Improvement of the alcoholic fermentation of grape juice with mixed cultures of Saccharomyces cerevisiae wild strains. Negative effect of Kloeckera apiculata

Four assays were designed to study the fermentative behaviour of two selected wild strains of Saccharomyces cerevislae when inoculated in sterilized grape juice either alone or in association. In addition, a wild strain of Kloeckera apiculata was employed for studying and characterizing the influence of this micro-organism on the fermentations when associated with Sacch. cerevisiae. Fermentation was improved when the two strains of Sacch. cerevisiae were grown together since the residual sugar fell to 0.12% and ethanol increased up to more than 11% (v/v). In this case, a proliferation of the two strains with successive exponential phases was observed. On the other hand, when Kloeckera apiculata was incorporated into the fermentations, these were incomplete with a residual sugar level of 1.64% and a final ethanol concentration of 6.4%.The authors are with the Cátedra de Microbiología, Facultad de Farmacia, Universidad de Santiago de Compostela, 15706 Santiago de Compostela, Spain. T.G. Villa is the corresponding author.     

Typing of Saccharomyces cerevisiae and Kloeckera apiculata strains from Aglianico wine

AIMS: Kloeckera apiculata and Saccharomyces cerevisiae yeast species are dominant, respectively, at the early and at the following stages of wine fermentation. In the present study, PCR fingerprinting and NTS region amplification and restriction were applied as techniques for monitoring yeast population performing Aglianico of Vulture grape must fermentation. METHODS AND RESULTS: Thirty S. cerevisiae and 30 K. apiculata strains were typed by PCR fingerprinting with (GAC)5 and (GTG)5 primers and by complete NTS region amplification followed by restriction with HaeIII and MspI enzymes. S. cerevisiae strains generated two patterns with (GAC)5 primer, while (GTG)5 primer yielded a higher genetic polymorphism. Conversely, in K. apiculata Aglianico wine strains (GAC)5 and (GTG)5 primers generated the same profile for all strains. Restriction analysis of the amplified NTS region gave the same profile for all strains within the same species, except for one strain of S. cerevisiae. CONCLUSIONS: The PCR fingerprinting technique was useful in discriminating at strain level S. cerevisiae, particularly with the primer (GTG)5. RFLP patterns generated from the NTS region of the two species can be more easily compared than the patterns resulting from PCR fingerprinting, thus RFLP is more suitable for the rapid monitoring of the species involved in different stages of fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: The molecular techniques used allow discrimination of S. cerevisiae at strain level and monitoring of the ratio of S. cerevisiae/K. apiculata during the fermentation process. Thus, their application can assure technological adjustments in a suitable time.     

Physiological variants of Saccharomyces cerevisiae and Kloeckera apiculata from palm wine and cashew juice

Physiological variants of Saccharomyces cerevisiae and Kloeckera apiculata have been identified in oil palm wine and cashew juice from Nigeria. Genomic DNA from the four S. cerevisiae variants had a % G + C of 36-41% while that of K. apiculata was 32.2%. Fermentation of cashew juice produced wine of alcoholic contents of 10% with S. cerevisiae, 8% with K. apiculata and 9.3% with both yeasts simultaneously.     

Physiological variants of Saccharomyces cerevisiae and Kloeckera apiculata from palm wine and cashew juice

Physiological variants of Saccharomyces cerevisiae and Kloeckera apiculata have been identified in oil palm wine and cashew juice from Nigeria. Genomic DNA from the four S. cerevisiae variants had a % G + C of 36–41% while that of K. apiculata was 32.2%. Fermentation of cashew juice produced wine of alcoholic contents of 10% with S. cerevisiae , 8% with K. apiculata and 9.3% with both yeasts simultaneously. and accepted 31 August 1989     

Metabolic characterization of Kloeckera apiculata strains from star fruit fermentation

A total of 37 strains of Kloeckera apiculata was isolated during the spontaneous fermentation of star fruit must. Each strain was differentiated from the others on the basis of its capacity to produce acetaldehyde, ethyl acetate, higher alcohols, acetoin and acetic acid. All the strains were characterized by the low production of higher alcohols and the high production of ethyl acetate, whereas consistent differences in the production of acetaldehyde, acetoin and acetic acid served to differentiate star fruit apiculate strains into six different phenotypes, present at different stages of the fermentation process. The metabolic strain diversity found can be interpreted as a natural consequence of environmental conditions, which influenced the frequency and selection of specific apiculate strains. From the biotechnological point of view the different metabolic biotypes represent an important source of strains for potential use as starter cultures for star fruit fermentation.     

Evaluation of stereoisomers of 2,3-butanediol and acetoin to differentiate Saccharomyces cerevisiae and Kloeckera apiculata wine strains

The (R)/(S) ratios of acetoin were always higher in wines obtained by Saccharomyces cerevisiae than in those obtained by Kloeckera apiculata. A significantly different behaviour was determined between the two species as regards contents and ratios of 2,3-butanediols: S. cerevisiae produced more (R,R)-2,3-butanediol (about 80%), whereas K. apiculata produced more meso-form (about 90%).     

Relations between elevated temperatures and fermentation behaviour of Kloeckera apiculata and Saccharomyces cerevisiae associated with winemaking in mixed cultures

One of the important factors affecting wine fermentation is temperature. The influence of elevated temperatures from 10 to 25 °C at 5 °C intervals on yeast growth and fermentation products were studied in mixed cultures of Kloeckera apiculata and Saccharomyces cerevisiae in grape juice. In the experiments carried out at 10 and 15 °C, K. apiculata grew and survived longer compared to trials conducted above 20 °C. In most cases, higher temperatures stimulated the production of higher alcohols but lowered the formation of esters and acetaldehyde. This revised version was published online in July 2006 with corrections to the Cover Date.     

The effects of temperature and pH on the ethanol tolerance of the wine yeasts, Saccharomyces cerevisiae, Candida stellata and Kloeckera apiculata

The influences of temperature and pH on the survival and growth of Saccharomyces cerevisiae, Candida stellata and Kloeckera apiculata were examined in the presence of ethanol concentrations between 2.5 and 15% v/v. At 15°C, the maximum concentrations of ethanol permitting the growth of S. cerevisiae, C. stellata and K. apiculata were 15%, 11% and 9%, respectively. These maximum concentrations were decreased at 10°C and 30°C. Cells of S. cerevisiae showed no loss in viability when incubated for 12 d at 10°C or 15°C in the presence of 15% ethanol but showed some loss at 30°C. Cells of C. stellata were tolerant of 12.5% ethanol at 10°C and 15°C but not at 30°C. Cells of K. apiculata were tolerant of 10–12.5% ethanol at 15°C but not at 10°C or 30°C. Sensitivity of the yeast cells to ethanol was marginally increased on decreasing the pH from 6-0 to 3–0.     

Optimized fermentation of grape juice by laboratory strains of Saccharomyces cerevisiae

Laboratory strains of yeast ( Saccharomyces cerevisiae ) based on S288C ferment grape juice relatively poorly. We show that slow fermentation appears to be inherent to this strain, because the original S288C isolate shows fermentation similar to current laboratory isolates. We demonstrate further that some auxotrophic mutations in the laboratory strain show reduced rates of fermentation in grape juice, with lysine auxotrophs particularly impaired compared with isogenic Lys⁺ strains. Supplementing lysine at a 10-fold higher concentration than recommended allowed yeast cultures to reach higher final cell densities and restored the fermentation rate of auxotrophic strains to those of the corresponding wild-type strains. However, even with the additional supplementation, the fermentation rates of S288C strains were still slower than those of a commercial wine yeast strain. Conditions were developed that enable auxotrophic laboratory strains derived from S288C to ferment grape juice to completion with high efficiency on a laboratory scale. Fermentation in media based on grape juice will allow the suite of molecular genetic tools developed for these laboratory strains to be used in investigations of complex ferment characteristics and products.     

Relationship between ethanol tolerance, lipid composition and plasma membrane fluidity in Saccharomyces cerevisiae and Kloeckera apiculata

Abstract The lipid composition of a strain of each of two yeasts, Saccharomyces csrevisiae and Kloeckera apiculata , with different ethanol tolerances, was determined for cells grown with or without added ethanol. An increase in the proportion of ergosterol, unsaturated fatty acid levels and the maintenance of phospholipid biosynthesis seemed to be responsible for ethanol tolerance. The association of ethanol tolerance of yeast cells with plasma membrane fluidity, measured by fluorescence anisotropy, is discussed. We propose that an increase in plasma membrane fluidity may be correlated with a decrease in the sterol: phospholipid and sterol: protein ratios and an increase in unsaturation index.     

Characterization of Kloeckera apiculata strains from the Friuli regionin Northern Italy

Forty-nine strains of Kloeckera apiculata, isolated from the Friuli region in Italy, were differentiated on the basis of fermentation behaviour and production of secondary compounds in two different grape musts at 18 °C. The isolates exhibited a controlled production of acetic acid, only in a few cases more that 1 g/l. In Moscato grape must the strains exhibited a more uniform behaviour for the production of higher alcohols, ethyl acetate and acetoin than in red grapes. In general, higher levels of ethanol, glycerol and acetic acid were produced in red grape must fermentation. Apiculate strains behaved differently in the two musts, with different metabolic phenotypes dominating the fermentation process. The existence of different metabolic phenotypes correlated with the must composition underlines the need to perform a selection of indigenous apiculate yeasts to obtain the desired consistent products.     

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     

Ethanol fermentation by nystatin-resistant strains of Saccharomyces cerevisiae

Nystatin-resistant mutants of haploid and polyploid strains of Saccharomyces cerevisiae were isolated by plating on gradient plates with increasing nystatin concentrations (60–3000 U/ml). Some of the mutants were defective in ergosterol biosynthesis, and produced zymosterol and cholestatetraenol-like sterols. Those mutants which do not form ergosterol produce less ethanol than the parent strains. They also had lower viability during fermentation of glucose solutions (8–13% vs. 33–47%). This became more pronounced in fermentations of higher concentrations of glucose. A nystatin-resistant but ergosterol-forming mutant had a similar fermentation capacity to the parent strain.     

The pentose phosphate pathway in the yeasts Saccharomyces cerevisiae and Kloeckera apiculata,an exercise in comparative metabolism for food and wine science students

Comparative cellular metabolism can be a difficult area of biochemistry to teach in the undergraduate laboratory class. Student practicals involving animal tissues generally require approval from animal ethic committees, and the relevance for students whose primary interest in biochemistry is in the area of food and wine sciences, is often questioned. In this report, we present an undergraduate practical exercise in which glucose catabolism via the pentose phosphate pathway is compared in two types of yeast with direct relevance to the wine and food industries, Saccharomyces cerevisiae and Kloeckera apiculata. The exercise is carried out as a demonstration to second year undergraduate students, studying metabolic biochemistry. It is of some value in that it illustrates comparative cellular metabolism in wine yeasts and introduces the students to the safe use of radioisotopes.     

Ethanol fermentation by nystatin-resistant strains of Saccharomyces cerevisiae

Nystatin-resistant mutants of haploid and polyploid strains of Saccharomyces cerevisiae were isolated by plating on gradient plates with increasing nystatin concentrations (60-3000 U/ml). Some of the mutants were defective in ergosterol biosynthesis, and produced zymosterol and cholestatetraenol-like sterols. Those mutants which do not form ergosterol produce less ethanol than the parent strains. They also had lower viability during fermentation of glucose solutions (8-13% vs. 33-47%). This became more pronounced in fermentations of higher concentrations of glucose. A nystatin-resistant but ergosterol-forming mutant had a similar fermentation capacity to the parent strain.     

Influence of killer strains of Saccharomyces cerevisiae on wine fermentation

The effect of killer strains of Saccharomyces cerevisiae on the growth of sensitive strains during must fermentation was studied by using a new method to monitor yeast populations. The capability of killer yeast strains to eliminate sensitive strains depends on the initial proportion of killer yeasts, the susceptibility of sensitive strains, and the treatment of the must. In sterile filtered must, an initial proportion of 2-6% of killer yeasts was responsible for protracted fermentation and suppression of isogenic sensitive strains. A more variable initial proportion was needed to get the same effect with non-isogenic strains. The suspended solids that remain in the must after cold-settling decreased killer toxin effect. The addition of bentonite to the must avoided protracted fermentation and the suppression of sensitive strains; however, the addition of yeast dietary nutrients with yeast cell walls did not, although it decreased fermentation lag.     

Xylulose fermentation by Saccharomyces cerevisiae and xylose-fermenting yeast strains

Xylulose fermentation by four strains of Saccharomyces cerevisiae and two strains of xylose-fermenting yeasts, Pichia stipitis CBS 6054 and Candida shehatae NJ 23, was compared using a mineral medium at a cell concentration of 10 g (dry weight)/l. When xylulose was the sole carbon source and fermentation was anaerobic, S. cerevisiae ATCC 24860 and CBS 8066 showed a substrate consumption rate of 0.035 g g cells^–1 h^–1 compared with 0.833 g g cells^–1 h^–1 for glucose. Bakers' yeast and S. cerevisiae isolate 3 consumed xylulose at a much lower rate although they fermented glucose as rapidly as the ATCC and the CBS strains. While P. stipitis CBS 6054 consumed both xylulose and glucose very slowly under anaerobic conditions, C. shehatae NJ 23 fermented xylulose at a rate of 0.345 g g cells^–1 h^–1, compared with 0.575 g g cells^–1 h^–1 for glucose. For all six strains, the addition of glucose to the xylulose medium did not enhance the consumption of xylulose, but increased the cell biomass concentrations. When fermentation was performed under oxygen-limited conditions, less xylulose was consumed by S. cerevisiae ATCC 24860 and C. shehatae NJ 23, and 50%–65% of the assimilated carbon could not be accounted for in the products determined.     

The effect of growth factors on anoxic chemostat cultures of two Saccharomyces cerevisiae strains

In anoxic chemostat cultures of Saccharomyces cerevisiae ATCC 4126 and CBS 8066 grown in a medium containing yeast extract, a sharp increase in the steady-state residual glucose concentration occurred at relatively low dilution rates, contrary to the expected Monod kinetics. However, supplementation with vitamins and amino acids facilitated efficient glucose uptake. This enhanced requirement for growth factors under anoxic conditions and at high growth rates could explain the exceptionally high apparent k _s values for S. cerevisiae reported in the literature.     

The effect of hexose ratios on metabolite production in Saccharomyces cerevisiae strains obtained from the spontaneous fermentation of mezcal

Mezcal from Tamaulipas (México) is produced by spontaneous alcoholic fermentation using Agave spp. musts, which are rich in fructose. In this study eight Saccharomyces cerevisiae isolates obtained at the final stage of fermentation from a traditional mezcal winery were analysed in three semi-synthetic media. Medium M1 had a sugar content of 100 g l^?1 and a glucose/fructose (G/F) of 9:1. Medium M2 had a sugar content of 100 g l^?1 and a G/F of 1:9. Medium M3 had a sugar content of 200 g l^?1 and a G/F of 1:1. In the three types of media tested, the highest ethanol yield was obtained from the glucophilic strain LCBG-3Y5, while strain LCBG-3Y8 was highly resistant to ethanol and the most fructophilic of the mezcal strains. Strain LCBG-3Y5 produced more glycerol (4.4 g l^?1) and acetic acid (1 g l^?1) in M2 than in M1 (1.7 and 0.5 g l^?1, respectively), and the ethanol yields were higher for all strains in M1 except for LCBG-3Y5, -3Y8 and the Fermichamp strain. In medium M3, only the Fermichamp strain was able to fully consume the 100 g of fructose l^?1 but left a residual 32 g of glucose l^?1. Regarding the hexose transporters, a high number of amino acid polymorphisms were found in the Hxt1p sequences. Strain LCBG-3Y8 exhibited eight unique amino acid changes, followed by the Fermichamp strain with three changes. In Hxt3p, we observed nine amino acid polymorphisms unique for the Fermichamp strain and five unique changes for the mezcal strains.