Dual laccase-tyrosinase based Sonogel-Carbon biosensor for monitoring polyphenols in beers

A biosensor based on the bi-immobilization of laccase and tyrosinase phenoloxidase enzymes has been successfully developed. This biosensor employs as the electrochemical transducer the Sonogel-Carbon, a novel type of electrode developed by our group. The immobilization step was accomplished by doping the electrode surface with a mixture of the enzymes, glutaricdialdehyde, and Nafion-ion exchanger, as protective additive. The response of this biosensor, denoted the dual Trametes versicolor laccase (La) and Mushroom tyrosinase (Ty) based Sonogel-Carbon, was optimized directly in beer real samples and its analytical performance with respect to five individual polyphenols was evaluated. The Lac-Ty/Sonogel-Carbon electrode responds to nanomolar concentrations of flavan-3-ols, hydroxycinnamic acids, and hydroxybenzoic acids. The limit of detection, sensitivity, and linear range for caffeic acid, taken as an example, were 26 nM, 167.53 nA M (-1), and 0.01-2 microM, respectively. In addition, the stability and reproducibility of the biosensor were also evaluated in beer samples. The Lac-Ty/sonogel-carbon electrode was verified as very stable in this matrix, maintaining 80% of its stable response for at least three weeks, with a RSD of 3.6% ( n = 10). The biosensor was applied to estimate the total polyphenol index in ten beer samples, and a correlation of 0.99 was obtained when the results were compared with those obtained using the Folin-Ciocalteau reagent.     

Electrochemical estimation of the polyphenol index in wines using a laccase biosensor

The use of a laccase biosensor, under both batch and flow injection (FI) conditions, for a rapid and reliable amperometric estimation of the total content of polyphenolic compounds in wines is reported. The enzyme was immobilized by cross-linking with glutaraldehyde onto a glassy carbon electrode. Caffeic acid and gallic acid were selected as standard compounds to carry out such estimation. Experimental variables such as the enzyme loading, the applied potential, and the pH value were optimized, and different aspects regarding the operational stability of the laccase biosensor were evaluated. Using batch amperometry at -200 mV, the detection limits obtained were 2.6 x 10(-3) and 7.2 x 10(-4) mg L(-1) gallic acid and caffeic acid, respectively, which compares advantageously with previous biosensor designs. An extremely simple sample treatment consisting only of an appropriate dilution of wine sample with the supporting electrolyte solution (0.1 mol L(-1) citrate buffer of pH 5.0) was needed for the amperometric analysis of red, rosé, and white wines. Good correlations were found when the polyphenol indices obtained with the biosensor (in both the batch and FI modes) for different wine samples were plotted versus the results achieved with the classic Folin-Ciocalteu method. Application of the calibration transfer chemometric model (multiplicative fitting) allowed that the confidence intervals (for a significance level of 0.05) for the slope and intercept values of the amperometric index versus Folin-Ciocalteu index plots (r = 0.997) included the unit and zero values, respectively. This indicates that the laccase biosensor can be successfully used for the estimation of the polyphenol index in wines when compared with the Folin-Ciocalteu reference method.     

Potential application of a glucose-transport-deficient mutant of Schizosaccharomyces pombe for removing gluconic acid from grape must

Musts from rotten grapes typically contain high levels of gluconic acid, which can raise severe problems in winemaking processes. In this work, the ability of the glucose-transport-deficient mutant YGS-5 of Schizosaccharomyces pombe to completely or partly remove gluconic acid from a synthetic glucose-containing medium and the potential use of this yeast strain for the same purpose in musts and wines were examined. Surprisingly, the S. pombe YGS-5 strain successfully removed 93% of the initial gluconic acid (2.5 gL(-1)) and 80% of the initial malic acid (1.0 gL(-1)) within 30 h after inoculation. Also, the yeast strain produced no volatile compounds other than those obtained in fermentations conducted with the wine yeast Saccharomyces cerevisiae. S. pombe YGS-5 could thus be used to remove gluconic acid present in musts from rotten grapes. On the basis of these results, various ways of using S. pombe YGS-5 to treat musts containing gluconic acid in order to solve the problems due to the high gluconic acid concentrations in botrytized grape must are proposed.     

Determination of phenolic compounds and ascorbic acid in different fractions of tomato by capillary electrophoresis with electrochemical detection

Tomato (Lycopersicon esculentum Mill.), one of the most important crops worldwide, contains different classes of substances with antioxidant properties such as carotenoids, vitamin C, and phenolics. A method based on capillary electrophoresis with electrochemical detection has been developed to analyze ascorbic acid and phenolics in the peel, pulp, and seeds of tomatoes. Operating in a wall-jet configuration, a 300 microm diameter carbon disk electrode was used as the working electrode, which exhibits a good response at +0.90 V (vs saturated calomel electrode) for the analytes. Under optimum conditions, the analytes were baseline separated within 20 min in a 50 mmol/L borate buffer (pH 8.7). Notably, excellent linearity was obtained over 3 orders of magnitude with detection limits (S/N=3) ranging from 1x10(-8) to 2x10(-7) g/mL for all analytes. This proposed method has been successfully applied to monitor the content of ascorbic acid and phenolics in real samples, and the assay results were satisfactory.     

Evidence for gluconic acid production by Enterobacter intermedium as an efficient strategy to avoid protozoan grazing

The effect of pure gluconic acid and of gluconic-acid-producing bacteria on the activity of three protozoan species, Colpoda steinii (a ciliate), Vahlkampfia sp. (an amoeba) and Neobodo designis (a flagellate), was determined in vitro and in soil microcosms. Pure gluconic acid was shown to mediate disappearance of active cells, due to encystment and/or death of protozoa, at 0.15 mM in saline medium. Similarly, the presence of gluconic acid inhibited excystment of the three protozoa tested. Enterobacter intermedium 60-2G (Wt), a gluconic acid-producing rhizobacterium, elicited the same effects on protozoa when co-cultured in the presence of 5 g L⁻¹ glucose. However, the effect was not observed when glucose was omitted from the medium. Similarly, a pqqA isogenic mutant strain, unable to produce gluconic acid from glucose, exhibited a reduced effect on protozoan activity. Rhizosphere-microcosm studies performed with wheat (Triticum aestivum L.) confirmed the reduced ability of the pqqA mutant to limit protozoa reproduction compared to the Wt strain. Since the sodium salt of gluconic acid did not cause any significant stress to protozoa and considering that addition of 50 mM Tris-Cl (pH 7.2) abolished the deleterious effect of gluconic acid, acidification of the medium appeared as the key factor that induced encystment/death of protozoa. We propose that production and excretion of gluconic acid should be considered an efficient mechanism evolved by bacteria to escape, tolerate or defend themselves against protozoan grazing in rhizosphere environments.     

Development of a new method, based on a bioreactor coupled with an L-lactate biosensor, toward the determination of a nonspecific inhibition of L-lactic acid production during milk fermentation

The development and characteristics of a bioreactor employing bacteria (Streptococcus thermophilus) encapsulated in Ca-alginate beads coupled with an L-lactate biosensor are reported. The biosensor comprises a carbon paste electrode modified with enzymes HRP (horseradish peroxidase), LOD (lactate oxidase), and FcH (ferrocene) as redox mediator. The measurement of L-lactate is based on the signal produced by H(2)O(2), the product of the enzymatic oxidation of L-lactate by LOD. The detection of H(2)O(2) is performed at the electrode surface via HRP/FcH at low operating potential (-100mV vs Ag/AgCl). Optimization studies were performed using the bioreactor in conjunction with an L-lactate electrode operating in a flow injection system to assess the ability of encapsulated bacteria to ferment carbohydrate solutions. The possibility of using the developed method to assess the fermentation capability of milk samples was evaluated. Bronopol (2-bromo-2-nitro propane-1,3-diol) was chosen to simulate the effect of an inhibitory agent of milk fermentation. The obtained results indicated that the evaluation of the amount of L-lactate amount produced through the bioreactor could be used as a measure of inhibition of lactic acid production in milk samples.     

Effect of Schizosaccharomyces pombe on aromatic compounds in dry sherry wines containing high levels of gluconic acid

Volatile compounds have been determined in control dry sherry wines and those supplemented with gluconic acid, which were inoculated with the Schizosaccharomyces pombe 1379 (ATCC 26760) yeast strain. These compounds were grouped, according to volatiles exhibiting the identical odor quality, into nine groups of the same odor character (aromatic series) as a way of establishing the aroma profile for the studied wines. Control and supplemented wines showed changes in the balsamic, spicy, roasty, and fruity aromatic series, and tasters judged the aroma as typical of wines subjected to biological aging. This fission yeast may be used as a treatment to reduce gluconic acid contents in wines obtained from rotten grapes, making feasible the incorporation of these wines into the biological aging process. In addition, this procedure may also help to accelerate the traditional biological aging in sherry winemaking due to the contribution of some specific compounds by S. pombe to the wine.     

Monitoring and estimation of the kinetics parameters in the binding process of tannic acid to bovine serum albumin with electrochemical quartz crystal impedance system

Combined measurements of piezoelectric quartz crystal impedance (PQCI) and electrochemical impedance (EI) were utilized in situ to monitor the adsorption of bovine serum albumin (BSA) onto the newly prepared Au colloid-modified electrode and study the binding process of tannic acid (TA) to BSA on the BSA-modified electrode surface. The time courses of the resonant frequency and the equivalent parameters of the sensor were simultaneously obtained during BSA adsorption and TA-BSA binding. Compared with the bare gold electrode, the Au colloid-modified gold electrode showed better biocompatibility, and the absorption capacity for BSA was increased by approximately 2.4 times. The observed frequency decrease was ascribed to the mass increase of the sensor surface resulting from the TA-BSA binding, which is believed to result mainly from the hydrogen bonding from FT-IR characterization. The maximal molar binding ratio of TA binding to immobilized BSA obtained from the frequency shift of the adsorbed BSA and TA was estimated to be 10.3:1. On the basis of the frequency decrease with time, the kinetics of the binding was quantitatively studied. By way of fitting the experimental data, the kinetics parameters, that is, binding and dissociation constant (k1, k(-1)), and the binding equilibrium constant (ka) were determined, giving values of 9.51 x 10(4) M(-1) s(-1), 3.15 s(-1), and 3.1 x 10(4) M(-1), respectively.     

Quenching mechanisms and kinetics of Trolox and ascorbic acid on the riboflavin-photosensitized oxidation of tryptophan and tyrosine

The effects of 0, 0.3, 0.6, and 0.9 mM Trolox and ascorbic acid on the singlet oxygen oxidation of tryptophan and tyrosine containing 25 ppm of riboflavin were determined by measuring tryptophan and tyrosine concentration by high-performance liquid chromatography analysis. The samples were stored in the a 1000 lx light storage box for 4 h at 30 degrees C. As the concentration of Trolox and ascorbic acid increased, the degradation of tryptophan and tyrosine decreased significantly at p < 0.05. Trolox reduced tryptophan and tyrosine degradation by quenching both singlet oxygen and excited triplet riboflavin, whereas ascorbic acid quenched singlet oxygen only. The total singlet oxygen quenchings of Trolox in the presence of tryptophan and tyrosine were 1.55 x 10(7) and 1.32 x 10(7) M(-1) s(-1), respectively. The total singlet oxygen quenchings of ascorbic acid in the presence of tryptophan and tyrosine were 1.16 x 10(7) and 1.10 x 10(7) M(-1) s(-1), respectively. Trolox was more effective than ascorbic acid in preventing the degradation of tryptophan and tyrosine.     

Determination of phenolic compounds in Perilla frutescens L. by capillary electrophoresis with electrochemical detection

A method based on capillary electrophoresis with electrochemical detection has been developed to analyze flavonoids and phenolic acids in Perilla frutescens L. for the first time. Catechin, ferulic acid, apigenin, luteolin, rosmarinic acid, and caffeic acid are major important active ingredients in the plant. Operated in a wall-jet configuration, a 300 microm diameter carbon-disk electrode was used as the working electrode, which exhibits a good response at 0.90 V (versus saturated calomel electrode) for the analytes. Under the optimum conditions, the analytes were baseline separated within 20 min in a 100 mmol/L borax buffer (pH 8.7). Notably, excellent linearity was obtained over 3 orders of magnitude with detection limits (S/N = 3) ranging from 2 x 10(-7) to 1 x 10(-6) g/mL for all analytes. This proposed method has been successfully applied to monitor the flavonoids and phenolic acids contents in the leaves and seeds of P. frutescens L. at different growth stages with relatively simple extraction procedures, and the assay results were satisfactory.     

Voltammetric determination of the antioxidant capacity in wine samples using a carbon nanotube modified electrode

A direct determination of gallic acid was achieved at a carbon paste electrode modified with carbon nanotubes under differential pulse voltammetry conditions. The values obtained for gallic acid were used to estimate the antioxidant properties of the wine sample based on gallic acid oxidation. The proposed method is based on the gallic acid oxidation process at a modified carbon paste electrode (MCPE) containing 30% (m/m) of carbon nanotubes monitored at 0.35 V versus Ag/AgCl (KCl 3 mol L(-1)). Using the optimized experimental conditions, the calibration curve for gallic acid was linear in the concentration range from 5.0 × 10(-7) to 1.5 × 10(-5) mol L(-1) with a detection limit of 3.0 × 10(-7) mol L(-1). The MCPE was successfully applied for the determination of the antioxidant capacity for red and white wine samples without interference of glucose and ascorbic acid, and the obtained results were compared with the standard spectrophotometric method.     

Sensitive determination of (-)-epigallocatechin gallate in tea infusion using a novel ionic liquid carbon paste electrode

This paper investigates the electrocatalytic oxidation of (-)-epigallocatechin gallate (EGCG), the main monomer flavanol found in green tea, with a novel ionic liquid, n-octylpyridinium hexafluorophosphate (OPFP) carbon paste electrode (CPE). Due to the natural viscosity and high conductivity of OPFP, this novel OPFP-CPE exhibited very attractive properties, such as high stability and electrochemical reactivity, low background current, and wide electrochemical window. Therefore, this electrode is a very good alternative to traditional chemically modified electrodes because the electrocatalytic effect can achieved without any further electrode modification. Comparative experiments were carried out using CPE and a glassy carbon electrode (GCE). With OPFP-CPE, highly reproducible and well-defined cyclic voltammograms were obtained for EGCG. Under optimal experimental conditions, the peak current of differential pulse voltammetry (DPV) response increased linearly with EGCG concentration over the range of 5.0 × 10(-7)-1.25 × 10(-5) M. The limit of detection (LOD) and the limit of quantification (LOQ) were 1.32 × 10(-7) and 4.35 × 10(-7) M, respectively. The method was applied to the determination of EGCG in green tea infusion samples, and the recovery of the spiked EGCG to the diluted (10-fold) tea extract was from 87.62 to 99.51%.     

Determination of phenolic constituents of biological interest in red wine by capillary electrophoresis with electrochemical detection

A simultaneous determination of trans-resveratrol, (-)-epicatechin, and (+)-catechin in red wine by capillary electrophoresis with electrochemical detection (CE-ED) is reported. The effects of the potential of the working electrode, pH and concentration of running buffer, separation voltage, and injection time on CE-ED were investigated. Under the optimum conditions, the analytes could be separated in a 100 mmol/L borate buffer (pH 9.2) within 20 min. A 300 microm diameter carbon disk electrode has a good response at +0.85 V (vs SCE) for all analytes. The response was linear over 3 orders of magnitude with detection limit (S/N = 3) ranging from 2 x 10(-7) to 5 x 10(-7) g/mL for all analytes. This method has been used for the determination of these analytes in red wine without enrichment, and the assay result was satisfactory.     

Determination of pharmacologically active ingredients in sweet potato (Ipomoea batatas L.) by capillary electrophoresis with electrochemical detection

Sweet potato (Ipomoea batatas L.), in which vitamin C, chlorogenic acid, caffeic acid, quercetin, and rutin are abundant, is one of the functional food products aimed at introducing human dietary ingredients that aid specific body functions in addition to being nutritious. A method based on capillary electrophoresis with electrochemical detection (CE-ED) to qualitatively and quantitatively determine the pharmacologically active ingredients in sweet potato has been developed by our group. The effects of working electrode potential, pH and concentration of running buffer, separation voltage, applied potential, and injection time on CE-ED were investigated. Under the optimum conditions, the analytes could be well-separated within 20 min at the separation voltage of 18 kV in a 60 mmol L(-1) Borax running buffer (pH 9.0). A good linear relationship was established between peak current and concentration of analytes over 2 orders of magnitude with detection limits (S/N = 3) ranging from 7.14 x 10(-7) to 2.88 x 10(-7) g mL(-1) for all target ingredients. The satisfactory results show that this method is very successful and effective for the analysis of real samples.     

Application of capillary electrophoresis to study phenolic profiles of honeybee-collected pollen

Honeybee-collected pollen is promoted as a health food with a wide range of nutritional and therapeutic properties. A high-performance capillary electrophoresis with amperometric detection method has been developed for the simultaneous determination of bioactive ingredients in 10 samples of honeybee-collected pollen in this work. Under the optimum conditions, 13 phenolic components can be well-separated or nearly baseline-separated (apigenin and vanillic acid peaks) within 29 min at the separation voltage of 14 kV in a 50 mM borax running buffer (pH 9.0), and adequate extraction was obtained with ethanol for the determination of the above 13 compounds. Recovery (94.1-104.0%), repeatability of the peak current (<5.4%), and detection limits (6.9 x 10(-7)-6.4 x 10(-9) g mL(-1)) for the method were evaluated. This procedure was successfully used for the analysis and comparison of the phenolic content of honeybee-collected pollen samples originating from different floral origins based on their electropherograms or "phenolic profiles".     

Role of beer lipid-binding proteins in preventing lipid destabilization of foam

The negative effect of fatty acids on the foam stability of beer has been assessed. Long-chain fatty acids are far more damaging than short-chain fatty acids on the foam stability of beer at the concentrations employed. Polypeptides have been isolated from an all malt beer by hydrophobic interaction chromatography. Using this technique five groups of polypeptides were isolated, group 1 being the least hydrophobic and group 5 the most hydrophobic, all of which exhibited similar polypeptide compositions by SDS-PAGE. All five hydrophobic polypeptide groups bound [(14)C]linoleic acid; however, group 5, the most hydrophobic group, bound the most linoleic acid. Groups 1 and 5 were titrated with cis-parinaric acid (CPA) to produce binding curves, which were compared with a binding curve obtained for bovine serum albumin (BSA). Groups 1 and 5 both produced binding curves that saturated at approximately 5.5 microM and 4 microM CPA and had association constants (K(a)) of 6.27 x 10(7) and 1.62 x 10(7) M(-1), respectively. In comparison, BSA produced a binding curve that saturated at 6 microM CPA and had a K(a) of 3.95 x 10(7) M(-1). Further investigation has shown that group 1 is pH sensitive and group 5 pH insensitive with respect to lipid binding. The lipid-binding activity of group 5 was also shown to be unaffected by ethanol concentration. Linoleic acid (5 microM) when added to beer resulted in unstable foam. Group 5 was added to the lipid-damaged beer and was shown to restore the foam stability to values that were obtained for the control beer. It has therefore been demonstrated that proteins isolated from beer have a lipid-binding capacity and that they can convey a degree of protection against lipid-induced foam destabilization.     

Free radical studies of ellagic acid, a natural phenolic antioxidant

Ellagic acid, a plant-derived polyphenol, inhibits gamma-radiation (hydroxyl radical) induced lipid peroxidation in rat liver microsomes in a dose- and concentration-dependent manner. Its antioxidant capacity has been estimated using the 1,1-diphenyl-2-picrylhydrazyl radical assay. To understand the actual mechanisms involved in antioxidant activity and the free radical scavenging ability,a nanosecond pulse radiolysis technique has been employed. The rate constants for the reactions of several reactive oxygen species and reactive nitrogen species such as hydroxyl, peroxyl, and nitrogen dioxide radicals have been found to be in the range of 10(6)-10(9) M(-1) s(-1). The ellagic acid radicals have been characterized by the absorption spectra and decay kinetics. Studies on the reactions of ellagic acid with the 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonate) radical and the radicals of ellagic acid with ascorbate have been used to estimate its one-electron reduction potential. Ellagic acid has also been found to be a good scavenger of peroxynitrite. Using stopped-flow reaction analyzer with absorption detection, the rate constant for this reaction has been determined to be 3.7 x 10(3) M(-1) s (-1). The electron spin resonance spectra of the oxidized ellagic acid radicals have been recorded by horseradish peroxidase and hydrogen peroxide method.     

Isolation and characterization of aminopeptidase (Jc-peptidase) from Japanese cedar pollen (Cryptomeria japonica)

An aminopeptidase, Jc-peptidase, was purified from Japanese cedar pollen by seven steps, including precipitation with ammonium sulfate, ion-exchange chromatography, gel filtration, hydrophobic interaction chromatography on phenyl-agarose, and high-performance liquid chromatography. Purified Jc-peptidease has a molecular weight of 42 kDa and hydrolyzes the synthetic substrates of L-phenylalanyl-4-methylcoumaryl-7-amide (Phe-MCA) with Km = 5 x 10(-5) M, Tyr-MCA with Km = 7 x 10(-4) M, Leu-MCA with Km = 1 x 10(-3) M, and Met-MCA with Km = 1 x 10(-3) M. Other MCA analogues such as Arg-MCA or Glu-MCA failed to serve as its substrates. The activity was inhibited in the presence of phebestin, [(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl-L-valyl]-L-phenylalanine, with Ki = 4.7 x 10(-5) M, or bestatin, [(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl]-L-leucine, with Ki = 1.1 x 10(-4) M. According to amino acid sequence analysis, the N-terminal amino group seems to be blocked. The physiological function of the aminopeptidase (Jc-peptidase) has not been clarified in vivo.     

Determination of trace hydroxyl radicals by flow injection spectrofluorometry and its analytical application

On the basis of the fluorescence increase of the reaction of ninhydrin with hydroxyl radicals, a new method for the determination of trace amounts of hydroxyl radicals by flow injection spectrofluorometry is presented. The introduction of flow injection analysis brought better reproducibility and avoided the effect of oxygen and other substances in the environment on the reaction of ninhydrin with hydroxyl radicals. Under optimum experimental conditions, the hydroxylated product of ninhydrin had excitation and emission maxima at 300 and 406 nm, respectively. The linear range was 2.60 x 10(-7) to 4.00 x 10(-5) M, and the limit of detection was 7.91 x 10(-8) M. A high analysis rate of 22 samples per hour was obtained. The proposed method has been applied successfully to the determination of scavenging effects of thiourea and vitamin C on hydroxyl radicals as well as to the evaluation of antioxidant capacities of some natural food.     

Release of D-xylose from wheat straw by acid and xylanase hydrolysis and purification of xylitol

Xylitol is a valuable sweetener produced from xylose-rich biomass. Our objective was to optimize conditions for maximum release of D-xylose from wheat straw by acid or enzyme hydrolysis with minimal release of other monosaccharides, and to purify xylitol from three other alditols. Ground straw was treated with 10 parts of 0.2-0.4 M sulfuric acid at 110-130 degrees C for 15-45 min or at reflux with 0.75-1.25 M sulfuric acid for 1.5-3 h. Under optimum conditions of either 0.3 M acid at 123 degrees C for 28 min or 1.0 M acid at 100 degrees C for 3 h, 18 or 19% of D-xylose plus approximately 6% other sugars were produced from straw (dry basis). A 16% yield of D-xylose plus 6% other sugars was obtained when hydrothermally (10% straw, 160 degrees C, 1 h) treated straw was incubated with a commercial xylanase. The lack of enzyme specificity for D-xylose release was attributed to the autohydrolysis of polysaccharides during the pretreatment plus slow hydrolysis of cellulose during enzyme digestion. Xylitol with a purity of 95% was obtained in 10% yield from straw after the reduction of an acid-hydrolyzate followed by fractional crystallization. Purification of the mixture of four alditols by open-column chromatography on a strongly basic anion-exchange resin in hydroxide form gave 7% xylitol crystals with a purity of 99%.