Glycerol and other fermentation products of apiculate wine yeasts

Ninety-six strains of apiculate wine yeasts were studied for their ability to produce glycerol, acetaldehyde, ethyl acetate, sulphur dioxide and hydrogen sulphide in synthetic medium. Hanseniaspora guilliermondii produced smaller quantities of glycerol, acetaldehyde and hydrogen sulphide than Kloeckera apiculata , whereas the production of ethyl acetate and sulphur dioxide was found to be similar. Strains characterized by different capacities and properties were found for both species. The existence of apiculate strains differing in secondary compound production is of technological interest, as these yeasts constitute potential flavour producers. Selected strains of apiculate yeasts might favour an enhanced flavour formation and yield desirable characteristics to the final product.     

On the question of vegetative reproduction in apiculate yeasts

Fluorescence microscopy, carbon replicas, ultrathin sections and metal-shadowed walls were used for studying changes during cell division of yeasts. Some common features and differences between multipolar budding, fission and the so-called bipolar budding of apiculate yeasts are specified. The conclusion was reached that in apiculate yeasts we are not dealing with a kind of budding but with a type of reproduction that is synonymous with multipolar budding and fission.     

Higher alcohol and acetic acid production by apiculate wine yeasts

P. ROMANO, G. SUZZI, G. COMI AND R. ZIRONI. 1992. Ninety-six strains of apiculate wine yeasts were investigated for their ability to produce higher alcohols and acetic acid in synthetic medium. Less isoamyl alcohol and more n -propanol and isobutanol were formed by Hanseniaspora guilliermondii than by Kloeckera apiculata. The latter produced twice as much acetic acid as H. guilliermondii. The production of higher alcohols and acetic acid was found to be a characteristic of individual strains and was statistically significant. In a multivariate analysis of higher alcohol production two main groupings were formed at 86%S, corresponding to the taxa H. guilliermondii and K. apiculata. Strains that produced low amounts (50 mg/1) of acetic acid, comparable with that of Saccharomyces cerevisiae, were found in both species of apiculate yeasts.     

葡萄酒酿酒酵母嗜果糖特性的评价方法

[目的]酿酒酵母的嗜果糖性是葡萄酒酵母选育工作的一项重要内容.建立评价菌体发酵果糖能力的方法,是葡萄酒酿酒酵母嗜果糖性研究的基础.[方法]以3株不同果糖发酵能力的酵母菌为研究对象,考察菌体在模拟葡萄汁培养基条件下,发酵情况与单糖利用之间的关系;并通过数学方程拟合单糖动力发酵曲线,得到发酵持续时间、葡萄糖浓度拟为0时的果糖浓度、果糖与葡萄糖曲线面积的差值等参数.[结果]这些参数可以反应出菌体的发酵速率和嗜果糖性.其中后两个参数能显著将3个菌株的嗜果糖特性区分开.[结论]为高果糖利周优良葡萄酒酿酒酵母菌株的筛选和构建,提供了较为全面、客观和有效的评价方法.     

Correlation between glucose/fructose discrepancy and hexokinase kinetic properties in different <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> wine yeast strains

Grape juice contains about equal amounts of glucose and fructose, but wine strains of Saccharomyces cerevisiae ferment glucose slightly faster than fructose, leading to fructose concentrations that exceed glucose concentrations in the fermenting must. A high fructose/glucose ratio may contribute to sluggish and stuck fermentations, a major problem in the global wine industry. We evaluated wine yeast strains with different glucose and fructose consumption rates to show that a lower glucose preference correlates with a higher fructose/glucose phosphorylation ratio in cell extracts and a lower K _m for both sugars. Hxk1 has a threefold higher V _max with fructose than with glucose, whereas Hxk2 has only a slightly higher V _max with glucose than with fructose. Overexpression of HXK1 in a laboratory strain of S. cerevisiae (W303–1A) accelerated fructose consumption more than glucose consumption, but overexpression in a wine yeast strain (VIN13) reduced fructose consumption less than glucose consumption. Results with laboratory strains expressing a single kinase showed that total hexokinase activity is inversely correlated with the glucose/fructose (G/F) discrepancy. The latter has been defined as the difference between the rate of glucose and fructose fermentation. We conclude that the G/F discrepancy in wine yeast strains correlates with the kinetic properties of hexokinase-mediated sugar phosphorylation. A higher fructose/glucose phosphorylation ratio and a lower K _m might serve as markers in selection and breeding of wine yeast strains with a lower tendency for sluggish fructose fermentation.     

Effect of glucose concentration on the rate of fructose consumption in native strains isolated from the fermentation of Agave duranguensis

Studies on hexose consumption by Saccharomyces cerevisiae show that glucose is consumed faster than fructose when both are present (9:1 fructose to glucose) in the medium during the fermentation of Agave. The objective of this work was to select strains of S. cerevisiae that consume fructose equal to or faster than glucose at high fructose concentrations by analyzing the influence of different glucose concentrations on the fructose consumption rate. The optimal growth conditions were determined by a kinetics assay using high performance liquid chromatography (HPLC) using 50?g of glucose and 50?g of fructose per liter of synthetic medium containing peptone and yeast extract. Using the same substrate concentrations, strain ITD-00185 was shown to have a higher reaction rate for fructose over glucose. At 75?g of fructose and 25?g of glucose per liter, strain ITD-00185 had a productivity of 1.02 gL^?1?h^?1 after 40?h and a fructose rate constant of 0.071?h^?1. It was observed that glucose concentration positively influences fructose consumption when present in a 3:1 ratio of fructose to glucose. Therefore, adapted strains at high fructose concentrations could be used as an alternative to traditional fermentation processes.     

The zymocidial activity of Tetrapisispora phaffii in the control of Hanseniaspora uvarum during the early stages of winemaking

Aims:  The yeast strain Tetrapisispora phaffii DBVPG 6706 (formerly Kluyveromyces phaffii ) secretes a killer toxin (Kpkt) that has antimicrobial activity against apiculate yeasts. The aim of this study was to evaluate the killer activity of Kpkt towards Hanseniaspora uvarum under winemaking conditions.
Methods and Results:  The zymocidial activity of Kpkt on H. uvarum was assayed in microfermentation trials inoculated with free and immobilized T. phaffii cells. The microbial evolution and fermentation profiles of the wines were evaluated to determine the effects of Kpkt on apiculate yeasts, in comparison with SO₂. The results indicate that the fungicidal activity of Kpkt against H. uvarum is stable for at least 14 days in wine, and the zymocin can control the proliferation of apiculate yeasts. The analytical composition of wines with the inoculum of T. phaffii immobilized cells did not differ from the wines with SO₂. In contrast to wines without this control of apiculate yeasts, an increase in ethyl acetate was seen.
Conclusions:  Tetrapisispora phaffii is an excellent candidate for the biological control of undesired proliferation of apiculate yeasts during the first steps of fermentation.
Significance and Impact of the Study:  Tetrapisispora phaffii cells in an immobilized form can be used as a biocontrol agent to reduce the need for SO₂ addition.     

Volatile metabolites produced in wine by mixed and sequential cultures of Hanseniaspora guilliermondii or Kloeckera apiculata and Saccharomyces cerevisiae

Summary Secondary products in wines obtained by pure, mixed and sequential cultures of Saccharomyces cerevisiae, Hanseniaspora guilliermondii or Kloeckera apiculata were studied. Consistent differences in the composition were determined in wines fermented by sequential cultures. When S. cerevisiae was added to musts partially fermented by apiculate yeasts, its metabolism was significantly affected. In particular it synthesized high amounts of n-propanol and metabolized high quantities of acetoin, produced by apiculate yeasts     

Comparison of glucose uptake kinetics in different yeasts.

The kinetics of glucose uptake were investigated in laboratory wild-type strains of Saccharomyces cerevisiae of differing genetic backgrounds, in other species of Saccharomyces, and in other yeasts, both fermentative and respiratory. All yeasts examined displayed more than one uptake system for glucose. Variations in apparent Km values, velocity of uptake, and effects of glucose concentration on carrier activity were observed. The three type strains for the species S. cerevisiae, Saccharomyces bayanus, and Saccharomyces carlsbergensis gave distinctive patterns, and each of the laboratory strains was similar to one or another of the type strains. Other fermentative yeasts (Pichia guillermondi and Pichia strasburgensis) regulated glucose uptake in a manner similar to that of Saccharomyces spp. Such was not true for the respiratory yeasts investigated, Pichia heedi and Yarrowia lipolytica, which did not demonstrate glucose repression of carrier activity; this finding suggests that this mechanism of control of transporter activity may be associated with fermentative ability.     

Characterization of kinetic parameters and the formation of volatile compounds during the tequila fermentation by wild yeasts isolated from agave juice

The production of aroma compounds during tequila fermentation using four native yeast strains isolated from agave juice was quantified at controlled (35 degrees C) and uncontrolled temperatures (room temperature) by gas chromatography (FID). Three of the four strains were identified as Saccharomyces cerevisiae (MTLI 1, MALI 1 and MGLI 1) and one as Kloeckera apiculata (MALI 2). Among the aroma compounds produced, acetaldehyde has the highest accumulation at the controlled temperature and before 50% of sugar was consumed. The S. cerevisiae strains produced ethyl acetate in almost the same quantity at a concentration of 5 mg/L and the K. apiculata produced six-times more (30 mg/L) than the S. cerevisiae strains, independent of the fermentation temperature. The rate and amount of 1-propanol, amyl alcohols and isobutanol production were affected by the type of yeast used. The K. apiculate strain produced 50% less of the higher alcohols than the Saccharomyces strains. The results obtained showed that indigenous isolated yeasts play an important role in the tequila flavor and suggest that mixtures of these yeasts may be used to produce tequila with a unique and desirable aroma.     

31P NMR and 13C NMR studies of recombinant Saccharomyces cerevisiae with altered glucose phosphorylation activities

Manipulation of cellular metabolism to maximize the yield and rate of formation of desired products may be achieved through genetic modification. Batch fermentations utilizing glucose as a carbon source were performed for three recombinant strains of Saccharomyces cerevisiae in which the glucose phosphorylation step was altered by mutation and genetic engineering. The host strain (hxk1 hxk2 glk) is unable to grow on glucose or fructose; the three plasmids investigated expressed hexokinase PI, hexokinase PII, or glucokinase, respectively, enabling more rapid glucose and fructose phosphorylation in vivo than that provided by wild-type yeast.Intracellular metabolic state variables were determined by ³¹P NMR measurements of in vivo fermentations under nongrowth conditions for high cell density suspensions. Glucose consumption, ethanol and glycerol production, and polysaccharide formation were determined by ¹³C NMR measurements under the same experimental conditions as used in the ³¹P NMR measurements. The trends observed in ethanol yields for the strains under growth conditions were mimicked in the nongrowth NMR conditions.Only the strain with hexokinase PI had higher rates of glucose consumption and ethanol production in comparison to healthy diploid strains in the literature. The hexokinase PII strain drastically underutilized its glucose-phosphorylating capacity. A regulation difference in the use of magnesium-free ATP for this strain could be a possible explanation. Differences in ATP levels and cytoplasmic pH values among the strains were observed that could not have been foreseen. However, cytoplasmic pH values do not account for the differences observed among in vivo and in vitro glucose phosphorylation activities of the three recombinant strains.     

Obtaining and selection of hexokinases-less strains of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> for production of ethanol and fructose from sucrose

Saccharomyces cerevisiae hexokinase-less strains were produced to study the production of ethanol and fructose from sucrose. These strains do not have the hexokinases A and B. Twenty-three double-mutant strains were produced, and then, three were selected for presenting a smaller growth in yeast extract–peptone–fructose. In fermentations with a medium containing sucrose (180.3 g L⁻¹) and with cell recycles, simulating industrial conditions, the capacity of these mutant yeasts in inverting sucrose and fermenting only glucose was well characterized. Besides that, we could also see their great tolerance to the stresses of fermentative recycles, where fructose production (until 90 g L⁻¹) and ethanol production (until 42.3 g L⁻¹) occurred in cycles of 12 h, in which hexokinase-less yeasts performed high growth (51.2% of wet biomass) and viability rates (77% of viable cells) after nine consecutive cycles.     

Biometric Study of Acetoin Production in Hanseniaspora guilliermondii and Kloeckera apiculata

Gas chromatographic analysis by direct injection of samples yielded quantitative data on acetoin content. Ninety-six strains of Hanseniaspora guilliermondii and Kloeckera apiculata were investigated for the ability to produce acetoin in synthetic medium and in must. High-level production of acetoin was found to be a characteristic of both species. In synthetic medium, the two species were not significantly different with respect to sugar utilization and ethanol or acetoin production. In grape must, the two species were significantly different (P = 0.001) in acetoin production and K. apiculata exhibited a significantly negative correlation between acetoin production and either sugar consumption or ethanol production. Use of selected apiculate yeasts in mixed cultures with Saccharomyces cerevisiae seems promising for optimization of wine bouquet.     

Glucose and fructose consumption in a phosphoglucoseisomeraseless mutant in Saccharomyces cerevisiae

Saccharomyces cerevisiae with a practically complete absence of phosphoglucoseisomerase activity when grown in fructose or glucose minimal medium showed different consumption of fructose and glucose during different periods of the culture. At the beginning of growth, cells had a great quantity of glucose available relative to their requirements and a large quantity of trehalose accumulated from ¹⁴C-glucose in comparison with the wild type strain. A second phase arises when the concentration of glucose in the medium was practically absent and the cells obtain glucose by mobilisation of stored glucose containing compounds. It is very likely that at this moment a balance rate between glucose 6-phosphate formation and consumption occurs. Finally cells reach conditions of glucose starvation and fructose consumption increases in this last stage. The different consumption of fructose throughout different periods of cell growth most probably indicates a strict regulation at the level of sugar uptake.Non Standard Abbreviation pgi phosphoglucoseisomerase     

Citric acid production by 2-deoxyglucose-resistant mutant strains of Aspergillus niger

Summary Many mutant strains showing resistance to 2-deoxy-d-glucose (DG) on minimal medium containing glycerol as a carbon source were induced from Aspergillus niger WU-2223L, a citric acid-producing strain. The mutant strains were classifiable into two types according to their growth characteristics. On the agar plates containing glucose as a sole carbon source, mutant strains of the first type showed good growth irrespective of the presence or absence of DG. When cultivated in shake cultures, some strains of the first type, such as DGR1–2, showed faster glucose consumption and growth than strain WU-2223L. The period for citric acid production shortened from 9 days for strain WU-2223L to 6–7 days for these mutant strains. The levels and yields of citric acid production of the mutant strains were almost the same as those of strain WU-2223L. The mutant strains of the second type, however, showed very slow or no growth on both the agar plates containing glucose and fructose as sole carbon sources. In shake cultures, mutant strains such as DGR2-8 showed decreased glucose consumption rates, resulting in very low production of citric acid.     

Automatic method for evaluating the activity of sourdough strains based on gas pressure measurements

A new method is proposed for the evaluation of the activity of sourdough strains, based on gas pressure measurements in closed air-tight reactors. Gas pressure and pH were monitored on-line during the cultivation of commercial yeasts and heterofermentative lactic acid bacteria on a semi-synthetic medium with glucose as the major carbon source. Relative gas pressure evolution was compared both to glucose consumption and to acidification and growth. It became obvious that gas pressure evolution is related to glucose consumption kinetics. For each strain, a correlation was made between maximum gas pressure variation and amount of glucose consumed. The mass balance of CO2 in both liquid and gas phase demonstrated that around 90% of CO2 was recovered. Concerning biomass production, a linear relationship was found between log colony-forming units/ml and log pressure for both yeasts and bacteria during the exponential phase; and for yeasts, relative gas pressure evolution also followed optical density variation.     

Effects of null mutations in the hexokinase genes of Saccharomyces cerevisiae on catabolite repression.

Saccharomyces cerevisiae has two homologous hexokinases, I and II; they are 78% identical at the amino acid level. Either enzyme allows yeast cells to ferment fructose. Mutant strains without any hexokinase can still grow on glucose by using a third enzyme, glucokinase. Hexokinase II has been implicated in the control of catabolite repression in yeasts. We constructed null mutations in both hexokinase genes, HXK1 and HXK2, and studied their effect on the fermentation of fructose and on catabolite repression of three different genes in yeasts: SUC2, CYC1, and GAL10. The results indicate that hxk1 or hxk2 single null mutants can ferment fructose but that hxk1 hxk2 double mutants cannot. The hxk2 single mutant, as well as the double mutant, failed to show catabolite repression in all three systems, while the hxk1 null mutation had little or no effect on catabolite repression.     

Effect of dietary carbohydrates on the in vitro epithelial adhesion of Candida albicans, Candida tropicalis, and Candida krusei

Adhesion to epithelial surfaces is considered as a critical step in the pathogenesis of oral candidosis. Therefore, the effects of the most commonly consumed dietary carbohydrates on the adhesion of Candida albicans, Candida tropicalis, and Candida krusei to monolayered HeLa cells were investigated. Adherence of C. albicans and C. tropicalis appeared significantly promoted by incubation in defined medium containing a high concentration (500 mM) of fructose, glucose, maltose, and sucrose (p < 0.001). C. albicans organisms grown in sucrose elicited maximal increase in adhesion, whereas adhesion of C. tropicalis and C. krusei was enhanced to the greatest extent when cultured in glucose. Maltose and fructose also promoted adherence of C. albicans and C. tropicalis (p < 0.001), but to a lesser extent than sucrose and glucose. On the other hand, sorbitol-grown yeasts demonstrated a marginal increase in adhesion (p > 0.01). Xylitol only significantly reduced adherence of C. albicans (p < 0.001). These results suggest that the frequent consumption of carbohydrates, such as sucrose, glucose, maltose, or fructose, might represent a risk factor for oral candidosis. The limitation of their consumption by substituting xylitol or sorbitol could be of value in the control of oral Candida colonization and infection.     

Short Communication: Characterization and succession of yeast populations associated with spontaneous fermentations during the production of Brazilian sugar-cane aguardente

A succession of yeasts was observed during fermentation of aguardente with Saccharomyces cerevisiae being the predominant species. Candida sake, Kluyveromyces marxianus var. drosophilarum and apiculate yeasts were also frequent. Transient yeast species were found in variable numbers, probably due to the daily addition of sugar-cane juice. Killer yeasts were isolated and may have a role in the exclusion of some transient and contaminant species.     

Phenotypic expression of Kluyveromyces lactis killer toxin against Saccharomyces spp.

The secretion of killer toxins by some strains of yeasts is a phenomenon of significant industrial importance. The activity of a recently discovered Kluyveromyces lactis killer strain against a sensitive Saccharomyces cerevisiae strain was determined on peptone-yeast extract-nutrient agar plates containing as the carbon source glucose, fructose, galactose, maltose, or glycerol at pH 4.5 or 6.5. Enhanced activity (50 to 90% increase) was found at pH 6.5, particularly on the plates containing galactose, maltose, or glycerol, although production of the toxin in liquid medium was not significantly different with either glucose or galactose as the carbon source. Results indicated that the action of the K. lactis toxin was not mediated by catabolite repression in the sensitive strain. Sensitivities of different haploid and polyploid Saccharomyces yeasts to the two different killer yeasts S. cerevisiae (RNA-plasmid-coded toxin) and K. lactis (DNA-plasmid-coded toxin) were tested. Three industrial polyploid yeasts sensitive to the S. cerevisiae killer yeast were resistant to the K. lactis killer yeast. The S. cerevisiae killer strain itself, however, was sensitive to the K. lactis killer yeast.