酵母菌在红葡萄酒酒精发酵串罐中稳定性研究

在生产条件下,对酵母菌在红葡萄酒酒精发酵串罐过程中的稳定性进行了研究。结果表明,在近1个月的时间内（相当于酵母菌细胞无性繁殖了200代）,串罐过程中的酵母菌细胞不仅能保持初始酵母菌的发酵活性和优良特性的稳定性,而且由于葡萄汁的选择作用,串罐用的酵母菌细胞的发酵活性比初始酵母菌的活性更强,因而其酒精发酵的启动和速度都更快。     

Ethanol Production and Maximum Cell Growth Are Highly Correlated with Membrane Lipid Composition during Fermentation as Determined by Lipidomic Analysis of 22 Saccharomyces cerevisiae Strains

Optimizing ethanol yield during fermentation is important for efficient production of fuel alcohol, as well as wine and other alcoholic beverages. However, increasing ethanol concentrations during fermentation can create problems that result in arrested or sluggish sugar-to-ethanol conversion. The fundamental cellular basis for these problem fermentations, however, is not well understood. Small-scale fermentations were performed in a synthetic grape must using 22 industrial Saccharomyces cerevisiae strains (primarily wine strains) with various degrees of ethanol tolerance to assess the correlation between lipid composition and fermentation kinetic parameters. Lipids were extracted at several fermentation time points representing different growth phases of the yeast to quantitatively analyze phospholipids and ergosterol utilizing atmospheric pressure ionization-mass spectrometry methods. Lipid profiling of individual fermentations indicated that yeast lipid class profiles do not shift dramatically in composition over the course of fermentation. Multivariate statistical analysis of the data was performed using partial least-squares linear regression modeling to correlate lipid composition data with fermentation kinetic data. The results indicate a strong correlation (R² = 0.91) between the overall lipid composition and the final ethanol concentration (wt/wt), an indicator of strain ethanol tolerance. One potential component of ethanol tolerance, the maximum yeast cell concentration, was also found to be a strong function of lipid composition (R² = 0.97). Specifically, strains unable to complete fermentation were associated with high phosphatidylinositol levels early in fermentation. Yeast strains that achieved the highest cell densities and ethanol concentrations were positively correlated with phosphatidylcholine species similar to those known to decrease the perturbing effects of ethanol in model membrane systems.     

Nitrogen demand of different yeast strains during alcoholic fermentation. Importance of the stationary phase

The nitrogen demand of industrial yeast strains were compared. Substantial differences were found between strains. These did not change regardless of the initial medium composition and added nitrogen source. To separately study growth and stationary phases, we ran fermentations with different nitrogen feeding profiles: a) exponentially fed fermentations with a long growth phase, and b) constant rate fermentations with nitrogen addition during the stationary phase. Differences between stains mostly appeared during the second phase. Measuring nitrogen requirements under such conditions would thus be an interesting complementary test when selecting new strains especially for enological purposes since most fermentation kinetics are nitrogen limited.     

Oxygen Response of the Wine Yeast Saccharomyces cerevisiae EC1118 Grown under Carbon-Sufficient,Nitrogen-Limited Enological Conditions

Discrete additions of oxygen play a critical role in alcoholic fermentation. However, few studies have quantitated the fate of dissolved oxygen and its impact on wine yeast cell physiology under enological conditions. We simulated the range of dissolved oxygen concentrations that occur after a pump-over during the winemaking process by sparging nitrogen-limited continuous cultures with oxygen-nitrogen gaseous mixtures. When the dissolved oxygen concentration increased from 1.2 to 2.7 μM, yeast cells changed from a fully fermentative to a mixed respirofermentative metabolism. This transition is characterized by a switch in the operation of the tricarboxylic acid cycle (TCA) and an activation of NADH shuttling from the cytosol to mitochondria. Nevertheless, fermentative ethanol production remained the major cytosolic NADH sink under all oxygen conditions, suggesting that the limitation of mitochondrial NADH reoxidation is the major cause of the Crabtree effect. This is reinforced by the induction of several key respiratory genes by oxygen, despite the high sugar concentration, indicating that oxygen overrides glucose repression. Genes associated with other processes, such as proline uptake, cell wall remodeling, and oxidative stress, were also significantly affected by oxygen. The results of this study indicate that respiration is responsible for a substantial part of the oxygen response in yeast cells during alcoholic fermentation. This information will facilitate the development of temporal oxygen addition strategies to optimize yeast performance in industrial fermentations.     

Effect of Yeast Hulls on Stuck and Sluggish Wine Fermentations: Importance of the Lipid Component

The effect of yeast hulls (yeast ghosts) on sluggish or stuck white wine fermentations was studied. The enhancing effect on yeast growth and fermentation rate displayed by the hulls was shown to be similar to the effect provided by lipid extract from the same hulls. Unsaturated fatty acids and sterols were incorporated into the yeast from lipid extracts during fermentation carried out under oxygen-limited conditions. Adsorption of toxic medium-chain fatty acid (decanoic acid) onto the yeast hulls took place through a dialysis membrane. However, when the hulls were placed inside a dialysis bag, the increase in yeast growth and fermentation rate seen when freely suspended hulls were used did not occur. Accordingly, the effect of yeast hulls in preventing stuck fermentations cannot be attributed only to the adsorption and consequent removal of medium-chain fatty acids from the juice.     

Fine measurement of ergosterol requirements for growth of Saccharomyces cerevisiae during alcoholic fermentation

Yeasts can incorporate a wide variety of exogenous sterols under strict anaerobiosis. Yeasts normally require oxygen for growth when exogenous sterols are limiting, as this favours the synthesis of lipids (sterols and unsaturated fatty acids). Although much is known about the oxygen requirements of yeasts during anaerobic growth, little is known about their exact sterol requirements in such conditions. We developed a method to determine the amount of ergosterol required for the growth of several yeast strains. We found that pre-cultured yeast strains all contained similar amounts of stored sterols, but exhibited different ergosterol assimilation efficiencies in enological conditions [as measured by the ergosterol concentration required to sustain half the number of generations attributed to ergosterol assimilation (P₅₀)]. P₅₀ was correlated with the intensity of sterol synthesis. Active dry yeasts (ADYs) contained less stored sterols than their pre-cultured counterparts and displayed very different ergosterol assimilation efficiencies. We showed that five different batches of the same industrial Saccharomyces cerevisiae ADY exhibited significantly different ergosterol requirements for growth. These differences were mainly attributed to differences in initial sterol reserves. The method described here can therefore be used to quantify indirectly the sterol synthesis abilities of yeast strains and to estimate the size of sterol reserves.     

Lipid nutrition of Saccharomyces cerevisiae in winemaking

Biosynthesis of cell membrane lipids is a crucial metabolic pathway for the growth and viability of eucaryotic microorganisms. In Saccharomyces cerevisiae, unsaturated fatty acids and ergosterol synthesis needs molecular oxygen. Stuck and sluggish fermentations are related to this aspect of metabolism and constitute a major problem in the wine industry. Anaerobiosis, when lipids are not available in the growth medium, highly stresses cells. They release lipid biosynthesis metabolites and soon cease to multiply. This paper describes an investigation of the nutritional role of exogenous lipids from inactivated yeast cells (IYCs). Fermentations were carried out in a nitrogen-rich synthetic medium similar to grape juice with glucose and fructose as carbon sources, without lipid sources, and in anaerobiosis. The effect of the addition of IYC was assessed. Cell growth, cell lipid composition, glucose and fructose consumption, and acetic acid production were measured during fermentation. Addition of IYC boosted cell growth and sugar consumption, whereas acetic acid production decreased. Biomass yield was influenced by ergosterol availability and increased when IYCs were added. Fatty acid composition of yeast cells was changed by IYC addition.     

Copper Tolerance and Biosorption of Saccharomyces cerevisiae during Alcoholic Fermentation

At high levels, copper in grape mash can inhibit yeast activity and cause stuck fermentations. Wine yeast has limited tolerance of copper and can reduce copper levels in wine during fermentation. This study aimed to understand copper tolerance of wine yeast and establish the mechanism by which yeast decreases copper in the must during fermentation. Three strains of Saccharomyces cerevisiae (lab selected strain BH8 and industrial strains AWRI R2 and Freddo) and a simple model fermentation system containing 0 to 1.50 mM Cu²⁺ were used. ICP-AES determined Cu ion concentration in the must decreasing differently by strains and initial copper levels during fermentation. Fermentation performance was heavily inhibited under copper stress, paralleled a decrease in viable cell numbers. Strain BH8 showed higher copper-tolerance than strain AWRI R2 and higher adsorption than Freddo. Yeast cell surface depression and intracellular structure deformation after copper treatment were observed by scanning electron microscopy and transmission electron microscopy; electronic differential system detected higher surface Cu and no intracellular Cu on 1.50 mM copper treated yeast cells. It is most probably that surface adsorption dominated the biosorption process of Cu²⁺ for strain BH8, with saturation being accomplished in 24 h. This study demonstrated that Saccharomyces cerevisiae strain BH8 has good tolerance and adsorption of Cu, and reduces Cu²⁺ concentrations during fermentation in simple model system mainly through surface adsorption. The results indicate that the strain selected from China’s stress-tolerant wine grape is copper tolerant and can reduce copper in must when fermenting in a copper rich simple model system, and provided information for studies on mechanisms of heavy metal stress.     

Evidence for the Existence of “Survival Factors” as an Explanation for Some Peculiarities of Yeast Growth, Especially in Grape Must of High Sugar Concentration

The retardation and arrest of fermentation, observed before the complete sugar consumption of high-sugar grape must, come from an inhibition of the yeast metabolism during its decline phase and are variable with the strain. The addition of nutritional growth factors stimulates the initial growth of the yeast but is ineffective in the decline phase. Some substances, known previously as yeast anaerobic growth factors (sterols, oleanolic acid, oxytocin), in some conditions (initially aerated grape must and aerobically cultivated yeast) act by increasing the viability of the resting cells and prolonging their fermentation activity. These substances have been named “survival factors.”     

Fermentation of soybean oil deodorizer distillate with Candida tropicalis to concentrate phytosterols and to produce sterols-rich yeast cells

Phytosterols have been recovered from the deodorizer distillate produced in the final deodorization step of vegetable oil refining by various processes. The deodorizer distillate contains mainly free fatty acids (FFAs), phytosterols, and tocopherols. The presence of FFAs hinders recovery of phytosterols. In this study, fermentation of soybean oil deodorizer distillate (SODD) with Candida tropicalis 1253 was carried out. FFAs were utilized as carbon source and converted into cellular components as the yeast cells grew. Phytosterols concentration in SODD increased from 15.2 to 28.43 % after fermentation. No significant loss of phytosterols was observed during the process. Microbial fermentation of SODD is a potential approach to concentrate phytosterols before the recovery of phytosterols from SODD. During SODD fermentation, sterols-rich yeast cells were produced and the content of total sterols was as high as 6.96 %, but its major sterol was not ergosterol, which is the major sterol encountered in Saccharomyces cerevisiae. Except ergosterol, other sterols synthesized in the cells need to be identified.     

Influence of grape treatment on the wine yeast populations isolated from spontaneous fermentations

AIM: To study the influence of different methods of grape treatment in wineries on the diversity of the yeast species in spontaneous fermentations. METHODS AND RESULTS: Grapes were crushed and pressed in three different ways followed by spontaneous fermentation. The same grape material picked and crushed aseptically directly in the vineyard served as control. Yeasts isolated at different stages of the fermentation were characterized by 5.8S-ITS-RFLP. Yeasts of the Saccharomyces sensu stricto complex were additionally analysed by microsatellite polymerase chain reaction fingerprinting. The diversity of yeast species isolated from winery fermentations was much greater than from the vineyard fermentation in respect to yeasts of the genus Saccharomyces as well as non-Saccharomyces. CONCLUSIONS: Oenonogical methods alter significantly the yeast diversity in spontaneous fermentations of grape juice. SIGNIFICANCE AND IMPACT OF THE STUDY: Managing spontaneous fermentations successfully depends not only on choosing the suitable grapes but also on the crushing and pressing techniques leading to different yeast populations.     

Evidence for the existence of "survival factors" as an explanation for some peculiarities of yeast growth, especially in grape must of high sugar concentration

The retardation and arrest of fermentation, observed before the complete sugar consumption of high-sugar grape must, come from an inhibition of the yeast metabolism during its decline phase and are variable with the strain. The addition of nutritional growth factors stimulates the initial growth of the yeast but is ineffective in the decline phase. Some substances, known previously as yeast anaerobic growth factors (sterols, oleanolic acid, oxytocin), in some conditions (initially aerated grape must and aerobically cultivated yeast) act by increasing the viability of the resting cells and prolonging their fermentation activity. These substances have been named "survival factors."     

Use of flow cytometry to monitor cell damage and predict fermentation activity of dried yeasts

Viable dried yeast is used as an inoculum for many fermentations in the baking and wine industries. The fermentative activity of yeast in bread dough or grape must is a critical parameter of process efficiency. Here, it is shown that fluorescent stains and flow cytometry can be used in concert to predict the abilities of populations of dried bakers' and wine yeasts to ferment after rehydration. Fluorescent dyes that stain cells only if they have damaged membrane potential (oxonol) or have increased membrane permeability (propidium iodide) were used to analyse, by flow cytometry, populations of rehydrated yeasts. A strong relationship (r2 = 0.99) was found between the percentages of populations staining with the oxonol and the degree of cell membrane damage as measured by the more traditional method of leakage of intracellular compounds. There were also were good negative relationships (r2 > or = 0.83) between fermentation by rehydrated bakers' or wine dry yeasts and percentage of populations staining with either oxonol or propidium iodide. Fluorescent staining with flow cytometry confirmed that factors such as vigour of dried yeast mixing in water, soaking before stirring, rehydration in water or fermentation medium and temperature of rehydration have profound effects on subsequent yeast vitality. These experiments indicate the potential of flow cytometry as a rapid means of predicting the fermentation performance of dried bakers' and wine yeasts.     

Nitrogen availability of grape juice limits killer yeast growth and fermentation activity during mixed-culture fermentation with sensitive commercial yeast strains.

The competition between selected or commercial killer strains of type K2 and sensitive commercial strains of Saccharomyces cerevisiae was studied under various conditions in sterile grape juice fermentations. The focus of this study was the effect of yeast inoculation levels and the role of assimilable nitrogen nutrition on killer activity. A study of the consumption of free amino nitrogen (FAN) by pure and mixed cultures of killer and sensitive cells showed no differences between the profiles of nitrogen assimilation in all cases, and FAN was practically depleted in the first 2 days of fermentation. The effect of the addition of assimilable nitrogen and the size of inoculum was examined in mixed killer and sensitive strain competitions. Stuck and sluggish wine fermentations were observed to depend on nitrogen availability when the ratio of killer to sensitive cells was low (1:10 to 1:100). A relationship between the initial assimilable nitrogen content of must and the proportion of killer cells during fermentation was shown. An indirect relationship was found between inoculum size and the percentage of killer cells: a smaller inoculum resulted in a higher proportion of killer cells in grape juice fermentations. In all cases, wines obtained with pure-culture fermentations were preferred to mixed-culture fermentations by sensory analysis. The reasons why killer cells do not finish fermentation under competitive conditions with sensitive cells are discussed.     

Relationship Between the Sterol Content of Yeast Cells and Their Fermentation Activity in Grape Must

In grape must of high sugar concentration, yeast growth, the viability rate of “resting” yeast cells, and fermentation activity were stimulated under certain conditions of aeration and temperature. This stimulation might be interpreted as being a result of the yeast cell sterol content. The addition of certain sterols to the fermenting medium was able to increase this sterol content. According to aeration conditions of the medium, which determined the sterol content of yeasts, the sterols added in the medium acted as (i) growth factors, (ii) fermentation inhibitors, and (iii) survival factors for the yeast.     

Production of higher alcohols,ethyl acetate,acetaldehyde and other compounds by 14Saccharomyces cerevisiae wine strains isolated from the same region (Salnés,N.W. Spain)

Fourteen strains of the yeastSaccharomyces cerevisiae were isolated from three wineries in the Salnés wine region (N.W. Spain) at the three different periods of the natural fermentation. Each wild yeast was screened for production of acetaldehyde, ethyl acetate, isobutanol,n-propanol, amylic alcohol and other important enological compounds during laboratory scale fermentations of grape juice. After 25 days at 20°C, the analytical results evidenced variations in the production of acetaldehyde (from 13.1 to 24.3 mg/l), isobutanol (from 27.7 to 51.1 mg/l), amyl alcohols (from 111 to 183 mg/l) and ethyl acetate (from 19.3 to 43.7 mg/l). Although isolated from the same wine region, differences in the wine composition were observed depending on the particular yeast strain used for the vinification experiments.     

Starvation Response of Saccharomyces cerevisiae Grown in Anaerobic Nitrogen- or Carbon-Limited Chemostat Cultures

Anaerobic starvation conditions are frequent in industrial fermentation and can affect the performance of the cells. In this study, the anaerobic carbon or nitrogen starvation response of Saccharomyces cerevisiae was investigated for cells grown in anaerobic carbon or nitrogen-limited chemostat cultures at a dilution rate of 0.1 h⁻¹ at pH 3.25 or 5. Lactic or benzoic acid was present in the growth medium at different concentrations, resulting in 16 different growth conditions. At steady state, cells were harvested and then starved for either carbon or nitrogen for 24 h under anaerobic conditions. We measured fermentative capacity, glucose uptake capacity, intracellular ATP content, and reserve carbohydrates and found that the carbon, but not the nitrogen, starvation response was dependent upon the previous growth conditions. All cells subjected to nitrogen starvation retained a large portion of their initial fermentative capacity, independently of previous growth conditions. However, nitrogen-limited cells that were starved for carbon lost almost all their fermentative capacity, while carbon-limited cells managed to preserve a larger portion of their fermentative capacity during carbon starvation. There was a positive correlation between the amount of glycogen before carbon starvation and the fermentative capacity and ATP content of the cells after carbon starvation. Fermentative capacity and glucose uptake capacity were not correlated under any of the conditions tested. Thus, the successful adaptation to sudden carbon starvation requires energy and, under anaerobic conditions, fermentable endogenous resources. In an industrial setting, carbon starvation in anaerobic fermentations should be avoided to maintain a productive yeast population.     

The nature of the nitrogen source added to nitrogen depleted vinifications conducted by a Saccharomyces cerevisiae strain in synthetic must affects gene expression and the levels of several volatile compounds

Nitrogen starvation may lead to stuck and sluggish fermentations. These undesirable situations result in wines with high residual sugar, longer vinification times, and risks of microbial contamination. The typical oenological method to prevent these problems is the early addition of ammonium salts to the grape juice, although excessive levels of these compounds may lead to negative consequences for the final product. This addition reduces the overall fermentation time, regardless of the time of addition, but the effect is more significant when nitrogen is added during the yeast exponential phase. In this work we analysed the effect of adding different nitrogen sources (ammonia, amino acids or a combination of both) under nitrogen depletion in order to understand yeast metabolic changes that lead to the adaptation to the new conditions. These studies were carried out in a synthetic must that mimics the composition of the natural must. Furthermore, we studied how this addition affects fermentative behaviour, the levels of several yeast volatile compounds in the final product, arginase activity, and the expression of several genes involved in stress response and nitrogen metabolism during vinification. We found that the nature of the nitrogen source added during yeast late exponential growth phase introduces changes to the volatile compounds profile and to the gene expression. On the other hand, arginase activity and the expression of the stress response gene ACA1 are useful to monitor nitrogen depletion/addition during growth of the wine yeast considered under our vinification conditions.     

Immobilization of yeast cells in PVA particles for beer fermentation

In this study, the potential of application of non-aggressive LentiKat^® technique for brewer's yeast immobilization on polyvinyl alcohol was assessed. High cell loads of about 10⁹ cells/ml were achieved by this procedure and immobilization procedure had no adverse effect on cell viability. The stability and activity of obtained immobilized biocatalyst was tested in the growth studies and fermentations. Immobilized cells exhibited high fermentation activity in both, laboratory and pilot-scale fermentations. In three successive gas-lift reactor fermentations the apparent attenuation of around 80% was reached after only 2 days, indicating good potential of immobilized cells for development of continuous primary beer fermentation. LentiKat^® particles showed high mechanical and fermentative stability, since they endured 30 days of operating time during 6-month period without significant change of cell activity, particle shape and particle size.