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15 p.-6 fig.-6 tab., Polyhydroxyalkanoates (PHAs) are polyesters of microbial origin that can be synthesized by prokaryotes from noble sugars or lipids and from complex renewable substrates. They are an attractive alternative to conventional plastics because they are biodegradable and can be produced from renewable resources, such as the surplus of whey from dairy companies. After an in silico screening to search for SS-galactosidase and PHA polymerase genes, several bacteria were identified as potential PHA producers from whey based on their ability to hydrolyse lactose. Among them, Caulobacter segnis DSM 29236 was selected as a suitable strain to develop a process for whey surplus valorization. This microorganism accumulated 31.5% of cell dry weight (CDW) of poly(3-hydroxybutyrate) (PHB) with a titre of 1.5gl-1 in batch assays. Moreover, the strain accumulated 37% of CDW of PHB and 9.3gl-1 in fed-batch mode of operation. This study reveals this species as a PHA producer and experimentally validates the in silico bioprospecting strategy for selecting microorganisms for waste re-valorization., PROGRAMA AYUDAS A EMPRESAS PARA LA EJECUCIÓN DE PROYECTOS DE I+D+i, Principado de Asturias. Referencia: IE09- 176.

Obesity and its related metabolic disorders, such as diabetes and cardiovascular disease, are major risk factors for morbidity and mortality in the world population. In this context, supplementation with the probiotic strain Bifidobacterium animalis subsp. lactis BPL1 (CECT8145) has been shown to ameliorate obesity biomarkers. Analyzing the basis of this observation and using the pre-clinical model Caenorhabditis elegans, we have found that lipoteichoic acid (LTA) of BPL1 is responsible for its fat-reducing properties and that this attribute is preserved under hyperglycaemic conditions. This fat-reducing capacity of both BPL1 and LTA-BPL1 is abolished under glucose restriction, as a result of changes in LTA chemical composition. Moreover, we have demonstrated that LTA exerts this function through the IGF-1 pathway, as does BPL1 strain. These results open the possibility of using LTA as a novel postbiotic, whose beneficial properties can be applied therapeutically and/or preventively in metabolic syndrome and diabetes-related disorders.

[EN] Here we report the toxicological evaluation of mesoporous silica particles (MSPs) in the nematode C. elegans. Specifically, we have investigated the effect of bare micro- (M0) and nano-sized (N0) MSPs, and their corresponding functionalized particles with a starch derivative (Glu-N) (M1 and N1, respectively) on C. elegans ageing parameters. The toxicity of MSPs, their impact on C. elegans lifespan, movement capacity, progeny and ability to survive upon exposure to acute oxidative stress were assessed. This study demonstrated that both size particles assayed (M0 and N0), labeled with rhodamine and monitored through fluorescence microscopy, are ingested by the nematode. Moreover, toxicity assays indicated that bare nano-sized particles (N0) have a negative impact on the C. elegans lifespan, reducing mobility and progeny production. By contrast, micro-sized particles (M0) proved innocuous for the nematodes. Furthermore, functionalization of nanoparticles with starch derivative reduced their toxicity in C. elegans. Thus, oral intake of N1 comparatively increased the mean lifespan and activity rates as well as resistance to oxidative stress. The overall findings presented here demonstrate the influence of MSP size and surface on their potential toxicity in vivo and indicate the silica-based mesoporous particles to be a potential support for encapsulation in oral delivery applications. Furthermore, the good correlation obtained between healthy aging variables and viability (mean lifespan) validates the use of C. elegans as a multicellular organism for nanotoxicology studies of MSPs., The authors wish to express their gratitude to the Spanish Government (MINECO Projects AGL2012-39597-C02-01, AGL2012-39597-C02-02, AGL2015-70235-C2-1, MAT2012-38429-C04-01 and MAT2015-64139-C4-1), the Generalitat Valenciana (Project PROMETEOII/2014/047) and Colombian Administrative Department of Science, Technology and Research which supported Ms. Acosta Scholarship. We would also like to thank the Institut de Ciencia dels Materials (ICMUV), the Microscopy Service of the Universitat Politecnica de Valencia and the microscopy service of IATA for technical support. We thank Roquette for the Glucidex samples.

The nematode Caenorhabditis elegans is a versatile and powerful model organism for animal experimental research and, despite being an invertebrate, displays remarkably similar molecular bases and conserved cellular pathways to those of humans. Oxidative stress is an etiological factor that influences numerous diseases, degenerative processes and aging. C. elegans has revealed as an opportune and feasible organism to investigate the antioxidant effects of different bioactives or complex food matrices, and a number of protocols have been developed by using different oxidative stressors. Carotenoids are recognized as quenchers and scavengers of reactive oxygen species, and many of their related health benefits attributed in the diet are tightly linked to their antioxidant properties. In this chapter, we report a simple and rapid assay to evaluate the protection capacity of pure carotenoids or complex carotenoid extracts against oxidative stress in the model system C. elegans. The protocol describes a representative feeding experiment by adding carotenoids to the nematode growth medium and after an incubation period, the C. elegans populations fed with carotenoids are exposed to an acute oxidative stress by using H2O2 as oxidative agent. The protection against oxidative stress is evaluated as the survival rate of the nematodes fed with the carotenoid prior to receiving oxidative treatment compared with the survival rate of control nematode population. In order to confirm the carotenoid intake by the nematodes during the feeding experiment a bioassimilation experiment is also reported.

Non-viable preparations of probiotics, as whole-cell postbiotics, attract increasing interest because of their intrinsic technological stability, and their functional properties, such as immune system modulation, gut barrier maintenance, and protection against pathogens. However, reports on Bifidobacteria-derived postbiotics remain scarce. This study aims to demonstrate the functional properties of a heat-treated (HT), non-viable, Bifidobacterium longum strain, CECT-7347, a strain previously selected for its anti-inflammatory phenotype and ability to improve biomarkers of intestinal integrity in clinical trials. The study used the nematode Caenorhabditis elegans and HT-29 cell cultures as eukaryotic model systems. Our results show that HT-CECT-7347 preserves the capacity to protect against oxidative stress damage, while it also reduces acute inflammatory response and gut-barrier disruption, and inhibits bacterial colonization, by activating pathways related to innate immune function. These findings highlight the interest of the ingredient as a novel postbiotic and pave the way to broaden the range of HT-CECT-7347 applications in gut health.

This randomized double-blind and controlled single-center clinical trial was designed to evaluate the effect of a 6-week intake of a probiotic product (1 capsule/day) vs. a placebo on an oxidative stress model of physical exercise (high intensity and duration) in male cyclists (probiotic group, n = 22, placebo, n = 21). This probiotic included three lyophilized strains (Bifidobacterium longum CECT 7347, Lactobacillus casei CECT 9104, and Lactobacillus rhamnosus CECT 8361). Study variables were urinary isoprostane, serum malondialdehyde (MDA), serum oxidized low-density lipoprotein (Ox-LDL), urinary 8-hydroxy-2’-deoxiguanosine (8-OHdG), serum protein carbonyl, serum glutathione peroxidase (GPx), and serum superoxide dismutase (SOD). At 6 weeks, as compared with baseline, significant differences in 8-OHdG (Δ mean difference −10.9 (95% CI −14.5 to −7.3), p &lt, 0.001), MDA (Δ mean difference −207.6 (95% CI −349.1 to −66.1, 0.05), and Ox-LDL (Δ mean difference −122.5 (95% CI −240 to −4.5), 0.05) were found in the probiotic group only. Serum GPx did not increase in the probiotic group, whereas the mean difference was significant in the placebo group (477.8 (95% CI 112.5 to 843.2), 0.05). These findings suggest an antioxidant effect of this probiotic on underlying interacting oxidative stress mechanisms and their modulation in healthy subjects. The study was registered in ClinicalTrials.gov (NCT03798821).

Bioconversion of biodiesel-derived waste glycerol into high-value products is proposed as a solution to improve economic viability of biorefineries. Several microorganisms are able to metabolize glycerol into 2,3-butanediol (2,3-BD), a promising bulk chemical with wide applications: solvent, fuel additive, and feedstock for synthetic rubber production, among them. In the present work, a wide screening of microorganisms present both into the waste water treatment system in a biodiesel industry and in culture collections was carried out in order to evaluate their potential as new 2,3-BD producer biocatalysts. Two microorganisms for 2,3-BD production from glycerol as sole carbon source have been selected, namely Raoultella planticola CECT 843 and Raoultella terrigena CECT 4519. Raoultella strains belong to the non‐pathogenic bacteria class (biosafety level 1). This genus has not been previously described as biocatalyst for the studied process. The influence of operational temperature, organic acid addition, and yeast extract concentration on 2,3-BD yield and productivity have been studied through Taguchi design methodology as well as initial glycerol concentration. Based on these results, the feasibility of the process employing pure glycerol and different samples of raw glycerol has been demonstrated.

Lactoferrin is a highly multifunctional glycoprotein involved in many physiological functions, including regulation of iron absorption and immune responses. Moreover, there is increasing evidence for neuroprotective effects of lactoferrin. We used Caenorhabditis elegans as a model to test the protective effects, both on phenotype and transcriptome, of a nutraceutical product based on lactoferrin liposomes. In a dose‐dependent manner, the lactoferrin‐based product protected against acute oxidative stress and extended lifespan of C. elegans N2. Furthermore, Paralysis of the transgenic C. elegans strain CL4176, caused by Aβ1‐42 aggregates, was clearly ameliorated by treatment. Transcriptome analysis in treated nematodes indicated immune system stimulation, together with enhancement of processes involved in the oxidative stress response. The lactoferrin‐based product also improved the protein homeostasis processes, cellular adhesion processes, and neurogenesis in the nematode. In summary, the tested product exerts protection against aging and neurodegeneration, modulating processes involved in oxidative stress response, protein homeostasis, synaptic function, and xenobiotic metabolism. This lactoferrin‐based product is also able to stimulate the immune system, as well as improving reproductive status and energy metabolism. These findings suggest that oral supplementation with this lactoferrin‐based product could improve the immune system and antioxidant capacity. Further studies to understand the molecular mechanisms related with neuronal function would be of interest.

The antiobesity effect of conjugated linoleic acid (CLA) has previously been described in different animal models. The aim of the present study was to investigate the effect of a commercial mixture (Tonalin) on Caenorhabditis elegans to assess their potential use for functional ingredient screenings. Body-fat reduction with Tonalin was demonstrated in wild-type strain N2. The 1 μg/mL dose was the most effective, either alone or added to a food matrix, and also significantly decreased triglyceride content in nematodes fed on the CLA mixture. Furthermore, the antiobesity effect was related to the CLA isomer trans-10, cis-12. Finally, the transcriptional study showed C. elegans fed with Tonalin (1 μg/mL) underwent an upregulation of energy metabolism, reproduction, protein metabolism and oxidative stress processes. In conclusion, the results presented here clearly correlate well with other animal studies, demonstrating the value of C. elegans as a useful model to evaluate antiobesity compounds/ingredients.

Polyphenol oxidase (PPO) catalyzes the oxidation of o-diphenols to their respective quinones which undergo autopolymerization and form dark pigments. The interaction of PPO with various substrates and effectors remains the focus of intensive investigations due to the enzyme's key role in pigments biosynthesis including animal melanogenesis and fruit/fungi enzymatic browning. In this study, the effect of a range of commonly used pesticides on the enzyme activity has been evaluated using the purified quince (Cydonia oblonga Miller) PPO. The biochemical analysis showed that, in the presence of high pesticide concentrations, the enzyme was competitively inhibited, particularly with benomyl, carbaryl, deltamethrine and parathion methyl for which inhibition constants (K(i)) were 8.3, 5.7, 12 and 4 microM, respectively. At lower pesticide concentrations (2-10 microM), however, the catecholase activity was significantly activated (p

Direct and sensitive PCR detection of contaminant microflora in cocoa extracts is affected by the quality of the template DNA. This study compares the efficacy of five different commercial DNA extraction methods, selective enrichment broths and use of glycolitic enzymes to obtain quality DNA for PCR detection of both fungi and bacteria in artificially inoculated cocoa extract samples. PCR-based methods were applied to detect contaminant microflora in cocoa extracts using as model organisms: Aspergillus nidulans, Bacillus subtilis, Escherichia coli and Salmonella enterica. The quality of the extracted DNA was assessed in terms of PCR inhibitor content with results indicating that the HighPure PCR template (Roche) kit was the best methodology under the conditions assayed. PCR protocols using this commercial kit and a combination of glycolitic enzymes and enrichment procedures gave a detection limit of 100 conidia/g and 100 cfu/g for filamentous fungi and bacteria, respectively. The selected extraction and PCR procedures were also tested to assess their suitability for detecting filamentous fungi and bacteria on an industrial scale. They were sensitive enough to detect fungal and bacterial contaminants within the legally required limits. The results obtained with the molecular approach were in agreement with those of standard microbiological tests but require a considerably shorter analysis time. Thus, the molecular approach provides a sensitive and rapid alternative to check for microbial contamination in cocoa extracts.

In the present study, ochratoxigenic mycobiota in cocoa beans was identified at species level by digestion of the ITS products using the endonucleases HhaI, NlaIII and RsaI. Of the 132 isolates of Aspergillus section Nigri collected from cocoa beans, 89 were identified as A. tubingensis, 27 as A. niger, 10 as A. tubingensis-like and 6 as A. carbonarius. No variation was observed between RFLP patterns (C, N, T1 and T2) described previously for grape isolates and those of the cocoa isolates analysed. With respect to OTA-producing fungi, a high percentage of black aspergilli (50.7%) was able to produce OTA. Additionally, most of the OTA-producing isolates were of moderate toxigenicity, producing amounts of OTA from 10 μg g−1 to 100 μg g−1. Percentages of OTA-producing isolates in the A. niger aggregate were higher than in other substrates, ranging from 30% to 51.7%. Furthermore, the detected levels of OTA production in the A. niger aggregate, particularly in A. tubingensis species was higher than in A. carbonarius, ranging from 0.7 μg g−1 to 120 μg g−1 (mean 24.55 μg g−1). Due to the high occurrence, percentage of ocratoxigenic isolates and their ability to produce OTA, isolates belonging to the A. niger aggregate could be considered as the main cause of OTA contamination in cocoa beans used for manufacturing cocoa products.

9p.-4 fig.-2 tab., A synthetic inducible operon (IbPSO) expressing alsS, ilvC, ilvD and kivD genes encoding a pathway capable to transform pyruvate into 2-isobutyraldehyde has been designed and two recombinant plasmids named pIZIbPSO and p424IbPSO were constructed. The IbPSO containing plasmids can generate in a single transformation event new recombinant isobutanol producer strains and are useful for testing as suitable hosts wild type bacteria in different culture media. In this way we found that Shimwellia blattae (p424IbPSO) was able to produce in flasks up to 6 g l−1 of isobutanol using glucose as carbon source. Moreover, for the first time, we have demonstrated that isobutanol can be produced from sucrose using Escherichia coli W (ATCC9367) transformed with pIZIbPSO. These robust recombinant strains were also able to produce isobutanol from a raw carbon source like hydrolysed lignocellulosic biomass., This work was supported by grants from Biopolis S.L. and Spanish Ministry of Science and Innovation BIOSOS CEN20091040 (CENIT-CDTI). We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).

The effects of combining enzyme treatments and heating protocols on pyrazine formation in cocoa powder extracts have been studied. Five commercial enzyme preparations containing protease and carbohydrase activities were initially assessed for their ability to release amino acids and reducing sugars, both of which are substrates for Maillard reactions. The enzyme preparation Flavourzyme was subsequently selected for further study given its ability to liberate both types of substrate. Cocoa powder solutions were treated with 2 doses of Flavourzyme and processed at 2 drying temperatures. The combination of the higher dose of the enzyme (12% w/w enzyme/substrate) and the higher drying temperature (150 °C) resulted in a synergistic increase in the concentration of methyl- and dimethylpyrazines. A statistically significant increase in the concentration of tetramethylpyrazine occurred in samples treated at 150 °C that was independent of the enzyme dose assayed. No significant changes in trimethylpyrazine content were detected in treated compared to untreated samples.

The mstE gene encoding a low affinity glucose transporter active during the germination of Aspergillus nidulans conidia on glucose medium has been identified. mstE expression also occurs in hyphae, is induced in the presence of other repressing carbon sources besides glucose, and is dependent on the function of the transcriptional repressor CreA. The expression of MstE and its subcellular distribution have been studied using a MstE-sGFP fusion protein. Concordant with data on mstE expression, MstE-sGFP is synthesized in the presence of repressing carbon sources, and fluorescence at the periphery of conidia and hyphae is consistent with MstE location in the plasma membrane. Deletion of mstE has no morphological phenotype but results in the absence of low affinity glucose uptake kinetics, the latter being substituted by a high affinity system.

d-Glucose uptake in germinating wild-typeAspergillus nidulansconidia is an energy-requiring process mediated by at least two transport systems of differing affinities for glucose: a low-affinity system (Km∼1·4 mM) and a high-affinity system (Km∼16 μM). The low-affinity system is inducible by glucose; the high-affinity system is subject to glucose repression effected by the carbon catabolite repressor CreA and is absent insorA3mutant conidia, which exhibit resistance tol-sorbose toxicity. An intermediate-affinity system (Km∼400 μM) is present insorA3conidia germinating in derepressing conditions.creAderepressed mutants show enhanced sensitivity tol-sorbose. The high-affinity uptake system appears to be responsible for the uptake of this toxic sugar.

An enzyme with α-L-rhamnoside activity was purified to homogeneity a culture filtrate of Aspergillus terreus after growth in a medium containing L-rhamnose as the sole carbon source. The biosynthesis of this enzyme was repressed by glucose. The enzyme had a molecular mass of 98 Kda on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an isoelectric point of 4.6 as determined by analytical isoelectric focusing. The pH and temperature optima for the enzyme were found to be 4.0 and 44°C, respectively. Using p-nitrophenyl-α-L-rhamnopyranoside as a substrate, the enzyme exhibited Michaelis-Menten kinetics with Km and Vmax values of 0.17 mM and 84 U/mg, respectively. The enzyme was inhibited competitively by L-rhamnoside activity, whereas this was inhibited by Hg 2+ and Mg 2+ Zn 2+ and Co 2+ stimulated the α-L- rhamnosidase activity, wheras this was inhibited by Hg 2+ and Cd 2+ . Ethanol (12% v/v) and glucose (21% w/v) decreased enzyme activity by approximately 20%, while this was not affected by SO 2 .

Expression of the Aspergillus nidulans xylanase gene xlnC is subject to regulation by carbon catabolite repression and ambient pH. In the presence of glucose, xlnC transcription is repressed by the zinc finger transcription factor CreA. These data have been confirmed using the extremely derepressed mutant creA d 30. Expression of xlnC is elevated at alkaline ambient pH and also in alkalinity-mimicking A. nidulans mutants.

Using a DNA fragment containing the Aspergillus niger abfB gene as a probe, the homologous Aspergillus nidulans gene, designated abfB, has been cloned from a genomic library containing size-selected HindIII fragments. The nucleotide sequence of the A. nidulans abfB gene shows strong homology with the A. niger abfB, Trichoderma reesei abf-1 and Trichoderma koningii alpha-L-arabinofuranosidase/beta-xylosidase genes. Regulation of abfB expression has been investigated in cultures induced with L-arabitol. The accumulation of abfB mRNA, total alpha-L-arabinofuranosidase activity and AbfB protein levels have been determined in a wild-type A. nidulans strain as well as in different mutant strains. These strains are affected either in their response to ambient pH (paIA1 and pacC(c)14 mutants), carbon catabolite repression (creA(d)4 mutant), the ability to utilize L-arabitol as a carbon source (araA1 mutant) or a combination of both latter genotypes (araA1 creA(d)4). The results obtained indicate that the expression of the A. nidulans abfB gene was higher at acidic pHs and was superinduced in this double mutant. Furthermore, disruption of the abfB gene demonstrated that in A. nidulans AbfB is the major p-nitrophenyl alpha-L-arabinofuranoside-hydrolysing activity but at least one minor activity is expressed, which is involved in the release of L-arabinose from polysaccharides.

Three Aspergillus nidulans endo-β-(1,4)-xylanases (X22, X24 and X34) have been produced in Saccharomyces cerevisiae laboratory strains. In fermenter cultivation, the resulting strains were able to secrete the heterologous xylanases when grown in minimal medium containing glucose as carbon source, yielding production levels between 0·5 and 1·0 U ml−1. From such a culture, the xylanases were purified in a single chromatographic step and then physico-chemically characterised. The yeast derived enzymes had the same enzymatic and biochemical properties as the fungal xylanases (ie molecular weights, pI values, pH and temperature optima, Km Michaelis constants and pH and temperature stabilities). The heterologously produced enzymes retained maximum activity in the presence of 10% (w/v) glucose or 10% (w/v) fructose, concentrations normally found in musts. However, ethanol at 12% (v/v) caused inhibitions of 20% for X22 and X24 and 48% for X34. © 1998 Society of Chemical Industry.

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.

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

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.

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.

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.

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.

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.

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

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.

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.

10 pages, 9 figures, 4 tables., Cocoa is a food rich in polyphenols, mainly the flavonoid procyanidins and flavan-3-ols. The improvement of the cardiovascular function in humans upon cocoa consumption has been specifically linked to the presence of flavan-3-ol derived metabolites in plasma, especially epicatechin glucuronide. In this context, a flavonoid-enriched cocoa-derived product could potentially exert stronger health benefits. The aim of the present study was to obtain a cocoa powder with a higher flavonoid content (mainly enriched in monomer compounds) and assess its flavonoid bioavailability in humans. For this purpose, an unfermented, nonroasted, and blanch-treated cocoa powder (A) was obtained. The powder contained four times more procyanidins than a conventional (B) cocoa powder. Powder A contained eight times more epicatechin and procyanidin B2 than powder B. Cocoa milk drinks were prepared with powder A (MDA) and B (MDB). The bioavailability of flavonoids in both drinks was assessed in a crossover intervention with healthy volunteers. The content of epicatechin glucuronide, the main metabolite detected in plasma, was five-fold higher upon consumption of MDA as compared with MDB. The urinary excretion of metabolites, mainly methyl epicatechin sulfate, was higher upon MDA consumption as compared with MDB, ranging from two- to 12-fold higher depending on the metabolite. These results, together with previous reports regarding the cardiovascular benefits linked to the presence of procyanidin metabolites in plasma, suggest that further clinical trials to validate the health benefits of a flavonoid-enriched cocoa powder are warranted.

The Aspergillus nidulans xylanase genes xlnA and xlnB are subject to regulation by ambient pH via the zinc finger transcription factor PacC. In the presence of d -xylose, xlnA is expressed under conditions of alkaline ambient pH while xlnB is expressed at acidic ambient pH. These data have been confirmed for acidity- and alkalinity-mimicking A. nidulans mutants.

An endo-β(1, 4)glucanase encoding gene from fungal origin has been expressed in a brewing yeast strain. The yeast transformation was carried out using a previously reported system based in the acquisition of cycloheximide resistance. The plasmid transferred to brewing yeast showed some changes in restriction pattern of the 2 μ portion after transformation while cycloheximide resistance marker and endoglucanase gene were not affected. The drug resistance phenotype showed by the recombinant yeast was highly stable in non-selective conditions. The endoglucanase enzyme was detected in cell-free culture medium and showed high activities in liquid and solid media.

Monoterpenes such as geraniol, linalool, and -terpineol present in grapes determine the varietal flavor properties of young quality wines made from Muscat varieties (for reviews, see references 19 and 21). Geraniol and linalool are considered to be the most important of the monoterpene alcohols, as they are present in greater concentrations and have lower flavor thresholds than other major wine monoterpenes. In particular, linalool is thought to be responsible for the grapelike aroma of wines produced from the Muscat variety. A large proportion of

A new PCR-based method has been developed to monitor inoculated wine fermentations. The method is based on the variation in the number and position of introns in the mitochondrial gene COX1. Oligonucleotide primers homologous to the regions flanking the Saccharomyces cerevisiae COX1 introns have been designed and tested for S. cerevisiae wine yeast strain differentiation. Four primers were selected for their subsequent use in a multiplex PCR reaction and have proved to be very effective in uncovering polymorphism in natural and commercial yeast strains. An important point is that the speed and simplicity of the technique, which does not require the isolation of DNA, allows early detection of the starter yeast strain throughout the fermentation process. The main advantage for the wineries is that the must sample can be used directly for the PCR reaction obtaining very fast results (in approximately 8 h). This allows the wine industries to intervene quickly if necessary.

The Aspergillus nidulans xlnB gene, which encodes the acidic endo-β-(1,4)-xylanase X 24 , is expressed when xylose is present as the sole carbon source and repressed in the presence of glucose. That the mutation creA d 30 results in considerably elevated levels of xlnB mRNA indicates a role for the wide-domain repressor CreA in the repression of xlnB promoter ( xlnBp ) activity. Functional analyses of xlnBp :: goxC reporter constructs show that none of the four CreA consensus target sites identified in xlnBp are functional in vivo. The CreA repressor is thus likely to exert carbon catabolite repression via an indirect mechanism rather than to influence xlnB expression by acting directly on xlnB .

We previously reported a study on the microbial flora of Alicante wines at different stages of vinification. On the basis of those data, we selected a Saccharomyces cerevisiae strain which is capable of producing high quality wine in pilot fermentation experiments with musts. The strain was characterized using several DNA fingerprinting techniques. These results open the way to use the strain as a dry yeast in order to perform controlled fermentations.

Resveratrol, a phenolic compound produced in grapes, exhibits properties that may contribute to the reduction of the incidence of coronary heart disease and other human health related processes. Recombinant yeast strains expressing the Aspergillus niger abfB gene encoding an α- l -arabinofuranosidase or the Candida molischiana bgIN gene encoding a β-glucosidase have been used in vinifications as tools to increase the resveratrol content of white wine. Glycosylated resveratrol isomers (trans- and cis-piceid) and free resveratrol isomers (trans- and cis-resveratrol) were detected and quantified in white wines. Wines fermented with the strain expressing BgiN showed an increased amount of total resveratrol derivatives, particularly the non-glycosylated forms.

An enzyme exhibiting alpha-L-rhamnosidase activity was purified by fractionating a culture filtrate of Aspergillus nidulans grown on L-rhamnose as the sole carbon source. The alpha-L-rhamnosidase was shown to be N-glycosylated and had a molecular mass of 102 kDa, of which approximately 7% was contributed by carbohydrate. The enzyme, optimally active at pH 4.5-6 and 60 degrees C, had an isoelectric point of 5. With rho-nitrophenyl-alpha-L-rhamnopyranoside as the substrate it showed Km and Vmax values of 0.27 mmol l-1 and 64.6 U mg-1, respectively. The enzyme was competitively inhibited by L-rhamnose (Ki 0.3 mmol l-1). Ca2+ (2 mmol l-1) stimulated the activity of the enzyme by 14%, whereas Mg2+ (2 mmol l-1) inhibited it by 63%. Substrate specificity studies showed the alpha-L-rhamnosidase to be active both on alpha-1,2 and alpha-1,6 linkages to beta-D-glucosides.

Fifty-four yeast strains belonging to the genera Candida, Dekkera, Hanseniaspora, Metschnikowia, Pichia, Rhodotorula, Schizosaccharomyces and Zygosaccharomyces, mainly isolated from grapes and wines, were screened for the production of beta-D-xylosidase activity. Beta-D-xylosidase activity was only detected in eight yeast strains belonging to the genera Hanseniaspora (H. osmophila and H. uvarum) and Pichia (P. anomala). Beta-D-xylosidase preparations active against p-nitrophenyl-beta-D-xyloside were characterised with respect to their optimal pH and temperature conditions. H. uvarum 11105 and 11107 and P. anomala 10320 beta-D-xylosidase preparations were active at pH and temperature ranges and at concentrations of glucose and ethanol usually found during winemaking processes.