Composition, ζ Potential, and Molar Mass Distribution of 20 Must and Wine Colloids from Five Different Cultivars Obtained during Four Consecutive Vintages.

Colloids are responsible for undesirable haze formation in wine. Here, we characterized 20 colloid batches after isolation by ultrafiltration of musts and wines from five cultivars obtained from four consecutive vintages. Polysaccharide and protein concentrations of the colloids ranged from 0.10 to 0.65 and 0.03 to 0.40 mg/L, respectively. Protein profiling in must and wine colloids by fast protein liquid chromatography (FPLC) and liquid chromatography-high-resolution tandem mass spectrometry (LC-HR-MS/MS) analyses indicated a lower number of proteins in wine than in must colloids. Molar mass distribution analyses revealed all colloids to consist of two carbohydrate- (424-33,390 and 48-462 kg/mol) and one protein-rich (14-121 kg/mol) fractions. The observed barely negative ζ potentials (-3.1 to -1.1 mV) in unstable wines unraveled that colloid instability might be partly related to their poor electrostatic repulsion in the wine matrix. ζ potentials of the colloids from pH 1 to 10 are also presented. Our data support future developments to eliminate haze-forming colloids from wine.

Immobilization of peroxidase on textile carrier materials and their application in the bleaching of colored whey.

Textiles represent promising support materials for enzymes. The goal of the present work was to investigate the immobilization of commercial peroxidase on a polyester needle felt and the repeated use in the gentle degradation of norbixin in whey from dairy cheese as a practical application. High enzyme loads were obtained by a 2-step immobilization procedure. First, the number of functional groups on the textile surface was increased by a modification with amino-functional polyvinylamine. Second, the enzyme was immobilized by using 2 types of crosslinking agents. Due to the iron content of peroxidase, inductively coupled plasma-optical emission spectrometry was used for the quantitative determination of the enzyme load on the textile. The enzyme activity was evaluated using common 2,2'-azino-di-(3-ethylbenzthiazoline-6-sulfonic acid) assay for peroxidases. By the variation of enzyme input and crosslinker concentration, a maximal enzyme load of 80 mg/g of textile was achieved, and a maximum specific activity of 57 U/g of textile. For the visualization of the enzyme on the fiber surface, fluorescence microscopy as well as scanning probe microscopy were used. The immobilized peroxidase showed significant activity, even after 50 reuse cycles. In addition, the potential of the new support and enzyme combination in commercial whey bleaching was demonstrated successfully on a 10-L scale.

Depolymerization of lignosulfonates by submerged cultures of the basidiomycete Irpex consors and cloning of a putative versatile peroxidase.

Lignosulfonates are abundantly available byproducts of the paper and pulping industry, and they therefore represent a promising feedstock for new sustainable processes. For industrial applications of lignosulfonates, their molecular weight distribution is a critical factor. In order to decrease the average molecular weight of lignosulfonates, Seventeen basidiomycetes were screened for their capability to depolymerize lignosulfonates from spent sulfite liquor (SSL) in surface and liquid cultures. Five basidiomycetes polymerized the lignosulfonates under the selected conditions. Only Irpex consors was found to efficiently degrade calcium lignosulfonates when SSL (0.5%, w/w) was used as the sole carbon and nitrogen source. The average molecular weight of the lignosulfonates was reduced from ∼26 to ∼4 kDa as determined by size exclusion chromatography (SEC) within two weeks. Various extracellular enzyme activities of I. consors were determined over the culture period. High peroxidase activities were correlating with a high degradation rate and the culture was harvested at the day of highest peroxidase activity. A putative versatile peroxidase was isolated by fast protein liquid chromatography (FPLC) and its encoding cDNA was cloned.

Production of cyathane type secondary metabolites by submerged cultures of Hericium erinaceus and evaluation of their antibacterial activity by direct bioautography.

BACKGROUND: Fungi of the phylum Basidiomycota are well-known to form a broad spectrum of biologically active secondary metabolites, especially low molecular weight compounds such as terpenoids. Hericium erinaceus produces various cyathane type diterpenoids including erinacines. However, no quantitative data and production kinetics have been reported on the biosynthesis of the erinacines C and P in submerged cultures. In the present study, the production of erinacine C was optimized, and the product formation kinetics as well as the antimicrobial activity were studied by high-performance liquid chromatography (HPLC), high-performance thin-layer chromatography (HPTLC) and direct bioautography. RESULTS: Oatmeal and Edamin® K were identified to be crucial media components for an efficient production of erinacine C. The highest concentrations of erinacine C were obtained in the optimized culture medium on the 9th culture day (approximately 260 mg L-1). The production of erinacine P was strongly time dependent. The maximum concentration of erinacine P of 184 mg L-1 was observed on the third culture day. Afterwards, the concentrations of erinacine P decreased while the concentrations of erinacine C steadily increased. Comparable results were obtained by HPTLC with UV detection and HPLC with diode-array detection (DAD) analyses. Direct bioautography allowed for an additional analysis of the antimicrobial activity of the secondary metabolites. CONCLUSIONS: The C and N sources oatmeal and Edamin® K induced the formation of erinacine C. Detailed product formation kinetics of the erinacines C and P have been reported for the first time. HPTLC combined with the Aliivibrio fischeri bioassay allowed for an instant detection of cyathane diterpenoids in crude extracts and for an evaluation of the antimicrobial activity of the secondary metabolites directly on the plate.

Endogenous boldenone-formation in cattle: alternative invertebrate organisms to elucidate the enzymatic pathway and the potential role of edible fungi on cattle's feed.

Although beta-boldenone (bBol) used to be a marker of illegal steroid administration in calves, its endogenous formation has recently been demonstrated in these vertebrates. However, research on the pathway leading to bBol remains scarce. This study shows the usefulness of in vivo invertebrate models as alternatives to vertebrate animal experiments, using Neomysis integer and Lucilia sericata. In accordance with vertebrates, androstenedione (AED) was the main metabolite of beta-testosterone (bT) produced by these invertebrates, and bBol was also frequently detected. Moreover, in vitro experiments using feed-borne fungi and microsomes were useful to perform the pathway from bT to bBol. Even the conversion of phytosterols into steroids was shown in vitro. Both in vivo and in vitro, the conversion of bT into bBol could be demonstrated in this study. Metabolism of phytosterols by feed-borne fungi may be of particular importance to explain the endogenous bBol-formation by cattle. To the best of our knowledge, it is the first time the latter pathway is described in literature.

Degradation of alpha-pinene oxide and [2H7]-2,5,6-trimethyl-hept-(2E)-enoic acid by Pseudomonas fluorescens NCIMB 11761.

When submerged cultured Pseudomonas fluorescens NCIMB 11761 was fed-batch supplemented with alpha-pinene oxide, a rapid formation of 2,6-dimethyl-5-methylene-hept-(2Z)-enal (I) (isonovalal) was observed. Biotransformation and isomerisation of (I) to the (2E)-isomer (II) (novalal) were enhanced by Lewatit OC 1064, a macroporous polystyrene adsorbent. Accelerated isomerisation in the presence of an amino donor (glycine) at pH 7.3 pointed to a merely chemical mechanism. A maximum yield of 48 g of aldehydesl(-1) was achieved, but quantitative analysis of the volatile fraction showed that the molar conversion of the pinene oxide substrate reached no more than 67%. To fill this gap of the mass balance, the acidic fraction was isolated. It contained several compounds which suggested a beta-oxidation-like catabolism starting from 2,6-dimethyl-5-methylene-hept-(2E)-enoic acid (III) (novalic acid). Using [2H7]-2,5,6-dimethyl-hept-(2E)-enoic acid as a conversion substrate and gas chromatography coupled to atomic emission detection and mass spectrometry a degradation pathway via labelled 3,4-dimethylpentenoic and methylpropanoic acids was evidenced. This pathway may play a predominant role in isoprenoid degradation by soil bacteria.

Cleavage of beta,beta-carotene to flavor compounds by fungi.

More than 50 filamentous fungi and yeasts, known for de novo synthesis or biotransformation of mono-, sesqui-, tri-, or tetraterpenes, were screened for their ability to cleave beta,beta-carotene to flavor compounds. Ten strains discolored a beta,beta-carotene-containing growth agar, indicating efficient degradation of beta,beta-carotene. Dihydroactinidiolide was formed as the sole conversion product of beta,beta-carotene in submerged cultures of Ganoderma applanatum, Hypomyces odoratus, Kuehneromyces mutabilis, and Trametes suaveolens. When mycelium-free culture supernatants from five species were applied for the conversions, nearly complete degradation of beta,beta-carotene was observed after 12 h. Carotenoid-derived volatile products were detected in the media of Ischnoderma benzoinum, Marasmius scorodonius, and Trametes versicolor. beta-Ionone proved to be the main metabolite in each case, whereas beta-cyclocitral, dihydroactinidiolide, and 2-hydroxy-2,6,6-trimethylcyclohexanone were formed in minor quantities. Using a photometric bleaching test, the beta,beta-carotene cleaving enzyme activities of M. scorodonius were partially characterized.

A labeling study to elucidate the biosynthesis of 4-(4-hydroxyphenyl)-butan-2-one (raspberry ketone) by Nidula niveo-tomentosa.

Submerged cells of the basidiomycete Nidula niveo-tomentosa, a microbial producer of 4-(4-hydroxyphenyl)-butan-2-one, were supplemented with (13)C-labeled L-phenylalanines and with [1-(13)C]glucose. Labeled transformation products were detected by a novel method of analyzing stable isotope-labeled metabolites, gas chromatography (GC) coupled to an atomic emission detector, and by GC-mass spectrometry. A benzoate moiety was side chain elongated according to the poly-beta-keto scheme. The presence of an acetyl coenzyme A-carboxylase inhibitor shifted the spectrum of products to benzyl compounds. Hence, the fungal pathway differs from the one established for plant tissues.

Scanning infrared radiometer for measuring the air-sea temperature difference.

We describe a vertically scanning infrared radiometer for measuring the air-sea temperature difference without disturbing the water skin layer. The radiometer operates with a single wavelength channel that is 1.1 mum wide, centered on 14.2 mum, on the short-wavelength edge of a CO(2) atmospheric absorption band. The resulting high atmospheric absorption enables calibration of the horizontal-viewing signal with an in situ air-temperature sensor. The signal at all other scan angles is measured relative to that at the horizontal, providing a differential air-sea temperature measurement that is nearly independent of calibration offsets that can be a problem with independent air- and water-temperature sensors. We show data measured on a ship in the Tropical Western Pacific Ocean during July 1999, which exhibit important discrepancies from in situ data using bulk air-and water-temperature sensors. These discrepancies illustrate important differences between bulk versus skin water temperature.

Purification, characterisation and cDNA sequencing of pyruvate decarboxylase from Zygosaccharomyces bisporus.

Cells of the wild-type yeast strain Zygosaccharomyces bisporus CBS 702 form alpha-hydroxy ketones from aromatic amino acid precursors during fermentation. Pyruvate decarboxylase (PDC, E.C. 4.1.1.1), the key enzyme of this biotransformation catalysing the non-oxidative decarboxylation of pyruvate and other 2-oxo-acids, was purified and characterised. The active enzyme is homotetrameric (alpha4) with a molecular mass of about 244 kDa. Activation of PDC by its substrate pyruvate results in a sigmoidal dependence of the reaction rate from substrate concentration (apparent Km value 1.73 mM; Hill coefficient 2.10). A cDNA library was screened using a PCR-based procedure, and a 1856 bp cDNA of PDC was identified and sequenced. The cDNA encodes a polypeptide of 563 amino acid residues (monomeric unit). Sequence alignments demonstrate high homologies (> 80%) to PDC genes from Saccharomyces cerevisiae, Kluyveromyces lactis and Kluyveromyces marxianus.

Generation of odorous acyloins by yeast pyruvate decarboxylases and their occurrence in sherry and soy sauce.

Volatile acyloins (alpha-hydroxy ketones) were obtained by condensing either aldehydes with pyruvate or 2-keto acids with acetaldehyde in a reaction catalyzed by yeast pyruvate decarboxylases (EC 4.1.1.1). Odor qualities and threshold values of 34 acyloins were evaluated, and 23 of them possessed distinct flavor properties. Sherry and soy sauce flavors were analyzed: 2-hydroxy-3-pentanone and 3-hydroxy-2-pentanone were identified in soy sauce for the first time; these and 2-hydroxy-5-methyl-3-hexanone and 3-hydroxy-1-phenyl-2-butanone were isolated from sherry for the first time. The biocatalytic efficiencies of crude pyruvate decarboxylase preparations from Zygosaccharomyces bisporus, Saccharomyces cerevisiae, Kluyveromyces lactis, and Kluyveromyces marxianus were compared. Product yields comparable to those of conversions with purified pyruvate decarboxylase demonstrated the suitability of crude enzyme extracts as cost-effective biocatalysts in acyloin formation. Conversion rates of >50% showed that the potential of this type of enzyme to catalyze the formation of aliphatic acyloins has been underestimated before.

Influence of chin tap force and bite force parameters on the duration of the electromyographic silent period.

Controversy exists as to whether chin tap force and bite force affect the duration of the electromyographic silent period following the jaw jerk reflex during isometric contraction of the elevator muscles of the mandible. This study demonstrates that statistical trends exist showing increased silent period duration for increased tap forces and decreased silent period duration with increased bite forces.

Toxicokinetic and toxicodynamic studies of beryllium.

The question of dose-effect relations of berylliosis was examined by measuring the Be-concentration in blood and urine, as well as the immunological behaviour of T-lymphocytes for a group of occupationally exposed men (smaller than 8 ng Be/m3, 4-6 h daily) and a non-exposed control group. Normal values 1.0 +/- 0.4 ng Be/g(n=10) in blood and 0.9 +/- 0.5 ng Be/g(n=10) in urine were found by an optimized flameless atomic absorption spectrometry method. The corresponding values for the exposed group (n=8) showed a mean increase of a factor of 4. The preferential enrichment of Be in the prealbumin and in the nu-globulin fractions was determined by a preparative isotachophoretic column with physiological pH conditions, especially developed for this application. The Be-specific stimulation of the T-lymphocytes of the exposed group was increased significantly. In blood and urine samples of exposed (2-40 mg Be/m3 as Be(NO3)2) Wistar-rats and guinea pigs Be values up to 36 ng Be/g in serum and up to 300 ng Be/g in urine were determined. After Be exposure, guinea pig serum showed a significant increase in the nu-globulins after the isotachophoretical separation of the serum proteins, which could not be correlated with increased Be-levels in this fraction. 70% of the total Be(10-100 ng/g serum) were detected in the prealbumin, only about 1% in the nu-globulin fraction. The same results were found in serum samples with in vitro addition of Be. The lymphocyte transformation rates for the exposed animals were increased. Skin tests were found to be positive in 70% for exposed rats and in 50-70% for guinea pigs.