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During the last few years, several novel foods produced by genetic engineering techniques have appeared on the market raising various social and ethical problems. Questions related to the safety, legislation and labelling of these novel foods and also the attitude of the consumer against them are discussed here.

A recombinant wine yeast strain expressing the Candidamolischiana bgln gene encoding a beta-glucosidase/anthocyanase under the control of the Saccharomyces cerevisiae actin gene promoter has been constructed. The corresponding protein, BGLN, was mainly located on the cell wall. BGLN was purified in a single chromotagraphic step, and different physicochemical and kinetic properties have been determined. BGLN showed maximum activity against the artificial substrate p-nitrophenyl beta-D-glucopyranoside. It also hydrolyzed salicin, p-nitrophenyl beta-D-xyloside, cellobiose, and arbutin to a lesser extent. Fructose and SO(2) did not affect enzyme activity, which was activated by ethanol, while glucose was a strong competitive inhibitor. The purified BGLN showed a novel anthocyanase decolorizing capability on red wines. This anthocyanase activity was readily observed during microvinification experiments. However, the physicochemical characteristics of the wines obtained with the recombinant wine yeast strain were indistinguishable from those obtained with the parental strain.

Cotyledon protoplasts of an albino Cucumis melo L. ‘Cantaloup Charentais’ somaclonal variant were electrofused with protoplasts of the wild species Cucumis anguria L. var. longipes (Hook. fil.) Meeuse. Selection of putative somatic hybrids was based on competence of the albino melon to grow and regenerate shoots together with the ability of the wild species to synthesise chlorophyll. The ITS region of C. anguria ribosomal DNA was sequenced to design species-specific primers, which allowed us to distinguish between parental lines and fusion products by PCR amplification. By using this method, all the organogenic lines characterised proved to be somatic hybrids. Three of sixteen selected lines produced shoots with albino and green sectors. Eleven lines remained green but shoots developed abnormally and did not produce roots in vitro. Two hybrid lines regenerated normal shoots but with a limited ability to produce roots in vitro and in vivo. Applicability of molecular characterisation to optimise the quick recovery of fusion products is discussed.

In the presence of L-arabitol as sole carbon source, Aspergillus terreus CECT 2663 produces three α-L-arabinofuranosidases (ABFs) named ABF1, ABF2, and ABF3, with molecular masses of 90 000, 82 000, and 78 500 Da, respectively. The synthesis of these enzymes is under carbon catabolite repression. Western blotting revealed that ABF2 is immunologically related to the α-L-arabino-furanosidase B previously isolated from Aspergillus niger. The three A. terreus proteins have been purified to homogeneity. They are acidic proteins with optimal pHs of 5.0 for ABF1 and ABF2 and 5.5 for ABF3 and optimal temperatures of 50, 60, and 65 °C, respectively. Kinetic constants for the purified enzymes on p-nitrophenyl α-L-arabinofuranoside (pNPA) as substrate have been determined. The three enzymes maintain elevated activities in the presence of ethanol or glucose at those concentrations normally present in must or wine.

A Trichoderma longibrachiatum transformant has been constructed constitutively expressing the homologous egl1 gene under the control of the Aspergillus nidulans glyceraldehyde phosphate dehydrogenase gene (gpdA) promoter. Consequently, the egl1 gene can be expressed in glucose-containing media. The egl1 product (EGL1) has been purified from such a medium. The physicochemical and kinetic properties of EGL1, which has both endoglucanase and xylanase activities, have been determined. Analysis of the effects of certain enological factors such as temperature, pH, and glucose, ethanol, and SO2 concentrations on the enzyme's activities indicates that it can be used in wine production. Keywords: Trichoderma longibrachiatum transformation; gpdA promoter; endoglucanase; purification; characterization; wine aroma

Los enfermos celiacos deben seguir de forma estricta una dieta libre de gluten. La base tecnologica de esta oferta se centra exclusivamente en la generacion de productos libres de dicha proteina o sus trazas, sin aportar ningun otro beneficio. Muy recientemente se ha desarrollado un suplemento lacteo denominado Proceliac que pretende cambiar esta tendencia en el diseno de productos para celiacos. La base de este producto es un probiotico denominado ES1 que tiene un fuerte efecto antiinflamatorio demostrado tanto en experimentos con celulas humanas en cultivo como en animales de experimentacion. Se ha evaluado la seguridad alimentaria de la bacteria ES1 siguiendo las reglas de la Organizacion Mundial de la Salud. Ademas se ha secuenciado su genoma para asegurar la ausencia de genes que codifiquen proteinas conflictivas. Finalmente se han llevado a cabo dos ensayos clinicos con adultos sanos y con ninos celiacos al comienzo de dieta libre de gluten con excelentes resultados que han revelado la capacidad de esta cepa probiotica para rebalancear la microbiota del tracto digestivo de los enfermos celiacos.

Numerousin vitroandin vivostudies conducted using different probiotic micro-organisms have demonstrated their ability to interfere with the growth and virulence of a variety of enteropathogens. The reported beneficial effects of the use of probiotics to complement antibiotic therapy or prevent diarrhoea or gastrointestinal infection in infants have increased in recent years. In the present study, we demonstrated the capacity of supernatants obtained from three novel probiotics (Lactobacillus paracaseiCNCM I-4034,Bifidobacterium breveCNCM I-4035 andLactobacillus rhamnosusCNCM I-4036) isolated from the faeces of breastfed infants to inhibit the growth of enterotoxigenic and enteropathogenic (EPEC) bacteria, such asEscherichia coli,SalmonellaandShigella. To assess their potential antimicrobial activity, the 17 and 24 h cell-free supernatants broth concentrates (10 × ) having 1, 2 or 4 % of the three probiotics were incubated with EPEC bacteria strains. After 17 h of co-culture, the supernatants were able to inhibit the growth ofE. coli,SalmonellaandShigellaup to 40, 55 and 81 %, respectively. However, the inhibitory capacity of some supernatants was maintained or completely lost when the supernatants (pH 3·0) were neutralised (pH 6·5). Overall, these results demonstrated thatL. paracaseiCNCM I-4034,B. breveCNCM I-4035 andL. rhamnosusCNCM I-4036 produce compounds that exhibited strain-specific inhibition of enterobacteria and have the potential to be used as probiotics in functional foods.

Bifidobacteria are common inhabitants of human gut and play a significant role in establishing a well-balanced intestinal microbiota. The strain Bifidobacterium longum CECT 7347 (ES1) has been demonstrated to ameliorate damage caused by gluten in celiac disease (CD), both In vitro and in a murine model. Studies suggest that administering this B. longum strain to supplement the gluten-free diet could provide an additional strategy, thereby improving the health status of patients. Here, we report an in-depth study of this strain, adopting a multidisciplinary strategy to demonstrate its safety according to FAO/WHO criteria for probiotic selection. Whole genome sequencing using a massive sequencing approach on the 454 platforms and annotation showed neither relevant virulence nor potential antibiotic resistance genes. It has been demonstrated that values of lactic acid isomer production, bile salt deconjugation and formation of biogenic amines, considered as specifi c traits to be evaluated according to FAO/ WHO, were very similar to levels previously reported in other Bifidobacteria. It has not shown acquired antibiotic resistance In vitro. Moreover, acute ingestion studies in immunocompetent and immunosuppressed BALB/c mouse models did not cause either mortality or morbidity in any group, and did not lead to signifi cant Bifi dobacterial organ translocation, even in the immunosuppressed group. Altogether, these results confi rm the safety status of the strain B. longum CECT 7347. The safety of strain CECT 7347, together with its previously reported functional role in ameliorating gluten- related damage in CD, would indicate it is a probiotic strain.

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Filosofía, Departamento de Filología Española. Fecha de lectura: 11-07-2013

ispartof: Plan Estratégico Provincia. Publicaciones Temáticas (Territorial Strategic Planning. Thematic Series) edition:1 ispartof: Seminario – Taller Desarrollo rural y agricultura familiar edition:1 location:Santa Fe, Argentina date:24-25 August 2011 edition: 1 nrpages: 155 status: published

Jiménez, Guillermo et al., Strain BCT-7112T was isolated in 1966 in Japan from a survey designed to obtain naturally occurring microorganisms as pure cultures in the laboratory for use as probiotics in animal nutrition. This strain, which was primarily identified as Bacillus cereus var toyoi, has been in use for more than 30 years as the active ingredient of the preparation TOYOCERIN®, an additive for use in animal nutrition (e.g. swine, poultry, cattle, rabbits and aquaculture). Despite the fact that the strain was initially classified as B. cereus, it showed significant genomic differences from the type strains of the B. cereus group that were large enough (ANI values below 92%) to allow it to be considered as a different species within the group. The polyphasic taxonomic study presented here provides sufficient discriminative parameters to classify BCT-7112T as a new species for which the name Bacillus toyonensis sp. nov. is proposed, with BCT-7112T (=CECT 876T; =NCIMB 14858T) being designated as the type strain. In addition, a pairwise comparison between the available genomes of the whole B. cereus group by means of average nucleotide identity (ANI) calculations indicated that besides the eight classified species (including B. toyonensis), additional genomospecies could be detected, and most of them also had ANI values below 94%. ANI values were on the borderline of a species definition only in the cases of representatives of B. cereus versus B. thuringiensis, and B. mycoides and B. weihenstephanensis. © 2013 Elsevier GmbH.

We previously described the isolation and characterization of three probiotic strains from the feces of exclusively breast-fed newborn infants: Lactobacillus paracasei CNCM I-4034, Bifidobacterium breve CNCM I-4035 and Lactobacillus rhamnosus CNCM I-4036. These strains were shown to adhere to intestinal mucus in vitro, to be sensitive to antibiotics and to resist biliary salts and low pH. In the present study, a multicenter, randomized, double-blind, placebo-controlled trial with 100 healthy volunteers in three Spanish cities was carried out to evaluate the tolerance, safety, gut colonization and immunomodulatory effects of these three probiotics. Volunteers underwent a 15-day washout period, after which they were randomly divided into 5 groups that received daily a placebo, a capsule containing one of the 3 strains or a capsule containing a mixture of two strains for 30 days. The intervention was followed by another 15-day washout period. Patients did not consume fermented milk for the entire duration of the study. Gastrointestinal symptoms, defecation frequency and stool consistency were not altered by probiotic intake. No relevant changes in blood and serum, as well as no adverse events occurred during or after treatment. Probiotic administration slightly modified bacterial populations in the volunteers’ feces. Intestinal persistence occurred in volunteers who received L. rhamnosus CNCM I-4036. Administration of B. breve CNCM I-4035 resulted in a significant increase in fecal secretory IgA content. IL-4 and IL-10 increased, whereas IL-12 decreased in the serum of volunteers treated with any of the three strains. These results demonstrate that the consumption of these three bacterial strains was safe and exerted varying degrees of immunomodulatory effects., Part of the research currently in progress in the authors' laboratory is funded by the company Hero Spain, S. A. through the grant #3582 managed by the Fundacion General Empresa-Universidad de Granada.

2 p., Clostridium saccharoperbutylacetonicum is one of the most important acetone-butanol-ethanol (ABE)-generating industrial microorganisms and one of the few bacteria containing choline in its cell wall. Here, we report the draft genome sequence of C. saccharoperbutylacetonicum strain N1-4 (6.6 Mbp; G+C content, 29.4%) and the findings obtained from the annotation of the genome., We acknowledge the financial support provided by the Ministry of Economy and Competitiveness Project BIOSOS (CENIT-E 2009) and by project Consolider CSD2007-00005.

Conditions for maximal batch culture production of extracellularα-L-rhamnosidase byAspergillus terreus have been investigated. Production of the enzyme appeared to be inducible by rhamnose and rutin, reaching a maximal level after an incubation period of 162 h when the fungus was grown at 37°C on either of these compounds as the carbon source and on ammonium phosphate as the nitrogen source. Nonionic surfactants did not enhanceα-L-rhamnosidase secretion. Under optimal conditions,A. terreus produced only oneα-L-rhamnosidase of approximate molecular weight 90 kDa (SDS-PAGE) and isoelectric point of 4.6. Onp-nitrophenyl-α-L-rhamnopyranoside as substrate, the enzyme showed pH and temperature optima of 6–8 and 45–50°C, respectively. Neither divalent cations nor ethylenediaminetetraacetate (EDTA) inhibited or stimulated enzyme activity. The enzyme was active at the concentrations of glucose found in must or of ethanol in wine.

The xlnC gene encoding the 34-kDa xylanase (X34) of Aspergillus nidulans (An) has been cloned and sequenced, as has its corresponding cDNA. xlnC contains nine introns and shows considerable similarity to the xynA and xylP xylanase-encoding genes of A. kawachii (Ak) and Penicillium chrysogenum (Pc), respectively. Analysis of xylanase production in An multicopy transformants showed elevated levels of X34 and increased total xylanase activity, but no elevated production of other xylanases. Northern analysis demonstrated transcriptional induction by xylan and repression by glucose.

Genes as POT1, HSP104 and SSA3, which are late expressed in laboratory culture conditions are expressed only during the first few days in microvinifications in wine yeast cells. This effect is probably due to the different growth conditions and leads to useless levels of enzyme activity for a reporter gene. However the ACT1 promoter, which is constitutively expressed in laboratory conditions, produces sufficient amounts of enzyme activity in late fermentation phases.

Using recombinant DNA techniques, an Aspergillus nidulans multicopy transformant for the gene xlnB coding for the minor X24 xylanase has been constructed. When grown on glucose as sole carbon source this transformant secretes 114 U of xylanase (mg protein)-1. In this culture condition, X24 is the only xylanase secreted and the predominant protein in the culture filtrate. This strategy has been used to purify the X24 enzyme to homogeneity. The purified xylanase showed a single band on sodium dodecyl sulphate/ polyacrylamide gel electrophoresis with a molecular mass of 24 kDa and had an isoelectric point of approximately 3.5. The enzyme was a non-debranching endo-1,4-β-xylan xylanohydrolase highly specific for xylans and showed optimal activity at pH 5.5 and 52°C. The X24 xylanase had a Michaelis constant, Km, of 12.43 mg oat spelt xylan ml-1 and a Vmax of 1639 μmol min-1 (mg protein)-1.

The taxonomy of the form-genus Keratinomyces (Trichophyton) within the group of the dermatophytes is based on morphological features which remain insufficient for the distinction of these anamorphic species. The three species included in the genus Keratinomyces, namely K. ajelloi, K. ceretanicus and K. longifusus were examined by means of their mitochondrial-like DNA diversity and compared to few other dermatophytes. The analysis of the mtDNA restriction fragments confirmed that the three species are different and well separate from the other dermatophytes.

Many winemakers use pure Saccharomyces cerevisiae cultures, sometimes isolated from their own region, to produce wine of a more reproducible quality. As most of the industrial yeast strains belong to the S. cerevisiae group, and cannot be distinguished and identified by classical microbiological methods, molecular techniques have recently been employed. Techniques such as mitochondrial DNA restriction analysis and chromosome electrophoretic analysis have been used to study the dynamics of both natural and inoculated wine fermentations. Natural fermentation is a complex process during which it is possible to observe sequential substitutions of many different S. cerevisiae strains as the process proceeds. However, in inoculated fermentations, the inoculated strain prepared from active dried yeast clearly dominates. This microbiological simplification, together with advances in biotechnology, opens the way for the genetic modification of active dried yeast and thus the construction of strains that express metabolic activities that have consistent effects on the organoleptic characteristics of the resulting wines.

β-xylosidase (EC 3.2.1.37) has been purified from Aspergillus nidulans mycelium grown on oat-spelt xylan as sole carbon source. Its pH optimum for activity was found to be 5.0 and the optimum temperature was 50 °C. Its molecular mass was estimated by gel filtration to be 180000. Using p-nitrophenyl-β-d-xylopyranoside as substrate, the Km and Vmax values have been found to be 1.1 mM and 25.6 μmol min−1(mg protein)−1, respectively. Enzyme activity was inhibited by Hg2+, Ag2+, and Cu2+ at a concentration of 1 × 10−3 M. The synthesis of β-xylosidase in A. nidulans is strongly induced by arabinose and xylose and is subject to carbon catabolite repression mediated by the cre A gene product.

A gene fusion between the Saccharomyces cerevisiae actin gene promoter and the cDNA of the Fusarium solani f. sp. pisi pelA gene has been constructed. This expression cassette has been introduced into the industrial wine yeast strain T73. The resulting recombinant strain is able to secrete active PELA enzyme into the culture medium. In preliminary microvinification experiments the wine produced by this pectinolytic strain is indistinguishable from wine produced using the non-transformed strain on the basis of the chemical analyses. Large scale fermentations need to be carried out in order to assess the effects on filtrability.

Nanoparticulated bimodal mesoporous silicas (NBS) have proved to constitute adequate supports in a variety of applications requiring enhanced accessibility to the active sites. Mass-transfer kinetics seems to be highly favoured in UVM-7-derived NBS materials. To understand the mass-diffusion phenomena throughout UVM-7-like supports requires well-grounded knowledge about their pore architecture. 3-D reconstructions of the UVM-7 mesostructure carried out by electron tomography reveal the existence of a true hierarchic connectivity involving both inter- and intra-nanoparticle pores. This connectivity makes self-supported nanoparticulated mesoporous bimodal carbon replicas of the supports feasible to obtaining by nanocasting. Both the temperature-induced mobility of gold nanodomains and the fast and efficient enzyme adsorption in UVM-7-like silicas are examples of non-constrained diffusion processes happening inside such an open network.

[EN] A previously validated constraint based model and possibilistic MFA have been used to design a simple estimator of protein production rate in Pichia pastoris cultures. A structured model of the yeast P. pastoris metabolism is used to predict the balance of key energetic equivalents such as ATP from available measurements, mainly substrate consumption, gases exchange rates and biomass specific growth. It has been shown that ATP flux can be related to biomass growth and protein productivity specific rates by linear regression. Cross-validation has been applied for robust parameter fitting on the basis of chemostat, steady-state experimental conditions. In this way, protein estimation can be integrated in the constraint-based model, and possibilistic protein productivity prediction can be given even if only a few extracellular rates are known. Complimentary estimation of biomass growth and intracellular rates are also shown in different lacking-data conditions, frequent in industrial environment. © 2012 Elsevier Ltd., This research has been partially supported by the Spanish Government (2nd and 4th authors are grateful to grants DPI2008-06880-C03-01 and Feder-Cicyt DPI2011-28112-C04-01). The authors are also grateful to the Company Biopolis for his support to this research.

International audience; Numerous studies have shown that resistance to oxidative stress is crucial to stay healthy and to reduce the adverse effects of aging. Accordingly, nutritional interventions using antioxidant food-grade compounds or food products are currently an interesting option to help improve health and quality of life in the elderly. Live lactic acid bacteria (LAB) administered in food, such as probiotics, may be good antioxidant candidates. Nevertheless, information about LAB-induced oxidative stress protection is scarce. To identify and characterize new potential antioxidant probiotic strains, we have developed a new functional screening method using the nematode Caenorhabditis elegans as host. C. elegans were fed on different LAB strains (78 in total) and nematode viability was assessed after oxidative stress (3 mM and 5 mM H(2)O(2)). One strain, identified as Lactobacillus rhamnosus CNCM I-3690, protected worms by increasing their viability by 30% and, also, increased average worm lifespan by 20%. Moreover, transcriptomic analysis of C. elegans fed with this strain showed that increased lifespan is correlated with differential expression of the DAF-16/insulin-like pathway, which is highly conserved in humans. This strain also had a clear anti-inflammatory profile when co-cultured with HT-29 cells, stimulated by pro-inflammatory cytokines, and co-culture systems with HT-29 cells and DC in the presence of LPS. Finally, this Lactobacillus strain reduced inflammation in a murine model of colitis. This work suggests that C. elegans is a fast, predictive and convenient screening tool to identify new potential antioxidant probiotic strains for subsequent use in humans.

An acidic xylanase from a culture filtrate of Aspergillus nidulans grown on oat-spelt xylan was purified to apparent homogeneity. The purified enzyme showed a single band on sodium dodecyl sulphate-polyacrylamide gel electrophoresis with a molecular mass of 34,000 Da and had an isoelectric point of approximately 3.4. The enzyme was a non-debranching endoxylanase highly specific for xylans. The xylanase showed an optimal activity at pH 6.0 and 56° C and had a Michaelis constant Km of 0.97 mg oat-spelt xylan (soluble fraction) ml and a maximed reaction velocity (Vmax) of 1,091 μmol min−1 (mg−1protein)−1. Using polyclonal antibodies raised against the purified enzyme, the regulation of its synthesis has been studied. The xylanase production is repressed by glucose and induced by oat-spelt xylan, arabinoxylan, 4-O-methylglucurono-xylan, birchwood xylan and xylose.

The induction of the synthesis of extracellular xylanases was investigated in the fungus Aspergillus nidulans using a number of compounds, including xylans of different origin, monosaccharides, xylooligosaccharides and xylose derivatives. Certain xylans (wheat arabinoxylan, oat spelt xylan, birchwood xylan and 4-O-methyl-D-glucurono-D-xylan) were found to be the most powerful inducers. Also, xylooligosaccharides such as xylobiose, xylotriose and xylotetraose served as inducers, their efficiency being directly related to their chain length. Xylose, on the contrary, was not a true inducer. Of the three endo-β-(1,4)-xylanases secreted by A. nidulans, that of 24 kDa was not under carbon catabolite repression, whereas the other two, of 22 and 34 kDa, were under glucose repression mediated by the creA gene product.

[ES] En este artículo presentamos los resultados derivados de un proyecto encaminado a la preparación y experimentación de un material multimedia dirigido a potenciar el aprendizaje autónomo en la adquisición de las competencias específicas de las prácticas de la asignatura Ecología, troncal en los estudios de la licenciatura en Biología de la Universitat de las Illes Balears (UIB). El objetivo fundamental radica en la progresiva adaptación al Espacio Europeo de Educación Superior (EEES) a partir de una mayor coordinación entre el profesorado y la obtención de nuevos recursos para la docencia. Los resultados obtenidos en la experimentación del material pueden considerarse significativamente positivos., [EN] In this paper we present the results arising from a project to the preparation and testing of a multimedia material to promoting autonomous learning in the acquisition of specific competences of the Ecology practices course, in Biology studies at the Universitat de les Illes Balears (UIB). The main objective lies in the gradual adaptation to the European Higher Education Area (EHEA) from greater coordination among teachers and the acquisition of new resources for teaching. The results obtained in the materials experimentation can be considered significantly positive., Esta experiencia ha sido subvencionada por el Vicerectorado de Ordenación Académica de la UIB en su “Convocatòria d’ajuts per a projectes d’innovació i millora de la qualitat docent. Any acadèmic 2008‐2009”.

Developing functional foods to improve the quality of life for elderly people has great economic and social impact. Searching for and validating ingredients with in vivo antioxidant effects is one of the key steps in developing this kind of food. Here we describe the combined use of simple biological models and transcriptomics to define the functional intracellular molecular targets of a polyphenol-enriched cocoa powder. Cocoa powder supplemented culture medium led to increased resistance to oxidative stress, in both the budding yeast Saccharomyces cerevisiae and the nematode Caenorhabditis elegans, and, in the latter, lifespan was also increased. These effects are fully dependent on the polyphenols present in the cocoa powder and on the sirtuins Hst3 (yeast) and SIR-2.1 (worm). The transcription factor DAF-16 also plays an important role in the case of the nematode, indicating that the insulin/IGF-1 (insulin-like growth factor) signaling pathway is related with the antioxidative effect of cocoa polyphenols. All in all, these results confirm that this polyphenol-enriched cocoa powder, with antioxidant activity, has great potential use as a functional food ingredient for elderly people. Furthermore, this work reveals the value of using simple biological models to screen for compounds that are of interest for the food and pharmacological industry.

Publisher Summary Both vines and wine yeasts have been subject to genetic improvement. Genetic engineering has begun to be applied to both vines and wine yeasts, although to varying extents and with differing outcomes. Genetic engineering relies on the ability to isolate genes from a genome and to then introduce them into cells through the use of genetic transformation techniques. It can be reasoned that any gene introduced into a yeast that is then inoculated in a winery fermenter, if expressed during fermentation, will lead to accumulation of the protein that it encodes as vinification advances and will therefore introduce the technological activity of interest. Extensive biochemical and physiological data have been accumulated over many years in studies of the growth of laboratory strains of S. cerevisiae in defined media and under controlled laboratory conditions. Furthermore, many of the genes associated with the metabolic generation of physicochemical or organoleptic properties that are relevant to winemaking have been cloned and sequenced. Since effective methods are available for the transformation of S. cerevisiae, including most of the wine strains studied to date, it has been possible to use all of this information to investigate the generation of improved wine yeasts through the use of genetic engineering.

Background: Insects are associated with microorganisms that contribute to the digestion and processing of nutrients. The European Corn Borer (ECB) is a moth present world-wide, causing severe economical damage as a pest on corn and other crops. In the present work, we give a detailed view of the complexity of the microorganisms forming the ECB midgut microbiota with the objective of comparing the biodiversity of the midgut-associated microbiota and explore their potential as a source of genes and enzymes with biotechnological applications. Methodological/Principal Findings: A high-throughput sequencing approach has been used to identify bacterial species, genes and metabolic pathways, particularly those involved in plant-matter degradation, in two different ECB populations (field-collected vs. lab-reared population with artificial diet). Analysis of the resulting sequences revealed the massive presence of Staphylococcus warneri and Weissella paramesenteroides in the lab-reared sample. This enabled us to reconstruct both genomes almost completely. Despite the apparently low diversity, 208 different genera were detected in the sample, although most of them at very low frequency. By contrast, the natural population exhibited an even higher taxonomic diversity along with a wider array of cellulolytic enzyme families. However, in spite of the differences in relative abundance of major taxonomic groups, not only did both metagenomes share a similar functional profile but also a similar distribution of non-redundant genes in different functional categories. Conclusions/Significance: Our results reveal a highly diverse pool of bacterial species in both O. nubilalis populations, with major differences: The lab-reared sample is rich in gram-positive species (two of which have almost fully sequenced genomes) while the field sample harbors mainly gram-negative species and has a larger set of cellulolytic enzymes. We have found a clear relationship between the diet and the midgut microbiota, which reveals the selection pressure of food on the community of intestinal bacteria. © 2011 Belda et al., The research was funded by the Spanish Ministerio de Ciencia e Innovacion, under grant agreement CIT-010000-2008-5 and by a MICINN (Ministerio de Ciencia e Innovacion) TIN2009-12359 ArtBioCom project. Arnau Montagud acknowledges Generalitat Valenciana grant BFPI/2007/283. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

A neutral endoxylanase from a culture filtrate of Aspergillus nidulans grown on oat spelt xylan was purified to apparent homogeneity. The purified enzyme showed a single band on SDS-PAGE with a molecular mass of 22,000 and had an isoelectric point of 6.4. The enzyme was a non-debranching endoxylanase highly specific for xylans and completely free from cellulolytic activity. The xylanase showed an optimum activity at pH 5.5 and 62°C and had a Km of 4.2 mg oat spelt xylan per ml and a Vmax of 710 μmol min−1 (mg protein)−1.

PMCID: PMC182369, A genetic transformation system for an industrial wine yeast strain is presented here. The system is based on the acquisition of cycloheximide resistance and is a direct adaptation of a previously published procedure for brewing yeasts (L. Del Pozo, D. Abarca, M. G. Claros, and A. Jiménez, Curr. Genet. 19:353-358, 1991). Transformants arose at an optimal frequency of 0.5 transformant per microgram of DNA, are stable in the absence of selective pressure, and produce wine in the same way as the untransformed industrial strain. By using this transformation protocol, a filamentous fungal beta-(1,4)-endoglucanase gene has been expressed in an industrial wine yeast under the control of the yeast actin gene promoter. Endoglucanolytic wine yeast secretes the fungal enzyme to the must, producing a wine with an increased fruity aroma.

A 0.85-kb HindIII mitochondrial DNA fragment of the dermatophytic fungus Trichophyton mentagrophytes has been sequenced. The fragment contains eight complete genes which corresponds to a tRNA gene cluster. From 5′ to 3′, the sequenced genes code for tRNAthr, tRNAglu, tRNAval, tRNAmet1, tRNAmet3, tRNAleu, tRNAala, and tRNAphe. This tRNA gene cluster is located downstream of the larger ribosomal RNA gene. The particularities ofthe sequenced genes and their comparison with other fungal tRNA mitochondrial genes are reported.

Helicobacter pylori is considered one of the major risk factors underlying the development of gastritis and gastric and duodenal ulcers. Moreover, 50% of the population carries this bacterium, and consequently, when it is detected, eradication of H. pylori is strongly recommended. Regarding the use of probiotics as functional agents, several studies have shown that there is a direct relationship between the addition of certain probiotic bacteria and in vitro inhibition of H. pylori ; however, in vivo studies showing bifidobacterial activity against H. pylori remain scarce. In this study, a Bifidobacterium bifidum strain which proved active in vitro against H. pylori has been isolated, with inhibition levels reaching 81.94% in the case of the supernatant and even 94.77% inhibition for supernatant purified by cationic exchange followed by an inverse phase. In vivo studies using a BALB/c mouse model have proved that this strain partially relieves damage to gastric tissues caused by the pathogen and also decreases the H. pylori pathogenicity ratio. This novel strain fulfills the main properties required of a probiotic (resistance to gastrointestinal juices, biliary salts, NaCl, and low pH; adhesion to intestinal mucus; and sensitivity to antibiotics). Furthermore, the absence of undesirable metabolites has been demonstrated, and its food safety status has been confirmed by acute ingestion studies in mice. In summary, the results presented here demonstrate that Bifidobacterium bifidum CECT 7366 can be considered a probiotic able to inhibit H. pylori both in vitro and in vivo .

Fermentation and roasting are the main causes of polyphenol degradation during the process for obtaining cocoa products. In the present study, a process for obtaining polyphenol-rich cocoa products on an industrial scale is described. The process avoids the fermentation and roasting steps and includes a step for the inactivation of the enzyme Polyphenol Oxidase (PPO), which helps preserve the polyphenol content present in the raw cocoa bean. In addition, our study evaluates the antioxidant capacity and characterizes the flavonoid profile of the polyphenol-rich cocoa products obtained from the natural polyphenolrich cocoa cake. Using different protocols, we have obtained three cocoa extracts with high polyphenol content, namely extracts A (167 mg/g), B (374 mg/g) and C (787 mg/g). The scavenging capacity of the extracts was measured as their ability to bleach the stable radicals DPPH and ABTS + while their antioxidant effect was evaluated with the FRAP assay. The results for A, B and C in the DPPH test expressed as Trolox equivalent (lmol)/mg dry weight of extract were 0.2, 1.4 and 3.0, respectively; in the ABTS test the results were 1.0, 4.7 and 9.8. The antioxidant capacity expressed as ascorbic acid equivalent (lmol)/mg dry weight of each product were 17.2, 76.1 and 207.7, respectively. The scavenging properties of cocoa powder against the superoxide anion, H2O2, HClO, and peroxynitrite were also determined. The IC50 (lg/mL) values in the hypoxanthine/xanthine oxidase test were 77.5, 12.3 and 10.3, for A, B and C, respectively, while as an HOCl scavenger the IC50 (lg/mL) values were 225.4, 73.2 and 21.5. As a peroxynitrite anion scavenger, only extract C had a relevant effect, with IC50 (lg/mL) values of 76.1 or 110.0 in the absence or presence of bicarbonate. None of the extracts tested showed activity in the hydrogen peroxide test, but B and C significantly increased the deoxyribose degradation in the absence of ascorbate. Likewise, none of the extracts inhibited the ferrous or copper chelating activity at 100 lg/mL, but they inhibited the lipid peroxidation in brain homogenates and human plasma through non-enzymatic generation systems, with extract C giving the best IC50 (lg/mL) values: 17.4 and 8.1 against lipid peroxidation in brain homogenates and human plasma, respectively. In conclusion, if the extractive protocol is well characterized, defined and optimized, cocoa could constitute a source of polyphenols for enriching foods, nutraceuticals and alimentary supplements., Facultad de Ciencias Médicas (FCM)

Podría decirse con todo derecho: ¡Cómo pretender celebrar la palabra con tan pocas palabras! Pero de eso se trata. Ni más, ni menos. Unas pocas palabras que recuperan la palabra sobre algunos escorzos de la realidad que me inquieta. Porque celebrar la palabra es una manera de recuperar nuestra palabra. La propia palabra, que aquí no requiere explicaciones, sino afirmaciones, unas pocas afirmaciones. (del prólogo del autor), Universidad Nacional de La Plata

Podría decirse con todo derecho: ¡Cómo pretender celebrar la palabra con tan pocas palabras! Pero de eso se trata. Ni más, ni menos. Unas pocas palabras que recuperan la palabra sobre algunos escorzos de la realidad que me inquieta. Porque celebrar la palabra es una manera de recuperar nuestra palabra. La propia palabra, que aquí no requiere explicaciones, sino afirmaciones, unas pocas afirmaciones. (del prólogo del autor)

Using filtration enrichment techniques, an Aspergillus terreus arginine auxotrophic strain which contains a mutation that abolishes ornithine transcarbamylase (OTCase) activity has been isolated. This mutant has been genetically transformed with the cloned Aspergillus nidulans OT-Case gene. Prototrophic transformants arose at a frequency of about 50 transformants per microgram of plasmid DNA. Southern blot analysis of DNA from the transformants showed that the transforming DNA was ectopically integrated at different locations in the A. terreus genome, often in multiple tandem copies. The transformants were phenotypically stable for several mitotic divisions and retained their capacity to produce extracellular enzymes.

Summary An extensive survey of different methods of yeast strain identification (classical microbiological tests, whole-cell protein electrophoresis, chromosomal patterns, DNA hybridization and mitochondrial DNA restriction analysis) has been carried out in order to differentiate, with industrial purposes, strains present in the Alicante wine ecosystem. Only chromosomal patterns and mitochondrial DNA (mtDNA) restriction analysis show differences between strains. Both techniques are very complex to be used in bio technological industries. For this reason, we have developed a new, simple, unexpensive and rapid method based on mtDNA restriction analysis.

When grown on Aspergillus-minimal medium, Aspergillus nidulans secreted a xylanolytic system containing the enzymes necessary for degradation of arabinoxylan. Xylanase (endo-1,4-β-d-xylan-hydrolase; EC 3.2.1.8) production was maximal for cultures at 37°C using oat-spelt xylan and casamino acids as respective carbon and nitrogen sources. SDS-PAGE and overlay techniques revealed the occurrence of two extracellular xylanases (Mr 22 900 and 34 300). Optimum activity was obtained when crude xylanase preparations were incubated for 10 min at pH 5.4 and 55°C. At these conditions the half-life of the xylanase preparation was 3 h.

The energy density (ED) of nine species of sub-Antarctic fishes was estimated by calorimetry. The fish, seven notothenioids, one atherinopsid and one galaxiid, represents some of the more abundant species in the ichthyofauna of the Beagle Channel. Principal-components analysis (PCA) of the ED of the different organs/tissues indicated that PC and PC accounted for 87% of the variability. Separation along PC 1 corresponded to differences in muscle and liver energy densities whereas separation along PC corresponded to differences in the ED of the gonads. Differences between species were significant except for P. sima. Inclusion of the gonadosomatic index (GSI) as an explanatory variable enabled us to establish the existence of energy transfer from muscle and liver to the gonads in ripe P.tessellata females. Total ED values varied between 4.21 and 6.26 kJg, the pelagic Odontesthes sp. being the species with the highest ED. A significant relationship between ED and muscle dry weight (DW) was found for all the species except P. tessellata. These data are the first direct estimates of ED of fishes from the Beagle Channel. Fil: Fernandez, Daniel Alfredo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Austral de Investigaciones Científicas; Argentina Fil: Lattuca, María Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Austral de Investigaciones Científicas; Argentina Fil: Boy, Claudia Clementina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Austral de Investigaciones Científicas; Argentina Fil: Pérez, Analía Fernanda. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Austral de Investigaciones Científicas; Argentina Fil: Ceballos, Santiago Guillermo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Austral de Investigaciones Científicas; Argentina Fil: Vanella, Fabián Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Austral de Investigaciones Científicas; Argentina Fil: Morriconi, Elba Rosa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Austral de Investigaciones Científicas; Argentina Fil: Malanga, Gabriela Fabiana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Austral de Investigaciones Científicas; Argentina Fil: Aureliano, Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Austral de Investigaciones Científicas; Argentina Fil: Rimbau, Sonia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Austral de Investigaciones Científicas; Argentina Fil: Calvo, Jorge. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Austral de Investigaciones Científicas; Argentina

Aspergillus terruss was transformed to hygromycin B resistance using a bacterial resistance gene under the control of Aspergillus nidulans regulatory sequences. Southern hybridization of transformants indicated that in most of the cases the vector DNA was integrated into the recipient chromosome in the form of tandem arrays. Transformants were mitotically stable in both selective and non-selective medium and retained their capacity to produce xylanase or glucoamylase activities.

This work examines ochratoxigenic mycobiota in grapes by ap-PCR analysis sequence analysis of the ITS and IGS regions and ability to produce OTA. A comparison was also made with many reference strains of Aspergillus section Nigri. Based on ap-PCR profiles, derived from two microsatellite primers, three main groups were obtained by UPGMA cluster analysis corresponding to A. carbonarius, A. niger and A. tubingensis. The cophenetic correlation values corresponding to ap-PCR UPGMA analysis revealed a higher genetic variability in A. niger and A. tubingensis than in A. carbonarius. In addition, no genotypical differences could be established between OTA producers and nonproducers in all species analysed. Phylogenetic relationships inferred from ITS and IGS sequences are, mostly, congruent with earlier works. A. niger and A. tubingensis strains were closely related, but not identical, and they clustered into two distinct groups within the A. niger aggregate. Sequence analysis also showed genetic divergences between strains of A. foetidus and the rest of the Aspergillus section Nigri. Additionally, the phylogenetic analysis was consistent in separating a new group of ochratoxigenic strains, frequently isolated from grapes, named A. tubingensis-like. All strains of A. carbonarius analysed by sequence analysis had identical ITS and IGS sequences confirming the lack of significant genetic variability within this important ochratoxigenic species.

The nucleotide sequences of the bglA and bglB genes encoding β-glucosidases from Bacillus polymyxa have been determined. Both genes contain coding regions of 1344 bp, corresponding to polypeptides with M r s of 51 643 and 51 547, respectively. Patterns of codon usage indicate that both genes are expressed at a low frequency. Previous data suggested that the proteins encoded by bglA and bglB were intra- and extracellular enzymes, respectively; however, neither of the two deduced amino acid sequences has N termini with the typical features of a leader peptide. The proteins encoded by bglA and bglB show remarkable homology to each other and to other β-glucosidases (Bgl) and β-galactosidases (βGal). On the basis of the observed homologies, we can define two groups of microbial Bgl: one of them, type I, including most bacterial Bgl, and type II, including enzymes from different yeast species and one from Clostridium thermocellum . Likewise, at least two groups of βGal can be distinguished: type I, including enzymes homologous to type-I Bgl, and type II, showing no homology to any of the previous groups.

The present study reports on the natural mycobiota occurring in cocoa beans, paying special attention to the incidence of fungal species that are potential producers of mycotoxins. The results show that predominant fungi were different species of the genus Aspergillus belonging to section Flavi and Nigri. Of the 214 strains of Aspergillus section Flavi collected from cocoa beans, 120 were identified as A. flavus and 94 as A. tamarii. Of Aspergillus section Nigri 138 strains were isolated, with 132 belonging to A. niger aggregate and 6 to A. carbonarius species. Potential ability to produce aflatoxins (AFs) B1, B2, G1 and G2, cyclopiazonic acid (CPA) and ochratoxin A (OTA) was studied by isolate culture followed by HPLC analysis of these mycotoxins in the culture extracts. Results indicated that 64.1% and 34.2% of the A. flavus strains produced AFs and CPA, respectively. Most of the A. flavus strains presented moderate toxigenicity with mean levels of AFs ranging from 100 ng g(-1) to 1000 ng g(-1). All the CPA-producing strains of A. flavus were highly toxigenic producing >30 microg g(-1) of CPA. Furthermore, 98% of A. tamarii strains produced CPA and over 50% of them were highly CPA toxigenic. With respect to OTA-producing fungi, a high percentage of black aspergilli strains (49.2%) were able to produce OTA. Additionally, most of the OTA-producing isolates were of moderate toxigenicity, producing amounts of OTA from 10 microg g(-1) to 100 microg g(-1). These results indicate that there is a possible risk factor posed by AFs, CPA and OTA contamination of cocoa beans, and consequently, cocoa products.

Grape musts contain a variety of terpenols that significantly affect wine aroma. The amounts of these metabolites depend on the grape variety, and many cultivars are non-aromatic. Yeasts like Saccharomyces cerevisiae cannot produce and excrete monoterpenes efficiently, mainly due to their lack of monoterpene synthases. By metabolic engineering we have modified the isoprenoid biosynthesis pathway in a wine yeast strain of S. cerevisiae expressing the Clarkia breweri S-linalool synthase gene. Under microvinification conditions, without compromising other desirable and useful fermentative traits, the recombinant yeast efficiently excreted linalool to levels exceeding the threshold of human perception. Bearing in mind the possibility of (co-)expressing other genes that encode enzymes leading to the production of various aroma compounds and the feasibility of controlling the levels of their expression, the potential of this achievement for future genetic manipulation of wine varietal aroma or for use in other alcoholic drinks seems very promising.