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.     

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.     

Influence of aeration on the physiological activity of flor yeasts.

The effect of periodic aeration on the physiological activity of a strain of Saccharomyces cerevisiae yeast during development of velum (flor) and biological aging of Sherry wine of the Fino type was investigated. L-Proline amino acid was the main nitrogen source for yeasts cells during the biological aging, and its exhaustion may be the cause of the production and consumption of other compounds that are involved in the aroma of wines. Aeration was found to increase adenylate energy charge, growth, and viability of the yeast cells. Also, it affected the intracellular redox equilibrium and the consumption and production of compounds including acetoin, acetaldehyde, higher alcohols, ethanol, glycerol, and acetic acid. Acetaldehyde reached its highest level after the second aeration, which coincided with the exhaustion of the nitrogen source in the medium. The enzyme activity of alcohol dehydrogenases I and II decreased immediately after each aeration, subsequently increasing once all of the dissolved oxygen in the wine had been consumed by yeast cells. Aldehyde dehydrogenase activity was detected only after the first aeration, and it may be related to the production and consumption of acetic acid in the wine.     

Chemical and biochemical transformations during the industrial process of sherry vinegar aging

The work described here concerns a study of the chemical and biochemical transformations in sherry vinegar during the different aging stages. The main factors that contribute to the nature and special characteristics of sherry vinegar are the raw sherry wine, the traditional process of acetic acid fermentation in butts (the solera system), and the physicochemical activity during the aging process in the solera system. A number of chemical and biochemical changes that occur during sherry vinegar aging are similar to those that take place in sherry wine during its biological activity process (where the wine types obtained are fino and manzanilla) or physicochemical activity process (to give oloroso wines). Significant increase in acetic acid levels was observed during the biological activity phase. In addition, the concentrations of tartaric, gluconic, succinic, and citric acids increased during the aging, as did levels of amino acids and acetoin. A color change was also produced during this stage. Glycerol was not consumed by acetic acid bacteria, and levels of higher alcohols decreased because of the synthesis of acetates. On the other hand, in the physicochemical phase the microbiological activity was lower. Concentrations of some cations increased because of evaporation of water through the wood. A color change was also produced in this stage. Concentrations of different amino acids decreased because of reaction with carbonyl compounds. A precipitation of potassium with tartaric acid was also observed.     

Interaction of yeasts with the products resulting from the condensation reaction between (+)-catechin and acetaldehyde

The condensation reaction between (+)-catechin and acetaldehyde was studied in model solutions in the presence and absence yeasts in order to evaluate its contribution to color changes in fermented drinks such as white wine. On the basis of the results, the yeasts retain the oligomers produced in the reaction, their retention ability increasing for higher polymerization degrees. As a result, the color of model solutions, measured as the absorbance at 420 nm, was found to decrease after the addition of yeasts. On the other hand, the yeasts exhibited no inhibitory effect on the condensation reaction, which took place at the same rate in their presence and absence. At acidity levels and reactant concentrations similar to those in wine, with acetaldehyde in high concentration as it is present in sherry wines, the reaction was found to occur very slowly. Taking into account that Yeasts are present during most of the winemaking process; consequently, they retain oligomers, and the studied reaction could mainly contribute to the alteration of the color of white wine after bottling.     

Gluconic acid,its lactones,and SO(2) binding phenomena in musts from botrytized grapes

Intramolecular gluconic acid esterification reactions led to the formation of two lactones, gamma- and delta-gluconolactone (glucono-1,4-lactone and glucono-1,5-lactone). The presence of the first has not yet been reported in must or wine. These lactones are in equilibrium with gluconic acid, gamma- and delta-gluconolactone representing, respectively, 5.8 and 4.1% of the acid level. Correlations between must SO(2) binding power, gluconic acid, and consequently its lactones are shown. The SO(2) affinity of a mixture containing this acid and gamma- and delta-gluconolactone was determined, and gluconic acid appeared to be indirectly responsible for approximately 8% of the bindable SO(2) in musts from botrytized grapes.     

Biological aging of sherry wines using pure cultures of two flor yeast strains under controlled microaeration

Sherry wines obtained after biological aging for an average of 0, 2, and 4 years were inoculated separately with the flor yeast strains Saccharomyces cerevisiae var. capensis and Saccharomyces bayanus and subjected to short, periodic microaeration to a dissolved oxygen concentration of 4 mg L(-1) after formation of the yeast film. A principal component analysis with the acetaldehyde, ethanol, volatile acidity, and glycerol concentrations obtained was performed. The first principal component was found to account for 49.5% of the overall variance and to be defined mainly by glycerol and ethanol. The second component accounted for 38.8% of the variance and was defined by volatile acidity and acetaldehyde. The conditions used in the tests allowed the biological aging of the wines to be substantially shortened. Thus, 42 days after flor-film formation by S. cerevisiae var. capensis, 0- and 2-year-old wines exhibited parameter values similar to those obtained for the wine aged for 4 years. The wines inoculated with S. bayanus exhibited high acetaldehyde concentrations and ethanol levels above 15% (v/v)-sherry wines with alcohol concentrations below 14.5% are undesirable-, so one need not exclude the sequential or simultaneous inoculation of S. bayanus together with S. cerevisiae var. capensis in order to improve the biological aging process.     

Gas chromatographic quantification of major volatile compounds and polyols in wine by direct injection

Methanol, propanol, isobutanol, isoamyl alcohol, 2-phenylethanol, acetaldehyde, 1,1-diethoxyethane, acetoin, ethyl acetate, ethyl lactate, and ethyl succinate and the polyols 2,3-butanediol (levo and meso forms) and glycerol were quantified by direct injection of wine samples. Linear responses over the usual concentration ranges for these compounds and r2 values from 0.9932 to 0.9998 were obtained. The confidence limits for the mean values ranged from 2.34% for diethyl succinate to 8.52% for 1,1-diethoxyethane, both at a probability level of 0.05. Relative errors ranged from 8 to 10% for the polyols and 1,1-diethoxyethane and were all less than 5% for alcohols and acetaldehyde. The proposed method is useful with a view to identifying relationships between alcoholic fermentation byproducts and controlling biological or chemical aging in wines.     

Influence of blending on the content of different compounds in the biological aging of sherry dry wines

Principal components analysis to examine the effect of blending (viz. the mixing and transfer of wine between cask rows in a "criaderas and solera" system) on metabolic activity in flor yeasts during biological aging of sherry dry wines was carried out. The variables used in the analysis were the wine compounds most deeply involved in the flor yeast metabolism, namely ethanol, acetaldehyde, glycerol, acetic acid, and l-proline. The greatest blending effect was found to be on the third and second "criadera", which are the stages where the yeasts show a high metabolic activity. The stages holding the oldest wine (viz. the first criadera and the solera) exhibited no differences before and after blending; therefore, the yeasts have a decreased biological activity in them and physical-chemical aging processes seemingly prevail over it.     

Study of some Saccharomyces cerevisiae strains for winemaking after preadaptation at low temperatures

Low-temperature fermentations (13 degrees C) are considered to improve wine aromatic profiles. However, because the risk of stuck and sluggish fermentations is high, these fermentations are not common. The aim of this paper was to analyze the effect of different preadaptation protocols in two commercial wine strains on the fermentation and some wine parameters. Preadaptation is understood to be the process between the rehydration of active dry yeast and the inoculation. In this study, it consisted of preparing a fermentation starter (addition of yeast grown at 25 degrees C) or inocula preadapted at low temperatures (as before, but grown at a fermentation temperature of 13 or 17 degrees C). These results were compared with those of rehydrated active dry yeast, and a commercial "cryotolerant" yeast was used as a reference. General fermentation kinetic parameters, yeast imposition, nitrogen consumption, and main wine products were analyzed. The results showed that the preadaptation of a yeast could improve the fermentation performance, although this improvement was strain-dependent. Low-temperature fermentations also had some general effects: reduction of acetic acid and fusel alcohol production and increased concentrations of glycerol. When the yeast performed better in fermentation because of preadaptation, nitrogen consumption was faster and the wine's "negative" attributes (acetic acid, fusel alcohols) were significantly reduced. Thus, in some strains, preadaptation could be an effective mechanism for improving low-temperature fermentation, which also significantly reduces detrimental wine attributes.     

Heterocyclic acetals from glycerol and acetaldehyde in Port wines: evolution with aging

In Port wine, isomers of glycerol and acetaldehyde acetals have been found at total contents ranging from 9.4 to 175.3 mg/L. During oxidative aging, the concentrations of the 5-hydroxy-2-methyl-1,3-dioxane and 4-hydroxymethyl-2-methyl-1,3-dioxolane isomers increased with time showing a linear correlation (r > 0.95). The flavor threshold for the mixture of the four isomers was evaluated in wine at 100 mg/L. Thus, it is expected that they contribute to "old Port wine" aroma in wines older than 30 years. Experiments with model solutions and wine clearly demonstrated that SO(2) combines with acetaldehyde and blocks the acetalization reaction.     

HPLC-DAD-ESI-MS/MS characterization of pyranoanthocyanins pigments formed in model wine

Formation of wine pyranoanthocyanins in model wine was monitored by HPLC-DAD-ESI-MS/MS, using red grape skin extracts and wine fermentation metabolites (acetaldehyde, pyruvic and acetoacetic acids, and diacetyl) and also hydroxycinnamic acids (p-coumaric, caffeic, ferulic, and sinapic acids). Pyruvic acid and acetaldehyde reacted fast, the first reaching high product yield and the second inducing mainly pigment polymerization. In contrast, acetoacetic acid and diacetyl reacted slowly with poor product yields. Hydroxycinnamic acids progressively reacted without apparent formation of polymeric pigments, the reaction rate and yield increasing as the number of hydroxy/methoxy groups did. Substituent at C-10 strongly affected the visible maximum absorbance wavelength, whereas B-ring substitution pattern or sugar acylation exerted little effect. The 10-methylpyranoanthocyanins formed from acetoacetic acid were also found as side products in the formation of 10-carboxypyranoanthocyanins. Finally, we report for the first time on UV-vis and MS spectral data of 10-acetylpyranoanthocyanins formed from diacetyl, and their occurrence in commercial red wines is suggested.     

Monitoring acetaldehyde concentrations during micro-oxygenation of red wine by headspace solid-phase microextraction with on-fiber derivatization

An analytical method was developed to quantify levels of acetaldehyde in wine samples. The method utilizes headspace solid-phase microextraction with on-fiber derivatization using O-(pentafluorobenzyl)hydroxylamine and quantification by gas chromatography with flame ionization detection. The technique showed good sensitivity and reproducibility in samples of Chardonnay, Petite Sirah, and Merlot wines containing acetaldehyde at levels below the sensory threshold (40-100 ppm). The method was used to monitor acetaldehyde concentrations during the micro-oxygenation of Merlot wine in a 141 L pilot-plant experiment and a 2400 L full-scale study. In both experiments, levels of acetaldehyde remained constant for several weeks before increasing at rates of the order of 1 ppm/day. Variations in the levels of acetaldehyde present are discussed within the context of the underlying chemical reactions.     

Response of the aroma fraction in sherry wines subjected to accelerated biological aging.

The effect of an acceleration assay, carried out with a periodic aeration and an increased surface/volume ratio, on various aroma compounds of "fino" Sherry wines aging under a veil of a pure culture of Saccharomyces cerevisiae race capensis G1 flor film yeast was studied. The results were subjected to multifactor analysis of variance, and the compounds simultaneously depending on acceleration conditions and aging time at p < 0.01 were subjected to principal component analysis. The first component, accounting for 86.14% of the overall variance, was mainly defined by acetaldehyde and its derivatives 1,1-diethoxyethane and acetoin. These compounds reached higher concentrations in accelerated aging wines in a shorter time than they did in control wines, and no browning problems were detected. Taking into account that these compounds can be used as indicators for biological aging of "fino" Sherry wines, the acceleration condition assayed can be applied to shorten the time of this process.     

The effect of SO2 on the production of ethanol,acetaldehyde, organic acids,and flavor volatiles during industrial cider fermentation

SO(2) is widely used in cider fermentation but also in other alcoholic beverages such as wine. Although the authorized limit is 200 ppm total SO(2), the International Organizations recommend its total elimination or at least reduction due to health concerns. Addition of SO(2) to apple juice at levels frequently used in industrial cidermaking (100 mg/L) induced significantly higher acetaldehyde production by yeast than that obtained without SO(2). Although the practical implications of acetaldehyde evolution under cidermaking conditions has been overcome by research and few data are available, this compound reached levels in two 2000 L bioreactors that may have prevented the occurrence of simultaneous alcoholic and malolactic fermentation. It was observed that malolactic fermentation had a positive effect promoting reduction of acetaldehyde levels in cider fermented with juice, SO(2)-treated or not. The addition of SO(2) clearly delayed malolactic fermentation comparing to the control, affecting not the onset of the malolactic fermentation but the rate of malic acid degradation. This compound, however, had a stimulatory effect on alcoholic fermentation.     

Integrated electrochemical gluconic acid biosensor based on self-assembled monolayer-modified gold electrodes. Application to the analysis of gluconic acid in musts and wines

An integrated amperometric gluconic acid biosensor constructed using a gold electrode (AuE) modified with a self-assembled monolayer (SAM) of 3-mercaptopropionic acid (MPA) on which gluconate dehydrogenase (GADH, 0.84 U) and the mediator tetrathiafulvalene (TTF, 1.5 micromol) were coimmobilized by covering the electrode surface with a dialysis membrane is reported. The working conditions selected were Eapp=+0.15 V and 25+/-1 degrees C. The useful lifetime of one single TTF-GADH-MPA-AuE was surprisingly long. After 53 days of continuous use, the biosensor exhibited 86% of the original sensitivity. A linear calibration plot was obtained for gluconic acid over the 6.0x10(-7) to 2.0x10(-5) M concentration range, with a limit of detection of 1.9x10(-7) M. The effect of potential interferents (glucose, fructose, galactose, arabinose, and tartaric, citric, malic, ascorbic, gallic, and caffeic acids) on the biosensor response was evaluated. The behavior of the biosensor in a flow-injection system in connection with amperometric detection was tested. The analytical usefulness of the biosensor was evaluated by determining gluconic acid in wine and must samples, and the results obtained were validated by comparison with those provided by using a commercial enzyme test kit.     

Role of carbonyl compounds in SO(2) binding phenomena in musts and wines from botrytized grapes

Carbonyl compounds play an important role in musts from botrytized grapes. Some of them, such as glyoxal and methylglyoxal, may explain a considerable part of bindable SO(2). Others, such as 2- and 5-oxogluconic acids, produced by gluconic acid oxidation in proportions respectively from 2.5 per 1 play an interesting role as SO(2) binding indicator. Finally, the levels of some compounds such as dihydroxyacetone, 5-oxofructose, and delta-gluconolactone in balance with gluconic acid are well correlated with SO(2) binding powers and also explain a large part of the bindable SO(2) in musts. During alcoholic fermentation, only dihydroxyacetone among these three compounds is metabolized by yeast. Thus, two compounds present in grapes, delta-gluconolactone and 5-oxofructose, with three yeast SO(2)-binding byproducts, ethanal, pyruvic, and 2-oxoglutaric acids, explain much of the SO(2) binding power in wines from botrytized grapes.     

戴尔有孢圆酵母调控晚采小芒森葡萄酒乙酸和香气

为降低甜型葡萄酒中的挥发酸,进一步增加其香气复杂度,该研究利用本土戴尔有孢圆酵母R12与酿酒酵母NX11424同时接种和顺序接种发酵,研究其对贺兰山东麓产区晚采小芒森葡萄酒乙酸及香气成分的调控。与酿酒酵母单菌发酵相比,本土戴尔有孢圆酵母与酿酒酵母按照5:1、20:1、50:1的不同菌体数量比例同时接种和间隔5d顺序接种发酵,均可以显著降低晚采小芒森葡萄酒的乙酸含量,其乙酸产率分别降低19.1%、21.2%、38.2%和48.9%。此外,按20:1比例同时接种发酵,可显著提高晚采小芒森葡萄酒中萜烯类和降异戊二烯类等品种香气物质含量,和乙酸异戊酯、丁酸乙酯及苯乙醇等发酵香气物质的含量。该研究表明合理的酿酒酵母接种发酵能有效降低高糖原料酿造葡萄酒挥发酸含量,为贺兰山东麓产区葡萄酒新产品的开发提供了的技术参考。     

Solubilization of zinc phosphate by a strain of Pseudomonas fluorescens isolated from a forest soil

 A strain of Pseudomonas fluorescens, able to solubilize zinc phosphate, was isolated from a forest soil. Colonies of the microorganism produced clear haloes on solid medium incorporating zinc phosphate, but only when glucose was provided as the carbon source. Solubilization of zinc phosphate occurred by both an increase in the H⁺ concentration of the medium, probably a consequence of ammonia assimilation, and the production of gluconic acid. High concentrations of gluconic acid were produced when P. fluorescens 3a was cultured in the presence of zinc phosphate. Although under some conditions gluconic acid is purportedly able to solubilize metals by the formation of chelates, no evidence of zinc chelation was obtained in our experiments. Furthermore, the increased Zn²⁺ concentration caused by the solubilization process resulted in the manifestation of toxic effects on the culture. A sample of the culture, sonicated to disrupt cells, still possessed the ability to produce gluconic acid from glucose, in the presence and absence of zinc phosphate. The lack of gluconic acid overproduction in cultures of P. fluorescens 3a which were not amended with zinc phosphate suggests that at least some of the glucose oxidation required for the zinc solubilization occurred as a result of the toxic stress caused by the high Zn²⁺ concentration. Received: 16 December 1997     

Rapid method for proline determination in grape juice and wine

Proline is typically the most abundant amino acid present in grape juice and wine. The amount present is influenced by viticultural and winemaking factors and can be of diagnostic importance. A method for rapid routine quantitation of proline would therefore be of benefit for wine researchers and the industry in general. Colorimetric determination utilizing isatin as a derivatizing agent has previously been applied to plant extracts, biological fluids, and protein hydrolysates. In the current study, this method has been successfully adapted to grape juice and wine and proved to be sensitive to milligram per liter amounts of proline. At sugar concentrations above 60 g/L, interference from the isatin-proline reaction was observed, such that proline concentrations were considerably underestimated in grape juice and dessert wine. However, the method was robust for the analysis of fermentation samples and table wines. Results were within ±10% agreement with data generated from typical HPLC-based analyses. The isatin method is therefore considered suitable for the routine analysis required to support research into the utilization or release of proline by yeast during fermentation.