Glycerol In Wine Research Articles

Development of a Disposable, Amperometric Glycerol Biosensor Based on a Screen-Printed Carbon Electrode, Modified with the Electrocatalyst Meldolas Blue, Coated with Glycerol Dehydrogenase and NAD+: Application to the Analysis of Wine Quality

This paper describes the design and development of a novel electrochemical biosensor for measuring glycerol in wine. Our initial detailed studies were aimed at deducing the optimum conditions for biosensor operation by conducting hydrodynamic voltammetric and amperometric studies. The resulting voltammograms revealed a maximum electrocatalytic current at 0.0 V vs. Ag/AgCl, which we used for all further studies. We also examined the effect of pH (8–10) on the amperometric responses of different glycerol concentrations over a range of 0.04 to 0.20 mM. Based on our findings, we propose that pH 9 would be suitable as the supporting electrolyte for further studies with the amperometric biosensor. The biosensor was constructed by immobilising 10 units of GLDH and 660 μg NAD+ onto the MB-SPCE surface using glutaraldehyde (GLA) as a cross-linking agent. Calibration studies were performed with glycerol over the 1.0–7.5 mM concentration range. Chronoamperometry was the electrochemical technique chosen for this purpose as it is convenient and can be performed with only 100 μL of sample directly deposited onto the biosensor’s surface. In the current study, we observed linear calibration plots with the above standard solutions using current measurements at a selection of sampling times along the chronoamperograms (30–340 s). We have evaluated the glycerol biosensor by carrying out an analysis of commercially available red wine. Overall, these findings will form a platform for the development of novel rapid technology for point-of-test evaluation of glycerol in the production and quality control of wine.

Development of a portable optical device with a multi-channel spectrometer sensor for quantification of glycerol in wine: a maker approach for on-site analysis.

In this work we present a novel and environmentally friendly approach for quantifying glycerol in wine samples using a portable optical device based on the maker concept and do-it-yourself (DIY) principles. This method offers significant advantages, including cost-effectiveness, reduced sample and reagent consumption, and the potential for integrating IoT (Internet of Things) technology. The chemical strategy involves the oxidation of glycerol using periodate, followed by the formation of the 3,5-diacetyl-1,4-dihydrolutidine (DDL) compound through a reaction with acetylacetone. The utilization of a cost-effective AS7341 color sensor as a detector enables accurate and sensitive detection of glycerol levels in wine samples. The optimized procedure demonstrates adequate analytical performance for glycerol determination in wine samples, encompassing a wide linear range (0.5 mg L-1 to 40.0 mg L-1), high correlation coefficient (r = 0.998), and low limits of detection (0.050 mg L-1). The method exhibits excellent precision, with the coefficient of variation estimated to be 0.1% for 10 independent measurements of a 20 mg L-1 solution. These features render it suitable not only for routine glycerol analysis in the wine industry, but also for addressing challenges related to wine adulteration and counterfeiting.

Impact of Yeast Breeding for Elevated Glycerol Production on Fermentative Activity and Metabolite Formation in Chardonnay Wine

Glycerol in wine originates mainly as a by-product during fermentation by yeast and is thought to add to the body and smooth mouth-feel. We evaluated the properties of Chardonnay wine produced using various wine yeast strains of Saccharomyces cerevisiae and hybrid strains that were bred to produce elevated glycerol concentrations in laboratory trial experiments. The wine yeast strains (commercial strains or strains from culture collections) produced a mean glycerol and ethanol concentration of 4.38 and 101.2 g/L (12.8% v/v; n=26) respectively, whereas the glycerol and ethanol concentrations in wine made using the hybrid strains was 7.18 g/L and 96.0 g/L (12.2% v/v; n=15). Considerable variability in the glycerol-producing ability of the wine yeast and hybrid strains was apparent. Coupled to the higher glycerol levels formed by the hybrid strains, acetic acid, volatile acidity, acetoin, acetaldehyde and 2,3-butanediol levels were higher than the levels produced by the wine yeast strains. The levels of some of these metabolites were strongly linked to elevated glycerol production. The hybrid strains fermented the Chardonnay grape juice more slowly than the wine yeast strains, but in most instances dryness was achieved. The concentrations of miscellaneous metabolites (alcohols, acids and esters) were in most instances similar in the wine made with the wine yeast strains and hybrid strains, indicating that the breeding of yeast to produce higher glycerol levels has a minor influence on the production of these compounds. In a wine production experiment one hybrid yeast strain producing elevated glycerol levels yielded a Chardonnay wine with a better or equivalent body than wine made with commercial wine yeast strains, although the aroma and general quality were worse. These results suggest that further breeding and selection might yield yeast strains for fermentation that improves the body of wine without impacting on the overall balance of wine.

Integrated multienzyme electrochemical biosensors for the determination of glycerol in wines

The construction and performance of integrated amperometric biosensors for the determination of glycerol are reported. Two different biosensor configurations have been evaluated: one based on the glycerol dehydrogenase/diaphorase (GDH/DP) bienzyme system, and another using glycerol kinase/glycerol-3-phosphate oxidase/peroxidase (GK/GPOx/HRP). Both enzyme systems were immobilized together with the mediator tetrathiafulvalene (TTF) on a 3-mercaptopropionic acid (MPA) self-assembled monolayer (SAM)-modified gold electrode by using a dialysis membrane. The electrochemical oxidation of TTF at +150 mV (vs. Ag/AgCl), and the reduction of TTF + at 0 mV were used for the monitoring of the enzyme reactions for the bienzyme and trienzyme configurations, respectively. Experimental variables concerning both the biosensors composition and the working conditions were optimized for each configuration. A good repeatability of the measurements with no need of cleaning or pretreatment of the biosensors was obtained in both cases. After 51 days of use, the GDH/DP biosensor still exhibited 87% of the original sensitivity, while the GK/GPOx/HRP biosensor yielded a 46% of the original response after 8 days. Calibration graphs for glycerol with linear ranges of 1.0 × 10 −6 to 2.0 × 10 −5 or 1.0 × 10 −6 to 1.0 × 10 −5 M glycerol and sensitivities of 1214 ± 21 or 1460 ± 34 μA M −1 were obtained with GDH/DP and GK/GPOx/HRP biosensors, respectively. The calculated detection limits were 4.0 × 10 −7 and 3.1 × 10 −7 M, respectively. The biosensors exhibited a great sensitivity with no significant interferences in the analysis of wines. The biosensors were applied to the determination of glycerol in 12 different wines and the results advantageously compared with those provided by a commercial enzyme kit.

Determination of Glycerol in Wines Using 31P‐NMR Spectroscopy

Abstract31P‐NMR spectroscopy was employed to detect and quantify glycerol in red wines from various regions of Greece. This novel analytical method was based on the derivatization of the hydroxyl groups of glycerol with 2‐chloro‐4,4,5,5‐tetramethyl dioxaphospholane, and identification of the phosphitylated compound on the basis of 31P chemical shifts. Quantification of glycerol in wines was accomplished by integration of appropriate signals in the 31P‐NMR spectrum and the use of the phosphitylated cyclohexanol as the internal standard. The method was reproducible (CV (%) = 2.35) and accurate (CV (%) = 1.34). Its applicability to glycerol quantification in wines was tested against a weighted amount of a glycerol‐model compound by linear regression analysis (R = 0.999; intercept = 0.074 ± 0.078; slope = 0.998 ± 0.003; p = 0.000). Furthermore, the NMR method was compared to the AOAC official method (HPLC) using the Bland and Altman statistical analysis. The distribution of the data points in the bias plot showed that 100% of the measurements of glycerol in 16 wine samples from various regions of Greece were within the limits of agreement of the two methods.

Kinetic Enzymatic Determination of Glycerol in Wine and Beer Using a Sequential Injection System with Spectrophotometric Detection

A sequential injection system for the automatic determination of glycerol in wine and beer was developed. The method is based on the rate of formation of NADH from the reaction of glycerol and NAD+ catalyzed by the enzyme glycerol dehydrogenase in solution. The determination of glycerol was performed between 0.3 and 3.0 mmol L(-1) (0.028 and 0.276 g L(-1)), and good repeatability was attained (rsd < 3.6%, n = 5) for all samples tested. The determination rate was 54 h(-1), the reagent consumption was only 0.75 micromol of NAD+ and 5.4 ng of enzyme per assay, and the waste production was 2.12 mL per assay. Results obtained for samples were in agreement with those obtained with the batch enzymatic method.

Identification and Quantification of Industrial Grade Glycerol Adulteration in Red Wine with Fourier Transform Infrared Spectroscopy Using Chemometrics and Artificial Neural Networks

Fourier transform infrared (FT-IR) single bounce micro-attenuated total reflectance (mATR) spectroscopy, combined with multivariate and artificial neural network (ANN) data analysis, was used to determine the adulteration of industrial grade glycerol in selected red wines. Red wine samples were artificially adulterated with industrial grade glycerol over the concentration range from 0.1 to 15% and calibration models were developed and validated. Single bounce infrared spectra of glycerol adulterated wine samples were recorded in the fingerprint mid-infrared region, 900-1500 cm(-1). Partial least squares (PLS) and PLS first derivatives were used for quantitative analysis (r2 = 0.945 to 0.998), while linear discriminant analysis (LDA) and canonical variate analysis (CVA) were used for classification and discrimination. The standard error of prediction (SEP) in the validation set was between 1.44 and 2.25%. Classification of glycerol adulterants in the different brands of red wine using CVA resulted in a classification accuracy in the range between 94 and 98%. Artificial neural network analysis based on the quick back propagation network (BPN) and the radial basis function network (RBFN) algorithms had classification success rates of 93% using BPN and 100% using RBFN. The genetic algorithm network was able to predict the concentrations of glycerol in wine up to an accuracy of r2 = 0.998.

Automatic flow procedure for the determination of glycerol in wine using enzymatic reaction and spectrophotometry

An automatic flow procedure for the determination of glycerol in wines by employing a flow system based on multicommutation and enzymatic reaction is described. Glycerol dehydrogenase was immobilized on aminopropyl glass beads and packed into a column that was coupled to the flow system. The NADH produced by the enzymatic reaction was monitored by spectrophotometry at 340 nm and its radiation absorption presented a relationship with glycerol concentration. The system manifold comprised a set of three-way solenoid valves controlled by a microcomputer, which was furnished with electronic interfaces and runs a software that was designed to carry out on-line sample dilution, reagent addition, and data acquisition. The procedure allows the determination of glycerol in wine samples without any prior pretreatment. The procedure presented as profitable features a linear response range between 2.0 and 10.0 g l −1 glycerol ( R=0.998), a detection limit of 0.006 g l −1 glycerol, a relative standard deviation of 1.8% ( n=14) for a typical wine sample presenting 5.3 g l −1 glycerol, a sampling throughput of 33 determinations per hour, and a NAD + consumption of 0.8 mg per determination. The results were compared with those obtained using a reference method and no significant difference at 90% confidence level was observed.

Simultaneous detection of ethanol, glucose and glycerol in wines using pyrroloquinoline quinone-dependent dehydrogenases based biosensors

Amperometric biosensors based on corresponding dehydrogenases have been developed for the determination of ethanol, glucose and glycerol. The enzymes have been integrated in redox hydrogels using an Os complex-modified non-conducting polymer employed as the electrochemical mediator and poly(ethyleneglycol)-diglycidyl ether (PEGDGE) as the cross-linking agent. The developed biosensors showed a sensitive response to ethanol, glucose and glycerol within the concentration range 2.5–250, 20–800, and 1–200 μM, detection limits of 1.2, 9 and 1 μM, and sensitivities of 220, 87 and 32 mA M −1 cm −2, respectively. The ethanol, glucose and glycerol content of several types of wine was determined with these biosensors, and the results were compared with those obtained by spectrophotometric methods. The developed biosensors have been successfully employed for simultaneous determination of all these substrates at the required sensitivity.

Glycerol Dehydrogenase Based Amperometric Biosensor for Monitoring of Glycerol in Alcoholic Beverages

Biosensors for measurement of glycerol in FIA were constructed using NAD+-dependent glycerol dehydrogenase (GlDH) either co-immobilized with phenazine methosulphate (PMS) or cross-linked to an Os-complex-modified poly(vinylimidazole) redox polymer (PVI13dmeOs) using poly(ethyleneglycole) diglycidilether (PEGDGE). The GlDH/PMS sensor was characterized by a linear range of 0.01–1 mM glycerol, a sensitivity of 1.83 mA/Mcm2, a detection limit (calculated as three times the signal-to-noise ratio) of 0.9 µM, and with 50% residual activity kept after 15 h of continuous operation at a sample throughput of 30 injections/h. The redox hydrogel-based biosensors showed the same dynamic range, but improved biosensors characteristics, i.e., a sensitivity of 4.79 mA/Mcm2, a detection limit of 0.1 µM, and a signal loss of only 20% after 15 h of operation in the same conditions. The optimized biosensor configurations were further used for analysis of glycerol in wine and the obtained results were compared with the ones obtained by spectrophotometrical experiments.

Automated multiple development method for determination of glycerol produced by wine yeasts

A rapid and efficient analytical method for the determination of glycerol in wines is described. This method utilizes high-performance thin layer chromatography (HPTLC) plates coupled with an automated multiple development system with an elution gradient based on acetonitrile–acetone–hexane on silica gel layers. The absence of clean-up procedures, sometimes only centrifugation, makes this method suitable also for the large-scale control of alcoholic beverages. In particular the capacity of different wine yeast species (Saccharomyces cerevisiae, Zygosaccharomyces bailii, Kloeckera apiculata and Saccharomycodes ludwigii) to produce glycerol was determined. Generally, the strains of S. cerevisiae produced elevated amounts of glycerol together with Z. bailii, whereas K. apiculata strains formed the lowest amounts of glycerol, exhibiting also a great strain variability.

Simultaneous Determination of Acetic Acid and Glycerol in Wines Using Amperometric Tri-Enzyme Electrodes

Simultaneous Determination of Acetic Acid and Glycerol in Wines Using Amperometric Tri-Enzyme Electrodes

Enzymatic determination of ethanol and glycerol by flow injection parallel multi-site detection

A flow injection method was developed for the sequential enzymatic determination of ethanol and glycerol in wines, using immobilised ethanol dehydrogenase and glycerol dehydrogenase, respectively. The enzymes were immobilised separately on alkylaminated controlled pore glass. A multi-site spectrophotometric detection system was used in parallel configuration to monitor the absorbance change in the two independent analytical channels. A 50-fold dilution of the samples was necessary before injection. The working range was between 0.05 and 0.5% (v/v) for the ethanol and between 0.03 and 0.3 g l −1 for the glycerol determination, with corresponding detection limits of 2×10 −3% (v/v) and 2×10 −3 g l −1. Relative standard deviations (R.S.D.) ( n=9) lower than 2.3% for the ethanol and 2.1% for the glycerol determination were found. For 13 samples of different types of table and Port wines, the results showed good agreement with the corresponding reference procedures; a two level recovery study also showed good accuracy for the developed methods. The sampling rate was 10 h −1, corresponding to 20 determinations per hour.

Simultaneous determination of ethanol and glycerol in wines by a flow injection-pervaporation approach with in parallel photometric and fluorimetric detection

A simultaneous method for the determination of ethanol and glycerol in wines based on the coupling of pervaporation-chemical derivatisation-photometric detection for ethanol and biochemical derivatisation-fluorimetric detection for glycerol is proposed. After separation by pervaporation the ethanol is collected in a K 2Cr 2O 7 acceptor stream and the Cr 3+ formed is driven to the spectrophotometer and monitored at 600 nm. The determination of glycerol is based on its oxidation by oxidised β-nicotinamide adenine dinucleotide (β-NAD +) catalysed by glycerol dehydrogenase immobilised on controlled-pore glass, the reduced form of the coenzyme (NADH) being spectrofluorimetrically monitored ( λ ex=340 nm, λ em=460 nm). The linear determination range is between 1–20% for ethanol and 2–8 g l −1 for glycerol, with RSDs 3 and 2%, respectively. The method applied to red and white Spanish wines, compares well with the official methods for these analytes.

Selective breeding of &lt;em&gt;Saccharomyces cerevisiae&lt;/em&gt; to increase glycerol levels in wine

&lt;p style="text-align: justify;"&gt;Glycerol in wines is at levels of 1 to 10 g/l and improves their body. To breed wine yeast for increased glycerol production, we crossed yeast isolated from natural fermentations with a commercial strain. The average glycerol concentration of 187 wine yeast strains was 4.2 g/l. Strain Ba25 had the highest glycerol production and a spore clone of Ba25 was crossed with one of Premier Cuvée, the best commercial strain, by spore-spore pairing and this was repeated three times. Several of the spore clones produced &amp;gt;15 g/l of glycerol. Glycerol and ethanol levels were inversely related.&lt;/p&gt;

The oenological characteristics of commercial dry yeasts

Twenty preparations of dry active yeast (18 Saccharomyces cerevisiae and two Saccharomyces bayanus) available in Italy were tested in white and red musts (Moscato, Albana and Sangiovese) to study their oenological characteristics (i.e. fermentation rate, total alcohol and acetic acid production). After the application of chemiometric techniques for descriptive analyses to the results of the oenological assessment, the yeasts were divided into three groups A, B and C. Group A had some strains which caused low values of dry extract and glycerol in wines. Yeasts in group B were less interesting in terms of their oenological characteristics. Yeasts in group C had mixed behaviour with positive and negative characteristics and their utilisation must be considered separately each time. The research has shown that technological investigation is necessary after a correct selection of yeasts to limit the number of strains for subsequent study, and to characterise suitable strains for different oenological practices.

Chemiluminometric method for the determination of glycerol in wine by flow-injection analysis with co-immobilized glycerol dehydrogenase/NADH oxidase

A flow-injection method for the determination of glycerol in wine is described. Glycerol dehydrogenase and NADH oxidase were co-immobilized on poly (vinyl alcohol) beads and incorporated in a flow-injection system. The hydrogen peroxide produced was detected chemiluminometrically via a luminol-hexacyanoferrate (III) reaction. Wine was diluted 1000-fold with water and sample solution (50 μ1) was injected into the carrier stream. The calibration graph was linear in the range 3 × 10 −7−3 × 10 −4 M; the detection limit was 7 × 10 −8 M and the sample throughout was 30 h −1 without carryover.

Flow-through spectrofluorimetric sensor for the determination of glycerol in wine

A flow-through sensor based on transient immobilization of NADH, the product of the reaction between glycerol and NAD+ catalysed by glycerol dehydrogenase, is proposed. The biocatalyst is immobilized on controlled-pore glass, where the enzymic reaction takes place, the product of the reaction being transiently retained and spectrofluorimetrically monitored on passage through a flow cell packed with a solid support. The proposed method affords a linear range between 0.3 and 5.0 µg ml–1 of glycerol, and a detection limit of 0.1 µg ml–1, with a relative standard deviation of less than 2%. The performance of the sensor was checked by applying it to the determination of glycerol in wine. The results obtained agree well with those provided by a conventional method based on HPLC.

A new Chemiluminometric Method for the Determination of Glycerol in Wine by Flow Injection Analysis with Immobilised Glycerol Dehydrogenase in Combination with NADH Oxidase

Abstract A new chemiluminometric method for the determination of glycerol in wine by FIA is presented. The method is based on the combination of immobilised glycerol dehydrogenase and NADH oxidase. It is compared with common methods of glycerol determinations in wine. Present address: MPI fur Biophysik, Abt. Biophys. Chem., Kennedyallee 70, D-60596 Frankfurt.

Determination of glycerol in wines by high-performance liquid chromatography: comparison with enzymatic method

A method for determining glycerol in wines by high-performance liquid chromatography has been developed using three 250 × 8 mm columns based on a sulphonated styrene-divinyl benzene polymer (SHODEX S-801/S,S-802/S) in series. This polymer combines cation-exchange mechanisms with molecular exclusion. The columns were thermostated at 75°C using water as the mobile phase. A refraction index detector was used and the samples were directly injected. The method is reproducible (coefficient of variation above 1%) and accurate. Linear regression analysis and Student's t-test for comparison of the mean show that the results are comparable with those obtained by an enymatic method (differences less than 4%).