two pulsotypes were common in different installations, detected before and after cleaning and disinfection, highlighting the importance of monitoring the presence of this pathogen in dry-cured ham processing environments.

Almost all soy isoflavones exist as glycosides, daidzin, genistin, and glycitin. We analyzed the capacity of 92 strains of lactic acid bacteria (LAB) and bifidobacteria with biotechnological interest to process the glycosylated isoflavones daidzin, genistin, and glycitin in their more bioavailable aglycones and their metabolites as dihydrodaidzein (DHD), O-desmethylangolensin, and equol. Representative strains of the four genera studied Lactobacillus, Enterococcus, Lactococcus, and Bifidobacterium were able to produce daidzein, genistein, and glycitein, with the exception of the lactobacilli, which did not produced glycitein in soy extracts. The production of the aglycone isoflavones could be correlated with the β-glucosidase activity of the strains. The isoflavone metabolism is limited to the glycoside hydrolysis in the most of these strains. Moreover, Enterococcus faecalis INIA P333 and Lactobacillus rhamnosus INIA P540 were able to transform daidzein in DHD. LAB and bifidobacteria studied in the present work have a great potential in the metabolism of isoflavones and could be selected for the development of functional fermented soy foods. © 2016 Taylor & Francis.

Bioprotective effect of reuterin-producing Lactobacillus reuteri INIA P572 was assessed in cheeses artificially contaminated with Listeria monocytogenes and Escherichia coli O157:H7 and ripened for 30 days. Reuterin production in cheese made with L. reuteri INIA P572 supplemented with 100 mM glycerol was confirmed up to 7 d of ripening with a maximum of 5.2 μmol/g at 1 day. L. reuteri was able to survive up to 5.37 log cfu/g in 30 d control cheese, although it was reduced to 2.59 log cfu/g when added simultaneously with glycerol. L. monocytogenes counts in control cheese were 4.31 log cfu/g after 30 days of ripening, whereas in cheese made with L. reuteri INIA P572 and 100 mM glycerol, this pathogen was only detectable by enrichment of 25 g samples from day 7 onwards. This antimicrobial strategy also diminished E. coli O157:H7 counts on day 1 by 5.53 log units with respect to control cheese. After 30 d, E. coli O157:H7 counts were 0.85 log cfu/g in control cheese, but the pathogen was not detected in 25 g cheese made with L. reuteri and 100 mM glycerol from day 7 onwards. Reuterin production in situ might be a feasible alternative to improve cheese safety that could also be exploited in the control of other undesirable microorganisms.

Pathogenic ability has been extensively studied in clinical enterococci, but to a lesser extent in community-derived ones. Most studies to date in enterococci from healthy infants have been focused on Enterococcus faecalis, despite the growing concern about nosocomial infections caused by E. faecium. In this work, we studied the antibiotic resistance and virulence determinants of 26 E. faecalis and 15 E. faecium intestinal isolates from Spanish healthy breastfed infants. Overall, commensal enterococci studied contained antibiotic resistance and virulence genes, although their patterns were not according to those described for antibiotic-resistant hospital-associated enterococci. None of the isolates was resistant to vancomycin, although the majority showed resistance to some antibiotics. E. faecalis isolates harbored considerably more virulence determinants than E. faecium isolates, but some genes linked to colonization were abundant in both species. Hemolysin activity was not detected in any of the isolates; and the gelatinase gene, when present, was silent in E. faecium, whereas gelatinase activity occurred in half of the E. faecalis isolates studied. These results suggest an ambivalent role of some virulence determinants as elements of pathogenesis.

Healthy effect of isoflavones consumption is attributed to their bioactive metabolites such as O-desmethylangolensin (O-DMA) produced by intestinal bacteria. Evidence from in vitro studies suggests that O-DMA has several cancer-related biological actions. Enterococcus spp. are indigenous species of the human gastrointestinal tract. Significant attention has been focused on Enterococcus faecium strains with probiotic and /or technological interest in foods. In this work, we describe the production of the bioactive isoflavones O-DMA and 6-hydroxy O-DMA by the strain E. faecium INIA P553. O-DMA was produced from dihydrodaidzein, daidzein, daidzin and formononetin, and 6-hydroxy O-DMA from dihydrogenistein, genistein and genistin with high efficiency. To our knowledge, it is the first bacterium with probiotic potential capable of producing O-DMA and 6-hydroxy O-DMA. Therefore, this strain shows potential applications in biotechnology in functional foods and as probiotic., This work was supported by project RM2012-00004-00-00 from the Spanish Ministry of Economy and Competitiveness (MINECO, Madrid, Spain). J.M. Landete has a postdoctoral contract with the research program “Ramón y Cajal” (MINECO).

Importance of bifidobacteria as part of the infant intestinal microbiota has been highlighted. Their acquisition is influenced by the mode of birth and the feed regime afterwards, with a special role of the maternal microbiota. The presence of the same shared bifidobacterial strains between breast milk and infant faeces in 14 mother-infant pairs was assessed by means of pulsed-field gel electrophoresis (PFGE) genotyping. Four shared strains of Bifidobacterium breve (2), Bifidobacterium longum subsp. infantis and B. longum subsp. longum were found in breast milk-infant faeces pairs. Two years later, a second survey yielded four shared strains of the species Bifidobacterium adolescentis, Bifidobacterium bifidum, B. longum subsp. longum and Bifidobacterium pseudocatenulatum. Moreover, a B. bifidum strain was found to be shared by the infant faeces of the first study and the mother faeces tested two years later, pointing out a long term persistence. Some of the selected bifidobacterial strains showed probiotic potential due to their survival to gastrointestinal conditions and their ability to form biofilms.

Isoflavones are usually found in nature in their glycosilated or methylated forms, and should be hydrolysed to become bioavailable and physiologically active. The deglycosylation of isoflavone C-glycosides and O-glycosides and the demethylase activity were studied in a selection of lactic acid bacteria (LAB) and bifidobacteria by assessing the degree of transformation of the pure precursor compounds, daidzin, genistin, puerarin, formononetin and biochanin A into daidzein or genistein. Only one Bifidobacterium strain and two Enterococcus strains hydrolysed the C-glycosidic bond of puerarin, while deglycosylation of O-glycosides daidzin and genistin was observed in all the tested strains. Demethylation of biochanin A and formononetin was observed in the most of LAB and bifidobacteria. Besides, the subsequent metabolites dihydrodaidzein and dihydrogenistein where produced by many of the strains via daidzein and genistein. In this work, we show the potential of LAB and bifidobacteria as part of functional foods because of their ability to transform plant isoflavones into their bioactive forms. © 2017 Elsevier Ltd

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. © 2017 José Mariá Landete et al.

An efficient method for genetic transformation of lactic acid bacteria (LAB) by electroporation is presented in this work. A comparative survey with other electrotransformation methods already published showed that the method proposed here yields the higher electrotransformation efficiency in the 12 LAB strains tested, which could make the method applicable to other LAB species or genera. © 2014 Elsevier B.V.

Bifidobacteria isolated from infant gut and breast milk exhibited different abilities to grow under microaerobic conditions, alone or in the presence of added catalase. In the present study, we demonstrated that some Bifidobacterium strains unable to grow under microaerobic conditions were cross-protected on solid media from oxidative stress by adjacent colonies of gut catalase-positive Staphylococcus epidermidis or Escherichia coli, but not by a catalase-deficient E. coli. The results of this study support the possible contribution of catalase-positive bacteria to the establishment of certain bifidobacteria in non-anaerobic human niches of the infant gastrointestinal tract or mammary gland. © 2015, Japanese Society of Microbial Ecology. All rights reserved.