Begoña Redruello

A fast, reliable, ultra high performance liquid chromatography method for the simultaneous determination of amino acids, biogenic amines and ammonium ions in cheese, using diethyl ethoxymethylenemalonate as a derivatising agent

Derivatisation treatment with diethyl ethoxymethylenemalonate followed by ultra-HPLC allowed the simultaneous quantification of 22 amino acids, 7 biogenic amines and ammonium ions in cheese samples in under 10 min. This is the fastest elution time ever reported for such a resolution. The proposed method shows good linearity (R(2)>0.995) and sensitivity (detection limit 0.08-3.91 μM; quantification limit <13.02 μM). Intra- and inter-day repeatability ranged from 0.35% to 1.25% and from 0.85% to 5.2%, respectively. No significant effect of the cheese matrix was observed.

Isolation and characterization of piscine osteonectin and downregulation of Its expression by PTH-related protein

The skeleton is the main source of osteonectin mRNA in adults of the seawater teleost sea bream Sparus auratus. It is expressed by cells forming the basement membrane of calcifying tissue indicating that, as in mammals, it may play a role in osteoblast differentiation. PTHrP induced downregulation of osteonectin mRNA in vitro in scales, a mineralizing tissue with bone‐like metabolism. This indicates a means to redirect calcium to activities such as vitellogenesis when this ion is in high demand.

Comparative analysis of the in vitro cytotoxicity of the dietary biogenic amines tyramine and histamine

Tyramine and histamine, the most toxic biogenic amines (BA), are often found in high concentrations in certain foods. Prompted by the limited knowledge of BA toxicity, and increasing awareness of the risks associated with high intakes of dietary BA, the in vitro cytotoxicity of tyramine and histamine was investigated. Tyramine and histamine were toxic for HT29 intestinal cell cultures at concentrations commonly found in BA-rich food, as determined by real-time cell analysis. Surprisingly, tyramine had a stronger and more rapid cytotoxic effect than histamine. Their mode of action was also different, while tyramine caused cell necrosis, histamine induced apoptosis. To avoid health risks, the BA content of foods should be reduced and legal limits established for tyramine.

Immunohistochemical detection of estrogen receptors in fish scales

Calcium mobilization from internal stores, such as scales, induced by 17beta-estradiol during sexual maturation in salmonids is well documented. This calcium mobilization from scales is proposed to be mediated by the estrogen receptor (ER). However, the ER subtypes involved and signaling mechanisms responsible for this effect remain to be fully characterized. In the present study, we have localized ERalpha, ERbetaa and ERbetab proteins in juvenile and adult sea bream (Sparus auratus) and Mozambique tilapia (Oreochromis mossambicus) scales by immunohistochemistry with sea bream ER subtype specific antibodies. The three ERs were detected in isolated or small groups of round cells, in the basal layer of the scales of both juvenile and adult fish and the localization and signal intensity varied with the species and age of the animals. The ERs may be co-localized in cells of the scale posterior region that expressed tartrate-resistant acid phosphatase (TRAP), a marker for osteoclasts. These results suggest that the calcium mobilizing action of 17beta-estradiol on fish scales is via its direct action on ERs localized in osteoclasts.

Equol status and changes in fecal microbiota in menopausal women receiving long-term treatment for menopause symptoms with a soy-isoflavone concentrate

The knowledge regarding the intestinal microbial types involved in isoflavone bioavailability and metabolism is still limited. The present work reports the influence of a treatment with isoflavones for 6 months on the fecal bacterial communities of 16 menopausal women, as determined by culturing and culture-independent microbial techniques. Changes in fecal communities were analyzed with respect to the women’s equol-producing phenotype. Compared to baseline, at 1 and 3 months the counts for all microbial populations in the feces of equol-producing women had increased strongly. In contrast, among the non-producers, the counts for all microbial populations at 1 month were similar to those at baseline, and decreased significantly by 3 and 6 months. Following isoflavone intake, major bands in the denaturing gradient gel electrophoresis (DGGE) profiles appeared and disappeared, suggesting important changes in majority populations. In some women, increases were seen in the intensity of specific DGGE bands corresponding to microorganisms known to be involved in the metabolism of dietary phytoestrogens (Lactonifactor longoviformis, Faecalibacterium prausnitzii, Bifidobacterium sp., Ruminococcus sp.). Real-Time quantitative PCR revealed that the Clostridium leptum and C. coccoides populations increased in equol producers, while those of bifidobacteria and enterobacteria decreased, and vice versa in the non-producers. Finally, the Atopobium population increased in both groups, but especially in the non-producers at three months. As the main findings of this study, (i) variations in the microbial communities over the 6-month period of isoflavone supplementation were large; (ii) no changes in the fecal microbial populations that were convincingly treatment-specific were seen; and (iii) the production of equol did not appear to be associated with the presence of, or increase in the population of, any of the majority bacterial types analyzed.

The putrescine biosynthesis pathway in Lactococcus lactis is transcriptionally regulated by carbon catabolic repression, mediated by CcpA.

Lactococcus lactis is the lactic acid bacterium most widely used by the dairy industry as a starter for the manufacture of fermented products such as cheese and buttermilk. However, some strains produce putrescine from agmatine via the agmatine deiminase (AGDI) pathway. The proteins involved in this pathway, including those necessary for agmatine uptake and conversion into putrescine, are encoded by the aguB, aguD, aguA and aguC genes, which together form an operon. This paper reports the mechanism of regulation of putrescine biosynthesis in L. lactis. It is shown that the aguBDAC operon, which contains a cre site at the promoter of aguB (the first gene of the operon), is transcriptionally regulated by carbon catabolic repression (CCR) mediated by the catabolite control protein CcpA.

Genetic and functional analysis of biogenic amine production capacity among starter and non-starter lactic acid bacteria isolated from artisanal cheeses

This work reports the capacity of 137 strains of starter and non-starter LAB belonging to nine species of the genera Lactobacillus, Lactococcus, Streptococcus and Leuconostoc (all isolated from artisanal cheeses) to produce histamine, tyramine, putrescine and β-phenylethylamine, the biogenic amines (BA) most commonly found in dairy products. Production assays were performed in liquid media supplemented with the appropriate precursor amino acid; culture supernatants were then tested for BA by (U)HPLC. In addition, the presence of key genes involved in the biosynthetic pathways of the target BA, including the production of putrescine via the agmatine deiminase pathway, was assessed by PCR. Twenty strains were shown to have genes involved in the synthesis of BA; these belonged to the species Lactobacillus brevis (4), Lactobacillus curvatus (3), Lactococcus lactis (11) and Streptococcus thermophilus (2). With the exception of the two S. thermophilus strains, all those possessing genes involved in BA production synthesized the corresponding compound. Remarkably, all the putrescine-producing strains used the agmatine deiminase pathway. Four L. brevis and two L. curvatus strains were found able to produce both tyramine and putrescine. There is increasing interest in the use of autochthonous LAB strains in starter and adjunct cultures for producing dairy products with ‘particular geographic indication’ status. Such strains should not produce BA; the present results show that BA production capacity should be checked by (U)HPLC and PCR.

The Relationship among Tyrosine Decarboxylase and Agmatine Deiminase Pathways in Enterococcus faecalis

Enterococci are considered mainly responsible for the undesirable accumulation of the biogenic amines tyramine and putrescine in cheeses. The biosynthesis of tyramine and putrescine has been described as a species trait in Enterococcus faecalis. Tyramine is formed by the decarboxylation of the amino acid tyrosine, by the tyrosine decarboxylase (TDC) route encoded in the tdc cluster. Putrescine is formed from agmatine by the agmatine deiminase (AGDI) pathway encoded in the agdi cluster. These biosynthesis routes have been independently studied, tyrosine and agmatine transcriptionally regulate the tdc and agdi clusters. The objective of the present work is to study the possible co-regulation among TDC and AGDI pathways in E. faecalis. In the presence of agmatine, a positive correlation between putrescine biosynthesis and the tyrosine concentration was found. Transcriptome studies showed that tyrosine induces the transcription of putrescine biosynthesis genes and up-regulates pathways involved in cell growth. The tyrosine modulation over AGDI route was not observed in the mutant Δtdc strain. Fluorescence analyses using gfp as reporter protein revealed PaguB (the promoter of agdi catabolic genes) was induced by tyrosine in the wild-type but not in the mutant strain, confirming that tdc cluster was involved in the tyrosine induction of putrescine biosynthesis. This study also suggests that AguR (the transcriptional regulator of agdi) was implicated in interaction among the two clusters.

The dietary biogenic amines tyramine and histamine show synergistic toxicity towards intestinal cells in culture

Tyramine and histamine are the biogenic amines (BA) most commonly found at high concentrations in food; they may even appear together at toxic concentrations. The present work examines, via real-time cell analysis, whether histamine and tyramine show synergistic toxicity towards intestinal cell cultures. Employing a constant equipotency ratio, their interaction was examined via the combination index (CI) method of Chou & Talalay. Co-treatment with tyramine and histamine was associated with a stronger cytotoxic effect than was treatment with either BA or on its own. Indeed, a synergistic interaction (CI<1) was observed in the range of concentrations found in foods. The results also show that histamine, at concentrations below the legal limit, increases the cytotoxicity of tyramine at concentrations frequently reached in some foods. The synergistic cytotoxicity of tyramine and histamine should be taken into account when establishing legal limits designed to ensure consumer safety.

Different metabolic features of Bacteroides fragilis growing in the presence of glucose and exopolysaccharides of bifidobacteria

Bacteroides is among the most abundant microorganism inhabiting the human intestine. They are saccharolytic bacteria able to use dietary or host-derived glycans as energy sources. Some Bacteroides fragilis strains contribute to the maturation of the immune system but it is also an opportunistic pathogen. The intestine is the habitat of most Bifidobacterium species, some of whose strains are considered probiotics. Bifidobacteria can synthesize exopolysaccharides (EPSs), which are complex carbohydrates that may be available in the intestinal environment. We studied the metabolism of B. fragilis when an EPS preparation from bifidobacteria was added to the growth medium compared to its behavior with added glucose. 2D-DIGE coupled with the identification by MALDI-TOF/TOF evidenced proteins that were differentially produced when EPS was added. The results were supported by RT-qPCR gene expression analysis. The intracellular and extracellular pattern of certain amino acids, the redox balance and the α-glucosidase activity were differently affected in EPS with respect to glucose. These results allowed us to hypothesize that three general main events, namely the activation of amino acids catabolism, enhancement of the transketolase reaction from the pentose-phosphate cycle, and activation of the succinate-propionate pathway, promote a shift of bacterial metabolism rendering more reducing power and optimizing the energetic yield in the form of ATP when Bacteroides grow with added EPSs. Our results expand the knowledge about the capacity of B. fragilis for adapting to complex carbohydrates and amino acids present in the intestinal environment.