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.     

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.     

The effects of temperature and pH on the growth of yeast species during the fermentation of grape juice

The effects of temperature and pH on the survival and growth of Saccharomyces cerevisiae, Kloeckera apiculata, Candida stellata, Candida krusei, Candida pulcherrima and Hansenula anomala were examined during mixed culture in grape juice. At 25°C, pH 3.0 and pH 3.5, S. cerevisiae dominated the fermentation and the other species died off before fermentation was completed. Saccharomyces cerevisiae also dominated the fermentation at 20°C but there was increased growth and survival of the other species. At 10°C the fermentation was dominated by the growth of both S. cerevisiae and K. apiculata and there was extended growth and survival of C. stellata and C. krusei. Juices fermented at 10°C exhibited ethanol concentrations between 7.4 and 13.4% and populations of K. apiculata, C. stellata and C. krusei in the range 10⁶-10⁸ cells/ml. However, these species produced maximum ethanol concentrations in the range 2.7–6.6% when grown as single cultures in grape juice.     

The effect of temperature on the growth of strains of Kloeckera apiculata and Saccharomyces cerevisiae in apple juice fermentation

The influence of temperature (10°C and 25°C) on the survival and growth of Saccharomyces cerevisiae and Kloeckera apiculata was examined in mixed and pure cultures during fermentation in apple juice. The growth reached by S. cerevisiae did not seem to be affected by temperature and the presence of K. apiculata . However, the growth and survival of K. apiculata , both in single and mixed cultures, were substantially enhanced at 10°C. The highest amount of ethyl acetate was produced by K. apiculata in pure culture at 10°C. Nevertheless, this concentration was lowest when both yeasts were fermented together at 10°C and 25°C.     

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.     

Growth of Natural Yeast Flora during the Fermentation of Inoculated Wines

The growth of yeasts that occur naturally in grape juice was quantitatively examined during the fermentation of four wines that had been inoculated with Saccharomyces cerevisiae. Although S. cerevisiae dominated the wine fermentations, there was significant growth of the natural species Kloeckera apiculata, Candida stellata, Candida colliculosa, Candida pulcherrima, and Hansenula anomala.     

The effect of acetic acid bacteria upon the growth and metabolism of yeasts during the fermentation of grape juice

The influence of species of Acetobacter and Gluconobacter upon growth of the wine yeasts Saccharomyces cerevisiae, Kloeckera apiculata and Candida stellata was examined during mixed culture in grape juice. Acetobacter pasteurianus, A. aceti and Gluconobacter oxydans grew in conjunction with yeasts during juice fermentation. As determined by viable counts, yeast growth was only slightly impaired by the presence of bacteria. However, as judged by the concentrations of glucose, fructose, ethanol, glycerol, acetaldehyde, ethyl acetate, iso -amyl alcohol and organic acids in the fermented juice, acetic acid bacteria significantly influenced the alcoholic fermentation by yeasts.     

The production of 2,3-butanediol as a differentiating character in wine yeasts

The capacity to produce 2,3-butanediol by 90 strains of four different species of wine yeasts (Kloeckera apiculata, Saccharomyces cerevisiae, Saccharomycodes ludwigii, Zygosaccharomyces bailii) was tested in grape must by automated multiple development HPTLC. The total amount of 2,3-butanediol produced varied between 23mg l–1 and 857.7mg l–1 according to the yeast species. S. cerevisiae and Z. bailii behaved similarly, producing elevated amounts of 2,3-butanediol. K. apiculata and Sc. ludwigii, in contrast, were low producers. When considerable amounts of 2,3-butanediol were found, little acetoin was present; the amounts of butanediol and acetoin were characteristic of the individual species.     

Nutrient utilization profile of Saccharomyces Cerevisiae from palm wine in tropical fruit fermentation

The nutrient utilization pattern of Saccharomyces cerevisiae from palm wine was studied using tropical fruits as substrate. Starter cultures were prepared by growing 15-18 h old stock cultures of the yeast in successively larger bottles containing pasteurized fruit must. Microvinification, substrate utilization and assay of yeast activity were performed. Soluble solute (SS) content of the juices ranged from 10-18 Brix. Pinapple must had the highest SS content (18 Brix) while pawpaw had a low SS value of 10 Brix. These SS values were low compared to that of grape juice. The wines produced from the fruit must had percentage alcohol levels ranging from 10.6 to 12.6. Volatile activity ranged from 0.25 to 0.32 while crude protein values ranged from 0.58 to 0.68%. Palm wine yeast and all the other yeast strains fermented and utilized the fruit must for growth with specific growth rates ranging from 0.18 to 0.22. Sugar loss in Brix was gradual for all the fruit musts from 20.0-24.0 Brix to a range of 4.8 to 6.0 Brix. Pineapple was highly preferred for tropical wine making. Mango, cashew and pawpaw had equal ranking for commercial scale fermentation though more sugar will be needed to ameliorate cashew and pawpaw than mango juice. Palm wine yeast (OW-11) compared favourably with the other wine yeasts (CBS 8066 and ATCC 4126) both in nutrient utilization pattern and growth performance. A high degree of adaptability was observed in palm wine yeast recommands it for industrial wine production.     

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.     

Evaluation of selective media for enumeration of yeasts during wine fermentation

The growth of individual species of yeasts during wine fermentations was measured by plating wine samples on malt extract, ethanol sulphite and lysine agars. Colonies of Saccharomyces cerevisiae dominated on plates of malt extract agar and sometimes masked the presence of other non- Saccharomyces species. Lysine agar suppressed the growth of S. cerevisiae and enabled the enumeration of non- Saccharomyces species such as Kloeckera apiculata, Candida stellata and Saccharomycodes ludwigii. The growth of non- Saccharomyces yeasts on ethanol sulphite agar was variable.     

Flocculation and cell surface characterization of Kloeckera apiculata from wine

AIMS: To characterize and analyze the flocculation phenomenon of Kloeckera apiculata mc1 from Argentinian wine to understand the cell-cell interaction pattern. METHODS AND RESULTS: Kloeckera apiculata mc1 possess intense cell-cell interactions in MYPG medium (0.5% malt extract, 1% yeast extract, 2% glucose, 2% peptone), pH 5.5 by shaking at 25 degrees C. Optimum flocculation is observed at pH 4.5 in the presence of 3 mmol l-1 Ca2+. The flocculation is induced by peptone and malt extract and not by yeast extract and is reversed by 50 mmol l-1 galactose or lactose. The flocculation is highly susceptible to pronase, chymotrypsine and proteases types IV and XXVII and is partially resistant to trypsin. The electronic microscopy shows that the cells are attached to each other along their sides by fine hair-like threads. CONCLUSIONS: The mechanism of flocculation of K. apiculata mc1 is mediated by protein-carbohydrate interaction, stabilized by Ca2+. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of selected pure yeast inocula of known ability is preferred to wine elaboration, therefore the indigenous flora must be avoided and the flocculation of K. apiculata could be an economic method to do it.     

双歧椰汁米酒实验室生产工艺的研究

目的 以优质糯米和椰汁为原料,通过双歧杆菌和酿酒酵母共生发酵研制出一种功能性的米酒.方法 采用正交试验设计的方法得到了双歧杆菌和酿酒酵母最佳的共生发酵条件.结果 最佳的共生发酵条件是:发酵温度35℃、双歧杆菌接种量3％、酿酒酵母接种量2％、发酵时间16 h,再通过添加30％酿酒酵母单独发酵产物之后,研制出双歧椰汁米酒.结论 采用此工艺生产的功能性米酒既有椰汁的果香味又有米酒的醇香味,口感柔和,易于接受.     

Peculiarities of flor strains adapted to Sardinian sherry-like wine ageing conditions

Saccharomyces cerevisiae flor yeasts, which are subjected to stressful conditions during wine ageing, exhibit a number of characteristics which distinguish them from non-flor S. cerevisiae wine strains. In the present work, 22 flor and 14 non-flor S. cerevisiae wine strains are compared, in order to elucidate other possible peculiarities of these yeasts. The results obtained demonstrate that in contrast to the homothallic nature of the non-flor strains, 77% of the flor strains exhibit two variants of a semi-homothallic life cycle. Moreover, the flor-forming ability is shown to be inversely correlated to spore viability and the utilisation of maltose and galactose.     

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.     

Interactions between Saccharomyces cerevisiae and malolactic bacteria: preliminary characterization of a yeast proteinaceous compound(s) active against Oenococcus oeni

AIMS: To investigate the occurrence and extent of Saccharomyces cerevisiae and Oenococcus oeni interactions. METHODS AND RESULTS: Interactions between S. cerevisiae and O. oeni were investigated by double-layer and well-plate assays showing the occurrence of specific interactions for each yeast-malolactic bacteria (MLB) coupling. Heat and protease treatments of synthetic grape juice fermented by the S. cerevisiae strain F63 indicated that the inhibitory activity exerted by this yeast on O. oeni is due to a proteinaceous factor(s) which exerts either bacteriostatic or bactericidal effect depending on concentration and affects malolactic fermentation in natural grape juice and wine. CONCLUSIONS: A proteinaceous factor(s) produced by a S. cerevisiae wine strain able to inhibit O. oeni growth and malic acid fermentation was characterized. SIGNIFICANCE AND IMPACT OF THE STUDY: The individuation, characterization and exploitation of yeast proteinaceous factor(s) exerting inhibitory activity on MLB may offer new opportunities for the management of malolactic fermentation.     

Microsatellite PCR profiling of Saccharomyces cerevisiae strains during wine fermentation

AIMS: Use of microsatellite PCR to monitor populations of Saccharomyces cerevisiae strains during fermentation of grape juice. METHOD AND RESULTS: Six commercial wine strains of S. cerevisiae were screened for polymorphism at the SC8132X locus using a modified rapid PCR identification technique. The strains formed four distinct polymorphic groups that could be readily distinguished from one another. Fermentations inoculated with mixtures of three strains polymorphic at the SC8132X locus were monitored until sugar utilization was complete, and all exhibited a changing population structure throughout the fermentation. CONCLUSIONS: Rapid population quantification demonstrated that wine fermentations are dynamic and do not necessarily reflect the initial yeast population structure. One or more yeast strains were found to dominate at different stages of the fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: The population structure of S. cerevisiae during mixed culture wine fermentation is dynamic and could modify the chemical composition and flavour profile of wine.     

关于四个酵母菌种对乙醇耐受性的研究

本文报道了从存活,生长,发酵能力和氢离子渗漏等角度研究和比较四种酵母菌(Zygosaccharomyces bailii NCYC 1427,Saccharomyces cerevisiae NCYC 431,Harisenula anomala NCYC 711和 Kloeckera apiculata NCYC 468)对乙醇的耐受性。乙醇对酵母在上述特性的抑制程度,取决于乙醇的浓度。Z．Bailii NCYC 1427是四个酵母中乙醇耐受性最强的。在2M的乙醇溶液中,其存活率与无乙醇的对照组一样高。在低于1.5M的乙醇中,其生长率高于 S．Cerevisiae NCYC 431,氢离子渗入细胞的速率增长则低于 S．Cerevisiae 和 H．Anomala。S．Cerevisiae NCYC 431对于乙醇抑制生长的效应耐受性很强,在低于IM的乙醇溶液中,其发酵能力下降得较为缓慢,存活率几乎没有变化。H．Anomala NCYC 711在生长、发酵能力和氢离子渗漏方面对乙醇的抑制作用均表现敏感,但其存活率变化却与S．Cerevisiae NCYC 431很相似。K．Apiculata NCYC 468在存活、生长和发酵能力方面对乙醇的效应是四个菌种中最敏感的。研究结果还表明,乙醇对生长、发酵能力和氢离子渗漏的作用比它对存活的影响大得多。环境条件也影响酵母对乙醇的耐受性。     

A Note on the Nature and Sequence of Yeasts during Fermentation of Apples grown in India

Six morphologically different groups of yeasts comprising Kloeckera apiculata. Candida krusei, Candida valida, Candida sorbosa, Metschnikowia pulcherrima and Saccharomyces chevalieri were isolated from fresh, fermenting and fermented apple juice. This is the first report on the isolation of C. valida, C. sorbosa and S. chevalieri from fermenting and fermented apple juice.     

Divergence in wine characteristics produced by wild and domesticated strains of Saccharomyces cerevisiae

The budding yeast Saccharomyces cerevisiae is the primary species used by wine makers to convert sugar into alcohol during wine fermentation. Saccharomyces cerevisiae is found in vineyards, but is also found in association with oak trees and other natural sources. Although wild strains of S. cerevisiae as well as other Saccharomyces species are also capable of wine fermentation, a genetically distinct group of S. cerevisiae strains is primarily used to produce wine, consistent with the idea that wine making strains have been domesticated for wine production. In this study, we demonstrate that humans can distinguish between wines produced using wine strains and wild strains of S. cerevisiae as well as its sibling species, Saccharomyces paradoxus. Wine strains produced wine with fruity and floral characteristics, whereas wild strains produced wine with earthy and sulfurous characteristics. The differences that we observe between wine and wild strains provides further evidence that wine strains have evolved phenotypes that are distinct from their wild ancestors and relevant to their use in wine production.