Interactions between yeast lees and wine polyphenols during simulation of wine aging: I. Analysis of remnant polyphenolic compounds in the resulting wines

Wine aging on yeast lees is a traditional enological practice used during the manufacture of wines. This technique has increased in popularity in recent years for the aging of red wines. Although wine polyphenols interact with yeast lees to a limited extent, such interactions have a large effect on the reactivity toward oxygen of wine polyphenolic compounds and yeast lees. Various domains of the yeast cell wall are protected by wine polyphenols from the action of extracellular hydrolytic enzymatic activities. Polysaccharides released during autolysis are thought to exert a significant effect on the sensory qualities of wine. We studied the chemical composition of polyphenolic compounds remaining in solution or adsorbed on yeast lees after various contact times during the simulation of wine aging. The analysis of the remnant polyphenols in the wine indicated that wine polyphenols adsorption on yeast lees follows biphasic kinetics. An initial and rapid fixation is followed by a slow, constant, and saturating fixation that reaches its maximum after about 1 week. Only very few monomeric phenolic compounds remained adsorbed on yeast lees, and no preferential adsorption of low or high polymeric size tannins occurred. The remnant condensed tannins in the wine contained fewer epigallocatechin units than the initial tannins, indicating that polar condensed tannins were preferentially adsorbed on yeast lees. Conversely, the efficiency of anthocyanin adsorption on yeast lees was unrelated to its polarity.     

Interactions between yeast lees and wine polyphenols during simulation of wine aging. II. Analysis of desorbed polyphenol compounds from yeast lees

In the first part of this work, the analysis of the polyphenolic compounds remaining in the wine after different contact times with yeast lees during simulation of red wine aging was undertaken. To achieve a more precise view of the wine polyphenols adsorbed on lees during red wine aging and to establish a clear balance between adsorbed and remnant polyphenol compounds, the specific analysis of the chemical composition of the adsorbed polyphenolic compounds (condensed tannins and anthocyanins) after their partial desorbtion from yeast lees by denaturation treatments was realized in the second part of the study. The total recovery of polyphenol compounds from yeast lees was not complete, since a rather important part of the initial wine colored polyphenols, especially those with a dominant blue color component, remained strongly adsorbed on yeast lees, as monitored by color tristimulus and reflectance spectra measurements. All anthocyanins were recovered at a rather high percentage (about 62%), and it was demonstrated that they were not adsorbed in relation with their sole polarity. Very few monomeric phenolic compounds were extracted from yeast lees. With the use of drastic denaturing treatments, the total recovery of condensed tannins reached 83%. Such tannins extracted from yeast lees exhibited very high polymeric size and a rather high percentage of galloylated residues by comparison with initial wine tannins, indicating that nonpolar tannins were preferentially desorbed from yeast lees by the extraction treatments.     

Effect of aging on lees and of three different dry yeast derivative products on Verdejo white wine composition and sensorial characteristics

A study was made of the effect of aging on lees and of three different commercial yeast derivative products of different composition and degree of purification on the phenolic compounds, color, proteins, polysaccharides, and sensorial characteristics of white wines. The results obtained showed that the lees and yeast derivative products can interact or adsorb some of the phenolic compounds present in wines, reducing their concentration. This reduction depends on the treatment applied, the phenolic compound analyzed, and the stage of vinification or aging process. The use of lees and yeast derivative products can reduce the color intensity and the browning of the wines immediately following treatment. The monosaccharide and polysaccharide content of yeast derivative products depends on the manufacturing process and degree of purification of the product, both of which have an influence on wine treatments. After 6 months in the bottle, both the aging on lees and the treatment with commercial yeast derivative products gave rise to wines with better sensorial characteristics than in the case of the control wines.     

Pear distillates from pear juice concentrate: effect of lees in the aromatic composition

Pear juice obtained from pear concentrate was fermented at room temperature using Saccharomyces cerevisiae (BDX, ENOFERM, France) as the fermentation microorganism. During the fermentation process, total sugars were measured. High performance liquid chromatography analyses were used to monitor the fermentation process and to characterize the pear wine. The pear wine obtained was distilled with its lees using three different equipments: a glass alembic (a glass pot still coupled to a glass column), a copper alembic, and a glass alembic with the addition of 5 g/L of copper shavings to the pot still. The same distillations were repeated with the wine without its lees (separated by decanting). Several distillation fractions were collected, up to a total of 500 mL of distillate. Gas chromatography was used to identify and quantify the volatile compounds in each fraction, and the methanol and ethanol contents. Based on these results, the heart fraction was defined. ANOVA tests were performed on the heart fractions to determine quantitative differences between some volatile compounds depending on the equipment used and the presence or absence of the wine lees. From this series of ANOVA tests, it can be concluded that the concentrations of the compounds that are considered to have a negative effect on the quality of the distillates (methanol, ethyl acetate, furfural) decrease or do not change when they are distilled in the presence of lees and in the copper alembic. In addition, the concentrations of the positive compounds (ethyl decanoate and ethyl-2-trans-4-cis-decadienoate) increase in the presence of lees for all of the equipment tested. So, it can be assumed that the distillation of pear wine with its lees in copper alembic leads to a better quality product.     

Influence of yeast macromolecules on sweetness in dry wines: role of the saccharomyces cerevisiae protein Hsp12

Yeast autolysis during lees contact influences the organoleptic properties of wines especially by increasing their sweet taste. Although observed by winemakers, this phenomenon is poorly explained in enology. Moreover, the compounds responsible for sweetness in wine remain unidentified. This work provides new insights in this way by combining sensorial, biochemical and genetic approaches. First, we verified by sensory analysis that yeast autolysis in red wine has a significant effect on sweetness. Moderate additions of ethanol or glycerol did not have the same effect. Second, a sapid fraction was isolated from lees extracts by successive ultrafiltrations and HPLC purifications. Using nano-LC-MS/MS, peptides released by the yeast heat shock protein Hsp12p were distinctly identified in this sample. Third, we confirmed the sweet contribution of this protein by sensorial comparison of red wines incubated with two kinds of yeast strains: a wild-type strain containing the native Hsp12p and a deletion mutant strain that lacks the Hsp12p protein (Δ°HSP12 strain). Red wines incubated with wild-type strain showed a significantly higher sweetness than control wines incubated with Δ°HSP12 strains. These results demonstrated the contribution of protein Hsp12p in the sweet perception consecutive to yeast autolysis in wine.     

Changes in the sotolon content of dry white wines during barrel and bottle aging

GC-MS in electron ionization mode (EI) was used as a simple, sensitive method for assaying sotolon [4,5-dimethyl-3-hydroxy-2(5) H-furanone] in various dry white wines. The impact of barrel-aging conditions, that is, whether yeast lees were present or not, on the formation of sotolon in dry white wines was then studied. The sotolon content was highest in dry white wines aged in new barrels without lees, often exceeding the perception threshold (8 microg/L). These results demonstrated that yeast lees were capable of minimizing the formation of sotolon in dry white wines during aging. The sotolon and oxygen contents of several bottle of the same white wine were also compared 7 years after bottling. At the range of dissolved oxygen concentrations generally measured, between 5 and 100 microg/L, the sotolon content remained below its perception threshold in wine. The perception threshold was exceeded only in wines with oxygen concentrations above 500 microg/L. The presence of dissolved oxygen in the wine samples analyzed also resulted in a decrease in their free sulfur dioxide content.     

Formulation of low-cost fermentative media for lactic acid production with Lactobacillus rhamnosus using vinification lees as nutrients

Lees coming from different steps in white wine and red wine vinification were characterized under physicochemical analyses to determine the content in carbon, nitrogen, ashes, solids in suspension, organic compounds, and minerals. Due to the hydrolytic activity of Lactobacillus strains, lees without autolysis treatments were used directly as the unique nutrient or in combination with corn steep liquor to carry out the glucose to lactic acid fermentation with Lactobacillus rhamnosus CECT-288. Time courses of glucose and lactic acid were modeled according to reported models. Using 20 g/L of lees coming from the white wine technology and re-collected after the second decanting step before distillation, as the only nutrient, the values achieved (P = 105.5 g/L, Q(P) = 2.470 g/L.h) were even higher than those obtained with the costly MRS broth (P = 104.3 g/L, Q(P) = 2.251 g/L.h).     

Evaluation of the combined effects of enzymatic treatment and aging on lees on the aroma of wine from Bombino bianco grapes

In this study, two different doses of commercial beta-glucanase enzyme preparation were tested to verify their effect on wines aged on lees. These wines were compared with two samples with no enzymatic treatment. The former was aged on lees (control), and the latter was readily filtered off from the yeast cell biomass (standard). Analysis of variance (one-way ANOVA), the Tukey test, and principal component analysis (PCA) were applied to all of the samples, which were analyzed for aroma composition, along with galacturonic acid, total acidity, pH, and color. Results showed a large number of statistically significant differences among samples. In general, wines treated with beta-glucanase were characterized by higher concentration of many volatile compounds. The presence of lees and even more the exogenous enzymatic action enhanced almost all volatile compounds. Besides the high presence of ethyl esters, it is worth mentioning the behavior of hexanol and trans-3-hexenol, which are strongly enhanced by the presence of lees and by enzymatic treatments.     

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.     

Characterization of the nitrogen compounds released during yeast autolysis in a model wine system

The nitrogen composition of wines aged with yeast for a long period of time, as in the case of sparkling wines, depends on the composition of the base wine and on the compounds released by the yeast. In this paper, the release of the different classes of nitrogen compounds during autolysis of one of the strains of yeast used in the manufacture of sparkling wines has been studied. The yeast, Saccharomyces bayanus, was suspended in a model wine buffer, pH 3.0 and 10% ethanol, and incubated at 30 degrees C. Samples of the autolysate were taken after 4, 24, 48, 72, 168, and 360 h of autolysis. An electrophoretic and chromatographic study was conducted of the proteins, peptides with molecular weights higher and lower than 700 Da, and amino acids released during the autolysis. Using SDS-PAGE, it was observed that it was predominantly polypeptides with molecular weights lower than 10 000 that were released. Through HPLC of the fraction lower than 10 000 Da, it was observed that it is polypeptides with molecular weights of between 10 000 and 700 Da that are released first and that these later break up to give rise to peptides with molecular weights lower than 700 Da, which in turn break down into amino acids. This indicates that the nature of the nitrogen compounds present in wines aged with yeast depends on the aging time, being less polymerized as the aging time increases.     

Fenhexamid adsorption behavior on soil amended with wine lees

The adsorption of fenhexamid (FEN) [N-(2,3-dichloro-4-hydroxyphenyl)-1-methylcyclohexanecarboxamide] on vineyard soil amended with wine lees (WL) produced by vinery was studied. The adsorption extent depends on WL fraction. The addition of the centrifuged solid lees (SWL) increases the FEN adsorption on soil. Most likely, the organic insoluble fraction formed mainly by dead fermentation yeasts is responsible for the observed increase. The adsorption measured on some deactivated yeasts of wine fermentation shows that Saccharomyces cerevisiae are the most active in FEN retention. On the other hand, the soil amendment with whole WL decreases considerably the fungicide adsorption. This opposite effect may be the result of FEN hydrophobic bonds with the dissolved organic matter of lees that keeps fungicide in solution. This hypothesis is substantiated by the increased FEN solubility in the supernatant of centrifuged wine lees (LWL). The results of soil column mobility confirm that the elution with LWL increases the mobility of FEN in soil.     

Saccharomyces cerevisiae mannoproteins that protect wine from protein haze: their release during fermentation and lees contact and a proposal for their mechanism of action

A fraction containing the mannoproteins released during fermentation from the winemaking strain of Saccharomyces cerevisiae, Maurivin PDM, was able to reduce the visible protein haze in white wine. This fraction of haze protective mannoprotein material (HPM) could be recovered by either ultrafiltration or ethanol precipitation. The kinetics of the release of both mannose- and glucose-containing polymers during the growth cycle of PDM were determined as a guide to the release of HPM. Active HPM was first detected in the culture supernatant when the cells were exponentially growing. HPM was also released into the medium under an environment simulating winemaking conditions by PDM cells during fermentation as well as during storage on yeast lees. Since the amounts of HPM released during fermentation are greater than those subsequently extracted from the cell wall, fermentation would be a more viable procedure than extraction from yeast cells for the commercial production of HPM. Yeast invertase, a mannoprotein with haze protective activity, was used as a model substrate to investigate the mechanism of haze protection. Invertase was found to reduce visible turbidity but not prevent protein precipitation. Invertase itself did not precipitate but remained soluble in the wine. On the basis of these observations, we propose that the mechanism of haze protection may be one of competition between HPM and wine proteins for unknown wine component(s), the latter being required for the formation of large insoluble aggregates of denatured protein. As the available concentration of these components decreases, due to the presence of HPM, the particle size of the haze decreases and thus visible turbidity declines.     

Phenolic compounds and browning in sherry wines subjected to oxidative and biological aging

The composition in hydroxybenzoic and hydroxycinnamic acids, hydroxycinnamic esters, tyrosol, syringaldehyde, and flavan-3-ol derivatives of three different types of sherry wine obtained by aging of the same starting wine under different conditions was studied. So-called "fino" wine was obtained by biological aging under flor yeasts, "oloroso" wine by oxidative aging, and "amontillado" wine by a first stage of biological aging followed by a second oxidative step. On the basis of the results, the wines subjected to oxidative aging exhibited higher phenol contents, in addition to scarcely polar compounds absorbing at 420 nm that were absent in the wines obtained by biological aging. Taking into account that flavan-3-ol derivatives play an important role in wine browning, a model catechin solution was inoculated with flor yeast which, contrary to the findings of other authors in the absence of yeasts, formed no colored compounds. This different behavior may account for the resistance to browning of pale sherry wines in the presence of flor yeasts.     

Determination of spherosomes from lees materials

To evaluate the effectiveness in adsorbing organochlorine compounds such as chloroform, dichloromethane, or benzene by lees materials, the determination of spherosomes from different lees materials was established by using a hemacytometer under an optical microscope. Rice bran, wheat bran, rapeseed, linseed, okara, and sakekasu were used for this investigation, and activated carbon was also used as a standard adsorbent material. The number of spherosomes varied from 1.82 x 10(10) particles/g for sakekasu to 4.95 x 10(10) particles/g for wheat bran. There was a high correlation between the removal efficiency in adsorbing organochlorine compounds such as chloroform, dichloromethane, or benzene by lees materials and the number of spherosomes from different lees materials.     

Release of lipids during yeast autolysis in a model wine system

The release of lipids during the aging of sparkling wines in contact with yeast can influence wine sensory attributes and, especially, foam characteristics. Model systems allow study of the autolysis process in a reasonable period of time compared to natural conditions, at which it can last several months. In this paper, the release of the different classes of lipids during the autolysis of three commercial yeast strains in a model wine medium has been monitored. Due to the absence of accurate quantitative methods, an HPLC method for separating and quantifying the different neutral and polar yeast lipid classes was developed. Lipids were eluted through a YMC PVA-Sil column with a complex solvent mixture. Detection was carried out with a light-scattering detector. The yeasts were suspended in the model wine buffer and incubated at 30 degrees C for up to 12 days. A release of triacylglycerols, 1,3-diacylglycerols, 2-monoacylglycerols, free fatty acids, sterol esters, and sterols was observed over the first 2 days, a period that corresponded to the maximum loss of yeast viability. A decrease in most of these lipids was observed from day 2, possibly indicative of the release of yeast hydrolytic enzymes due to the breakdown of the cell wall. Phospholipids were not detected in any of the autolysates. The mean lipid content in the autolysates as a percentage of the total lipid content in the yeasts was 8.6% for sterol esters, 3.8% for sterols, 2% for triacylglycerols, and <2% for 1,3-diacylglycerols and free fatty acids.     

Recovery of squalene from wine lees using ultrasound assisted extraction-a feasibility study

The present work is a systematic approach for valorization of wine lees regarding the recovery of squalene, a bioactive lipid. Such a study is presented for the first time in literature. Separate examination of squalene content in "light" and "heavy" lees from different vinification processes by RP-HPLC demonstrated that these waste streams can be used as a source for this lipid, despite variations due to technological or genetic effects. Next, ultrasound assisted extraction of squalene from the "industrial waste" (the mixture of wine lees generated from different wines) using n-hexane was optimized with the aid of response surface methodology (independent variables: sonication duration and duty cycles). Autolysis was monitored through optical microscopy. Squalene yield (0.6 ± 0.08 g SQ/kg dry lees) was comparable to that of recently examined potential sources (0.2-0.35 g SQ/kg dry olive pomace and 0.06 g SQ/kg olive leaves).     

Influence of wine turbidity on the accumulation of volatile compounds from the oak barrel

In this work the influence of the turbidity of a red wine aged in oak barrels on the accumulation of volatile compounds has been studied. From the results obtained, it may be concluded that the wine turbidity had an effect on the concentration of many of the oak volatile compounds, especially furanic and phenolic compounds. In all cases the concentrations of furanic aldehydes and vanillin were significantly higher (p < 0.01) in the filtered wine than in the unfiltered wine. However, in the unfiltered wine the concentrations of gamma-butyrolactone and eugenol were higher. This difference in the concentrations would be due to the different compositions of the wines and the binding of compounds to some components of the lees. Likewise, it has been observed that the composition of the lees is fundamental in the binding of volatile compounds to the lees as the fermentative lees behave differently from the lees from the wine aging in barrels.     

Comparison of components released by fermented or active dried yeasts after aging on lees in a model wine

Comparison of different components released during autolysis with fermented or active dried yeast, which has never been reported previously, is related in this paper. Three autolysates were elaborated with Saccharomyces cerevisiae in a model wine (pH 3.5) at 30 or 18 degrees C. Composition of the autolysate appears to depend on both the growth medium and the physiological state of the yeast. The autolysate obtained from active dried yeast presents a higher total nitrogen concentration (a factor of 1.5-1.9 for the fermented yeast autolysate), a greater proportion of free amino acids (42 vs 16-25%), the lowest proportion of oligopeptides (25 vs 31-37%) and polypeptides (27 vs 45-34%), and an absence of nucleosides. Distribution of amino acids in peptides and proteins is relatively homogeneous in the autolysates but shows significant differences in free amino acids. Parietal macromolecules (mannoproteins and glucans) are present in greater quantities in autolysates elaborated from fermented yeast. The influence of the different fractions of these autolysates (<0.5, 0.5-1, 1-10, and >10 kDa) on bacterial growth has been investigated.     

Changes in nitrogen compounds in must and wine during fermentation and biological aging by flor yeasts.

Urea, ammonium, and free amino acid contents were quantified in a must from Vitis vinifera cv. Pedro Ximenez grapes and in fermented wine and after a short aging of this wine by Saccharomyces cerevisiae race capensis yeast under variable oxygen availability conditions. The previous compounds were also determined in a wine in which the nitrogen source was depleted by the same race of flor yeast (old wine) and also following the addition of ammonium ion, L-glutamic acid, and L-proline. Under specific conditions such as low oxygen level and the absence of some nutrients, the yeasts release some amino acids including L-threonine, L-tryptophan, L-cysteine, and L-methionine to the medium. These amino acids must originate primarily in a de novo synthesis from ethanol that regenerates NAD(P)+. On the basis of these results, the yeasts may be able to use amino acids not only as nitrogen sources but also as redox agents to balance the oxidation-reduction potential under conditions of restricted oxygen, when electron transport along the respiratory chain may be hindered or limited.