SELECTIVE SACCHAROMYCES MEDIA CONTAINING ERGOSTEROL AND TWEEN 80

A mixture (ET80) of ergosterol and Tween 80 stimulated the anaerobic growth on solid media of almost all strains of Saccharomyces tested. Yeasts from other genera have only produced micro-colonies. It is possible to detect Saccharomyces yeasts after 40 h anaerobic incubation and subsequently to estimate total infection after a further 24 h aerobic incubation. Saccharomyces contaminants can be differentiated from other yeasts and from bacteria within 24 h by a simple staining procedure using malachite green. Anaerobic incubation for 4 days on ET80-supplemented crystal violet agar is selective for wild Saccharomyces only, thus allowing a clear differentiation between lysine-positive and lysine-negative yeasts. This medium is suitable for use with membrane filter-discs.     

Physiological requirements for growth and competitiveness of Dekkera bruxellensis under oxygen-limited or anaerobic conditions

The effect of glucose and oxygen limitation on the growth and fermentation performances of Dekkera bruxellensis was investigated in order to understand which factors favour its propagation in ethanol or wine plants. Although D. bruxellensis has been described as a facultative anaerobe, no growth was observed in mineral medium under complete anaerobiosis while growth was retarded under severe oxygen limitation. In a continuous culture with no gas inflow, glucose was not completely consumed, most probably due to oxygen limitation. When an air/nitrogen mixture (O₂‐content ca. 5%) was sparged to the culture, growth became glucose‐limited. In co‐cultivations with Saccharomyces cerevisiae, ethanol yields/g consumed sugar were not affected by the co‐cultures as compared to the pure cultures. However, different population responses were observed in both systems. In oxygen‐limited cultivation, glucose was depleted within 24 h after challenging with S. cerevisiae and both yeast populations were maintained at a stable level. In contrast, the S. cerevisiae population constantly decreased to about 1% of its initial cell number in the sparged glucose‐limited fermentation, whereas the D. bruxellensis population remained constant. To identify the requirements of D. bruxellensis for anaerobic growth, the yeast was cultivated in several nitrogen sources and with the addition of amino acids. Yeast extract and most of the supplied amino acids supported anaerobic growth, which points towards a higher nutrient demand for D. bruxellensis compared to S. cerevisiae in anaerobic conditions. Copyright © 2012 John Wiley & Sons, Ltd.     

Contribution of wild yeasts to the formation of volatile compounds in inoculated wine fermentations

The aim of this work was to study the contribution of wild yeasts to the volatile composition of wine in inoculated fermentations. To do so, Parellada must, sterilized and inoculated with Saccharomyces cerevisiae strain Na33 (pure inoculated fermentation), inoculated Parellada must (mixed inoculated fermentation) and Parellada must that fermented with its wild yeasts (control fermentation) were used. From the results obtained in the pure inoculated fermentation it can be seen that S. cerevisiae produced appreciable quantities of isoamyl acetate, ethyl hexanoate, ethyl octanoate, and ethyl decanoate. However, the wild yeasts also contributed to the synthesis of esters since the total concentration of these substances was higher in the mixed inoculated fermentation than in the pure inoculated fermentation. 2-Phenylethyl acetate was only synthesized by wild yeasts when they did not compete with S. cerevisiae. The concentration of total alcohols was similar in the three samples; the important production of isobutanol and 2-phenylethanol in the control fermentation is noteworthy. As regards the acids, the greatest concentration corresponded to the mixed inoculated fermentation. The wild yeasts contributed to the synthesis of these compounds to a significant extent and S. cerevisiae synthesized appreciable amounts of short-chain fatty acids.     

The influence of pre‐fermentative practices on the dominance of inoculated yeast starter under industrial conditions

The influence of pre‐fermentative practices on the growth dynamics of a ‘natural’ starter culture with specific phenotype (H₂S^?) concurrently with wild yeast populations was evaluated under winery conditions. Different clarification procedures and added SO₂ strongly influenced species and cell numbers isolable at the pre‐fermentation stage. Independent treatments of must with sulphite addition or vacuum‐filtering clarification caused a 30‐fold reduction in viable cells. Clarification procedures, enhanced by the selective effect of SO₂ addition, induced the appearance of Saccharomyces cerevisiae ‘wild’ yeasts. Correct application of the inoculum generally guarantees the dominance of fermentation by starter cultures. However, inoculated fermentations using unclarified white and red musts exhibited a consistent presence and persistence of non‐Saccharomyces and/or Saccharomyces ‘wild’ yeasts during fermentation. The extent and composition of the initial wild microflora at the start of fermentation may affect the presence and persistence of wild Saccharomyces and non‐Saccharomyces yeasts during guided fermentations under commercial conditions. The above findings confirm the results of previous works carried out at laboratory‐ or pilot‐scale level. Furthermore, they suggest a clear correlation between the modality of pre‐fermentative practices and the presence and persistence of ‘wild’ yeasts during fermentation. © 2002 Society of Chemical Industry     

Performance of wild Saccharomyces and Non-Saccharomyces yeasts as starter cultures in dough fermentation and bread making

The use of wild Saccharomyces and non-Saccharomyces yeasts might result in bread with different and attractive sensory characteristics. This study aimed to evaluate the performance of Saccharomyces and non-Saccharomyces yeasts as starter culture in dough fermentation to bread making and the physicochemical parameters and aromatic profile of bread. All 26 wild yeasts strains isolated from Brazilian Cerrado fruit and tree bark were osmotolerant, and 19.4% were able to ferment maltose. Candida tropicalis ART101.3 and Saccharomyces cerevisiae SC5952 had the best growth capacity under high concentrations of glucose and maltose. Also, they were resistant to lyophilisation. Kinetic parameters of bioreactor cultivations showed high cell growth and lower generation time with 10 g L⁻¹ maltose. Bread produced with C. tropicalis ART101.3 and the control bread had similar physicochemical properties and acceptance of consumers. Bread with S. cerevisiae SC5951 had a lower specific volume and a different colour than control bread; however, the consumers found no significant difference. More than 70% of the consumers demonstrated purchase intention of bread produced with both wild yeasts. The present study shows the potential of native Cerrado yeasts to be used and exploited in industrial processes and contributes to the diversification of bread starter cultures.     

Effect of dimorphic regulation on heterologous glucose oxidase production by Mucor circinelloides

The production of Aspergillus niger glucose oxidase (GOX) and native amylase by the recombinant M. circinelloides KFA199 strain under conditions of dimorphic growth was investigated. The recombinant KFA199 strain was compared to its parental ATCC 1216b strain and a wild‐type CBS 232.29 strain under similar morphology‐controlled conditions. Cultivation in Vogel's medium supplemented with ergosterol/Tween‐80 and sparged with nitrogen gas was most suitable for yeast‐like biomass production under anaerobic conditions. Anaerobic growth was characterized by high levels of ethanol formation and linear growth rates of 0.24–0.05/h, indicating metabolic stress. Subsequent to anaerobic growth, cultures were shifted to aerobic conditions to induce aerobic mycelial growth. GOX produced by the recombinant KFA199 after the shift to aerobic conditions was poorly secreted and accumulated intracellularly to 0.56 U/ml_culture. Amylase production by the KFA199, ATCC12b and CBS 232.29 strains was determined during growth on starch after the shift to aerobic culture. Growth‐associated amylase production by the ATCC 1216b (0.63 U/ml_culture) and wild‐type CBS 232.29 (0.33 U/ml_culture) strains was substantially higher than by the recombinant KFA199 strain (0.07 U/ml_culture), which may be related to the leucine auxotrophy of the transformation host, or genetic changes induced during transformation of the KFA199 strain. Copyright © 2010 John Wiley & Sons, Ltd.     

Differentiation between fermenting and spoilage yeasts in wine by total free fatty acid analysis

Differentiation between fermenting and spoilage yeasts in wine was estimated by cellular fatty acid profiles. Forty-two strains of yeasts representing 17 genera were grown on a defined liquid medium for 48 h at 25°C in a rotary shaker. After saponification of yeast cells, free fatty acid extracts were analysed by gas chromatography. Multivariate analysis was performed by Principal Components Analysis (PCA) to define clusters of fatty acids and yeasts. Strains were characterised especially by long-chain fatty acids, palmitic (C16) to linolenic (C18:3) acid under aerobic culture. Nevertheless, most of Saccharomyces cerevisiae and also Dekkera bruxellensis (former names D intermedia and Brettanomyces lumbicus) synthesized medium-chain fatty acids, octanoic (C8) acid to dodecanoic (C12) acid. With this method it was possible to differentiate fermenting grape yeasts such as S cerevisiae from spoilage yeasts on the basis of the absence of linoleic (C18:2) and linolenic (C18:3) acids. However, the method seemed unreliable for the identification of strains, more particularly those of species of S cerevisiae.     

Profile of volatile compounds during papaya juice fermentation by a mixed culture of Saccharomyces cerevisiae and Williopsis saturnus

This study investigated the formation and utilization of volatile compounds during papaya juice fermentation by a mixed culture of Saccharomyces cerevisiae and Williopsis saturnus. Time-course papaya juice fermentations were carried out using pure cultures of S. cerevisiae var. bayanus R2 and W. saturnus var. mrakii NCYC2251 and a mixed culture of the two yeasts at a ratio of 1:1000 (R2:NCYC2251). Changes in S. cerevisiae cell population, Brix, sugar consumption and pH were similar in the mixed culture and in the S. cerevisiae monoculture. There was an early growth arrest of W. saturnus in the mixed culture fermentation. A range of volatile compounds were produced during fermentation including fatty acids, alcohols, aldehydes and esters and some volatile compounds including those initially present in the juice were utilized. The mixed culture fermentation of S. cerevisiae and W. saturnus benefited from the presence of both yeasts, with more esters being produced than the S. cerevisiae monoculture and more alcohols being formed than the W. saturnus monoculture. The study suggests that papaya juice fermentation with a mixed culture of S. cerevisiae and W. saturnus may be able to result in the formation of more complex aroma compounds and higher ethanol level than those using single yeasts.     

Nadh: Ubiquinone oxidoreductase in obligate aerobic yeasts

The strictly aerobic yeasts Candida pinus, Cryptococcus albidus, Rhodotorula minuta, Rhodotorula mucilaginosa and Trichosporon beigelii possess mitochondrial NADH dehydrogenases with significant features of the NADH:ubiquinone oxidoreductase (complex I). These species show in all growth phases and under standard cultivation conditions, NADH dehydrogenases of approximately 700 kDa, which are sensitive to rotenone, a specific inhibitor of this complex. Identical results were obtained with the weakly fermenting C. pinus. The facultatively fermenting yeasts Saccharomyces cerevisiae and Kluyveromyces marxianus do not possess the 700 kDa-complex and are insensitive to rotenone. In S. cerevisiae, a rotenone-insensitive NADH dehydrogenase of about 500–600 kDa is detected only in stationary phase cells. As in Neurospora crassa, upon incubation of the obligately aerobic yeast R. mucilaginosa with chloramphenicol, an intermediate NADH dehydrogenase of approximately 350 kDa was formed, which was insensitive to rotenone.     

Improving the performance of industrial ethanol‐producing yeast by expressing the aspartyl protease on the cell surface

The yeasts used in fuel ethanol manufacture are unable to metabolize soluble proteins. The PEP4 gene, encoding a vacuolar aspartyl protease in Saccharomyces cerevisiae, was either secretively or cell‐surface anchored expressed in industrial ethanol‐producing S. cerevisiae. The obtained recombinant strains APA (expressing the protease secretively) and APB (expressing the protease on the cell wall) were studied under ethanol fermentation conditions in feed barley cultures. The effects of expression of the protease on product formation, growth and cell protein content were measured. The biomass yield of the wild‐type was clearly lower than that of the recombinant strains (0.578 ± 0.12 g biomass/g glucose for APA and 0.582 ± 0.08 g biomass/g glucose for APB). In addition, nearly 98–99% of the theoretical maximum level of ethanol yield was achieved (relative to the amount of substrate consumed) for the recombinant strains, while limiting the nitrogen source resulted in dissatisfactory fermentation for the wild‐type and more than 30 g/l residual sugar was detected at the end of fermentation. In addition, higher growth rate, viability and lower yields of byproducts such as glycerol and pyruvic acid for recombinant strains were observed. Expressing acid protease can be expected to lead to a significant increase in ethanol productivity. Copyright © 2010 John Wiley & Sons, Ltd.     

Determinants of flocculence of brewer's yeast during fermentation in wort

Ability of Saccharomyces cerevisiae MPY3 cells to flocculate during fermentation in wort was found to be triggered after growth limitation by oxygen shortage and to coincide with a sharp increase in cell surface hydrophobicity of the cells. Presence of oxygen in the pitching wort influenced final cell number, flocculence of the cells and cell surface hydrophobicity. Flocculation ability of cells grown in air-depleted pitching wort was hampered, concomitant with a decrease in final cell number and in final cell surface hydrophobicity. Addition of ergosterol and Tween 80 to air-depleted wort restored normal growth of the cells as well as flocculation ability and the increase in cell surface hydrophobicity. The same parameters increased in value after addition of ergosterol and Tween 80 to a fermentation with air-saturated pitching wort. Hydrophobicity of a non-flocculent mutant of S. cerevisiae strain MPY3, fermenting in air-saturated pitching wort, did not increase at cell division arrest. These results support the hypothesis that cell surface hydrophobicity is a major determinant for yeast cells to become flocculent, and suggest that shortage of sterols and unsaturated fatty acids precedes flocculence under brewing conditions.     

Studies on acetate ester production by non-Saccharomyces wine yeasts

A double coupling bioreactor system was used to fast screen yeast strains for the production of acetate esters. Eleven yeast strains were used belonging to the genera Candida, Hanseniaspora, Metschnikowia, Pichia, Schizosaccharomyces and Zygosacharomyces, mainly isolated from grapes and wine, and two wine Saccharomyces cerevisiae strains. The acetate ester forming activities of yeast strains belonging to the genera Hanseniaspora (Hanseniaspora guilliermondii and H. uvarum) and Pichia (Pichia anomala) showed different substrate specificities and were able to produce ethyl acetate, geranyl acetate, isoamyl acetate and 2-phenylethyl acetate. The influence of aeration culture conditions on the formation of acetate esters by non-Saccharomyces wine yeast and S. cerevisiae was examined by growing the yeasts on synthetic microbiological medium. S. cerevisiae produced low levels of acetate esters when the cells were cultured under highly aeration conditions, while, under the same conditions, H. guilliermondii 11104 and P. anomala 10590 were found to be strong producers of 2-phenylethyl acetate and isoamyl acetate, respectively.     

Effect of pure and mixed cultures of the main wine yeast species on grape must fermentations

Mixed inoculation of non-Saccharomyces yeasts and S. cerevisiae is of interest for the wine industry for technological and sensory reasons. We have analysed how mixed inocula of the main non-Saccharomyces yeasts and S. cerevisiae affect fermentation performance, nitrogen consumption and volatile compound production in a natural Macabeo grape must. Sterile must was fermented in triplicates and under the following six conditions: three pure cultures of S. cerevisiae, Hanseniaspora uvarum and Candida zemplinina and the mixtures of H. uvarum:S. cerevisiae (90:10), C. zemplinina:S. cerevisiae (90:10) and H. uvarum:C. zemplinina:S. cerevisiae (45:45:10). The presence of non-Saccharomyces yeasts slowed down the fermentations and produced higher levels of glycerol and acetic acid. Only the pure H. uvarum fermentations were unable to finish. Mixed fermentations consumed more of the available amino acids and were more complex and thus better able to synthesise volatile compounds. However, the amount of acetic acid was well above the admissible levels and compromises the immediate application of mixed cultures.     

Isolation and Characterization of Shochu Yeasts with Superior Brewing Ability from Shochu Mashes

Seven yeasts with superior ability in terms of alcohol fermentation and aromatic ingredient generation were isolated from 272 wild yeasts obtained from shochu mashes of shochu breweries. These seven yeasts were examined for their fermentation ability with rice and sweet potato using small scale of fermentation tests. Moreover, their thermotolerance was evaluated by growth tests and fermentation tests with barley koji. Among the isolated seven yeasts, the shochu yeast “MF062” was superior in the characteristics of fast fermentation, high alcohol production, aromatic ingredient generation and thermotolerance. It was named the “Heisei Miyazaki Yeast”. From the results of analyses of physiological and genetic characteristics of MF062, it was shown to be a Saccharomyces cerevisiae, but it showed different characteristics from the industrial yeasts used in shochu production. Using a sweet potato shochu brewing test, with 60 kg of raw materials and yeast MF062, it was confirmed that a high alcohol production yield and a high quality shochu could be obtained.     

Effects of mediums on fermentation behaviour and aroma composition in pure and mixed culture of Saccharomyces cerevisiae with Torulaspora delbrueckii

The nutrient status and composition in mediums have a significant effect on yeast metabolism and phenotypic characteristics in wine fermentation. In this study, the effects of three frequently used mediums, including synthetic grape juice (SGJ), grape juice without grape pericarp and seeds (GJ) and grape must with grape pericarp and seeds (GMPS), on yeast fermentation behaviour and aroma compounds produced by pure and mixed culture of Saccharomyces cerevisiae T73 with Torulaspora delbrueckii TD20 were investigated after alcoholic fermentation. The results showed that high fermentation activities and cell population were always found in GJ medium irrespective of inoculated approach. More esters and higher alcohols were produced in GMPS medium fermented by pure S. cerevisiae, while SGJ medium had increased levels of fatty acids. Consistent with previous literatures, the mixed fermentation of T. delbrueckii and S. cerevisiae produced more acetate esters and fatty acids than the pure culture of S. cerevisiae, while this enological trait was only found in SGJ and GJ, not in GMPS. Our results highlighted that more attention should be paid to the fermentation medium when evaluating the enological and aromatic properties of selected yeasts used in industrial winemaking. In this regard, the combined use of GJ and GMPS medium might be a suitable choice.     

Aerobic industrial processing of Empeltre cv. natural black olives and product characterisation

The fermentation of natural black olives is currently carried out under anaerobic conditions. This work investigates the aerobic fermentation of this product in tanks containing 16 000 kg of Empeltre olives during two seasons. The microorganisms in brines were yeasts in all cases, whereas lactic acid bacteria growth was only observed in some tanks. Consequently, the pH during fermentation was in most cases higher than recommended 4.3 units. However, the Empeltre olive brines displayed bactericidal effect against Salmonella enterica, Sthapylococcus aureus, Escherichia coli and Listeria monocytogenes, with a 5 log population drop in one or 24 h depending on the brine age so that these olive brines are a harsh environment for the growth of these pathogens. It was found HyEDA as the major phenolic compound in both brine and olive pulp at the beginning of fermentation that slowly hydrolysed into hydroxytyrosol that reached a concentration up to 1500 mg kg⁻¹.     

Monitoring of Brewing Yeast Propagation Under Aerobic and Anaerobic Conditions Employing Flow Cytometry

The vitality and viability of industrial strains of Saccharomyces cerevisiae was monitored during pilot plant experiments simulating yeast propagation under aerobic and anaerobic conditions. Industrial wort of 12°P original gravity was used as a growth substrate for yeast propagation. The work was carried out with three widely used Czech lager yeast industrial strains: strains 2, 7 and 95. Cell cycle, cell size, granularity, glycogen content, DNA and protein content were analyzed by flow cytometry. Significantly higher specific growth rates, higher content of yeast glycogen, earlier G2/M phase cells maximum, and faster cell protein creation was observed under aerobic conditions compared to anaerobic. Strains 7 and 95 showed losses in flocculation ability after aerobic propagation compared to anaerobic propagation. Under either aerobic or strictly anaerobic conditions, only strain 2 did not show a significant loss in flocculation ability.     

Adaptive response and tolerance to weak acids in Saccharomyces cerevisiae boulardii: a metabolomics approach

Weak acids inhibit the growth of probiotics, such as Saccharomyces boulardii. We explored the tolerance of S. boulardii to different weak acids. S. boulardii had better fermentation ability under lactic acid conditions compared with acetic and butyric acid conditions; however, the budding of S. boulardii was significantly stronger than that of Saccharomyces cerevisiae under acetic acid conditions. Although the surface structure of S. boulardii was destroyed, it produced more daughter cells. S. boulardii metabolites were also significantly different from S. cerevisiae under acidic stress. The growth of S. boulardii under weak acid conditions differed significantly from that of S. cerevisiae. S. boulardii-mediated fingerprints under weak acid conditions were identified as latent biomarkers, related to fructose and mannose metabolism, tricarboxylic acid cycle, and the glycolysis pathway. Identified biomarkers will aid in the genetic engineering of S. boulardii and other Saccharomyces strains for improved acid resistance and biomass yield.     

MICROBIOLOGICAL ASPECTS OF SPONTANEOUS WORT FERMENTATION IN THE PRODUCTION OF LAMBIC AND GUEUZE

Lambic and Gueuze are special Belgian beers obtained by spontaneous fermentation. Micro-organisms involved in this fermentation were counted and differentiated using several selective growth media. Micro-organisms were also isolated from samples of Lambic of different age and originating from different casks and brews and identified by classical tests. The following general pattern of microbial development was observed. After 3 to 7 days the fermentation started with the development of wort Enterobacteriaceae and strains of Kloeckera apiculata. These organisms were overgrown after 3 to 4 weeks by strains of Saccharomyces cerevisiae and S. bayanus. These were responsible for the main fermentation, lasting for 3 to 4 months. This was followed by a strong bacterial activity. This period was dominated by the growth of strains of Pediococcus cerevisiae. These reached their maximal numbers during the summer months and were responsible for a fivefold increase in lactic acid concentration. In some casks they caused ropiness. After the main fermentation period Lambic is very sensitive to spoilage by acetic acid bacteria of the genus Acetomonas. The presence of air may be the determining factor for their development. After 8 months a new increase in yeast cells was noted. These belonged now mainly to the genus Brettano-myces bruxellensis and Br. lambicus. They caused a further slow decrease in residual extract and the appearance of special flavours. Oxidative yeasts of the genera Candida, Cryptococcus, Torulopsis and Pichia were also detected and may be responsible for the formation of a flim on the beer surface after the main fermentation.     

Selected non-Saccharomyces wine yeasts in controlled multistarter fermentations with Saccharomyces cerevisiae

Non-Saccharomyces yeasts are metabolically active during spontaneous and inoculated must fermentations, and by producing a plethora of by-products, they can contribute to the definition of the wine aroma. Thus, use of Saccharomyces and non-Saccharomyces yeasts as mixed starter cultures for inoculation of wine fermentations is of increasing interest for quality enhancement and improved complexity of wines. We initially characterized 34 non-Saccharomyces yeasts of the genera Candida, Lachancea (Kluyveromyces), Metschnikowia and Torulaspora, and evaluated their enological potential. This confirmed that non-Saccharomyces yeasts from wine-related environments represent a rich sink of unexplored biodiversity for the winemaking industry. From these, we selected four non-Saccharomyces yeasts to combine with starter cultures of Saccharomyces cerevisiae in mixed fermentation trials. The kinetics of growth and fermentation, and the analytical profiles of the wines produced indicate that these non-Saccharomyces strains can be used with S. cerevisiae starter cultures to increase polysaccharide, glycerol and volatile compound production, to reduce volatile acidity, and to increase or reduce the total acidity of the final wines, depending on yeast species and inoculum ratio used. The overall effects of the non-Saccharomyces yeasts on fermentation and wine quality were strictly dependent on the Saccharomyces/non-Saccharomyces inoculum ratio that mimicked the differences of fermentation conditions (natural or simultaneous inoculated fermentation).