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Characterization by electron paramagnetic resonance techniques of several variants of Anabaena flavodoxin, where the naturally occurring FMN cofactor is substituted by different analogs, makes it possible to improve the details of the spin distribution map in the isoallosazine ring in its semiquinone state. The analyzed variants were selected to monitor the effects of intrinsic changes in the flavin ring electronic structure, as well as perturbations in the apoflavodoxin-flavin interaction, on the spin populations. When these effects were analyzed together with the functional properties of the different flavodoxin variants, a relationship between spin population and biochemical parameters, as the reduction potential, could be envisaged.

Bacterial dioxygenase systems are multicomponent enzymes that catalyze the initial degradation of many environmentally hazardous compounds. In Sphingopyxis granuli strain TFA tetralin dioxygenase hydroxylates tetralin, an organic contaminant. It consists of a ferredoxin reductase (ThnA4), a ferredoxin (ThnA3) and a oxygenase (ThnA1/ThnA2), forming a NAD(P)H-ThnA4-ThnA3-ThnA1/ThnA2 electron transport chain. ThnA3 has also a regulatory function since it prevents expression of tetralin degradation genes (thn) in the presence of non-metabolizable substrates of the catabolic pathway. This role is of physiological relevance since avoids gratuitous and wasteful production of catabolic enzymes. Our hypothesis for thn regulation implies that ThnA3 exerts its action by diverting electrons towards the regulator ThnY, an iron-sulfur flavoprotein that together with the transcriptional activator ThnR is necessary for thn gene expression. Here we analyze electron transfer among ThnA4, ThnA3 and ThnY by using stopped-flow spectrophotometry and determination of midpoint reduction potentials. Our results indicate that when accumulated in its reduced form ThnA3 is able to fully reduce ThnY. In addition, we have reproduced in vitro the regulatory circuit in the proposed physiological direction, NAD(P)H-ThnA4-ThnA3-ThnY. ThnA3 represents an unprecedented way of communication between a catabolic pathway and its regulatory system to prevent gratuitous induction.

In this work, a variety of biochemical properties of Enterococcus faecium CECT 8849, which had been isolated from breast milk, were analyzed. Its acidifying capacity and proteolytic activity were low but, in contrast, remarkable peptidase and esterase activities were observed. Ethanol and 3-hydroxy-2-butanone were the most abundant volatile compounds found in experimental model cheese manufactured with E. faecium CECT 8849. This strain inhibited the growth of several Listeria monocytogenes and Listeria innocua strains in vitro. Enterocin A and B structural genes were detected in E. faecium CECT 8849. Model fermented milk and cheeses were manufactured from milk inoculated or not with L. innocua CECT 8848 (2.5–3 log10 colony forming units mL−1) using E. faecium CECT 8849 or Lactococcus lactis ESI 153 as starter cultures. Although E. faecium CECT 8849 controlled Listeria growth in both dairy models, it led to lower reduction in Listeria counts when compared with L. lactis ESI 153. © 2016, Springer-Verlag Berlin Heidelberg.

Reuterin has a high potential as a food preservative due to both its chemical characteristics and its antimicrobial activity against food-borne pathogens and spoilage bacteria. However, there is a lack of information about its toxicity and its capacity to interfere with the metabolism of drugs by inhibiting cytochrome P450 (CYP) activity. The results of this study indicated that reuterin exhibited a moderate cytotoxicity in the human hepatoma cell line HepG2 according to assays measuring three different endpoints in the same set of cells. Reuterin was much less toxic than acrolein and only four times more toxic than diacetyl, a generally recognized as safe flavoring compound. In vitro experiments utilizing human liver microsomes showed that reuterin presents low possibility of displaying in vivo drug interactions by inhibition of CYP3A4, CYP2D6, and CYP2C9. Therefore, reuterin can be considered a promising food biopreservative, although additional toxicology research is needed before permission for use can be granted. © 2016 Elsevier Ltd

The effect of reuterin, lactoperoxidase system (LPS) and lactoferrin (LF) combined with high hydrostatic pressure (HHP) on the characteristics of sliced cooked ham during 35 days at 4 and 10 °C were investigated. Reuterin and LPS inhibited the growth of total microorganisms during 35 days at 4 and 10 °C, whereas a regrowth at 10 °C was observed when HHP was applied. Combined treatments kept total viable counts below 1.5 log cfu/g after 35 days at 10 °C. Regarding the effect of treatments on colour of cooked ham, LPS alone or in combination with HHP slightly affected L∗, a∗and b∗values, but these changes tended to attenuate during storage. Likely, slight differences were registered in shear strength values among control and treated cooked ham. The accumulation of volatile compounds was reduced in cooked ham treated with LPS and LF in combination with HHP, even under abuse temperature conditions (10 °C). Industrial relevance LPS applied in combination with HHP was the most effective treatment at reducing the growth of total microorganisms in refrigerated cooked ham with minor changes in its characteristics. The antimicrobial activity of such combined treatment against food-borne pathogens, which has also been reported in RTE foods, points to its usefulness to assure a safe product of sensory characteristics similar to those of untreated cooked ham. © 2016 Elsevier Ltd. All rights reserved.

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. © 2016, Springer Science+Business Media Dordrecht.

Phytoestrogens are plant-derived polyphenols with a structure similar to human estrogens. The three main groups of phytoestrogens, isoflavones, ellagitannins, and lignans, are transformed into equol, urolithins, and enterolignans, respectively, by bacteria. These metabolites have more estrogenic/antiestrogenic and antioxidant activities than their precursors, and they are more bioavailable. The aim of this study was to analyze the metabolism of isoflavones, lignans and ellagitannins by gut microbiota, and to study the possible correlation in the metabolism of these three groups of phytoestrogens. In vitro fermentation experiments were performed with feces samples from 14 healthy adult volunteers, and metabolite formation was measured by HPLC-PAD and HPLC-ESI/MS. Only the microbiota of one subject produced equol, while most of them showed production of O-desmethylangolensin (O-DMA). Significant inter-subject differences were observed in the metabolism of dihydrodaidzein and dihydrogenistein, while the glucoside isoflavones and their aglycones showed less variability, except for glycitin. Most subjects produced urolithins M-5 and E. Urolithin D was not detected, while uroltithin B was found in half of the individuals analyzed, and urolithins A and C were detected in two and four subjects, respectively. Enterolactone was found in all subjects, while enterodiol only appeared in five. Isoflavone metabolism could be correlated with the metabolism of lignans and ellagitannins. However, the metabolism of ellagitannins and lignans could not be correlated. This the first study where the metabolism of the three groups together of phytoestrogen, isoflavones, lignans, and ellagitannins by gut microbiota is analyzed. © 2016 by the authors; licensee MDPI.

BACKGROUND High hydrostatic pressure (HHP) combined with reuterin and lactoperoxidase system (LPS) has exerted antimicrobial activity against Listeria monocytogenes in cold-smoked salmon at chilled temperatures. Therefore the purpose of this work was to evaluate the effect of HHP combined with reuterin, LPS and lactoferrin (LF) on the survival of Salmonella enterica subsp. enterica serovar Enteritidis and Escherichia coli O157H7 in cold-smoked salmon stored at 4 and 10°C. RESULTS Salmonella Enteritidis and E. coli O157H7 were reduced more than 3 log colony-forming units (CFU) g-1 by the pressure treatment (450 MPa/5min). LPS slightly diminished pathogen levels throughout storage, whereas no effect was recorded when reuterin or LF was added. The Salmonella population was below the detection limit (<1 log CFU g-1) during the storage of HHP-treated smoked salmon at 4 and 10°C. The antimicrobial activity of HHP against E. coli O157H7 was increased when 450 MPa was applied in combination with LPS in cold-smoked salmon at 4 and 10°C. CONCLUSION HHP at 450 MPa/5min inactivated S. Enteritidis in cold-smoked salmon and in combination with LPS would be useful as a hurdle technology approach against E. coli O157H7, even under mild temperature abuse conditions. © 2015 Society of Chemical Industry.

Bacterial dioxygenase systems are multicomponent enzymes that catalyze the initial degradation of many environmentally hazardous compounds. In Sphingopyxis granuli strain TFA tetralin dioxygenase hydroxylates tetralin, an organic contaminant. It consists of a ferredoxin reductase (ThnA4), a ferredoxin (ThnA3) and a oxygenase (ThnA1/ThnA2), forming a NAD(P)H-ThnA4-ThnA3-ThnA1/ThnA2 electron transport chain. ThnA3 has also a regulatory function since it prevents expression of tetralin degradation genes (thn) in the presence of non-metabolizable substrates of the catabolic pathway. This role is of physiological relevance since avoids gratuitous and wasteful production of catabolic enzymes. Our hypothesis for thn regulation implies that ThnA3 exerts its action by diverting electrons towards the regulator ThnY, an iron-sulfur flavoprotein that together with the transcriptional activator ThnR is necessary for thn gene expression. Here we analyze electron transfer among ThnA4, ThnA3 and ThnY by using stopped-flow spectrophotometry and determination of midpoint reduction potentials. Our results indicate that when accumulated in its reduced form ThnA3 is able to fully reduce ThnY. In addition, we have reproduced in vitro the regulatory circuit in the proposed physiological direction, NAD(P)H-ThnA4-ThnA3-ThnY. ThnA3 represents an unprecedented way of communication between a catabolic pathway and its regulatory system to prevent gratuitous induction

This is the peer reviewed version of the following article: [Ferreira, P., Hernández-Ortega, A., Lucas, F., Carro, J., Herguedas, B., Borrelli, K. W., Guallar, V., Martínez, A. T. and Medina, M. (2015), Aromatic stacking interactions govern catalysis in aryl-alcohol oxidase. FEBS J, 282: 3091–3106. doi:10.1111/febs.13221], which has been published in final form at [10.1111/febs.13221]. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving." http://olabout.wiley.com/WileyCDA/Section/id-820227.html The version posted may not be updated or replaced with the final published version (the Version of Record)., Aryl-alcohol oxidase (AAO, EC 1.1.3.7) generates H2O2 for lignin degradation at the expense of benzylic and other π system-containing primary alcohols, which are oxidized to the corresponding aldehydes. Ligand diffusion studies on Pleurotus eryngii AAO showed a T-shaped stacking interaction between the Tyr92 side chain and the alcohol substrate at the catalytically competent position for concerted hydride and proton transfers. Bi-substrate kinetics analysis revealed that reactions with 3-chloro- or 3-fluorobenzyl alcohols (halogen substituents) proceed via a ping–pong mechanism. However, mono- and dimethoxylated substituents (in 4-methoxybenzyl and 3,4-dimethoxybenzyl alcohols) altered the mechanism and a ternary complex was formed. Electron-withdrawing substituents resulted in lower quantum mechanics stacking energies between aldehyde and the tyrosine side chain, contributing to product release, in agreement with the ping–pong mechanism observed in 3-chloro- and 3-fluorobenzyl alcohol kinetics analysis. In contrast, the higher stacking energies when electron donor substituents are present result in reaction of O2 with the flavin through a ternary complex, in agreement with the kinetics of methoxylated alcohols. The contribution of Tyr92 to the AAO reaction mechanism was investigated by calculation of stacking interaction energies and site-directed mutagenesis. Replacement of Tyr92 by phenylalanine does not alter the AAO kinetic constants (on 4-methoxybenzyl alcohol), most probably because the stacking interaction is still possible. However, introduction of a tryptophan residue at this position strongly reduced the affinity for the substrate (i.e. the pre-steady state Kd and steady-state Km increase by 150-fold and 75-fold, respectively), and therefore the steady-state catalytic efficiency, suggesting that proper stacking is impossible with this bulky residue. The above results confirm the role of Tyr92 in substrate binding, thus governing the kinetic mechanism in A, This work was supported by the BIO2013-42978-P (to MM), BIO2011-26694 (to ATM), “Juan de la Cierva” (to FL) and CTQ2010-18123 (to VG) Grants of the Spanish Ministry of Economy and Competitiveness (MINECO) and by the INDOX (KBBE-2013-7-613549, to ATM) and PELE (ERC-2009-Adg 25027, to VG) European projects., Peer Reviewed, Postprint (author's final draft)

In the photosynthetic electron transfer (ET) chain, two electrons transfer from photosystem I to the flavin- dependent ferredoxin-NADP+ reductase (FNR) via two sequential independent ferredoxin (Fd) electron carriers. In some algae and cyanobacteria (as Anabaena), under low iron conditions, flavodoxin (Fld) replaces Fd as single electron carrier. Extensive mutational studies have characterized the protein–protein interaction in FNR/Fd and FNR/Fldcomplexes.Interestingly,eventhoughFd and Fldsharethe interaction site on FNR,individual residueson FNR do not participate to the same extent in the interaction with each of the protein partners, pointing to different electron transfer mechanisms. Despite of extensive mutational studies, only FNR/Fd X-ray structures from Anabaena and maize have been solved; structural data for FNR/Fld remains elusive. Here, we present a multiscale modelling approach including coarse-grained and all-atom protein–protein docking, the QM/MM e-Pathway analysis and electronic coupling calculations, allowing for a molecular and electronic comprehensive analysis of the ET process in both complexes. Our results, consistent with experimental mutational data, reveal the ET in FNR/Fd proceeding through a bridge-mediated mechanism in a dominant protein–protein complex, where transfer of the electron is facilitated by Fd loop-residues 40– 49. In FNR/Fld, however, we observe a direct transfer between redox cofactors and less complex specificity than in Fd; more than one orientation in the encounter complex can be efficient in ET., Work was supported by computational time from the Barcelona Supercomputer Center and funds from theSpanishMinistry ofEconomy and Competitiveness through the projects CTQ2013-48287 (to V.G.) and BIO2013-42978-P (to M.M.) and a Beatriu de Pinos grant BP-B-00252 from the Catalan Government (to S.S.)., Peer Reviewed, Postprint (author's final draft)

The food industry seeks alternatives to satisfy consumer demands of safe foods with a long shelf-life able to maintain the nutritional and organoleptic quality. The application of antimicrobial compounds-producing protective cultures may provide an additional parameter of processing in order to improve the safety and ensure food quality, keeping or enhancing its sensorial characteristics. In addition, strong evidences suggest that certain probiotic strains can confer resistance against infection with enteric pathogens. Several mechanisms have been proposed to support this phenomenon, including antimicrobial compounds secreted by the probiotics, competitive exclusion, or stimulation of the immune system. Recent research has increasingly demonstrated the role of antimicrobial compounds as protective mechanism against intestinal pathogens and therefore certain strains could have an effect on both the food and the gut. In this aspect, the effects of the combination of different strains keep unknown. The development of multistrain probiotic dairy products with good technological properties and with improved characteristics to those shown by the individual strains, able to act not only as protective cultures in foods, but also as probiotics able to exert a protective action against infections, has gained increased interest. © 2015 Juan L. Arqués et al.

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.

The antimicrobial effect of high hydrostatic pressure (HHP) processing combined with reuterin, lactoperoxydase system (LPS) and lactoferrin (LF) on the survival of Listeria monocytogenes, Salmonella enterica subsp. enterica serovar Enteritidis and Escherichia coli O157H7 in sliced cooked ham stored under strict refrigeration temperature (4°C) and mild temperature abuse conditions (10°C) was investigated. One day after treatment, L.monocytogenes counts in HHP at 450MPa for 5min were 0.8log units lower, but a recovery was observed with counts not significantly different to those observed in control after 35d. S. Enteritidis and E.coli O157H7 levels were reduced around 5 log cfu/g by the pressure treatment (450MPa/5min) and the numbers of these pathogens did not increase significantly during the 35d of storage at 4°C. The individual application of reuterin and LPS influenced the survival of the three pathogens studied, extending the lag phase of L.monocytogenes and diminishing S. Enteritidis and E.coli levels throughout storage, whereas no effect was recorded when LF was added. When reuterin or LPS were applied in combination with HHP there was a synergistic antimicrobial effect against L.monocytogenes, avoiding at 4°C the recovery observed with individual treatments. These combined treatments also kept the levels of S. Enteritidis and E.coli O157H7 below the detection limit (<1log unit) in cooked ham stored at 4 and 10°C during 35d. The results obtained in the present work suggest that HHP at 450MPa for 5min in combination with LPS or reuterin would be useful as a hurdle technology approach against L.monocytogenes, S. Enteritidis and E.coli O157H7 in cooked ham. © 2014 Elsevier Ltd.

The genes involved in the glycerol metabolism, glycerol dehydratase (gdh) and two propanediol dehydrogenases (pdh30 and pdh1734), were analyzed in different reuterin- and non-reuterin-producing lactobacilli of biotechnological interest. All the reuterin-producing lactobacilli expressed the gdh, pdh30 and pdh1734, except Lb. coryniformis CECT 5711 which did not contain pdh30. Reuterin production levels in Lb. coryniformis CECT 5711 were much lower than those in reuterin-producing Lb. reuteri. A positive relationship between cobalamin production levels and reuterin production levels was observed in all reuterin-producing lactobacilli tested. Intriguingly, when Lb. coryniformis CECT 5711 was supplemented with cobalamin, a seven times increase in reuterin production was observed. On the other hand, Lb. brevis ESI38 that possess and express gdh, pdh30 and pdh1734, was unable to produce reuterin or cobalamin. To study the role of pdh30 during glycerol metabolism, the gene disruption mutant Lb. brevis INIA ESI38pORI28-pdh30 was constructed. HPLC analysis of the glycerol fermentation products showed an involvement of the pdh30 in the 3-hydroxypropionic acid (3-HP) biosynthesis. However, Lb. coryniformis, that lack pdh30, showed the higher levels of 3-HP, indicating other catalytic mechanisms to produce 3-HP in this strain. The 1,3-propanediol peak was detected in the Lb. reuteri and Lb. coryniformis chromatograms, but not in Lb. brevis, which also confirm divergences in Lactobacillus glycerol metabolism. © 2015, Springer-Verlag Berlin Heidelberg.

This study revealed the possibility of manufacturing prefried lamb nuggets from low-value cuts (flank) and evaluated the effect of formulation (50:50 vs. 20:80 of leg/flank cuts) and freezing (−20 ℃ for two months) on different quality parameters. Frying process produced a decrease of water content, an increase in lipid oxidation indicators, and an intense browning of nuggets surface. The use of flank up to a 75% of the total meat in the formula led to higher fat contents and higher polyunsaturated fatty acid proportions in fried nuggets. This made them more prone to get oxidized during the freezing storage and subsequent final frying. Nuggets with a higher proportion of flank also showed lower shear force values. However, nuggets with both formulations showed similar sensory acceptance. Freezing storage of lamb meat nuggets for two months increased the levels of lipid oxidation indicators, but again not to a level high enough to influence the sensory perception by consumers. So that, frozen nuggets from both formulations showed similar consumer acceptance to freshly produced ones with quite good scores (3.46–3.86 out of 5). Thus, low-value lamb cuts are suitable for being processed into highly acceptable prefried frozen lamb nuggets, which constitutes an opportunity for adding value to the whole lamb value chain.

In a previous article, we demonstrated that histones (H1 or histone octamers) interact with negatively charged bilayers and induce extensive aggregation of vesicles containing phosphatidylinositol-4-phosphate (PIP) and, to a lesser extent, vesicles containing phosphatidylinositol (PI). Here, we found that vesicles containing PIP, but not those containing PI, can undergo fusion induced by histones. Fusion was demonstrated through the observation of intervesicular mixing of total lipids and inner monolayer lipids, and by ultrastructural and confocal microscopy studies. Moreover, in both PI- and PIP-containing vesicles, histones caused permeabilization and release of vesicular aqueous contents, but the leakage mechanism was different (all-or-none for PI and graded release for PIP vesicles). These results indicate that histones could play a role in the remodeling of the nuclear envelope that takes place during the mitotic cycle.

Riboflavin kinases (RFKs) catalyse the phosphorylation of riboflavin to produce FMN. In most bacteria this activity is catalysed by the C-terminal module of a bifunctional enzyme, FAD synthetase (FADS), which also catalyses the transformation of FMN into FAD through its N-terminal FMN adenylyl transferase (FMNAT) module. The RFK module of FADS is a homologue of eukaryotic monofunctional RFKs, while the FMNAT module lacks homologyto eukaryotic enzymes involved in FAD production. Previously, the crystal structure of Corynebacterium ammoniagenes FADS (CaFADS) was determined in its apo form. This structure predicted a dimer-of-trimers organization with the catalytic sites of two modules of neighbouring protomers approaching each other, leading to a hypothesis about the possibility of FMN channelling in the oligomeric protein. Here, two crystal structures of the individually expressed RFK module of CaFADS in complex with the products of the reaction, FMN and ADP, are presented. Structures are complemented with computational simulations, binding studies and kinetic characterization. Binding of ligands triggers dramatic structural changes in the RFK module, which affect large portions of the protein. Substrate inhibition and molecular-dynamics simulations allowed the conformational changes that take place along the RFK catalytic cycle to be established. The influence of these conformational changes in the FMNAT module is also discussed in the context of the full-length CaFADS protomer and the quaternary organization.

Ferredoxin-NADP+ reductase (FNR) catalyzes the electron transfer from ferredoxin to NADP+ via its flavin FAD cofactor. To get further insights in the architecture of the transient complexes produced during the hydride transfer event between the enzyme and the NADP+ coenzyme we have applied NMR spectroscopy using Saturation Transfer Difference (STD) techniques to analyze the interaction between FNRox and the oxidized state of its NADP+ coenzyme. We have found that STD NMR, together with the use of selected mutations on FNR and of the non-FNR reacting coenzyme analogue NAD+, are appropriate tools to provide further information about the the interaction epitope.

Ferredoxin-NADP+ reductase (FNR) is the structural prototype of a family of FAD-containing reductases that catalyze electron transfer between low potential proteins and NAD(P)+/H, and that display a two-domain arrangement with an open cavity at their interface. The inner part of this cavity accommodates the reacting atoms during catalysis. Loops at its edge are highly conserved among plastidic FNRs, suggesting that they might contribute to both flavin stabilization and competent disposition of substrates. Here we pay attention to two of these loops in Anabaena FNR. The first is a sheet-loop-sheet motif, loop102-114, that allocates the FAD adenosine. It was thought to determine the extended FAD conformation, and, indirectly, to modulate isoalloxazine electronic properties, partners binding, catalytic efficiency and even coenzyme specificity. The second, loop261-269, contains key residues for the allocation of partners and coenzyme, including two glutamates, Glu267 and Glu268, proposed as candidates to facilitate the key displacement of the C-terminal tyrosine (Tyr303) from its stacking against the isoalloxazine ring during the catalytic cycle. Our data indicate that the main function of loop102-114 is to provide the inter-domain cavity with flexibility to accommodate protein partners and to guide the coenzyme to the catalytic site, while the extended conformation of FAD must be induced by other protein determinants. Glu267 and Glu268 appear to assist the conformational changes that occur in the loop261-269 during productive coenzyme binding, but their contribution to Tyr303 displacement is minor than expected. Additionally, loop261-269 appears a determinant to ensure reversibility in photosynthetic FNRs

Ferredoxin-nicotinamide-adenine dinucleotide phosphate (NADP+) reductase (FNR) catalyses the production of reduced nicotinamide-adenine dinucleotide phosphate (NADPH) in photosynthetic organisms, where its flavin adenine dinucleotide (FAD) cofactor takes two electrons from two reduced ferredoxin (Fd) molecules in two sequential steps, and transfers them to NADP+ in a single hydride transfer (HT) step. Despite the good knowledge of this catalytic machinery, additional roles can still be envisaged for already reported key residues, and new features are added to residues not previously identified as having a particular role in the mechanism. Here, we analyse for the first time the role of Ser59 in Anabaena FNR, a residue suggested by recent theoretical simulations as putatively involved in competent binding of the coenzyme in the active site by cooperating with Ser80. We show that Ser59 indirectly modulates the geometry of the active site, the interaction with substrates and the electronic properties of the isoalloxazine ring, and in consequence the electron transfer (ET) and HT processes. Additionally, we revise the role of Tyr79 and Ser80, previously investigated in homologous enzymes from plants. Our results probe that the active site of FNR is tuned by a H-bond network that involves the side-chains of these residues and that results to critical optimal substrate binding, exchange of electrons and, particularly, competent disposition of the C4n (hydride acceptor/donor) of the nicotinamide moiety of the coenzyme during the reversible HT event.

Lactobacillus reuteri INIA P579 was used for the production and purification of reuterin. The purity of reuterin was assessed by high resolution electrospray ionization mass spectrometry (HRESIMS) and nuclear magnetic resonance (NMR) spectroscopy. After purification, reuterin concentration obtained was 1.3M. The inhibitory activity using Escherichia coli K12 as indicator strain was estimated to be 510 AU/ml. Survival curves in tryptic soy broth revealed that reuterin required to inhibit the growth of three Listeria monocytogenes strains was in the range of 2-4 AU/ml. Purified reuterin (10 AU/g) significantly reduced the growth of L.monocytogenes in cold-smoked salmon kept under moderate or strong temperature abuse conditions. After 15d at 8°C, cold-smoked salmon with added reuterin exhibited L.monocytogenes counts 2.0 log CFU/g lower than control smoked salmon with no reuterin added. At 30°C, reuterin also controlled the growth of the pathogen, with counts 1.4 and 0.9 log CFU/g lower than those observed in control smoked salmon after 24 and 48h, respectively. The addition of purified reuterin might be used as a hurdle technology to improve the safety and extend the shelf-life of lightly preserved seafood products such as cold-smoked salmon. © 2014 Elsevier Ltd.

The effect of reuterin and high hydrostatic pressure (HHP) processing at 450 MPa for 5 min on the inactivation of Listeria monocytogenes and the characteristics of cold-smoked salmon during 35 days at 4 and 10 °C were investigated. The growth rate of the pathogen was reduced by reuterin addition and a synergistic antimicrobial effect against L. monocytogenes was recorded when the biopreservative was applied in combination with HHP at 450 MPa for 5 min. This combined treatment prevented the pathogen recovery observed with individual treatments and delayed the spoilage of smoked salmon maintaining total viable counts under 3.5 log units during 35 days of storage at 4 °C. All treatments assayed induced changes in lightness (L*) and redness (a*), resulting in a brighter appearance of smoked salmon, whereas no modifications were recorded in shear strength values immediately after treatments. Moreover, reuterin and HHP treatments, individually or in combination, avoided the formation of biogenic amines during the 35 days of storage at 4 and 10 °C. The addition of reuterin in combination with HHP at 450 MPa for 5 min might be applied as a hurdle technology to improve the safety and extend the shelf life of lightly preserved seafood products, such as cold-smoked salmon. © 2014 Springer Science+Business Media New York.

Cheeses have been proposed as a good alternative to other fermented milk products for the delivery of probiotic bacteria to the consumer. The objective of this study was to assess the survival of two Lactobacillus salivarius strains (CECT5713 and PS2) isolated from human milk during production and storage of fresh cheese for 28 days at 4°C. The effect of such strains on the volatile compounds profile, texture, and other sensorial properties, including an overall consumer acceptance, was also investigated. Both L. salivarius strains remained viable in the cheeses throughout the storage period and a significant reduction in their viable counts was only observed after 21 days. Globally, the addition of the L. salivarius strains did not change significantly neither the chemical composition of the cheese nor texture parameters after the storage period, although cheeses manufactured with L. salivarius CECT5713 presented significantly higher values of hardness. A total of 59 volatile compounds were identified in the headspace of experimental cheeses, and some L. salivarius-associated differences could be identified. All cheeses presented good results of acceptance after the sensory evaluation. Consequently, our results indicated that fresh cheese can be a good vehicle for the two L. salivarius strains analyzed in this study. © 2014 Nivia Cárdenas 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.