Strategies to achieve significant physiological concentrations of bioactive phytoestrogens in plasma.

The benefits to health attributed to the intake of phytoestrogens (PEs) have been demonstrated in previous studies with significant physiological concentrations of bioactive PEs, such as genistein, equol, enterolignans and urolithins in plasma. However, the achievement of high bioactive PE levels in plasma is restricted to a select population group, mainly due to the low intake of plant PEs and/or the absence, or inhibition, of the microbiota capable of producing these bioactive forms. In this study, the intake of plant PEs, the concentration of bioactive PEs in plasma, the ability of the intestinal microbiota to produce bioactive PEs, as well as the different mechanisms used by GRAS bacteria to increase the level of bioactive PEs were evaluated concluding that the use of GRAS bacteria bioactive PE producers and the development of fermented foods enriched in bioactive PEs in addition to a high intake of plant PEs and taking care of the intestinal microbiota, are some of the different strategies to achieve significant physiological concentrations of bioactive PEs in the intestine and, subsequently, in plasma and targets organs which are essential to improve menopausal symptoms or reduce the risk of some pathologies such as breast and colon cancer, or cardiovascular disease.

Flavone, flavanone and flavonol metabolism from soybean and flaxseed extracts by the intestinal microbiota of adults and infants.

BACKGROUND: Flaxseed and soybean are an important source of lignans and flavonoids. Previously, the metabolism of isoflavones and lignans from soybean and flaxseed extracts by the microbiota of adult individuals (n = 14) and infants (n = 23) was analyzed. Thus, the present study aimed to examine the metabolism of flavones, flavanones and flavonols, as well as the production of phenolic acids, by the intestinal microbiota of these individuals. RESULTS: Concentrations of aglycones of flavonoids, such as herbacetin, quercetin, quercetagetin, myricetin, kaempferol, apigenin and luteolin, increased for most of individuals as a consequence of deglycosylation reactions. On the other hand, a diminution in the antioxidant activity and phenolic compound concentration and an increase in the concentration of 3,4-dihydroxyphenylacetic acid, 2-(4-hydroxyphenyl)-propionic acid, protocatechuic acid and catechol was also observed. CONCLUSION: The present study found that deglycosylation reactions were the main reactions and accelerated the formation of more bioavailable flavonoids, with greater biological activity, in most of the individuals. However, other reactions also occurred, including the total or partial catabolism of flavonoids. © 2021 Society of Chemical Industry.

Bacterial metabolism as responsible of beneficial effects of phytoestrogens on human health.

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.

Natural and engineered promoters for gene expression in Lactobacillus species.

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.

Expression of a ß-glucosidase in bacteria with biotechnological interest confers them the ability to deglycosylate lignans and flavonoids in vegetal foods.

Lignans and flavonoids are found in plants in their glycosylated forms and need to be hydrolyzed to aglycones to become bioavailable. Putative ß-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 ß-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.

Short communication: Labeling Listeria with anaerobic fluorescent protein for food safety studies.

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.

Optimization of reuterin production in cheese by Lactobacillus reuteri.

Cheeses manufactured from pasteurized milk supplemented with glycerol and reuterin-producing Lactobacillus reuteri INIA P572 as adjunct to the commercial starter culture were analysed in order to optimize a biopreservation strategy. The highest reuterin concentration determined by a colorimetric assay was detected on day 1 in cheeses with 100-500 mM glycerol. The presence of reuterin was confirmed by a direct detection technique as HPLC. Cheeses made with L. reuteri and 200 or 500 mM glycerol showed a red tendency in color in comparison with control. The results with purified reuterin suggested that the development of slightly rosy colour in cheese was related to some compound produced/overproduced when higher levels of glycerol were present in cheese, but not due to reuterin. Application of L. reuteri INIA P572 as adjunct to the commercial starter with 100 mM glycerol led to such a reuterin concentration in cheese that could control undesirable microorganisms, avoiding the presence of color-changing compounds.

Fluorescent reporter systems for tracking probiotic lactic acid bacteria and bifidobacteria.

In the last two decades, there has been increasing evidence supporting the role of the intestinal microbiota in health and disease, as well as the use of probiotics to modulate its activity and composition. Probiotic bacteria selected for commercial use in foods, mostly lactic acid bacteria and bifidobacteria, must survive in sufficient numbers during the manufacturing process, storage, and passage through the gastro-intestinal tract. They have several modes of action and it is crucial to unravel the mechanisms underlying their postulated beneficial effects. To track their survival and persistence, and to analyse their interaction with the gastro-intestinal epithelia it is essential to discriminate probiotic strains from endogenous microbiota. Fluorescent reporter proteins are relevant tools that can be exploited as a non-invasive marker system for in vivo real-time imaging in complex ecosystems as well as in vitro fluorescence labelling. Oxygen is required for many of these reporter proteins to fluoresce, which is a major drawback in anoxic environments. However, some new fluorescent proteins are able to overcome the potential problems caused by oxygen limitations. The current available approaches and the benefits/disadvantages of using reporter vectors containing fluorescent proteins for labelling of bacterial probiotic species commonly used in food are addressed.

Use of anaerobic green fluorescent protein versus green fluorescent protein as reporter in lactic acid bacteria.

Lactic acid bacteria (LAB) are commonly used in the production of fermented and probiotic foods. Development of molecular tools to discriminate the strains of interest from the endogenous microbiota in complex environments like food or gut is of high interest. Green fluorescent protein (GFP)-like chromophores strictly requires molecular oxygen for maturation of fluorescence, which restrict the study of microorganisms in low-oxygen environments. In this work, we have developed a noninvasive cyan-green fluorescent based reporter system for real-time tracking of LAB that is functional under anoxic conditions. The evoglow-Pp1 was cloned downstream from the promoters D-alanyl-D-alanine carboxypeptidase and elongation factor Tu of Lactobacillus reuteri CECT925 using pNZ8048 and downstream of the lactococcal P1 promoter using pT1NX. The classical gfp was also cloned in pT1NX. These recombinant expression vectors were electroporated into Lactococccus, Lactobacillus, and Enterococcus strains with biotechnological and/or probiotic interests to assess and compare their functionality under different conditions of oxygen and pH. The expression was analyzed by imaging and fluorometric methods as well as by flow cytometry. We demonstrate that reporter systems pNZ:TuR-aFP and pT1-aFP are two versatile molecular markers for monitoring LAB in food and fecal environments without the potential problems caused by oxygen and pH limitations, which could be exploited for in vivo studies. Production of the fluorescent protein did not disturb any important physiological properties of the parental strains, such as growth rate, reuterin, or bacteriocin production.

Analysis of gene expression of bifidobacteria using as the reporter an anaerobic fluorescent protein.

OBJECTIVES: To determine the effectiveness of evoglow-Pp1 as a reporter to study gene expression in bifidobacteria. To choose a strong and constitutive promoter to track fluorescently labelled bifidobacteria in environments under anaerobic conditions. RESULTS: The elongation factor P (EF-P) promoter from Bifidobacterium longum CECT 4551 produced the highest emission of fluorescence signal and was therefore able to produce the highest gene expression of the promoters studied. The promoters from B. longum CECT 4551 showed different fluorescence signal intensities which, in descending order, were: EF-P, initiation factor IF-2, elongation factor G, elongation factor Tu, elongation factor Nus A, elongation factor Ts and 30S ribosomal protein S12. CONCLUSIONS: The consistency of the methods employed (fluorescence imaging system, fluorescence microscopy, fluorimetry and flow cytometry) showed that the construction pNZ:Prom.GFPana contained the anaerobic fluorescent protein evoglow-Pp1 could be exploited as a tool for analysing the gene expression in bifidobacteria strains.

Genome Sequence of the Reuterin-Producing Strain Limosilactobacillus reuteri INIA P572.

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.

Probiotic and Functional Properties of Limosilactobacillus reuteri INIA P572.

Limosilactobacillus reuteri INIA P572 is a strain able to produce the antimicrobial compound reuterin in dairy products, exhibiting a protective effect against some food-borne pathogens. In this study, we investigated some probiotic properties of this strain such as resistance to gastrointestinal passage or to colonic conditions, reuterin production in a colonic environment, and immunomodulatory activity, using different in vitro and in vivo models. The results showed a high resistance of this strain to gastrointestinal conditions, as well as capacity to grow and produce reuterin in a human colonic model. Although the in vitro assays using the RAW 264.7 macrophage cell line did not demonstrate direct immunomodulatory properties, the in vivo assays using a Dextran Sulphate Sodium (DSS)-induced colitic mice model showed clear immunomodulatory and protective effects of this strain.

The role of two families of bacterial enzymes in putrescine synthesis from agmatine via agmatine deiminase.

Putrescine, one of the main biogenic amines associated to microbial food spoilage, can be formed by bacteria from arginine via ornithine decarboxylase (ODC), or from agmatine via agmatine deiminase (AgDI). This study aims to correlate putrescine production from agmatine to the pathway involving N-carbamoylputrescine formation via AdDI (the aguA product) and N-carbamoylputrescine amidohydrolase (the aguB product), or putrescine carbamoyltransferase (the ptcA product) in bacteria. PCR methods were developed to detect the two genes involved in putrescine production from agmatine. Putrescine production from agmatine could be linked to the aguA and ptcA genes in Lactobacillus hilgardii X1B, Enterococcus faecalis ATCC 11700, and Bacillus cereus ATCC 14579. By contrast Lactobacillus sakei 23K was unable to produce putrescine, and although a fragment of DNA corresponding to the gene aguA was amplified, no amplification was observed for the ptcA gene. Pseudomonas aeruginosa PAO1 produces putrescine and is reported to harbour aguA and aguB genes, responsible for agmatine deiminase and N-carbamoylputrescine amidohydrolase activities. The enzyme from P. aeruginosa PAO1 that converts N-carbamoylputrescine to putrescine (the aguB product) is different from other microorganisms studied (the ptcA product). Therefore, the aguB gene from P. aeruginosa PAO1 could not be amplified with ptcA-specific primers. The aguB and ptcA genes have frequently been erroneously annotated in the past, as in fact these two enzymes are neither homologous nor analogous. Furthermore, the aguA, aguB and ptcA sequences available from GenBank were subjected to phylogenetic analysis, revealing that gram-positive bacteria harboured ptcA, whereas gram-negative bacteria harbour aguB. This paper also discusses the role of the agmatine deiminase system (AgDS) in acid stress resistance.

Fluorescent detection of nisin by genetically modified Lactococcus lactis strains in milk and a colonic model: Application of whole-cell nisin biosensors.

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.

Virulence and Antibiotic Resistance of Enterococci Isolated from Healthy Breastfed Infants.

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.

Expression of plasminogen activator inhibitors type 1 and type 3 and urokinase plasminogen activator protein and mRNA in breast cancer.

BACKGROUND: The urokinase plasminogen activator (uPA) system has been involved in cancer cell invasion and in metastasis. uPA activity is controlled by its principal inhibitor, the PA inhibitor type-1 (PAI-1), but it can also be inhibited by PAI-3. Increased levels of uPA and PAI-1 are known to be associated with a poor prognosis in breast cancer. To our knowledge this is the first study of the expression and role of PAI-3 in human breast cancer tissue. MATERIALS AND METHODS: Protein and mRNA levels were evaluated for uPA, PAI-1 and PAI-3 in breast cancer tissues from 70 different patients. The localization of antigen and mRNA of these proteins was studied by immunohistochemistry and in situ hybridization, respectively. RESULTS: No significant differences were observed for PAI-3 mRNA or protein levels between the nodal status groups or the different post-surgical tumor-node-metastasis (pTNM) stages. However, uPA and PAI-1 mRNA and antigen levels significantly increased at the pTNM stage and in node-positive patients. PAI-3 antigen levels were significantly higher in early relapse-free patients, whereas PAI-1 antigen levels were significantly higher in patients who suffered a relapse. PAI-3 protein and mRNA were localized in stromal cells. PAI-1 and uPA protein were detected in cancer, endothelial and stromal cells and their mRNA mainly in stromal cells. CONCLUSIONS: Our results indicate that PAI-3 is expressed in human breast cancer tissues, and that elevated levels of PAI-3 could be a positive prognostic factor in this disease. A potential mechanism for the contribution of PAI-3 to a positive long-term outcome may involve suppression of tumor invasion through protease inhibition in stroma.

Genetic engineering as a powerful tool to improve probiotic strains.

Over the last decade, there has been increasing interest in the use of probiotic microorganisms. However, certain doubts have arisen around probiotics, because of the beneficial effects of these microorganisms are not clear yet, and in many occasions those beneficial effects have not been proven. Therefore, it would be of interest if these probiotic strains were able to acquire new attributes to allow them improve and increase their beneficial characteristics. Genetic engineering can be used for human applications; for instance, the resistance to antibiotics is removed and the probiotic bacteria are modified in its own DNA. This process can be achieved by: (1) the use of food-grade vectors derived from lactic acid bacteria and/or bifidobacteria cryptic plasmids, (2) the genes integration or deletion in the chromosome of the probiotic strain, by site-specific recombination using the attP/integrase system, or by homologous recombination, using either suicide vectors, (3) using the clustered regularly interspaced short palindromic repeats (CRISPR) and the CRISPR-associated (Cas) nuclease. Through genetic engineering, the knowledge of probiotic strains as well as its use could be improved, and the doubts about probiotics could be crumped.

Biogenic amines in wines from three Spanish regions.

One hundred and sixty-three wines from La Rioja, Utiel-Requena, and Tarragona were analyzed to determine if there were any differences in the concentrations of six biogenic amines that are found in these three regions. The influence of grape variety, type of vinification, wine pH, malolactic fermentation, and storage in bottle on biogenic amine concentrations was studied. Results show important differences in putrescine and histamine concentrations among regions, varieties of grape, and type of wine; differences were less appreciable for the remaining biogenic amines studied. Low pH prevented biogenic amine formation. Malolactic fermentation and short storage periods in bottle (3-6 months) showed increases in histamine concentration, whereas longer periods of storage led to a general decrease in histamine. Several strains of lactic acid bacteria were isolated in this work, and their ability to form biogenic amines was assayed in synthetic media, grape must, and wine. Grape varieties, different types of winemaking, pH, and lactic acid bacteria may be responsible for the differences observed in the biogenic amine concentrations of the wines analyzed.

Distribution of calcitonin gene-related peptide-like immunoreactivity in the brain of the lizard Podarcis hispanica.

The present work studies the distribution of calcitonin gene-related peptide-immunoreactive (CGRP-li) neurons and fibers in the brain of a reptile, the lizard Podarcis hispanica. CGRP-li perikarya were not present in the telencephalon. In the thalamus, CGRP-li perikarya were restricted to the posteromedial and posterolateral nuclei. In the hypothalamus, CGRP-li cells were found mainly in the supramammillary and mammillary nuclei. In the midbrain and brainstem, CGRP-li cells appeared in the ventral tegmental area, the parabrachial nucleus, and the motor nuclei of the III-VII, IX, X, and XII cranial nerves. Motoneurons of the ventral horn of the spinal cord were also immunoreactive for CGRP. CGRP-li fibers were seen in the telencephalic hemispheres, where a dense plexus of reactive fibers appeared in the septum and in the lateral striatoamygdaloid transition area. From the latter, CGRP-li fibers entered the posterior dorsal ventricular ridge, the cell layer and deep stratum of the ventral lateral cortex, and various amygdaloid nuclei. Parts of the striatum (nucleus accumbens) and pallidum also displayed CGRP-li innervation. In the diencephalon, CGRP-li innervation was observed in parts of the dorsal thalamus and in the periventricular and medial hypothalamus. The pretectum and deep layers of the optic tectum also showed CGRP-li fibers, and numerous CGRP-li fibers were observed in the midbrain central gray, tegmentum, and pons. Some of the sensory fibers of the trigeminal, vagal, and spinal nerves were also CGRP-li. These results show that the distribution of CGRP-li structures in the reptilian brain is similar to that described for other vertebrates and suggest that the thalamotelencephalic CGRPergic projections appear to be conserved among amniote vertebrates.

Anaerobic green fluorescent protein as a marker of Bifidobacterium strains.

Some strains of Bifidobacterium are considered as probiotics and are being added as adjunct culture in food products due to their potential in maintaining a healthy intestinal microbial balance. However, despite these benefits, bifidobacteria still remain poorly understood at the genetic level compared with other microorganisms of industrial interest. In this work, we have developed a non-invasive green fluorescent based reporter system for real-time tracking of Bifidobacterium species in vivo. The reporter vector pNZ:Tu-GFPana is based on the pNZ8048 plasmid harboring a bifidobacterial promoter (elongation factor Tu from Bifidobacterium longum CECT 4551) and a fluorescent protein containing a flavin-mono-nucleotide-based cofactor (evoglow-Pp1) which is fluorescent under both aerobic and anaerobic conditions. pNZ:Tu-GFPana was constructed and found to stably replicate in B. longum CECT 4551 and in the intestinal strain Bifidobacterium breve INIA P734. The subsequent analysis of these strains allowed us to assess the functionality of this plasmid. Our results demonstrate the potential of pNZ:Tu-GFPana as a real-time reporter system for Bifidobacterium in order to track the behavior of this probiotic species in complex environments like food or intestinal microbiota, and to estimate their competition and colonization potential.