Isabel Cuesta

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.

Quantitative detection and identification of tyramine-producing enterococci and lactobacilli in cheese by multiplex qPCR

Tyramine is the most abundant biogenic amine in fermented dairy products, in which it is produced through the microbial enzymatic decarboxylation of tyrosine. This activity has been detected in a variety of lactic acid bacteria mainly belonging to the genera Enterococcus and Lactobacillus. This paper describes a culture-independent qPCR method, based on the specific amplification of the tdc gene, for the detection, quantification and identification of bacteria with the ability to produce tyramine. This method was found to be specific and to show a wide dynamic range, thus allowing the quantification of these tdc+ bacterial groups among the complex microbiota of cheese. tdc qPCR was used to follow the development of tdc+ microbiota during the manufacture of a blue-veined cheese (Cabrales) made from raw milk. In this type of cheese, tdc+ enterococci seem to be responsible for the high concentrations of tyramine detected. The method was also used to identify and quantify tdc+ enterococci and lactobacilli in 18 commercially available cheeses. Different types and numbers of these microorganisms were found. Their relationships with the concentration of tyramine and technological factors are discussed.

Conformational rearrangements of SV40 large-T antigen during early replication events

The Simian virus 40 (SV40) large tumor antigen (LTag) functions as the replicative helicase and initiator for viral DNA replication. For SV40 replication, the first essential step is the assembly of an LTag double hexamer at the origin DNA that will subsequently melt the origin DNA to initiate fork unwinding. In this study, we used three-dimensional cryo-electron microscopy to visualize early events in the activation of DNA replication in the SV40 model system. We obtained structures of wild-type double-hexamer complexes of LTag bound to SV40 origin DNA, to which atomic structures have been fitted. Wild-type LTag was observed in two distinct conformations: In one conformation, the central module containing the J-domains and the origin binding domains of both hexamers is a compact closed ring. In the other conformation, the central module is an open ring with a gap formed by rearrangement of the N-terminal regions of the two hexamers, potentially allowing for the passage of single-stranded DNA generated from the melted origin DNA. Double-hexamer complexes containing mutant LTag that lacks the N-terminal J-domain show the central module predominantly in the closed-ring state. Analyses of the LTag C-terminal regions reveal that the LTag hexamers bound to the A/T-rich tract origin of replication and early palindrome origin of replication elements are structurally distinct. Lastly, visualization of DNA density protruding from the LTag C-terminal domains suggests that oligomerization of the LTag complex takes place on double-stranded DNA.

Comparison of molecular dynamics and superfamily spaces of protein domain deformation

BackgroundIt is well known the strong relationship between protein structure and flexibility, on one hand, and biological protein function, on the other hand. Technically, protein flexibility exploration is an essential task in many applications, such as protein structure prediction and modeling. In this contribution we have compared two different approaches to explore the flexibility space of protein domains: i) molecular dynamics (MD-space), and ii) the study of the structural changes within superfamily (SF-space).ResultsOur analysis indicates that the MD-space and the SF-space display a significant overlap, but are still different enough to be considered as complementary. The SF-space space is wider but less complex than the MD-space, irrespective of the number of members in the superfamily. Also, the SF-space does not sample all possibilities offered by the MD-space, but often introduces very large changes along just a few deformation modes, whose number tend to a plateau as the number of related folds in the superfamily increases.ConclusionTheoretically, we obtained two conclusions. First, that function restricts the access to some flexibility patterns to evolution, as we observe that when a superfamily member changes to become another, the path does not completely overlap with the physical deformability. Second, that conformational changes from variation in a superfamily are larger and much simpler than those allowed by physical deformability. Methodologically, the conclusion is that both spaces studied are complementary, and have different size and complexity. We expect this fact to have application in fields as 3D-EM/X-ray hybrid models or ab initio protein folding.

Assessment of intestinal microbiota modulation ability of Bifidobacterium strains in in vitro fecal batch cultures from preterm neonates.

Microbial colonization of the infant gut is essential for the development of the intestine and the immune system. The intestinal microbiota of full-term breast-fed infants is considered as the health standard for newborns. A culture medium containing formula milk was designed, which allowed a balanced growth of intestinal microorganisms and was used to perform fecal batch cultures from preterm babies. Sixteen Bifidobacterium strains and fructooligosaccharides (FOS) were tested for their ability to modulate in vitro the intestinal microbiota. The production of short chain fatty acids (SCFA) was measured by Gas Chromatography and the levels of some anaerobe (Bifidobacterium and Bacteroides groups) and facultative anaerobes (Enterobacteriaceae, Enterococcaceae, Weissella group, and Klebsiella pneumoniae) were determined by quantitative PCR. Results were referred to a fecal negative control culture without microorganisms or FOS added. Strains that in fecal cultures counteracted better the aberrancies previously found in feces of preterm babies, as compared with full-term breast-fed infants, were selected. The three Bifidobacterium bifidum strains tested in this work promoted the most suitable shifts in SCFA and in the ratio of variables facultative anaerobes to anaerobes. Two Bifidobacterium breve strains complied with the requirement for facultative anaerobes and anaerobes and one of them also promoted a suitable shift of SCFA. Bifidobacteria behaved similarly as FOS regarding the microbial profiles in fecal cultures but the production of lactic and acetic acid was much lower. B. breve and B. bifidum strains selected represent promising candidates for their assessment in more complex in vitro and in vivo models.

Technological characterization and survival of the exopolysaccharide-producing strain Lactobacillus delbrueckii subsp. lactis 193 and its bile-resistant derivative 193+ in simulated gastric and intestinal juices.

The capacity of lactic acid bacteria to produce exopolysaccharides (EPS) conferring microorganisms a ropy phenotype could be an interesting feature from a technological point of view. Progressive adaptation to bile salts might render some lactobacilli able to overcome physiological gut barriers but could also modify functional properties of the strain, including the production of EPS. In this work some technological properties and the survival ability in simulated gastrointestinal conditions of Lactobacillus delbrueckii subsp. lactis 193, and Lb. delbrueckii subsp. lactis 193+, a strain with stable bile-resistant phenotype derived thereof, were characterized in milk in order to know whether the acquisition of resistance to bile could modify some characteristics of the microorganism. Both strains were able to grow and acidify milk similarly; however the production of ethanol increased at the expense of the aroma compound acetaldehyde in milk fermented by the strain 193+, with respect to milk fermented by the strain 193. Both microorganisms produced a heteropolysaccharide composed of glucose and galactose, and were able to increase the viscosity of fermented milks. In spite of the higher production yield of EPS by the bile-resistant strain 193+, it displayed a lower ability to increase viscosity than Lb. delbrueckii subsp. lactis 193. Milk increased survival in simulated gastric juice; the presence of bile improved adhesion to the intestinal cell line HT29-MTX in both strains. However, the acquisition of a stable resistance phenotype did not improve survival in simulated gastric and intestinal conditions or the adhesion to the intestinal cell line HT29-MTX. Thus, Lb. delbrueckii subsp. lactis 193 presents suitable technological properties for the manufacture of fermented dairy products; the acquisition of a stable bile-resistant phenotype modified some properties of the microorganism. This suggests that the possible use of bile-resistant derivative strains should be carefully evaluated in each specific application considering the influence that the acquisition of a stable bile-resistant phenotype could have in survival ability in gastric and intestinal conditions and in technological properties.

Bacteroides fragilis metabolises exopolysaccharides produced by bifidobacteria

BackgroundBacteroides fragilis is the most frequent species at the human intestinal mucosal surface, it contributes to the maturation of the immune system although is also considered as an opportunistic pathogen. Some Bifidobacterium strains produce exopolysaccharides (EPS), complex carbohydrate polymers that promote changes in the metabolism of B. fragilis when this microorganism grows in their presence. To demonstrate that B. fragilis can use EPS from bifidobacteria as fermentable substrates, purified EPS fractions from two strains, Bifidobacterium longum E44 and Bifidobacterium animalis subsp. lactis R1, were added as the sole carbon source in cultures of B. fragilis DSMZ 2151 in a minimal medium. Bacterial counts were determined during incubation and the evolution of organic acids, short chain fatty acids (SCFA) and evolution of EPS fractions was analysed by chromatography.ResultsGrowth of B. fragilis at early stages of incubation was slower in EPS than with glucose, microbial levels remaining higher in EPS at prolonged incubation times. A shift in metabolite production by B. fragilis occurred from early to late stages of growth, leading to the increase in the production of propionate and acetate whereas decrease lactate formation. The amount of the two peaks with different molar mass of the EPS E44 clearly decreased along incubation whereas a consumption of the polymer R1 was not so evident.ConclusionsThis report demonstrates that B. fragilis can consume some EPS from bifidobacteria, with a concomitant release of SCFA and organic acids, suggesting a role for these biopolymers in bacteria-bacteria cross-talk within the intestine.

A novel UHPLC method for the rapid and simultaneous determination of daidzein, genistein and equol in human urine

This work reports on a novel method involving reverse-phased ultra-high performance liquid chromatography (UHPLC) plus a spectrophotometric photodiode array/fluorescence (FLR) detection system for determining the concentration of equol and major soy isoflavones (daidzein and genistein) in human urine. The proposed method was validated in terms of its linearity, sensitivity, accuracy (recovery) and precision (intra- and inter-day repeatability). The isoflavone profiles of urine samples from a group of menopausal women following oral soy isoflavone supplementation were determined and compared. Screening for equol-producer status was accomplished with high sensitivity (detection limit of the FLR detector 2.93nM). The method involves a short chromatographic run time compared to conventional HPLC methods while allowing for the simultaneous and reliable quantification of daidzein, genistein and equol in human urine. It also allows for the rapid screening of multiple urine samples when testing for equol production status and checking patient adherence to isoflavone treatment regimens.

Glucolytic fingerprinting reveals metabolic groups within the genus Bifidobacterium: an exploratory study.

Microorganisms of the genus Bifidobacterium are inhabitants of diverse niches including the digestive tract of humans and animals. The species Bifidobacterium adolescentis, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium breve and Bifidobacterium longum have qualified presumption of safety status granted by EFSA and several strains are considered probiotic, and are being included in functional dairy fermented products. In the present work we carried out a preliminary exploration of general metabolic characteristics and organic acid production profiles of a reduced number of strains selected from these and other species of the genus Bifidobacterium. The use of resting cells allowed obtaining metabolic fingerprints without interference of metabolites accumulated during growth in culture media. Acetic acid was the most abundant organic acid formed per mol of glucose consumed (from 1.07 ± 0.03 to 1.71 ± 0.22 mol) followed by lactic acid (from 0.34 ± 0.06 to 0.90 ± 0.12 mol), with moderate differences in production among strains; pyruvic, succinic and formic acids were also produced at considerably lower proportions, with variability among strains. The acetic to lactic acid ratio showed lower values in stationary phase as regard to the exponential phase for most, but not all, the microorganisms; this was due to a decrease in acetic acid molar proportions together with increases of lactic acid proportions in stationary phase. A linear discriminant analysis allowed to cluster strains into species with 51-100% probability, evidencing different metabolic profiles, according to the relative production of organic acids from glucose by resting cells, of microorganisms collected at the exponential phase of growth. Looking for a single metabolic marker that could adequately discriminate metabolic groups, we found that groups established by the acetic to lactic acid ratio fit well with differences previously evidenced by the discriminant analysis. The proper establishment of metabolic groups within the genus Bifidobacterium could help to select the best suited probiotic strains for specific applications.

A proteomic approach towards understanding the cross talk between Bacteroides fragilis and Bifidobacterium longum in coculture

A better understanding of the interactions among intestinal microbes is needed to decipher the complex cross talk that takes place within the human gut. Bacteroides and Bifidobacterium genera are among the most relevant intestinal bacteria, and it has been previously reported that coculturing of these 2 microorganisms affects their survival. Therefore, coculturing of Bifidobacterium longum NB667 and Bacteroides fragilis DSMZ2151 was performed with the aim of unravelling the mechanisms involved in their interaction. To this end, we applied proteomic (2D-DIGE) analyses, and by chromatographic techniques we quantified the bacterial metabolites produced during coincubation. Coculture stimulated the growth of B. longum, retarding that of B. fragilis, with concomitant changes in the production of some proteins and metabolites of both bacteria. The combined culture promoted upregulation of the bifidobacterial pyruvate kinase and downregulation of the Bacteroides phosphoenolpyruvate carboxykinase - 2 enzymes involved in the catabolism of carbohydrates. Moreover, B. fragilis FKBP-type peptidyl-prolyl cis-trans isomerase, a protein with chaperone-like activity, was found to be overproduced in coculture, suggesting the induction of a stress response in this microorganism. This study provides mechanistic data to deepen our understanding of the interaction between Bacteroides and Bifidobacterium intestinal populations.