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.     

Genetic determinants of the release of mannoproteins of enological interest by Saccharomyces cerevisiae

Cell wall mannoproteins released by Saccharomyces cerevisiae during wine fermentation and aging have recently attracted the attention of enologists and researchers in enology due to their positive effect over a number of technological and quality properties of the wines, including protein and tartaric stability, aroma and color stability, astringency, mouthfeel, malolactic fermentation, or foam properties of sparkling wines. This work has investigated the effect of deletions involving genes related to cell wall biogenesis on the release of mannoproteins, as well as the effect of the released mannoproteins on wine protein stability. When available, the phenotypes have been studied in different genetic backgrounds, in haploid or diploid strains, and in homo- or heterozygosis. Strains deleted for GAS1, GPI7, or KNR4 release higher amounts of mannoproteins and polysaccharides to the medium. These increased amounts of mannoproteins and polysaccharides lead to a stronger stability of Sauvignon Blanc wines against protein haze.     

Roles of grape thaumatin-like protein and chitinase in white wine haze formation

Grape chitinase was found to be the primary cause of heat-induced haze formation in white wines. Chitinase was the dominant protein in a haze induced by treating Sauvignon blanc wine at 30 °C for 22 h. In artificial wines and real wines, chitinase concentration was directly correlated to the turbidity of heat-induced haze formation (50 °C for 3 h). Sulfate was confirmed to have a role in haze formation, likely by converting soluble aggregates into larger visible haze particles. Thaumatin-like protein was detected in the insoluble fraction by SDS-PAGE analysis but had no measurable impact on turbidity. Differential scanning calorimetry demonstrated that the complex mixture of molecules in wine plays a role in thermal instability of wine proteins and contributes additional complexity to the wine haze phenomenon.     

Sulfate--a candidate for the missing essential factor that is required for the formation of protein haze in white wine

Protein haze formation in white wine is dependent on the presence of both wine protein and other unknown wine components, termed factor(s) X. The ability to reconstitute protein haze upon heating artificial model wine solutions (500 mg/L thaumatin, 12% ethanol, 4 g/L tartaric acid) to which candidate components were added was employed to identify factor(s) X. No protein haze was formed in the absence of additives. The individual or combined addition of caffeic acid, caftaric acid, epicatechin, epigallocatechin-O-gallate, gallic acid, or ferulic acid at typical white wine concentrations did not generate protein haze. However, PVPP fining of commercial wines resulted in a reduction in protein haze, suggesting that phenolic compounds may play a modulating role in haze formation. To elucidate the nature of the unknown factor(s) wine was fractionated and fractions were back-added to model wine and tested for their essentiality. Wine fractions were generated by ultrafiltration, reverse-phase chromatography, and mixed-mode anion-exchange and reverse-phase chromatography. The only purified fraction containing the essential component(s) was free of phenolic compounds, and analysis by mass spectrometry identified sulfate anion as the dominant component. Reconstitution with KHSO4 using either commercially available thaumatin or wine proteins confirmed the role of sulfate in wine protein haze formation. The two main wine proteins, thaumatin-like protein and chitinase, differed in their haze response in model wines containing sulfate. Other common wine anions, acetate, chloride, citrate, phosphate, and tartrate, and wine cations, Fe(2+/3+) and Cu(+/2+), when added at typical white wine concentrations were not found to be essential for protein haze formation.     

Effect of different strains of Saccharomyces cerevisiae on production of volatiles in Napa Gamay wine and Petite Sirah wine

Napa Gamay grapes were fermented with four different strains of the yeast Saccharomyces cerevisiae (VL1, MI16, Fermirouge, and RA17). Petite Sirah grapes were fermented with seven different strains of the same yeast (BM45, Fermirouge, RA17, NI, CX3079, A350, and A796). Volatile compounds formed in the wines were analyzed by gas chromatography/mass spectrometry. Volatile compounds found in both wines were alcohols, esters, and acids, as well as some miscellaneous compounds. Isoamyl alcohol was the compound found in the highest relative amount with all four yeast strains in the Napa Gamay wines, followed by 2-phenyl ethanol, monoethyl succinate, and hexanoic acid. The relative amounts of isoamyl alcohol ranged from 30.84% (VL1) to 43.28% (RA17). Major volatile compounds found in Petite Sirah wines were isoamyl alcohol, 2-phenyl ethanol, 2-hydroxy ethyl propanoate, monoethyl succinate, and octanoic acid. The several esters, including 2-hydroxyethyl propanoate, may contribute to the fruity flavor of Petite Sirah wines. Overall, the S. cerevisiae yeast strains used to ferment Napa Gamay grapes and Petite Sirah grapes produced the same major components, with certain variations in formation levels.     

Alkali extraction of beta-d-glucans from Saccharomyces cerevisiae cell wall and study of their adsorptive properties toward zearalenone

The isolated cell wall of Saccharomyces cerevisiae has some capacity to adsorb zearalenone (affinity near 30%) and reduce the bioavailability of toxins in the digestive tract. The adsorption process was quantified in vitro, and the data obtained when plotted with Hill's equation indicated a cooperative process. The model showed that the adsorption capacity was related to the yeast cell wall composition. This work focused on the role of various beta-d-glucan types in the efficacy of zearalenone adsorption by yeast cell wall and sought to elucidate some of the adsorption mechanisms. Zearalenone was mixed at 37 degrees C with a constant quantity of alkali-soluble or alkali-insoluble beta-d-glucans isolated from yeast cell walls, and the amount of adsorbed zearalenone was measured. Given that the alkali solubility of beta-d-glucans is a determining factor for their three-dimensional conformation and that the alkali-insoluble fraction had a greater affinity (up to 50%) than the alkali-soluble fraction ( approximately 16%), it was concluded that the three-dimensional structure strongly influences the adsorption process. The alkali insolubility of beta-d-glucans led to the formation of single and/or triple helices, which have been identified as the most favorable structures for zearalenone adsorption efficacy. The beta(1,3)-d-glucan and beta(1,6)-d-glucan compositions of the two alkali-extracted fractions and their involvement in the adsorption process are discussed.     

Saccharomyces cerevisiae oxidative response evaluation by cyclic voltammetry and gas chromatography-mass spectrometry

This study is focused on the evaluation of the impact of Saccharomyces cerevisiae metabolism in the profile of compounds with antioxidant capacity in a synthetic wine during fermentation. A bioanalytical pipeline, which allows for biological systems fingerprinting and sample classification by combining electrochemical features with biochemical background, is proposed. To achieve this objective, alcoholic fermentations of a minimal medium supplemented with phenolic acids were evaluated daily during 11 days, for electrochemical profile, phenolic acids, and the volatile fermentation fraction, using cyclic voltametry, high-performance liquid chromatography-diode array detection, and headspace/solid-phase microextraction/gas chromatography-mass spectrometry (target and nontarget approaches), respectively. It was found that acetic acid, 2-phenylethanol, and isoamyl acetate are compounds with a significative contribution for samples metabolic variability, and the electrochemical features demonstrated redox-potential changes throughout the alcoholic fermentations, showing at the end a similar pattern to normal wines. Moreover, S. cerevisiae had the capacity of producing chlorogenic acid in the supplemented medium fermentation from simple precursors present in the minimal medium.     

高压脉冲电场对酿酒酵母蛋白质及DNA的影响

为探究高压脉冲电场（PEF）对微生物的钝化机理,本实验主要采用Lowry法、SDS-PAGE电泳、APIZYM试剂盒、流式细胞仪等方法测定PEF处理对酿酒酵母蛋白质含量、胞内酶活性和DNA等的影响。结果显示：PEF处理后,酿酒酵母胞内蛋白质含量显著降低(p<0.05),SDS-PAGE 电泳图分析发现酿酒酵母胞内蛋白谱图不清晰,且有小分子和 60 KDa 以上大分子蛋白谱带缺失,酿酒酵母细胞内 13 种酶的活性均有不同程度的下降,细胞DNA含量降低,部分DNA变性。结论：PEF钝化微生物与其胞内酶失活、蛋白质和DNA发生外泄或变性有关。     

Effect of fungicide residues on the aromatic composition of white wine inoculated with three Saccharomyces cerevisiae strains

The effects of three fungicide residues (cyprodinil, fludioxonil, and pyrimethanil) on the aromatic composition (acids, alcohols, and esters) of Vitis vinifera white wines (var. Airén) inoculated with three Saccharomyces cerevisiae strains (syn. bayanus, cerevisiae, and syn. uvarum) are studied. The aromatic exponents were extracted and concentrated by adsorption-thermal desorption and were determined by gas chromatography using a mass selective detector. The addition of the three fungicides at different doses (1 and 5 mg/L) produces significant differences in the acidic fraction of the aroma, especially in the assays inoculated with S. cerevisiae, although the final contents do not exceed the perception thresholds. The lower quality wines, according to isomeric alcohol content [(Z)-3-hexen-1-ol and 3-(methylthio)propan-1-ol] are those obtained by inoculation with S. cerevisiae(syn. bayanus) and addition of cyprodinil. The addition of fungicides in the assays inoculated with S. cerevisiae (syn. bayanus) produces an increase in the ethyl acetate and isoamyl acetate contents, which causes a decrease in the sensorial quality of the wine obtained.     

Biocatalytic production of dihydrocoumarin from coumarin by Saccharomyces cerevisiae

Natural dihydrocoumarin, which is of great interest in the flavor industry, was biotechnologically produced from pure coumarin or tonka bean meal with Pseudomonas orientalis, Bacillus cereus, and various Saccharomyces cerevisiae strains. Coumarin was shown to be converted to melilotic acid, which yielded dihydrocoumarin upon distillation during purification. About 1.0 g/L product was obtained from 25 g/L tonka beans with S. cerevisiae within 147 h. This dihydrocoumarin thus fulfills all of the criteria of a natural raw material and can be used as a natural flavoring in accordance with U.S. and European Union regulations.     

Comparative analysis of the effects of locally used herbicides and their active ingredients on a wild-type wine Saccharomyces cerevisiae strain

Herbicides are released to the environment with potential ecotoxicological risks for mammals. Yeast is a good model to elucidate toxicity mechanisms. We investigated how three commercial herbicides (Proper Energy, Pointer, and Silglif) and their active ingredients (respectively, fenoxaprop-P-ethyl, tribenuron methyl, and glyphosate) can affect biological activities of an oenological Saccharomyces cerevisiae strain, which may be resident on grape vineyards of the same geographical areas where herbicides are used. The use of commercial grade herbicides employed in Italy allowed us to reproduce the same conditions applied in crops; at the same time, assaying pure single active compounds made it possible to compare the effects obtained with commercial formulations. Interestingly, we found that while pure active compounds affect cell growth and metabolism at a lower extent, commercial preparations have a significant major negative influence on yeast biology.     

Maceration enzymes and mannoproteins: a possible strategy to increase colloidal stability and color extraction in red wines

Different strategies were adopted to achieve increases in color stability in Tempranillo wines: (i) addition of maceration enzymes directly to the must, (ii) addition of commercial mannoproteins to the must, and (iii) inoculation of must with yeast overexpressed of mannoproteins. The addition of enzymes favored color extraction, and the wines obtained presented higher values of wine color, color intensity, bisulfite-stable color, and visually enhanced color intensity. The enzyme hydrolytic activity produced an increase in the acid polysaccharide content and polyphenol index and yielded to wines with more astringency, tannin, and length. Added mannoproteins had clearer effects on the analyzed parameters than yeast. Contrary to what may be thought, mannoproteins did not maintain the extracted polyphenols in colloidal dispersion and neither ensured color stability. These compounds clearly modified the gustative structure of the wines, enhancing the sweetness and roundness.     

Yeast (Saccharomyces cerevisiae) protein concentrate: preparation, chemical composition, and nutritional and functional properties

The main objective of the present study was to compare the composition and functional and nutritional properties of whole yeast cells (WY) from an ethanol distillery with those of a phosphorylated protein concentrate (PPC) prepared from the same cells. Comparisons were also made of PPC with texturized soy protein (TSP) and soy protein isolate (SPI), both acquired in the local market. Yeast (Saccharomyces cerevisiae) is a rich source of protein, soluble fiber, and some minerals. Saturated fatty acids predominated over monounsaturated and polyunsaturated in both WY and PPC. The functional properties of PPC were similar to those of SPI and TSP. Both soy products and PPC replaced 20 or 40% chuck roll protein without affecting the emulsion properties of the meat products. Amino acid scoring was high for both WY and PPC; digestibility was higher (90%) for PPC and lower (68%) for WY. The protein nutritive value of PPC did not differ from that of casein and was significantly higher than that for WY.     

Red wine antioxidants bind to human lipoproteins and protect them from metal ion-dependent and -independent oxidation

Plant-derived polyphenols may exert beneficial effects on atherosclerosis and cardiovascular diseases, in part, because of their antioxidant properties. In this study we compared the effects of unbound (free) and lipoprotein-associated red wine components on in vitro antioxidant protection of human low-density lipoprotein (LDL). Preincubation of LDL (1 mg protein/mL) with 0-2.5% (v/v) red wine for 3 h at 37 degrees C followed by gel filtration to remove unbound red wine components resulted in a dose-dependent, up to 4-fold increase in LDL-associated antioxidant capacity (measured as Trolox equivalents). Similar results were obtained with high-density lipoprotein (HDL) and bovine serum albumin (BSA). Furthermore, LDL was subjected to oxidation by copper and aqueous peroxyl radicals (2,2'-azobis[2-amidinopropane] dihydrochloride, AAPH). Under both types of oxidative stress, LDL-associated and free red wine components significantly decreased oxidation of the lipoprotein's protein moiety (assessed by tryptophan fluorescence) and lipid moiety (assessed by thiobarbituric acid-reactive substances and conjugated dienes). Similar protective effects of red wine components were observed against HDL oxidation. In contrast, red wine exerted a pro-oxidant effect on copper-induced oxidation of BSA tryptophan residues, while protecting them from AAPH-induced oxidation. Ascorbate strongly enhanced the protective effect of red wine against copper-induced LDL oxidation, and had an additive effect against AAPH-induced oxidation. Our data indicate that red wine components bind to LDL and HDL and protect these lipoproteins from metal ion-dependent and -independent protein and lipid oxidation.     

Antioxidant protection of resveratrol and catechin in Saccharomyces cerevisiae

Moderate consumption of red wine reduces the risk of heart disease and extends lifespan, but the relative contribution of wine polyphenols to these effects is unclear. In this work, the capacity of resveratrol and catechin to protect the eukaryotic microorganism Saccharomyces cerevisiae against oxidative stress caused by different agents, hydrogen peroxide, carbon tetrachloride, and cadmium, was evaluated. Under all stress conditions, both polyphenols increased tolerance, although their protection was more evident under peroxide exposure. By using mutant strains deficient in specific antioxidant defense systems (superoxide dismutases, catalase, or glutathione), it was observed that increased H2O2 tolerance produced by both polyphenols was associated with catalase, as well as the rise in survival rates caused by resveratrol under CCl4. The acquisition of tolerance was correlated with a reduction in lipid peroxidation, indicating that the antioxidant property of resveratrol and catechin involves protection against membrane oxidation.     

Qualitative and quantitative evaluation of the genomic DNA extracted from GMO and non-GMO foodstuffs with four different extraction methods

The presence of DNA in foodstuffs derived from or containing genetically modified organisms (GMO) is the basic requirement for labeling of GMO foods in Council Directive 2001/18/CE (Off. J. Eur. Communities 2001, L1 06/2). In this work, four different methods for DNA extraction were evaluated and compared. To rank the different methods, the quality and quantity of DNA extracted from standards, containing known percentages of GMO material and from different food products, were considered. The food products analyzed derived from both soybean and maize and were chosen on the basis of the mechanical, technological, and chemical treatment they had been subjected to during processing. Degree of DNA degradation at various stages of food production was evaluated through the amplification of different DNA fragments belonging to the endogenous genes of both maize and soybean. Genomic DNA was extracted from Roundup Ready soybean and maize MON810 standard flours, according to four different methods, and quantified by real-time Polymerase Chain Reaction (PCR), with the aim of determining the influence of the extraction methods on the DNA quantification through real-time PCR.     

Phosphorus in wine: comparison of atomic absorption spectrometry methods

Phosphorus in wine may be determined directly or indirectly by atomic absorption spectrometry. The direct method uses the carbon rod atomizer as the excitation source and a phosphorus hollow cathode lamp. In the indirect determination, one measures the amount of molybdenum that will complex with phosphorus in the wine. Both nitrous oxide-acetylene and air-acetylene flames are suitable as atomization sources in this indirect method. The resultant data have been compared with those from the AOAC colorimetric method (11.032-11.034). A 2-sample comparison test showed the results to be insignificantly different at the 95% confidence limits.     

瘤胃真菌与酿酒酵母仿生共培养提升秸秆发酵产乙醇量

玉米秸秆中具有较高的纤维素、半纤维素含量,是一种具有稳定产率、可集中处理、可代替木材作制浆原料的生物质材料。为了研究厌氧微生物与酵母共培养预处理玉米秸秆的产物,该研究模拟反刍动物消化玉米秸秆的过程,从羊瘤胃液中分离出厌氧真菌（Pecoramyces sp.）。以玉米秸秆茎皮碎为底物,与厌氧真菌、酿酒酵母菌S1145在39℃进行共培养72 h,分析发酵对秸秆茎皮降解及其代谢产物的影响。结果表明,在瘤胃真菌作用下,添加不同量的酿酒酵母可对代谢产物中乙醇含量产生影响,其中添加5 mL酿酒酵母时产生的乙醇含量最高,占总代谢产物比例为32.09%,相对于未添加酿酒酵母的对照组,乙醇含量提高了23.04百分点。研究表明,在厌氧真菌与酿酒酵母共培养预处理玉米秸秆茎皮的过程中,添加酿酒酵母可提高乙醇产量,为玉米秸秆高效资源化处理和生物质燃料生产提供了一种可靠的方法。     

Comparative study of protein stabilization in white wine using zirconia and bentonite: physicochemical and wine sensory analysis

A semi-industrial application of the continuous stabilization of white wine protein using a column packed with zirconia was studied and compared to the traditional bentonite treatment using a Macabeu white wine. Physicochemical and wine sensory properties were evaluated using a rating system and triangle tests. Continuous protein stabilization was analyzed in three residence times, and the equivalent of 300 BV of wine was used for both treatments. Wine protein content was reduced by 21%, 40%, and 42% using the continuous process with residence times of 7.5, 15, and 30 min, respectively, and by 61.4% using the bentonite treatment. The wines obtained from the packed column were protein stable up to 25, 75, and 175 BV for residence times of 7.5, 15, and 30 min, respectively. The amount of polyphenol removed was less than 10%, and similar amounts were removed from the wine regardless of residence time, while 20.6% of polyphenol was removed using bentonite. The physicochemical and sensory properties of wine treated with bentonite were similar to those of wine treated with zirconia.     

Aroma extracts from oyster Crassostrea gigas: comparison of two extraction methods.

The study of the aroma of oysters is of great economic interest in France because it enables their organoleptic quality to be verified. The aim of this study is to optimize the extraction methods of the volatile compounds of oysters Crassostrea gigas in order to obtain an extract with an odor as close as possible to that of the original oysters'. Oyster aroma is rarely studied, and its sensory profile has not been investigated to date. Two extraction methods were studied: vacuum hydrodistillation carried out at 20 degrees C with noncrushed oyster using ultrapure water and dynamic headspace carried out using noncrushed oyster during a 30 min purge. They were compared with regard to their sensory characteristics by a panel of seven judges, all trained in seafood aroma recognition. This study has shown that vacuum hydrodistillation is the better method to obtain an extract closest in aroma to the oyster reference.