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Biomedical subjects

Ralf G Berger

Publications and source records attributed to Ralf G Berger.

At least 19 recordsLinked to original sources
PubMed2006

Odor-active alcohols from the fungal transformation of alpha-farnesene.

Submerged microbial cultures were screened for their potential to oxifunctionalize alpha-farnesene. The major oxidation product in all transforming cultures, 3,7,11-trimethyldodeca-1,3(E),5(E)10-tetraen-7-ol, showed a pleasant citrus-like odor and peak concentrations of 170 mg L-1. An Aspergillus niger isolate from mango generated another two terpene alcohols identified as diastereomeric menth-1-en-3-[2-methyl-1,3-butadienyl]-8-ol, a new natural compound with an apricot-like odor. The regiospecifity of the oxygen attack with concurrent lack of stereoselectivity suggested that the initial step of the bioconversion resembled the chemical autoxidation starting with the generation of an intermediate resonance-stabilized carbon-centered radical or carbocation.

Alcohols↗
PubMed2005

Laccases of Pleurotus sapidus: characterization and cloning.

Peanut shells, a major waste stream of food processing, served as a renewable substrate for inducing the production of laccases by basidiomycetes. Of 46 surface cultures examined, 29 showed laccase activity under the experimental conditions. The edible fungus Pleurotus sapidus was selected as the most active producer, immobilized on the shells, and cultivated in the fed-batch mode. A continuous rise in laccase activity was found, indicating the inducibility of laccase secretion by the peanut shells and the reusability of the mycelium. Two laccase isoenzymes were purified by decoupled 2-D electrophoresis, and amino acid sequence information was obtained by electrospray ionization tandem mass spectrometry. cDNAs of the corresponding gene and another laccase were cloned and sequenced using a PCR-based screening of a synthesized P. sapidus cDNA library. Data bank searches against public databases returned laccases of P. ostreatus and P. sajor-caju as the best hits. The potential use of laccases by the food industry is discussed.

Amino Acid Sequence↗
PubMed2005

Tweezing-adsorptive bubble separation. Analytical method for the selective and high enrichment of metalloenzymes.

A novelly developed tweezing-adsorptive bubble separation (ABS) method for the enrichment of metalloenzymes (laccase C and horseradish peroxidase) is introduced. The method is based on the chelation of the enzymes' active center and can also be applied for analysis. N-(2-acetamido)iminodiacetic acid served as a chelator and was synthesized with an octyl unit to become ADA-C8. Laccase was enriched 13.3-fold (66.31% recovery) and HPOX 17.8-fold (85.34%) without a significant loss of enzymatic activity. To prove that the entire enzyme is tweezed at the active center, ABS trials were done using ADA-C8 already complexed with Cu2+ and Fe3+. As only marginal enrichment occurred (ER laccase, 0.17; ER HPOX, 0.44), no chelating effect was concluded. It was determined how the chelation toward the active center was directed by applying other chelators such as EDTA, NTA, N,N-dimethylaminoglycine, oxalic acid, malonic acid, adipinic acid, and tripropylamine, which are similar in structure to ADA-C8. The results concluded that the chelation is 3-fold coordinated on the type 1 copper center of laccase, whereas that of HPOX only 1-fold at Fe3+ and additionally at the cationic amino acid arginine, which is also located at the active center. Tweezing-ABS has been proven to selectively and effectively enrich metalloenzymes.

Absorption↗
PubMed2005

Characteristic volatiles from young and aged fruiting bodies of wild Polyporus sulfureus (Bull.:Fr.) Fr.

The volatile compounds of fresh fruiting bodies of wild Polyporus sulfureus (Bull.:Fr.) Fr. growing on oak trees were isolated by continuous liquid-liquid extraction (CLLE) and investigated by high-resolution gas chromatography-mass spectrometry (HRGC-MS) on two GC columns of different polarity (DB-5 and ZB-WAX), and by gas chromatography-olfactometry (GC-O). A total of 40 major volatile compounds from the young samples were identified and semiquantified. Five odorous compounds were determined to be responsible for the characteristic flavor of young Polyporus sulfureus: 1-octen-3-one, 1-octen-3-ol, 3-methylbutanoic acid, phenylethanol, and phenylacetic acid. Four volatiles investigated by GC-O and detected by GC-MS were determined as the characteristic odorants of aged species: 2-methylpropanoic acid, butanoic acid, 3-methylbutanoic acid, and phenylacetic acid. The comparative results revealed that the volatile composition of the fruiting bodies even from the same fungal species may greatly vary with its host, location, and age.

Chromatography, Gas↗
PubMed2005

The secretome of Pleurotus sapidus.

Due to their unique capability to attack lignified biopolymers, extracellular enzymes of white-rot fungi enjoy an increasing interest in various fields of white biotechnology. The edible fungus Pleurotus sapidus was selected as a model organism for the analysis of the secretome by means of 2-DE. For enzyme production, the fungus was grown in submerged cultures either on peanut shells or on glass wool as a carrier material. Identification of the secreted enzymes was performed by tryptic digestion, ESI-MS/MS ab initio sequencing, and homology searches against public databases. The spectrum of secreted enzymes comprised various types of hydrolases and lignolytic enzymes of the manganese peroxidase/versatile peroxidase family. While peptidases were secreted mainly by the cultures grown on peanut shells, versatile peroxidase type enzymes dominated in the cultures grown on glass wool.

Amino Acid Sequence↗
PubMed2005

Foam fractionation of exo-lipases from a growing fungus (Pleurotus sapidus).

Active lipases were isolated from the culture supernatant of the basidiomycetous fungus Pleurotus sapidus by foam fractionation. The pH value, the gas flow, and the foaming period were systematically varied to optimize the transport of the enzymes into the foam phase. On a 70-mL scale, a maximum recovery of hydrolytic activity of 95% was obtained at pH 7.0 and 60 mL N2 min(-1) after 50 min. The procedure, with minor modifications, was also applicable to native pellet cultures of P. sapidus. The same recovery of 95% was achieved, with purification and enrichment factors of 11.6 and 28.0, respectively.

Extracellular Space↗
PubMed2005

An extracellular carboxylesterase from the basidiomycete Pleurotus sapidus hydrolyses xanthophyll esters.

An extracellular enzyme capable of efficient hydrolysis of xanthophyll esters was purified from culture supernatants of the basidiomycete Pleurotus sapidus. Under native conditions, the enzyme exhibited a molecular mass of 430 kDa, and SDS-PAGE data suggested a composition of eight identical subunits. Biochemical characterisation of the purified protein showed an isoelectric point of 4.5, and ideal hydrolysis conditions were observed at pH 5.8 and 40 degrees C. Partial amino acid sequences were derived from N-terminal Edman degradation and from mass spectrometric ab initio sequencing of internal peptides. An 1861-bp cDNA containing an open reading frame of 1641 bp was cloned from a cDNA library that showed ca. 40% homology to Candida rugosa lipases. The P. sapidus carboxylesterase represents the first enzyme of the lipase/esterase family from a basidiomycetous fungus that has been characterised at the molecular level.

Amino Acid Sequence↗
PubMed2004

Bioconversion of (+)-valencene in submerged cultures of the ascomycete Chaetomium globosum.

Submerged cultures of the ascomycete Chaetomium globosum oxidised the exogenous sesquiterpene (+)-valencene to nootkatone via the stereoselective generation of alpha-nootkatol. Inhibition experiments suggested that the first introduction of oxygen, the rate-limiting step of the bioconversion, may have been catalysed by a cytochrome-P450-monooxygenase. However, nootkatone was not the final metabolite: further flavour-active and inactive, non-volatile oxidation products were identified. (+)-Valencene and the flavour-active mono-oxyfunctionalised transformation products, alpha-nootkatol, nootkatone, and valencene-11,12-epoxide accumulated preferably inside the fungal cells. Di- and poly-oxygenated products, such as nootkatone-11,12-epoxide, were found solely in the culture medium, indicating an active transport of these metabolites into the extracellular compartment during (+)-valencene detoxification. These metabolic properties may have contributed to the high tolerance of the fungus towards the exogenous hydrocarbon.

Biotechnology↗
PubMed2004

In vitro study of the influence of physiological parameters on dynamic in-mouth flavour release from liquids.

Influences of shear rate (surface extension), airflow, in-mouth headspace volume, synthetic saliva and human epithelial cells (modelling mucosa) on the initial dynamic flavour release from liquids were analysed. Simulating physiological mouth parameters, initial dynamic flavour release experiments over a time period of 30 s were carried out using a proven mouth model apparatus. Flavour compounds of different chemical classes were dissolved in water or in aqueous starch hydrolysate in concentrations typically present in food ( micro g/l to mg/l). Forced by increasing shear rates the enlargement of the gas-liquid interface (vortex formation) caused an increased release of flavour molecules. The release of less soluble compounds was reduced by increasing shear forces due to an improved dissolution. Increasing volumetric airflow rates resulted generally in higher release rates and in a change of pattern of release kinetics. Maximum flavour release was found at a ratio of 1:1 for in-mouth headspace and liquid volume. Neither addition of saliva alone nor the combination of saliva and mucosa showed significant influence on in-mouth flavour release from liquids in the model mouth.

Chromatography, Gas↗
PubMed2004

Volatile release from liquids: a comparison of in vivo APCI-MS, in-mouth headspace trapping and in vitro mouth model data.

In-mouth volatile release from flavoured water was followed using atmospheric pressure chemical ionization-mass spectrometry (APCI-MS) or using a hand-held, computer-controlled device based on sequential trapping of flavours on Tenax traps. The present results verify recent in vitro data obtained with a sophisticated, fully computerized mouth model apparatus and confirm its validity for the simulation of in-mouth dynamic volatile release. In-nose APCI-MS measurements showed considerable person-to-person variability in non-trained individuals during drinking due to subconscious control of muscles during swallowing and subsequent breathing. Data showed a 'swallow breath' volume reaching the nasal cavity from the throat, not from the mouth cavity. Flavour enriched air from the mouth was shown to be transported to the nose (via exhalation) immediately after the swallowing event, but the dynamic process of volatile equilibration between residuals of the swallowed liquid and the exhaled air predominantly determined volatile in-nose concentration. Owing to its dynamic character, the process of volatile equilibration and release in the throat upon exhalation should be similar to the in-mouth process studied in the present work. A full mechanical simulation of retronasal volatile transport, however, will remain difficult.

Beverages↗
PubMed2003

Dynamic flavor release from sucrose solutions.

The initial dynamic flavor release from sucrose solutions was modeled. Modeling was based on the theoretical hydration behavior of sucrose, theoretical physicochemical data of flavor volatiles, and process parameters of a headspace apparatus used for model validation. The rate-limiting factor determining the initial flavor release was the hydration of sucrose, which in turn depends on the molarity of sucrose in the solution and, therefore, on the actual amount of nonbound water. Improved solubility of the more hydrophilic compounds due to their orientation toward the hydration shells of the sugar molecules was considered. The viscosity of nonassociated water forming the microregion for mass transfer of volatiles was considered instead of the bulk solution viscosity. Experimental validation of the model by real-time measurements of dynamic flavor release using foodlike flavor concentrations confirmed the above theory. Increasing sucrose concentrations resulted predominantly in increased flavor release, and bulk solution viscosity showed no effect.

Chemical Phenomena↗
PubMed2003

Lipid molarity affects liquid/liquid aroma partitioning and its dynamic release from oil/water emulsions.

Initial dynamic flavor release from oil/water emulsions containing different TAG phases was studied using a computerized apparatus and thermodesorption GC. A significant influence of lipid molarity on liquid/liquid partitioning and release of some flavor compounds was found. The release of the least hydrophobic compounds was not affected by any type of lipid. Hydrophobic compounds showed a positive correlation between their release and decreasing molarity of the lipid phase, that is, with increasing number of lipid molecules; only the most hydrophobic compounds did not show such a correlation. A strong linear correlation between low-melting TAG/water partition coefficients and lipid phase molarity was validated by volatile partition data of C6, C11, and C16 alkane/water systems. Lipid phase transition from the liquid to solid state did not affect flavor partitioning and release. Neither experimental nor theoretical octanol/water partition coefficients agreed with experimental TAG/water and alkane/water partition coefficients.

Emulsions↗
PubMed2003

A peroxidase from Lepista irina cleaves beta,beta-carotene to flavor compounds.

Extracellular liquid of the edible fungus Lepista irina was found to effectively degrade beta,beta-carotene, beta-lonone, beta-cyclocitral, dihydroactinidiolide, and 2-hydroxy-2,6,6-trimethylcyclohexanone were formed as volatile breakdown products of beta,beta-carotene with mycelium-free culture supernatants, whereas beta-apo-10'-carotenal was identified as non-volatile degradation product. The key enzyme catalyzing the oxidative cleavage of beta,beta-carotene was purified with an overall yield of 63% and a purification factor of 43. Biochemical characterization showed a molecular mass of 50.5 kDa and an isoelectric point of 3.75. Fastest beta,beta-carotene degradation occurred at 34 degrees C and pH values between 3.5 and 4. Degenerate oligonucleotides were derived from N-terminal and internal amino acid sequences. By means of PCR-based cDNA-library screening a 1284 bp cDNA was identified which showed great overall similarity to Pleurotus eryngii polyvalent peroxidases. The obtained sequence contains an open reading frame of 1083 nucleotides, encoding a polypeptide of 361 amino acids. A 30 amino acid signal peptide was identified upstream of the N-terminal sequence of the mature enzyme. The L. irina versatile peroxidase represents the first microbial enzyme capable of carotenoid degradation that has been characterized on a molecular level, proving the participation of extracellular enzymes of white rot fungi in biotic carotenoid degradation processes.

Amino Acid Sequence↗
PubMed2002

Computerized apparatus for measuring dynamic flavor release from liquid food matrices.

A fully computer-controlled apparatus was designed. It combines a glass reactor with a temperature-controlled hood, in which headspace volatiles are captured. Flavored liquids can be introduced into the reactor and exposed to conditions of temperature, air flow, shear rate, and saliva flow as they occur in the mouth. As the reactor is completely filled before measurements are started, creation of headspace just before sampling start prevents untimely flavor release resulting in real time data. In the first 30 s of flavor release the concentrations of the volatiles can be measured up to four times by on-line sampling of the dynamic headspace, followed by off-line trapping of the samples on corresponding Tenax traps and analysis using GC-TDS-FID. Flavor compounds from different chemical classes were dissolved in water to achieve concentrations typically present in food (micrograms to milligrams per liter). Most of the compounds showed constant release rates, and the summed quantities of each volatile of three 10 s time intervals correlated linearly with time. The entire method of measurement including sample preparation, release, sampling, trapping, thermodesorption, and GC analysis showed good sensitivity [nanograms (10 s)(-1)] and reproducibility (mean coefficient of variation = 7.2%).

Chromatography, Gas↗
PubMed2002

Modeling dynamic flavor release from water.

A mathematical model derived from the convective mass transfer theory was developed to predict dynamic flavor release from water. A specific mass transfer correlation including a new term for volatile permeability was applied. The model was entirely based on physicochemical constants of flavor compounds and on some parameters of an apparatus used for validation. The model predicted a linear pattern of release kinetics during the first 30 s and large differences of absolute release for individual compounds. Both calculated and experimentally determined release profiles of a test mixture of flavors showed good agreement.

Kinetics↗
PubMed2002

Production of coconut aroma by fungi cultivation in solid-state fermentation.

The production of 6-pentyl-alpha-pyrone (6-PP), an unsaturated D-lactone with a strong coconut-like aroma was studied and compared with liquid and solid substrates. A fungi strain that produces coconut aroma compound was selected. The liquid medium of the submerged culture was used to impregnate a solid support of sugarcane bagasse in SSF (Solid State Fermentation). This substrate was adequate for growth and aroma production; the concentration obtained using SSF was higher than using liquid fermentation process. In the present work, it is demonstrated that, by solid-state-fermentation process, it is possible to produce 6-PP. The amount of 6-PP produced using a solid state substrate, following a 5 d culture, was 3 mg/g dry matter. Therefore, the amount of 6-PP produced during solid-state-fermentation process is higher than that reported in literature for submerged process.

Cocos↗
PubMedDate not supplied

Regio- and stereoselective fungal oxyfunctionalisation of limonenes.

Selective transformations of limonene by asco- and basidiomycetes were investigated. On the shake flask scale, Penicillium citrinum hydrated R-(+)-limonene to a-terpineol [83% regioselectivity (rs), more than 80 mg l(-1) product yield], and Gongronella butleri catalysed the terminal oxidation to yield perillyl alcohol (60% rs, 16 mg l(-1)). On the laboratory bioreactor scale, Penicillium digitatum produced a peak concentration of 506 mg a-terpineol l(-1) in the fed-batch mode, equivalent to a theoretical yield of 67%, and no volatile by-products were found. Fusarium proliferatum transformed R-(+)-limonene enantiospecifically to cis-(+)-carveol (98.6% ee, more than 35 mg l(-1) product yield) and S-(-)-limonene predominantly to trans-(-)-carveol (96.3% ee). Pleurotus sapidus selectively dehydrogenised the accumulating trans-(-)-carveol to the corresponding enantiopure R-(-)-carvone. The results show that a careful selection of strain and bioprocess parameters may improve both the yield and the optical purity of a desired product.

Ascomycota↗