Search

2 Pág. Departamento de Tecnología de Alimentos​​ (INIA), 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., This work was supported by project RTA2017-00002-00-00 from the Spanish Ministry of Science and Innovation.

Bioengineering of probiotics allows the improvement of their beneficial characteristics. In this work, we develop a molecular tool that would allow the activation of desirable traits in probiotics once they reach the intestine. The activity of upstream regions of bile-inducible genes of Lactobacillus casei BL23 and Lactobacillus plantarum WCFS1 was analyzed using plasmids encoding an anaerobic fluorescent protein as reporter. The promoter P16090 from Lb. casei BL23 was selected and its bile induction confirmed in Lb. casei BL23, Lb. plantarum WCFS1, and in Lactobacillus rhamnosus and Lactobacillus reuteri strains. However, the induction did not occur in Lactococcus lactis MG1363 or Bifidobacterium strains. Studies with different bile compounds revealed the importance of cholic acid in the bile induction process. Induction of fluorescence was also confirmed for transformed Lb. casei BL23 under simulated colonic conditions and in the presence of intestinal microbiota. The developed vector, pNZ:16090-aFP, constitutes a promising tool suitable for the expression of genes of interest under intestinal conditions in probiotic strains of the species Lb. casei, Lb. plantarum, Lb. rhamnosus, and Lb. reuteri.

11 Pág., Lacticaseibacillus paracasei INIA P272 and Lacticaseibacillus rhamnosus INIA P344, isolated from breast-fed infants, are two promising bacterial strains for their use in functional foods according to their demonstrated probiotic and technological characteristics. To better understand their probiotic characteristics and evaluate their safety, here we report the draft genome sequences of both strains as well as the analysis of their genetical content. The draft genomes of L. paracasei INIA P272 and L. rhamnosus INIA P344 comprise 3.01 and 3.26 Mb, a total of 2994 and 3166 genes and a GC content of 46.27 % and 46.56 %, respectively. Genomic safety was assessed following the EFSA guidelines: the identification of both strains was confirmed through Average Nucleotide Identity, and the absence of virulence, pathogenic and antibiotic resistance genes was demonstrated. The genome stability analysis revealed the presence of plasmids and phage regions in both genomes, however, CRISPR sequences and other mechanisms to fight against phage infections were encoded. The probiotic abilities of both strains were supported by the presence of genes for the synthesis of SCFA, genes involved in resistance to acid and bile salts or a thiamine production cluster. Moreover, the encoded exopolysaccharide biosynthesis genes could provide additional protection against the deleterious gastrointestinal conditions, besides which, playing a key role in adherence and coaggregation of pathogenic bacteria together with the high number of adhesion proteins and domains encoded by both genomes. Additionally, the bacteriocin cluster genes found in both strains, could provide an advantageous ability to compete against pathogenic bacteria. This genomic study supports the probiotic characteristics described previously for these two strains and satisfies the safety requirements to be used in food products., This work was supported by projects PID2020-11960RB-I00 from the Spanish Ministry of Science and Innovation, and RMP2015-00001-00-00 from the National Institute for Agricultural and Food Research and Technology (INIA) and the Spanish Agency for Research (AEI-MCIU) co-financed by the European Regional Development Fund

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.

Departamento de Tecnología de los Alimentos, The isoflavones daidzin and genistin, present in soybeans, can be transformed by the intestinal microbiota into equol and 5-hydroxy-equol, compounds with enhanced availability and bioactivity, although these are only produced by a fraction of the population. Hence, there is an interest in the production of these compounds, although, to date, few bacteria with biotechnological interest and applicability in food have been found able to produce equol. In order to obtain lactic acid bacteria able to produce equol, the daidzein reductase (dzr), dihydrodaidzein reductase (ddr), tetrahydrodaidzein reductase (tdr) and dihydrodaidzein racemase (ifcA) genes, from Slackia isoflavoniconvertens DSM22006, were cloned into the vector pNZ:TuR, under a strong constitutive promoter (TuR). Lactococcus lactis MG1363, Lacticaseibacillus casei BL23, Lactiplantibacillus plantarum WCFS1, Limosilactobacillus fermentum INIA 584L and L. fermentum INIA 832L, harbouring pNZ:TuR.tdr.ddr, were able to produce equol from dihydrodaidzein, while L. fermentum strains showed also production of 5-hydroxy-equol from dihydrogenistein. The metabolization of daidzein and genistein by the combination of strains harbouring pNZ:TuR.dzr and pNZ:TuR.tdr.ddr showed similar results, and the addition of the correspondent strain harbouring pNZ:TuR.ifcA resulted in an increase of equol production, but only in the L. fermentum strains. This pattern of equol and 5-hydroxy-equol production by L. fermentum strains was also confirmed in cow's milk supplemented with daidzein and genistein and incubated with the different combination of strains harbouring the constructed plasmids. Bacteria generally recognized as safe (GRAS), such as the lactic acid bacteria species used in this work, harbouring these plasmids, would be of value for the development of fermented vegetal foods enriched in equol and 5-hydroxy-equol., This work was supported by project RTA2017-00002-00-00 from the Spanish Ministry of Science, Innovation and Universities, 7 Pág.

Departamento de Tecnología de Alimentos​​ (INIA), The draft genome sequence of Bifidobacterium breve INIA P734, a strain shared by mother and child, is reported. It consists of 50 contigs, with 2,391,925 bp, 2,099 genes, and a G+C content of 58.8%. The genome analysis revealed the absence of antibiotic resistance and pathogenicity-related genes., This work was supported by project no. RTA2017-00002-00-00 from the Spanish Ministry of Science and Innovation and RMP2015-00001-00-00 from the National Institute for Agricultural and Food Research and Technology (INIA) and the Spanish Agency for Research (AEI-MCIU), cofinanced by the European Regional Development Fund., 2 Pág.

9 Pág. Departamento de Tecnología de Alimentos​​, Genes involved in the transport and catabolism of carbohydrates are usually controlled through the binding of the catabolite control protein A (CcpA) to the catabolite-responsive elements (cre) of target genes in Gram-positive bacteria. In this work, we show how the elimination of the cre sites in Lactobacillus casei BL23 promoters induced by sorbitol (PgutF), maltose (PmalL), and myo-inositol (PiolT) allowed the induction of gene expression in media supplemented with sorbitol, maltose, and myo-inositol, respectively, even in the presence of glucose. This was studied using plasmids encoding the anaerobic fluorescent protein evoglow-Pp1 as a reporter. In addition, gutF cre site was introduced into a bile inducible promoter (P16090) and into the constitutive promoter of the elongation factor P (PEf-P) of L. casei BL23. The existence of the cre site blocked gene expression in the P16090 inducible promoter in the presence of glucose, while it had no influence on the expression of the PEf-P constitutive one. These results demonstrated that the introduction or elimination of cre sites in inducible promoters allows the control and modification of their heterologous genetic expression, showing how the cre site, the transcriptional regulator, and CcpA interact to control gene expression in inducible genes. KEY POINTS: • Cre sequences regulate gene expression in inducible promoters in L. casei BL23. • Cre sites do not affect gene expression in constitutive promoters in L. casei BL23. • Cre sequences could control heterologous genic expression in lactobacilli., This work was supported by RTA2017-00002-00-00 project from the Ministry of Economy and Competitiveness (Spain).

Aims The aim of this study was the coproduction in a single strain of the Gram-negative bacteriocin colicin V with other bacteriocins from lactic acid bacteria (LAB). Methods and Results Colicin V was expressed in Lactococcus and Enterococcus strains by replacing the colicin V leader peptide by the leader peptide and promoter of d-alanyl-d-alanine carboxypeptidase from Lactobacillus reuteri CECT925 in pNZ8048 (pNZLR-colV). The antimicrobial activity of colicin V against the indicator organism Escherichia coli DH5α in transformed strains was checked by agar diffusion assay and SDS-PAGE analysis. Conclusions Lactococcus and Enterococcus transformed with pNZLR-colV were able to coproduce colicin V at high levels together with other LAB bacteriocins such as nisin A, nisin Z, lacticin 481 or enterocins A and B, obtaining broad-spectrum activity strains with large potential applications. Significance and Impact of the study The construction showed in this work could be used for the heterologous expression of other bacteriocins active against Gram-negative bacteria or wide-spectrum bacteriocins from LAB. © 2017 The Society for Applied Microbiology

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.

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. © 2017 American Dairy Science Association

Technological processes in the dairy industry and the further passage through the gastrointestinal tract could impair viability and functionality of probiotic bifidobacteria. In the present work, the growth in milk of nine bifidobacterial strains shared by mother and child, their survival to freeze-drying and cold storage, and their behavior in a model cheese were investigated. All the strains exhibited high stability to the technological conditions studied when compared with two commercial strains.Bifidobacterium breveINIA P734 andBifidobacterium bifidumINIA P671 as adjunct cultures maintained high stability during manufacture and ripening of cheese. Both strains showed, at the end of ripening period, resistance to simulated gastrointestinal conditions. Moreover, their presence did not affect negatively the quality of cheese.B. breveINIA P734 andB. bifidumINIA P671 could be considered as potential candidates for their use in cheese as adjunct cultures.

The aim of this study was to test the compatibility of two selected Lactobacillus strains with different prebiotic carbohydrates in order to develop a synbiotic cheese. Initially, the growth of the probiotic lactobacilli with the prebiotic formulations was monitored in optimized growth medium and in milk. Afterwards, semi-hard cheeses were manufactured with the selected combination of Lb. paracasei INIA P272 and fructo-oligosaccharides (FOS). Results showed that the addition of FOS did not affect Lb. paracasei viability in the cheese after 28 days of ripening, but a higher viability of this strain was achieved in the presence of FOS when cheese was tested in a colonic model. Thus, the inclusion of FOS in the cheese could have a beneficial effect by increasing the probiotic strain viability in the host.

Phytoestrogens (isoflavones, ellagitannins, and lignans) are polyphenols with an estrogenic/antiestrogenic function present in fruits, vegetables, and foods derived from them. Some microorganisms are capable of transforming isoflavones, ellagitannins, and lignans into equol, urolithins, and enterolignans, respectively. These compounds are more bioavailable, they have more estrogenic/antiestrogenic and antioxidant activity than their precursors, and they are associated with beneficial effects for human health. Extracts of soy- bean and lignans and ellagic acid were incubated with feces, or bacteria isolated from feces, and later the phytoestrogens were extracted twice with diethyl ether and twice with ethyl acetate, and the solvents were evaporated at room temperature under a N2. Subsequently, phytoestrogens were detected by HPLC-PAD and chromatographic peaks were identified by HPLC-ESI/M, and they were confirmed by comparison of retention times and characteristics of UV spectra with those of standards. Analysis of phytoestrogens by means of HPLC- PAD allowed the simultaneous detection of isoflavones, ellagitannins, and lignans metabolized, or not, by intestinal microbiota with high sensitivity (LOD: equol 0.11 mg/L; urolithin A 0.17 mg/L, enterodiol 0.15 mg/L). Assays were linear up to at least 20 mg/L in all standards with a high correlation (equol >0.999; urolithin A >0.998, enterodiol >0.999). Therefore, the described method constitutes an affordable and easy alternative method for the detection of phytoestrogen metabolism. Moreover, this method could be applied to the detection of isoflavones, lignans, and ellagitannins in food., This work was supported by RM2012-00004-00-00. J.M. Landete has a postdoctoral contract with the research program “Ramón y Cajal” (MINECO, Spain).

Bioprotective effect of reuterin-producing Lactobacillus reuteri INIA P572 was assessed in cheeses artificially contaminated with Listeria monocytogenes and Escherichia coli O157:H7 and ripened for 30 days. Reuterin production in cheese made with L. reuteri INIA P572 supplemented with 100 mM glycerol was confirmed up to 7 d of ripening with a maximum of 5.2 μmol/g at 1 day. L. reuteri was able to survive up to 5.37 log cfu/g in 30 d control cheese, although it was reduced to 2.59 log cfu/g when added simultaneously with glycerol. L. monocytogenes counts in control cheese were 4.31 log cfu/g after 30 days of ripening, whereas in cheese made with L. reuteri INIA P572 and 100 mM glycerol, this pathogen was only detectable by enrichment of 25 g samples from day 7 onwards. This antimicrobial strategy also diminished E. coli O157:H7 counts on day 1 by 5.53 log units with respect to control cheese. After 30 d, E. coli O157:H7 counts were 0.85 log cfu/g in control cheese, but the pathogen was not detected in 25 g cheese made with L. reuteri and 100 mM glycerol from day 7 onwards. Reuterin production in situ might be a feasible alternative to improve cheese safety that could also be exploited in the control of other undesirable microorganisms.

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.

Importance of bifidobacteria as part of the infant intestinal microbiota has been highlighted. Their acquisition is influenced by the mode of birth and the feed regime afterwards, with a special role of the maternal microbiota. The presence of the same shared bifidobacterial strains between breast milk and infant faeces in 14 mother-infant pairs was assessed by means of pulsed-field gel electrophoresis (PFGE) genotyping. Four shared strains of Bifidobacterium breve (2), Bifidobacterium longum subsp. infantis and B. longum subsp. longum were found in breast milk-infant faeces pairs. Two years later, a second survey yielded four shared strains of the species Bifidobacterium adolescentis, Bifidobacterium bifidum, B. longum subsp. longum and Bifidobacterium pseudocatenulatum. Moreover, a B. bifidum strain was found to be shared by the infant faeces of the first study and the mother faeces tested two years later, pointing out a long term persistence. Some of the selected bifidobacterial strains showed probiotic potential due to their survival to gastrointestinal conditions and their ability to form biofilms.

Control and quantification of effector molecules such as heavy metals, toxins or other target molecules is of great biotechnological, social and economic interest. Microorganisms have regulatory proteins that recognize and modify the gene expression in the presence or absence of these compounds (effector molecules) by means of binding to gene sequences. The association of these recognizing gene sequences to reporter genes will allow the detection of effector molecules of interest with high sensitivity. Once investigators have these two elements–recognizing gene sequences and reporter genes that emit signals–we need a suitable vehicle to introduce both elements. Here, we suggest lactic acid bacteria (LAB) and bifidobacteria as promising carrier microorganisms for these molecular biosensors. The use of fluorescent proteins as well as food-grade vectors and clustered regularly interspaced short palindromic repeats (CRISPR) are indispensable tools for introducing biosensors into these microorganisms. The use of these LAB and bifidobacteria would be of special interest for studying the intestinal environment or other complex ecosystems. The great variety of species adapted to many environments, as well as the possibility of applying several protocols for their transformation with recognizing gene sequences and reporter genes are considerable advantages. Finally, an effort must be made to find recognizable gene sequences. © 2017 by the authors. Licensee MDPI, Basel, Switzerland.

Iron is a central micronutrient needed by all living organisms. Competition for iron in the intestinal tract is essential for the maintenance of indigenous microbial populations and for host health. How symbiotic relationships between hosts and native microbes persist during times of iron limitation is unclear. Here, we demonstrate that indigenous bacteria possess an iron-dependent mechanism that inhibits host iron transport and storage. Using a high-throughput screen of microbial metabolites, we found that gut microbiota produce metabolites that suppress hypoxia-inducible factor 2α (HIF-2α) a master transcription factor of intestinal iron absorption and increase the iron-storage protein ferritin, resulting in decreased intestinal iron absorption by the host. We identified 1,3-diaminopropane (DAP) and reuterin as inhibitors of HIF-2α via inhibition of heterodimerization. DAP and reuterin effectively ameliorated systemic iron overload. This work provides evidence of intestine-microbiota metabolic crosstalk that is essential for systemic iron homeostasis.

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.

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.

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. © 2017, Association of Food Scientists & Technologists (India).

Age-related degeneration gives rise to a number of pathologies, many of them associated with imbalances of the microbiota and the gut-associated immune system. Thus, the intestine is considered a key target organ to improve the quality of life in senescence. Gut microbiota can have a powerful impact in the deterioration linked to aging by its nutritional and immunomodulatory activity. Reduced numbers of beneficial species and low microbial biodiversity in the elderly have been linked with pathogenesis of many diseases. A healthy lifestyle with an elderly customized diet including probiotics can contribute to reducing the chronic proinflammatory status and other age-related pathologies. Beneficial effects of probiotic lactic acid bacteria and bifidobacteria to alleviate some of these disorders based on their immunomodulatory properties as well as their capacity to produce bioactive metabolites from dietary phytoestrogens are summarized. On one hand, the preservation of gut barrier integrity and an increased ability to fight infections are the main reported immune benefits of probiotics. On the other hand, the intake of a diet rich in phytoestrogens along with the presence of selected probiotic bacteria may lead to the production of equol, enterolignans, and urolithins, which are considered protective against chronic diseases related to aging. © 2017 José Mariá Landete et al.

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.

While many compounds have been reported to change in laboratory based drought-stress experiments, little is known about how such compounds change, and are significant, under field conditions. The Pisum sativum L. (pea) leaf metabolome has been profiled, using 1D and 2D NMR spectroscopy, to monitor the changes induced by drought-stress, under both glasshouse and simulated field conditions. Significant changes in resonances were attributed to a range of compounds, identified as both primary and secondary metabolites, highlighting metabolic pathways that are stress-responsive. Importantly, these effects were largely consistent among different experiments with highly diverse conditions. The metabolites that were present at significantly higher concentrations in drought-stressed plants under all growth conditions included proline, valine, threonine, homoserine, myoinositol, γ-aminobutyrate (GABA) and trigonelline (nicotinic acid betaine). Metabolites that were altered in relative amounts in different experiments, but not specifically associated with drought-stress, were also identified. These included glutamate, asparagine and malate, with the last being present at up to 5-fold higher concentrations in plants grown in field experiments. Such changes may be expected to impact both on plant performance and crop end-use.

Summary Background Changes in the composition of the gut microbiota have been implicated in the pathogenesis of allergic disorders, suggesting beneficial interactions between the intestinal immune system and specific bacterial strains. Lactobacilli are naturally present within the complex gastrointestinal microbiota of humans and they are currently present in many probiotic supplements. Objective We sought to investigate the role that Lactobacillus casei Shirota (LcS) may play in modulating seasonal allergic rhinitis (SAR). Methods The study format was double-blinded, placebo-controlled with 10 SAR sufferers in each group. We have documented and compared changes in immune status arising through the daily ingestion of a milk drink with or without live LcS, over a period of 5 months. Pre-, peak- and post-grass pollen season blood samples were collected for determination of plasma total IgE and grass pollen-specific IgG and IgE levels by an enzyme immunoassay. At the same time, cytokine levels were determined by flow cytometric bead array technology following culture of peripheral blood mononuclear cells for 6 days in the presence or absence of specific grass pollen antigens. Results Volunteers treated with LcS showed a significant reduction in levels of antigen-induced IL-5, IL-6 and IFN-γ production compared with volunteers supplemented with placebo. Meanwhile, levels of specific IgG increased and IgE decreased in the probiotic group. Conclusion Changes in antigen-induced production of cytokines were observed in patients treated with probiotics. These data show that probiotic supplementation modulates immune responses in allergic rhinitis and may have the potential to alleviate the severity of symptoms.

Heterologous production of the antilisterial bacteriocin pediocin PA-1 in lactococci is an attractive objective to increase the safety of dairy products. In a previous paper, we developed a system for the heterologous production of the bacteriocin pediocin PA-1 in pediocin-resistant lactococcal hosts through a leader exchange strategy. The system was based on 3 genes, 1 encoding the fusion between the lactococcin A leader and propediocin PA-1, and the other 2 encoding the lactococcin A secretion machinery. In this study, we investigated whether the addition of the pediocin PA-1 immunity gene (pedB) to this system has any effect on pediocin production. Introduction of the plasmid(s) carrying the genes described above into nisinproducing and non-nisinproducing lactococcal hosts led to a significant increase in the production of pediocin compared with the equivalent pedB-devoid systems. In addition, we obtained a nisin-producing strain with the ability to secrete pediocin PA-1 at a level equivalent to that of the parental strain Pediococcus acidilactici 347, which represents a notable improvement over our previous systems.

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

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., This work was supported by the project RM2012-00004-00-00

Inhibitory activity of reuterin (β-hydroxypropionaldehyde) combined with the antimicrobial peptide nisin or the lactoperoxidase system (LPOS) against food-borne Gram-negative pathogens in milk refrigerated at 4 and 8 °C was investigated. At 4 °C, reuterin (8 AU/ml) and LPOS were bactericidal against Salmonella enterica, Campylobacter jejuni, Aeromonas hydrophila and Yersinia enterocolitica, whereas the only effect recorded for nisin was a slight inhibition of Escherichia coli O157H7. At 8 °C, reuterin was bactericidal against all the Gram-negative pathogens studied. Same results were attained with LPOS, except for its effect on Y. enterocolitica which was only bacteriostatic. The combination of reuterin with nisin did not enhance the antimicrobial effect of reuterin. A strong synergistic bactericidal activity of reuterin in combination with LPOS on E. coli O157H7 and S. enterica was observed in milk at 4 °C, and against all the Gram-negative bacteria assayed in milk refrigerated at 8 °C. The application of both antimicrobials would be a useful means to inhibit pathogenic microorganisms, which may be present in milk due to postpasteurization contamination. © 2007 Springer-Verlag.

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 pNZProm.GFPana contained the anaerobic fluorescent protein evoglow-Pp1 could be exploited as a tool for analysing the gene expression in bifidobacteria strains. © 2015, Springer Science+Business Media Dordrecht.

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. © 2015, Springer-Verlag Berlin Heidelberg., This work was supported by projects RTA2010-00116-00-00 and RM2012-0004-00-00 from the Spanish Ministry of Economy and Competitiveness (MINECO). J.M. Landete has a postdoctoral contract with the research program “Ramón y Cajal” (MINECO, Spain).

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.

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.

Reuterin (β-hydroxypropionialdehyde) is a broad-spectrum antimicrobial substance produced by some strains of Lactobacillus reuteri during anaerobic fermentation of glycerol. Some of these strains are able to survive and produce reuterin in cheese and yogurt when added as adjuncts to the starter. Similarly, in fermented dairy foods, other inhibitory compounds such as lactic acid and diacetyl are produced during fermentation. In this work, we studied the combined effect of reuterin and diacetyl under different pH conditions against Escherichia coli O157H7, Salmonella Enteritidis, and Listeria monocytogenes. Results from agar spot assays showed that the antimicrobial activity of reuterin-producing strains against the gram-negative bacteria tested was enhanced as the concentration of diacetyl increased to 50. mg/kg, and was higher under acidic conditions (pH 5.0) for the 3 pathogenic strains. The combination of reuterin and diacetyl had an additive effect against L. monocytogenes only at diacetyl concentrations of 50. mg/kg and pH 5.0. In addition, growth kinetics studies showed that the combination of 1 activity unit (AU)/mL of reuterin with 100. mg/kg diacetyl increased the lag time of the 3 pathogens. In milk, synergistic antimicrobial activity was observed with the combination of 1. AU/mL reuterin and 50 or 100. mg/kg of diacetyl on the gram-negative strains tested, and with 1. AU/mL reuterin and 100. mg/kg of diacetyl on L. monocytogenes. The greatest inhibition of the 3 pathogens was achieved in acidified milk at pH 5.0 with reuterin (1. AU/mL) and diacetyl (100. mg/kg). Based on these results, the combination of reuterin and diacetyl in acidified dairy products could be a promising strategy to control food pathogens in these products. © 2014 American Dairy Science Association.

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 1.3 M. 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 15 d 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 48 h, 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.

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. © 2014 Elsevier B.V., This work was supported by projects RTA2010-00116-00-00 and RM2010-00008-00-00. J.M. Landete has a postdoctoral contract with the research program “Ramón y Cajal” (MINECO, Spain). The authors thank the valuable help of Concepción Revilla from INIA (Spain), and of V. Monedero and M. Zuñiga from IATA-CSIC (Spain).

In situ reuterin production during the manufacture and storage of two dairy model systems elaborated from milk supplemented with an optimized concentration of glycerol (50 mM) and inoculated with a commercial starter and with different reuterin-producing Lactobacillus reuteri strains was investigated. L. reuteri was able to survive and to produce reuterin in cheese and yogurt models. The highest reuterin production was achieved by L. reuteri INIA P572 and INIA P579, which displayed reuterin concentrations up to 5.5 mM in cheese and up to 1.5 mM in yogurt. The addition of reuterin-producing L. reuteri and glycerol to milk reduced the viable counts of the cheese starter from day 10 onwards, while did not influence the counts of the yogurt starter, compared to control dairy models without L. reuteri. Strains L. reuteri INIA P572 and INIA P579 could be promising candidates in the development of bioprotective cultures to control pathogenic microorganisms in dairy products due to the potentially inhibitory concentrations of reuterin achieved in situ. © 2013 Elsevier Ltd.

The protective and probiotic characteristics of intestinal lactobacilli, bifidobacteria and enterococci isolated from breast-fed infant faeces were investigated. Fifty-two selected isolates were identified at the species level and further characterized for their antimicrobial and safety features. Resistance to simulated gastric digestion and adherence to Caco-2 cells were also studied in the selected strains . Lactobacillus paracasei INIA P272, . Lactobacillus rhamnosus INIA P344, . L. rhamnosus INIA P426, . Lactobacillus mucosae INIA P459, . Bifidobacterium pseudolongum INIA P2, . Bifidobacterium breve INIA P18, . Enterococcus faecium INIA P445 and . Enterococcus faecalis INIA 127 due to their tolerance to GIT (gastrointestinal tract) conditions and interesting antimicrobial properties compared to the other strains investigated. A considerable diversity in the probiotic and antimicrobial properties among breast-fed infant lactic acid bacteria and bifidobacteria isolates was recorded. Some of the selected strains could be promising candidates to be exploited in the development of infant formulas or novel food products. © 2012 Elsevier Ltd.

Antimicrobial activity of reuterin in combination with different bacteriocins from lactic acid bacteria against food-borne pathogens in milk was investigated. A strong synergistic effect of reuterin in combination with nisin, lacticin 481 or enterocin AS-48 on Listeria monocytogenes was observed. Only nisin increased the antimicrobial activity of reuterin against Staphylococcus aureus. Bactericidal activity of reuterin towards Escherichia coli O157H7, Salmonella enterica, Yersinia enterocolitica, Aeromonas hydrophila and Campylobacter jejuni was not enhanced significantly by the addition of any of the bacteriocins investigated. The synergism of reuterin and nisin against L. monocytogenes and S. aureus was also found at refrigeration temperatures, where the pathogens were completely inactivated. Refrigerated milk treated with both natural antimicrobials would mean a feasible system to control pathogenic contaminants. © 2010 Elsevier Ltd.

We have demonstrated that direct antigen sampling of bacteria by intestinal dendritic cells (DCs) is accompanied by a rapid migration of CD11c+CX3CR1+MHCII+CD8alpha; CD11b-DCs into the intestinal lumen upon exposure to non-invasive δSPI1-Salmonella. Importantly, intraluminal DCs internalized Salmonella but were not able to cross the epithelium to return into tissue, thus showing that these DCs do not function as antigen-presenting cells and participate in the conventional regulation of immune responses to intestinal pathogens. Here we show that the presence of the chemokine receptor CX3CR1, that plays a vital role in DC-mediated antigen sampling and clearance in the gut, is also instrumental for the transepithelial migration of DCs. The latter observation, along with the notion that CX3CR1-deficient mice displayed higher susceptibility to Salmonella infection compared to wild type mice raises the possibility that Salmonella-induced migration of bacteria-capturing DCs into the lumen may be an important mechanism of mucosal defence and clearance. © 2010 Landes Bioscience.

Background & Aims Intestinal dendritic cells (DCs) sample bacteria, such as Salmonella , by extending cellular processes into the lumen to capture bacteria and shuttle them across the epithelium; however, direct evidence of bacteria-loaded DCs travelling back into the tissue is lacking. We hypothesized that sampling is paralleled by migration of DCs into the lumen prior to or following the internalization of Salmonella . Methods The small intestine and the colon of BALB/c and C57BL/6 mice were challenged with noninvasive Salmonella enterica serovar Typhimurium SL1344-Δ Salmonella pathogenicity island (SPI) 1 or Escherichia coli DH5α by using isolated loops or oral administration by gavage. Transepithelial migration of DCs was documented by immunohistochemistry, microscopy, and flow cytometry. The role of flagellin was determined by using flagellin (ΔfliC ΔfljB)- and SPI1-SPI2 (ΔSPI1 ΔssrA)-deficient Salmonella , flagellated E coli K12, and MyD88 mice. Results Salmonella ΔSPI1 induced migration of CD11c + CX 3 CR1 + MHCII + CD11b − CD8α − DCs into the small intestine, whereas flagellin- and SPI1-SPI2-deficient Salmonella , soluble flagellin, and E coli DH5α or flagellated K12, failed to do so. DC migration did not occur in the colon; it was not observed in MyD88 mice, and intraluminal DCs internalized Salmonella but did not cross the epithelium to return into tissues. Finally, DC migration was not linked to Salmonella -induced damage of the epithelium. Conclusions DC-mediated sampling of Salmonella is accompanied by flagellin- and MyD88-dependent migration of Salmonella -capturing DCs into the intestinal lumen. We suggest that the rapid intraluminal migration of Salmonella -capturing DCs may play a role in the protection of the intestinal mucosa against bacterial infection.

The inhibitory activity of nisin (N), reuterin (R), and the lactoperoxidase system (LPS), added individually or in combination, against Listeria monocytogenes and Staphylococcus aureus was investigated in "cuajada" (curdled milk), a semisolid dairy product manufactured in Spain. Cuajada was manufactured from UHT skim milk separately inoculated with L. monocytogenes and Staph, aureus, each at approximately 4 log cfu/mL, and held under conditions of temperature abuse (10°C). On d 3, a synergistic bactericidal activity was observed for the combinations of biopreservatives assayed, with L. monocytogenes counts of only 0.30 log cfu/mL in cuajada made with N + R + LPS vs. 8.31 log cfu/mL in control cuajada. After 12 d, L. monocytogenes could not be detected in cuajada made with added N + LPS or N + R + LPS. Staphylococcus aureus was more resistant than L. monocytogenes to biopreservatives added individually. On d 3, the synergistic effect of the 3 biopreservatives against Staph, aureus resulted in counts of 3.03 log cfu/mL in cuajada made with N + R + LPS vs. 6.40 in control cuajada. After 12 d, Staph, aureus counts were 2.61 log cfu/mL in cuajada made with N + R + LPS, whereas they ranged from 6.11 to 7.70 log cfu/ mL in control cuajada and in cuajada made with other combinations of biopreservatives. The most pronounced decrease in pathogen counts was achieved by the triple combination N + R + LPS, which acted synergistically on the inactivation of L. monocytogenes and Staph, aureus in cuajada over 12 d at 10°C. The treatment combining these 3 natural biopreservatives at low concentrations, within the hurdle concept of food preservation, might be a useful tool to control the growth of pathogenic microorganisms in nonacidified dairy products. © American Dairy Science Association, 2008.

Antigen-specific T cell-mediated apoptosis of dendritic cells (DCs) represents a unique down-regulatory mechanism that prevents the continuous activation of T cells by antigen-loaded DCs; this regulatory mechanism is impaired in allergy and as a consequence a large proportion of DCs tends to escape apoptosis following cognate interaction with CD4(+) T cells. However, the biological relevance of greater numbers of apoptosis-resistant DCs to the development of allergic IgE-mediated reactions remained to be determined. Here, we sought to investigate the in vitro and in vivo regulatory features of apoptosis-resistant DCs and to assess their role in host sensitization. Freshly isolated CD11c(+/hi)B220(-)DCs from ovalbumin (OVA)-sensitized, OVA-immunized and naïve Balb/c mice were cultured with OVA-specific T cells and levels of T cell-mediated DCs apoptosis assessed by flow cytometry. Surviving apoptosis-resistant DCs were then recovered and subsequently co-cultured with OVA-specific CD62L(hi)CD44(low) naïve T cells or passively transferred into naive syngenic recipients. In vitro profile of DC and T cell lymphokine production, chemokine receptors expression and in vivo, post-adoptive DC transfer T helper (T(H)) and IgE responses were assessed. Apoptosis-resistant DCs showed differential regulatory properties compared to their freshly isolated counterpart independent of the sensitization status of the donor. When co-cultured with naïve OVA-specific T cells, apoptosis-resistant DCs from either sensitized or immunized mice induced T cells that produced increased levels of IL-4 and reduced levels of IFN-gamma and showed increased expression of T(H)-2 related CCR4 and CCR8 chemokine receptors. Finally, adoptive transfer of apoptosis-resistant DCs, induced higher levels of OVA-specific IgE responses in absence of antigen challenge in syngenic recipients compared to freshly isolated DCs from both sensitized and immunized mice. These data would suggest that sensitization-associated increased numbers of apoptosis-resistant T cell-activating DCs contribute to the generation/maintenance of IgE-mediated allergic reactions.

La Serena cheeses, made from Merino ewes' raw milk, were high-pressure (HP)-treated at 300 or 400 MPa for 10 min at 10 °C, on days 2 or 50 of ripening. Cheeses treated by HP on day 2 showed higher pH values than control cheese on day 3, but cheeses treated by HP on days 2 or 50 and control cheese had similar pH values on day 60. Breakdown of caseins was delayed by HP treatment of cheeses on day 2. Cheeses treated by HP on day 2 showed higher levels of hydrophilic peptides, lower levels of hydrophobic peptides, lower hydrophobic peptides hydrophilic peptides ratios, and higher total contents of free amino acids than those of control cheese. HP treatment of cheese on day 50 scarcely affected proteolysis of 60-day-old cheeses. Fracturability, hardness and elasticity values of cheeses treated by HP on day 2 were higher than those of control cheese and of cheeses treated on day 50. Cheeses treated at 400 MPa on day 2 received the lowest scores for quality of taste from panellists, whereas the rest of HP-treated cheeses did not differ from control cheese. © 2007 Elsevier Ltd. All rights reserved.

La Serena cheeses made from raw Merino ewe's milk were high-pressure (HP) treated at 300 or 400 MPa for 10 min on d 2 or 50 after manufacture. Ripening of HPtreated and control cheeses proceeded until d 60 at 8°C. Volatile compounds were determined throughout ripening, and analysis of related sensory characteristics was carried out on ripe cheeses. High-pressure treatments on d 2 enhanced the formation of branched-chain aldehydes and of 2-alcohols except 2-butanol, but retarded that of n-aldehydes, 2-methyl ketones, dihydroxy-ketones, n-alcohols, unsaturated alcohols, ethyl esters, propyl esters, and branched-chain esters. Differences between HP-treated and control cheeses in the levels of some volatile compounds tended to disappear during ripening. The odor of ripe cheeses was scarcely affected by HP treatments on d 2, but aroma quality and intensity scores were lowered in comparison with control cheese of the same age. On the other hand, HP treatments on d 50 did not influence either the volatile compound profile or the sensory characteristics of 60d-old cheese. © American Dairy Science Association, 2007.

La Serena cheese, a Spanish variety made from Merino ewes' raw milk, has a high pH value, low salt content, and high moisture, conditions that are all favorable for growth and survival of contaminating microorganisms, including pathogens. To improve its microbiological quality and safety, high-pressure treatments at 300 or 400 MPa for 10 min at 10°C were applied to 2 batches of La Serena cheese on d 2 or 50 of ripening. Cheese treated on d 2 at 300 MPa showed viable aerobic counts that were 0.99 log units lower than those for control cheese on d 3 and showed counts of enterococci, coagulase-positive staphylococci, gram-negative bacteria, and coliforms that were 2.05, 0.49, 3.14, and 4.13 log units lower, respectively, than control cheese. For cheese treated on d 2 at 400 MPa, the respective reductions in counts were 2.02, 2.68, 1.45, 3.96, and 5.50 log units. On d 60, viable aerobic counts in cheese treated on d 50 at 300 MPa were 0.50 log units lower than those in control cheese, and counts of enterococci, gram-negative bacteria, and coliforms were 1.37, 2.30, and 4.85 log units lower, respectively. For cheese treated on d 50 at 400 MPa, the respective reductions in counts were 1.29, 1.98, 4.47, and >5 log units. High-pressure treatments at 300 or 400 MPa on d 2 or 50 reduced significantly the counts of undesirable microorganisms, improving the microbiological quality and safety of La Serena cheese immediately after treatment and at the end of the ripening period. © American Dairy Science Association, 2006.