Urethanase of Bacillus licheniformis sp. isolated from mouse gastrointestine.

Trace levels of urethane, a cancer causing chemical, were detected in many kinds of wine, sherry, whisky, brandy and sake. Urethane formation from urea and ethanol in sake can be prevented by the treatment of acid urease, which is produced by Lactobacillus fermentum, but urethane, once formed, is very difficult to decompose. In order to keep the safety of alcoholic beverages, enzymatic removal of urethane has become an urgent problem. We found that Bacillus licheniformis sp., isolated from mouse gastrointestine, decomposed urethane to ethanol and ammonia. The enzyme showed higher urethanase activity at an acidic condition than at a neutral condition, and was resistant against ethyl alcohol of high concentrations. However, the enzyme had a low affinity to urethane for the industrial removal of the compound from alcoholic beverages.     

Urethanases for the Enzymatic Hydrolysis of Low Molecular Weight Carbamates and the Recycling of Polyurethanes

Enzymatic degradation and recycling can reduce the environmental impact of plastics. Despite decades of research, no enzymes for the efficient hydrolysis of polyurethanes have been reported. Whereas the hydrolysis of the ester bonds in polyester-polyurethanes by cutinases is known, the urethane bonds in polyether-polyurethanes have remained inaccessible to biocatalytic hydrolysis. Here we report the discovery of urethanases from a metagenome library constructed from soil that had been exposed to polyurethane waste for many years. We then demonstrate the use of a urethanase in a chemoenzymatic process for polyurethane foam recycling. The urethanase hydrolyses low molecular weight dicarbamates resulting from chemical glycolysis of polyether-polyurethane foam, making this strategy broadly applicable to diverse polyether-polyurethane wastes.     

Purification and Characterization of a Urethanase from Penicillium variabile

Urethanase produced by Penicillium variabile was purified through ultrasonication, concentration by polyethylene glycol 20,000, and Superdex G-200 gel filtration chromatography. The molecular weight of urethanase was determined to be around 96 kDa by gel filtration. The purified enzyme showed a single band in SDS-PAGE with the molecular weight of ~13.7 kDa, which suggests that the enzyme has a multimeric structure composed of the same subunits. Peptide map fingerprinting analysis was then carried out by MALDI/TOF-TOF MS. Within the known sequences in NCBI, glucosamine-6-phosphate deaminase and 6-phosphogluconate dehydrogenase get high score as compared with urethanase. Sequence analysis informs that N-terminal sequence of urethanase is GTNTADNDAA. The Minchaelis constant (K _m) and maximum reaction rate (V _m) of urethanase are 27.2 mmol/L and 156.25 μmol/L min, respectively.     

Simple method for the simultaneous quantification of medium-chain fatty acids and ethyl hexanoate in alcoholic beverages by gas chromatography-flame ionization detector: development of a direct injection method

Free medium-chain fatty acids (MCFAs) can negatively influence the fermentation process and taste quality in alcoholic beverages. Ethyl hexanoate is important in providing a fruit-like flavour to drinks, particularly in Japanese sake. In this study, we developed a direct injection method for a gas chromatography-flame ionization detector following the semi-purification of chemical components, such as esters, alcohols and MCFAs in alcoholic beverages. Evaluation of MCFAs by this method gave a limit of detection on the order of sub-ppm and relative standard deviations less than 10% in standard solution. Good repeatability and recovery rates against MCFAs and ethyl hexanoate were also obtained in non-distilled real alcoholic beverages. Because this method enabled us to simultaneously quantify the concentrations of MCFAs and ethyl hexanoate, the proportion of ester against MCFAs was proposed as a quality control index. This method could be suitable for routine analysis in the alcohol beverage industry.     

Chromatographic study of the chemical composition and potential toxicity of spirits and alcoholic beverages

The composition of vodkas, rectified food alcohols, cognacs, cognac spirits, and other strong alcoholic beverages was studied by chromatography and compared with the composition of industrial (synthetic and hydrolysis) ethyl alcohols, adulterated cognacs, and homemade spirits (samogon) from the illicit market. It was found that the majority of the test alcohol-containing liquids were close to commercial alcoholic beverages in terms of chemical composition and toxic properties. The samples containing ethylene glycol and enriched components of the head fraction of rectification should be considered most toxic.     

Isolation and Characterization of Urethanase from Penicillium variabile and Its Application to Reduce Ethyl Carbamate Contamination in Chinese Rice Wine

A strain with urethanase activity was isolated from mouse gastrointestine. By combination of morphological characterization of the colony, hyphae, and spore and the sequence analysis of its rDNA ITS, the strain was determined as Penicillium variabile and named as P. variabile JN-A525. The enzymatic properties of urethanase from P. variabile JN-A525 were further studied. The optimum temperature and pH value of urethanase are of 50 °C and 6.0, respectively. The enzyme maintains stability when the temperature is below 50 °C and the pH is in the range of 7.0–10.0. The enzyme also exhibits ethanol tolerance. It can remove ethyl carbamate from Chinese rice wine without the change of flavor substances in the wine.     

Immobilized Rhodotorula mucilaginosa: A Novel Urethanase-Producing Strain for Degrading Ethyl Carbamate

Rhodotorula mucilaginosa, producing the ethyl carbamate (EC)-degrading enzyme, urethanase, was newly isolated from the Chinese rice wine making process. It removed 80 % of EC when it was incubated with 5.0 g/L EC. It grew and stably produced urethanase, with pH ranging from 7.0 to 3.0. In addition, urethanase production by R. mucilaginosa was systematically optimized. Glucose, yeast extract, peptone, and inoculum size were selected with the Plackett–Burman design. They were further optimized via uniform design and determined to be 24.6 g/L, 2.5 g/L, 23.1 g/L, and 65.8 mL/500 mL, respectively. Urethanase activity reached 4,340.0 U/L in the optimal fermentation condition. Furthermore, cell immobilization of R. mucilaginosa in calcium alginate/chitosan was applied to improve cell resistance to environmental stresses. The immobilized cells removed 51.6 % of EC in commercial rice wine, which was 10 times more than that of the free cells. It indicated that the immobilized R. mucilaginosa was effective for degrading EC.     

Determination of biogenic amines in alcoholic beverages by ion chromatography with suppressed conductivity detection and integrated pulsed amperometric detection

The determination of biogenic amines in alcoholic beverages is important to assess the potential risks associated with the consumption of high concentrations of these compounds. In addition, product storage conditions and the length of storage can cause the formation of biogenic amines that reduce product quality. We report a new method using cation-exchange chromatography with either suppressed conductivity, integrated pulsed amperometry, UV, or a combination of these detection techniques to determine biogenic amines in alcoholic beverages. The main objective was to provide a direct comparison between IPAD and suppressed conductivity detection for determining biogenic amines in alcoholic beverages. Suppressed conductivity is the simplest detection approach for determining putrescine, cadaverine, histamine, agmatine, phenylethylamine, spermidine, and spermine with good sensitivity (0.004-0.08 mg/l) and was used to evaluate the influence of storage time and conditions on the evolution of biogenic amines in alcoholic beverages. Integrated pulsed amperometric detection (IPAD) detects more biogenic amines than suppressed conductivity detection, enabling the detection of dopamine, tyramine, and serotonin. Tyramine was simultaneously determined by UV detection and IPAD to provide confirmation and ensure the accuracy of the analytical results. The linearity of biogenic amine responses was within 0.1-20 mg/l and peak area precisions were 0.24-4.97% for IPAD, suppressed conductivity-IPAD, and UV detection. The sensitivity for the 10 biogenic amines using the 3 detection techniques varied considerably from 0.004-1.1 mg/l and recoveries were within 85-122%.     

Advanced solvent signal suppression for the acquisition of 1D and 2D NMR spectra of Scotch Whisky

A simple and robust solvent suppression technique that enables acquisition of high‐quality 1D ¹H nuclear magnetic resonance (NMR) spectra of alcoholic beverages on cryoprobe instruments was developed and applied to acquire NMR spectra of Scotch Whisky. The method uses 3 channels to suppress signals of water and ethanol, including those of ¹³C satellites of ethanol. It is executed in automation allowing high throughput investigations of alcoholic beverages. On the basis of the well‐established 1D nuclear Overhauser spectroscopy (NOESY) solvent suppression technique, this method suppresses the solvent at the beginning of the pulse sequence, producing pure phase signals minimally affected by the relaxation. The developed solvent suppression procedure was integrated into several homocorrelated and heterocorrelated 2D NMR experiments, including 2D correlation spectroscopy (COSY), 2D total correlation spectroscopy (TOCSY), 2D band‐selective TOCSY, 2D J‐resolved spectroscopy, 2D ¹H, ¹³C heteronuclear single‐quantum correlation spectroscopy (HSQC), 2D ¹H, ¹³C HSQC‐TOCSY, and 2D ¹H, ¹³C heteronuclear multiple‐bond correlation spectroscopy (HMBC). A 1D chemical‐shift‐selective TOCSY experiments was also modified. The wealth of information obtained by these experiments will assist in NMR structure elucidation of Scotch Whisky congeners and generally the composition of alcoholic beverages at the molecular level.     

Determination of ethanol in alcoholic beverages by high-performance liquid chromatography-flame ionization detection using pure water as mobile phase

A method for the determination of ethanol in alcoholic beverages by high-performance liquid chromatography-flame ionization detection (HPLC-FID) was developed. An FID system could be directly connected to an HPLC system using pure water as a mobile phase. In a durability test using triacontylsilyl (C30)-silica gel stationary phase for 96 h, no significant change in the retention time of four alcohol compounds was observed. So the HPLC separation of alcoholic beverages was carried out on the C30-silica gel stationary phase. On application to the analysis of six kinds of alcoholic beverages, ethanol could be determined accurately by the proposed method.     

Rapid Detection of Vanillin in Alcoholic Beverages Using Stabilized Polymerized Lipid Film Based Biosensors

This work reports a technique for the rapid detection of vanillin in alcoholic beverages using stabilized lipid membrane based biosensors. Microporous filters composed of glass fibers (nominal pore sizes 0.7 and 1.0 μm) were used as supports for the polymerization of the lipid film and stabilization of these devices. The lipid film is formed on the filter by polymerization prior its use. Methacrylic acid was the functional monomer, ethylene glycol dimethacrylate was the crosslinker and 2,2′‐azobis‐(2‐methylpropionitrile) was the initiator. The response towards vanillin of the present stabilized lipid membrane biosensors composed of phosphatidylcholine was investigated. The stabilized lipid membranes provided artificial ion gating events in the form of transient signals and can be used again after storage in air. This has allowed the practical use of the technique for chemical sensing based on lipid film for the rapid detection of vanillin in wines and alcoholic beverages.     

Investigation into the structural composition of hydroalcoholic solutions as basis for the development of multiple suppression pulse sequences for NMR measurement of alcoholic beverages

An eight‐fold suppression pulse sequence was recently developed to improve sensitivity in ¹H NMR measurements of alcoholic beverages [Magn. Res. Chem. 2011 (49): 734–739]. To ensure that only one combined hydroxyl peak from water and ethanol appears in the spectrum, adjustment to a certain range of ethanol concentrations was required. To explain this observation, the structure of water–ethanol solutions was studied. Hydroalcoholic solutions showed extreme behavior at 25% vol, 46% vol, and 83% vol ethanol according to ¹H NMR experiments. Near‐infrared spectroscopy confirmed the occurrence of four significant compounds (‘individual’ ethanol and water structures as well as two water–ethanol complexes of defined composition – 1 : 1 and 1 : 3). The successful multiple suppression can be achieved for every kind of alcoholic beverage with different alcoholic strengths, when the final ethanol concentration is adjusted to a range between 25% vol and 46% vol (e.g. using dilution or pure ethanol addition). In this optimum region, an individual ethanol peak was not detected, because the ‘individual’ water structure and the 1 : 1 ethanol–water complex predominate. The nature of molecular association in ethanol–water solutions is essential to elucidate NMR method development for measurement of alcoholic beverages. The presented approach can be used to optimize other NMR suppression protocols for binary water–organic solvent mixtures, where hydrogen bonding plays a dominant role. Copyright © 2014 John Wiley & Sons, Ltd.     

Optically specific detection of D- and L-lactic acids by a flow-injection dual biosensor system with on-line microdialysis sampling.

A flow-injection dual biosensor system with microdialysis sampling is proposed for the simultaneous determination of D-lactic and L-lactic acids. The dialysate from the microdialysis tube is delivered to a sample loop of the six-way autoinjector and then automatically injected into the flow-injection line with a dual enzyme electrode arranged in perpendicular to the flow direction. The dual enzyme electrode is constructed by hybridizing a poly(1,2-diaminobenzene) film into two sensing parts which respond selectively to D-lactic and L-lactic acids, respectively, without any cross-reactivity. The proposed flow-injection analysis method can be successfully applied to the simultaneous determination of D,L-lactic acids in alcoholic beverages.     

Development of enzyme technology and enzyme engineering in China

Enzymes have been utilized in China for thousands of years for the production of various foods and alcoholic beverages. Today China manufactures and uses enzymes for not only the traditional areas of application, but is expanding the use of enzymes for a variety of nonfood areas. This report describes the present state of the art of enzyme manufacture and application in China today.     

HPLC determination of 2-furaldehyde and 5-hydroxymethyl-2-furaldehyde in alcoholic beverages

A method for the determination of 2-furaldehyde (F) and 5-hydroxymethyl-2-furaldehyde (HMF) in alcoholic beverages by high performance liquid chromatography (HPLC) is described. A C-18 column and an acetonitrile-water mobile phase with isocratic elution were used. UV detection at 280 nm was carried out. The method was validated according to the EURACHEM guidelines. The procedure offers a high specificity and detection limits of the order of 0.005 μg mL^− 1. Recoveries of 94-103% were obtained from spiked samples at different levels with both analytes. Intermediate precision calculated for a period of 2 months was + 0.8% for HMF and + 0.4% for F. The method was applied to the analysis of alcoholic beverages like bourbon, rum, brandy, liquors and aperitif wines.     

Solid-phase microextraction for the enantiomeric analysis of flavors in beverages

Solid-phase microextraction combined with gas chromatographic/mass spectrometric analysis and separation on a chiral cyclodextrin stationary phase was a rapid, reliable technique for profiling chiral aroma compounds in flavored alcoholic beverages. Several enantiomeric terpenes, esters, alcohols, norisoprenoids, and lactones were identified in berry-, peach-, strawberry-, and citrus-flavored wine and malt beverages (wine coolers). Using this technique, we were able to confirm the addition of synthetic flavoring to several beverages, consistent with label designations.     

Dual electrode electrochemical detector for HPLC

Summary A method for determination of phenolic compounds in distilled alcoholic beverages has been developed. After separation by reversed phase chromatography these compounds are detected coulometrically in a dual electrode detector. The hydrodynamic electrochemical behaviour of the substances in oxidative and reductive mode was investigated. For quantitative determination phenolic compounds are oxidized at the first working electrode (+0.65 V); then the oxidation products are reduced at the second working electrode (0.0 V). The current due to these processes is recorded. By the high selectivity of the detection mode matrix interferences can be eliminated in several alcoholic beverages. In this way qualitative information is improved. The detection limits of phenolic acids and aldehydes are between 0.01 and 1 ng (S/N=3).

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Dualelektroden-Detektor für die HPLCBestimmung von phenolischen Verbindungen in Spirituosen

     

Breeding an Amylolytic Yeast Strain for Alcoholic Beverage Production

A starch-utilizing, yeast-like fusant was successfully created from fused protoplasts of Schizosaccharomyces pombe and Monascus anka, and the feasibility of using this fusant as a new strain for alcoholic beverage development was reported. The new fusant utilized various carbon sources more efficiently than its parent cells did. Rice koji prepared separately by cultivating the fusant and its parental strains on rice was compared to explore the effect of yeast strain on the production of α-amylase, glucoamylase, and acid protease that are crucial in wine making using cereal grains. It was found that the fusant produced greater levels of the above-mentioned enzymes than its parental strain does. Consequently, the usage of this fusant in the alcoholic fermentation of polished rice was found to reduce approximately 50% consumption of added glucoamylase than when its parental strain was used. Besides, at the end of fermentation, the fusant yeast resulted in a mash with distribution of flavor components very different from that produced by its parental strains. Thus, the fusant can be used as a new yeast strain for creating novel alcoholic beverages.     

Surface-enhanced Raman scattering for quantitative detection of ethyl carbamate in alcoholic beverages

Ethyl carbamate, a by-product of fermentation and storage with widespread occurrence in fermented food and alcoholic beverages, is a compound potentially toxic to humans. In this work, a new approach for quantitative detection of ethyl carbamate in alcoholic beverages, based on surface-enhanced Raman scattering (SERS), is reported. Individual silver-coated gold nanoparticle colloids are used as SERS amplifiers, yielding high Raman enhancement of ethyl carbamate in three kinds of alcoholic beverages (vodka, Obstler, and white rum). The characteristic band at 1,003 cm^-1, which is the strongest and best reproducible peak in the SERS spectra, was used for quantitative evaluation of ethyl carbamate. The limit of detection, which corresponds to a signal-to-noise ratio of 3, was 9.0?×?10^-9 M (0.8 μg?·?L^-1), 1.3?×?10^-7 M (11.6 μg?·?L^-1), and 7.8?×?10^-8 M (6.9 μg?·?L^-1), respectively. Surface-enhanced Raman spectroscopy offers great practical potential for the in situ assessment and identification of ethyl carbamate in the alcoholic beverage industry.     

A novel strategy to verification of adulteration in alcoholic beverages based on Schlieren effect measurements and chemometric techniques

A novel strategy to evaluation of adulteration in alcoholic beverages based on the measurement of the Schlieren effect using an automated FIA system with photometric detection is proposed. The assay is based on the Schlieren effect produced when beverage samples are injected in a single-line FIA system that uses water as carrier stream and a light-emitting diode-phototransistor photometer controlled by microcomputer as detector. The flow system presents limited mixing conditions which make possible to create gradients of refractive index (Schlieren effect) in the injected sample zone. These gradients are reproducible, characteristic of each alcoholic beverage and undergo specific modifications when adulterations with water or ethanol are imposed. Schlieren effect data of brandies, cachaças, rums, whiskies and vodkas were treated by SIMCA to elaborate class models applied in the evaluation of alcoholic beverages adulteration. Samples of the original matrix of each sort of beverages were adulterated in laboratory by adding water, methanol and ethanol in levels of 5% and 10% (v/v). These samples were used as test set to validate SIMCA class models. The verification of authenticity using Schlieren effect measurements presented good results making possible to identify 100% of the beverages samples adulterated in laboratory and 93% of the actual adulterated alcoholic beverages with confidence levels of 95%. As principal advantage, the automated system does not use reagents to carry out the analysis.