Bioactivation of Phytoestrogens: Intestinal Bacteria and Health.

Phytoestrogens are polyphenols similar to human estrogens found in plants or derived from plant precursors. Phytoestrogens are found in high concentration in soya, flaxseed and other seeds, fruits, vegetables, cereals, tea, chocolate, etc. They comprise several classes of chemical compounds (stilbenes, coumestans, isoflavones, ellagitannins, and lignans) which are structurally similar to endogenous estrogens but which can have both estrogenic and antiestrogenic effects. Although epidemiological and experimental evidence indicates that intake of phytoestrogens in foods may be protective against certain chronic diseases, discrepancies have been observed between in vivo and in vitro experiments. The microbial transformations have not been reported so far in stilbenes and coumestans. However, isoflavones, ellagitanins, and lignans are metabolized by intestinal bacteria to produce equol, urolithins, and enterolignans, respectively. Equol, urolithin, and enterolignans are more bioavailable, and have more estrogenic/antiestrogenic and antioxidant activity than their precursors. Moreover, equol, urolithins and enterolignans have anti-inflammatory effects and induce antiproliferative and apoptosis-inducing activities. The transformation of isoflavones, ellagitanins, and lignans by intestinal microbiota is essential to be protective against certain chronic diseases, as cancer, cardiovascular disease, osteoporosis, and menopausal symptoms. Bioavailability, bioactivity, and health effects of dietary phytoestrogens are strongly determined by the intestinal bacteria of each individual.

Synthesis of a heterogeneous artificial metallolipase with chimeric catalytic activity.

A solid-phase strategy using lipase as a biomolecular scaffold to produce a large amount of Cu(2+)-metalloenzyme is proposed here. The application of this protocol on different 3D cavities of the enzyme allows creating a heterogeneous artificial metallolipase showing chimeric catalytic activity. The artificial catalyst was assessed in Diels-Alder cycloaddition reactions and cascade reactions showing excellent catalytic properties.

Production and characterization of a tributyrin esterase from Lactobacillus plantarum suitable for cheese lipolysis.

Lactobacillus plantarum is a lactic acid bacterium that can be found during cheese ripening. Lipolysis of milk triacylglycerols to free fatty acids during cheese ripening has fundamental consequences on cheese flavor. In the present study, the gene lp_1760, encoding a putative esterase or lipase, was cloned and expressed in Escherichia coli BL21 (DE3) and the overproduced Lp_1760 protein was biochemically characterized. Lp_1760 hydrolyzed p-nitrophenyl esters of fatty acids from C2 to C16, with a preference for p-nitrophenyl butyrate. On triglycerides, Lp_1760 showed higher activity on tributyrin than on triacetin. Although optimal conditions for activity were 45°C and pH 7, Lp_1760 retains activity under conditions commonly found during cheese making and ripening. The Lp_1760 showed more than 50% activity at 5°C and exhibited thermal stability at high temperatures. Enzymatic activity was strongly inhibited by sodium dodecyl sulfate and phenylmethylsulfonyl fluoride. The Lp_1760 tributyrin esterase showed high activity in the presence of NaCl, lactic acid, and calcium chloride. The results suggest that Lp_1760 might be a useful tributyrin esterase to be used in cheese manufacturing.

Technological and safety properties of lactic acid bacteria isolated from Spanish dry-cured sausages.

Technological and safety-related properties were analyzed in lactic acid bacteria isolated from Spanish dry-cured sausages in order to select them as starter cultures. In relation to technological properties, all the strains showed significative nitrate reductase activity; Lactobacillus plantarum, Lactobacillus paracasei and 52% of the Enterococcus faecium strains showed lipolytic activity and only Lactobacillus sakei strains (43%) were able to form biofilms. Related to safety aspects, E. faecium strains were the most resistant to antibiotics, whereas, L. sakei strains were the most sensitive. In relation to virulence factors, in the E. faecium strains analyzed, only the presence of efaA gene was detected. The analysis of biogenic amine production showed that most E. faecium strains and L. sakei Al-142 produced tyramine. In conclusion, L. paracasei Al-128 and L. sakei Al-143 strains possess the best properties to be selected as adequate and safe meat starter cultures.

An amperometric affinity penicillin-binding protein magnetosensor for the detection of ß-lactam antibiotics in milk.

The preparation, characterization and performance evaluation of an amperometric affinity disposable magnetosensor, based on the use of a recombinant penicillin-binding protein (PBP) and screen-printed carbon electrodes (SPCEs), for the specific detection and quantification of ß-lactam antibiotic residues in milk are reported. The PBP was immobilized onto His-Tag-Isolation-modified magnetic beads (His-Tag-Isolation-MBs), and a direct competitive assay using a tracer with horseradish peroxidase (HRP) for the enzymatic labeling was performed. The amperometric response obtained at -0.20 V vs. the Ag pseudo-reference electrode of the SPCE after the addition of H2O2 in the presence of hydroquinone (HQ) was used as the transduction signal. The developed methodology showed very low detection limits (in the low ppb level) for the 6 antibiotics tested in untreated milk samples, and a good selectivity against other antibiotic residues frequently detected in milk and dairy products. Due to the bioreceptor employed, this methodology was able to detect only the active form of ß-lactam antibiotics with high affinities for both penicillins and cephalosporins. Moreover, the analysis took only 30 min.

Characterization of coagulase-negative staphylococci isolated from Spanish dry cured meat products.

Technological and safety-related properties were analyzed in a coagulase-negative staphylococci (CNS) collection isolated from Spanish dry-cured meat products in order to use them as starter cultures. The highest nitrate reductase and proteolytic activity was showed by Staphylococcus carnosus and Staphylococcus equorum. Only a few strains were able to form biofilms and the presence of the ica gene was analyzed on them. In relation to antibiotic resistance, all S. carnosus and most of the S. equorum strains were sensitive to the antibiotics tested and the presence of the blaZ gene in the ß-lactamic resistant strains was studied. Biogenic amines were produced by 25% of the strains analyzed being all the S. carnosus strains tyramine producers. Taking into account the studied properties, two S. equorum strains could be selected as adequate and safe potential starter cultures for the elaboration of meat products.

Production of biogenic amines by lactic acid bacteria and enterobacteria isolated from fresh pork sausages packaged in different atmospheres and kept under refrigeration.

The occurrence of in vitro amino acid activity in bacterial strains associated with fresh pork sausages packaged in different atmospheres and kept in refrigeration was studied. The presence of biogenic amines in decarboxylase broth was confirmed by ion-exchange chromatography and by the presence of the corresponding decarboxylase genes by PCR. From the 93 lactic acid bacteria and 100 enterobacteria strains analysed, the decarboxylase medium underestimates the number of biogenic amine-producer strains. 28% of the lactic acid bacteria produced tyramine and presented the tdc gene. All the tyramine-producer strains were molecularly identified as Carnobacterium divergens. Differences on the relative abundance of C. divergens were observed among the different packaging atmospheres assayed. After 28 days of storage, the presence of argon seems to inhibit C. divergens growth, while packing under vacuum seems to favour it. Among enterobacteria, putrescine was the amine more frequently produced (87%), followed by cadaverine (85%); agmatine and tyramine were only produced by 13 and 1%, respectively, of the strains analysed. Packing under vacuum or in an atmosphere containing nitrogen seems to inhibit the growth of enterobacteria which produce simultaneously putrescine, cadaverine, and agmatine. Contrarily, over-wrapping or packing in an atmosphere containing argon seems to favour the growth of agmatine producer-enterobacteria. The production of putrescine and cadaverine was associated with the presence of the corresponding amino acid decarboxylase genes. The biogenic amine-producer strains were included in a wide range of enterobacterial species, including Kluyvera intermedia, Enterobacter aerogenes, Yersinia kristensenii, Serratia grimesii, Serratia ficaria, Yersinia rodhei, Providencia vermicola and Obesumbacterium proteus.

Evaluation of exopolysaccharide production by Leuconostoc mesenteroides strains isolated from wine.

Exopolysaccharide (EPS)-producing lactic acid bacteria are responsible for the alteration of wine and other fermented beverages. The potential to produce EPS was investigated for Leuconostoc mesenteroides strains isolated from Spanish grape must and wine. Most strains were able to produce EPS from sucrose containing media. Based on their EPS-producing phenotype and on their EPS monosaccharide composition, the L. mesenteroides strains analyzed could be arranged in 2 groups. One group comprises mucoid strains producing a glucan polymer, and the other group includes strains producing a fructan polymer. The presence of a glucosyltransferase encoding gene in the glucan producing L. mesenteroides strains was assayed by PCR. Two primer sets, PF1-PF8 and GTFF-GTFR, were used to amplify internal fragment of known glucosyltransferase genes. None of the glucan-producing strains gave a positive amplicon by the primer sets used. Therefore, new tools need to be developed to broaden the range of potentially spoiling agents detected by PCR in fermented beverages.

Biogenic amine production by Gram-positive bacteria isolated from Spanish dry-cured "chorizo" sausage treated with high pressure and kept in chilled storage.

We studied the production of biogenic amines by 200 strains of lactic acid bacteria and staphylococci isolated during chilled storage from samples of Spanish dry-cured "chorizo" sausage treated with high-pressure. The presence of biogenic amines in a decarboxylase synthetic broth was confirmed by ion-exchange chromatography. ß-phenylethylamine was the biogenic amine more frequently produced (22.5%), followed by tyramine (7.5%). In tyramine producer-strains the presence of a tyrosine decarboxylase gene was confirmed by PCR. Among lactic acid bacteria, the production of tyramine was mainly related to the species Lactobacillus curvatus. Most of the L. curvatus strains were also ß-phenylethylamine-producers. In relation to staphylococci, tyramine-production was mainly associated to Staphylococcus carnosus strains. The S. carnosus strains analysed in this study produced ß-phenylethylamine or ß-phenylethylamine and tyramine simultaneously. RAPD-PCR results indicated that the biogenic amine-producer S. carnosus population changes along storage independently of the high-pressure treatment.

Molecular cloning and functional characterization of a histidine decarboxylase from Staphylococcus capitis.

AIMS: Histamine intoxication is probably the best known toxicological problem of food-borne disease. A histamine-producing Staphylococcus capitis strain has been isolated from a cured meat product. The aim of this study was to gain deeper insights into the genetic determinants for histamine production in Staph. capitis. METHODS AND RESULTS: The nucleotide sequence of a 6446-bp chromosomal DNA fragment containing the hdcA gene encoding histidine decarboxylase (HDC) has been determined in Staph. capitis IFIJ12. This DNA fragment contains five complete and two partial open reading frames. Putative functions have been assigned to gene products by sequence comparison with proteins included in the databases. The hdcA gene has been expressed in Escherichia coli resulting in HDC activity. The presence of a functional promoter (Phdc) located upstream of hdcA has been demonstrated. Insertion of the histamine biosynthetic locus in Staph. capitis seems to be associated with a noticeable genome reorganization. CONCLUSIONS: Among the staphylococcal species analysed in this study only Staph. capitis strains produce histamine. The hdcA gene cloned from Staph. capitis encodes a functional HDC that produce histamine from the amino acid histidine. SIGNIFICANCE AND IMPACT OF THE STUDY: The identification of the DNA region involved in histamine production in Staph. capitis will allow further work in order to avoid histamine production in foods.

Gene organization of the ornithine decarboxylase-encoding region in Morganella morganii.

AIMS: The production of putrescine is a relevant property related to food quality and safety. Morganella morganii is responsible for putrescine production in fresh fish decomposition. The aim of this study was to gain deeper insights into the genetic determinants for putrescine production in M. morganii. METHODS AND RESULTS: The 6972 bp DNA region showed the presence of three complete and two partial open reading frames all transcribed in the same direction. The second and third genes putatively coded for an ornithine decarboxylase (SpeF) and a putrescine-ornithine antiporter (PotE), respectively, and constituted an operon. The speF gene has been expressed in Escherichia coli HT414, an ornithine decarboxylase defective mutant, resulting in ornithine decarboxylase activity. The genetic organization of the SpeF-PotE-encoding region in M. morganii is different to that of E. coli and several Salmonella species. CONCLUSIONS: The speF gene cloned from M. morganii encodes a functional ornithine decarboxylase involved in putrescine production. Phylogenetic tree based on 16S rDNA showed that ornithine decarboxylase activity is not related to a specific phylogenetic tree branch in Enterobacteriaceae. SIGNIFICANCE AND IMPACT OF THE STUDY: The identification of the DNA region involved in putrescine production in M. morganii will allow additional research on their induction and regulation in order to minimize putrescine production in foods.

Complete nucleotide sequence and structural organization of pPB1, a small Lactobacillus plantarum cryptic plasmid that originated by modular exchange.

A small cryptic plasmid designated pPB1 was isolated from Lactobacillus plantarum BIFI-38 and its complete 2899 bp nucleotide sequence was determined. Sequence analysis revealed four putative open reading frames. Based on sequence analysis two modules could be identified. First, the replication module consisted of a sequence coding for a replication protein (RepB) and its corresponding target site, and two putative repressor proteins (RepA and RepC). Sequence analysis indicated the possible synthesis of an antisense RNA that might regulate RepB production. A putative lagging-strand initiation site was also found, suggesting that pPB1 replicates via a rolling circle mechanism. The second module of pPB1 consisted of a sequence coding for a putative mobilization protein and its corresponding oriT site. Since the nucleotide sequence of the replication module showed 94.5% identity to the similar region on the Leuconostoc lactis plasmid pCI411, and the nucleotide sequence of the mobilization module had 97.5% identity to L. plantarum plasmid pLB4, it is concluded that pPB1 originated by modular exchange between two such plasmids by homologous recombination. Putative recombination sites where crossover might have taken place were also identified.

Molecular characterization of the pneumococcal teichoic acid phosphorylcholine esterase.

A search to identify proteins with high affinity for choline-containing pneumococcal cell walls (choline-binding proteins) has permitted the localization, cloning, sequencing, and overexpression of a gene (pce), coding for a protein (Pce) that liberates phosphorylcholine from purified cell walls of Streptococcus pneumoniae. The pce gene of the pneumococcal strain R6 encodes a protein of 627 amino acids with a predicted Mr of 72,104. Pce can remove a maximum of 20% phosphorylcholine residues from the cell wall teichoic acid. In silico analysis of Pce shows a modular organization of the enzyme where the choline-binding domain, involved in cell wall substrate recognition, and the catalytic domain are located at the carboxy- and amino-terminal moieties of the protein, respectively. Remarkably, a long tail of 85 amino acids follows the carboxy-terminal domain, a structural feature that had not been described for the published choline-binding proteins. The carboxy-terminal moiety of Pce is assembled by 10 repeated motifs, and the protein has also a cleavable signal peptide of 25 amino acids that renders after its cleavage a mature protein of 69,426 Da (602 amino acids). The pce gene has been expressed in Escherichia coli, and Pce was active when assayed on pneumococcal walls. We have also found that the signal peptide of Pce was functional in E. coli. Biochemical analysis suggested that Pce is the teichoic acid phosphorylcholine esterase of S. pneumoniae that had been biochemically characterized previously. Construction of two pce pneumococcal mutants (R6D and M31D) by insertion-duplication mutagenesis revealed that these mutants grew at a doubling-time similar to those of the parental strains of the wild-type R6 and the lytA-mutant M31, respectively. R6D and M31D were morphologically indistinguishable from the parental strains when whole-mounted cells were observed under the electron microscope and exhibited levels of competence for genetic transformation slightly lower than those reported for R6 and M31.