Kloeckera Apiculata Research Articles

NATURAL MICROFLORA OF WINE GRAPE BERRIES

The diversity of yeasts and bacterial species on grapes has been investigated in vineyards worldwide. For winemaking are very important three groups of microorganisms. First two includes acetic acid and lactic acid bacteria; they live and grow normally on grape surface. The third group includes more than 20 detectable genera of yeasts. There are three principal genera found on grapes Hanseniaspora uvarum (Kloeckera apiculata), Metschnikowia pulcherrima (Candida pulcherrima), and Candida stellata or new descripted Candida zemplinina. Aim of this study was investigate of number of three major groups of microorganisms which are important for grapes and winemaking. The number of bacteria on Acetobacter agar (AA) ranged from 1.76 log CFU/mL to 2.80 log CFU/mL. Lactic acid bacteria were counted on MRS agar and the number of detectable colonies ranged from 0.48 log CFU/mL to 2.06 log CFU/mL. Sabouraud dextrose agar (SDA) was used for cultivation of yeast and the number of yeasts ranged from 2.47 log CFU/mL to 2.76 log CFU/mL. For identification of yeast species were used different types of agar media with acid base indicator bromocresol green. Identified 10 yeasts species includes to genus: Candida, Metschnikowia, Pichia, Kluyveromyces, Hanseniaspora, Hansenula, Candida, Debaromyces, Rhodotorula and Saccharomyces. We identified only few bacterial species includes to genus Lactobacillus, Pediococcus, Gluconobacter and Acetobacter.

Mechanisms of action for 2-phenylethanol isolated from Kloeckera apiculata in control of Penicillium molds of citrus fruits.

BackgroundGreen and blue mold decay, caused by Penicillium digitatum and P. italicum, respectively, are important postharvest diseases of citrus. Biocontrol by microbes is an alternative to synthetic fungicide application. In this study, the antagonistic yeast strain Kloeckera apiculata 34–9 was used to investigate the action mechanisms involved in the biocontrol of postharvest diseases.ResultsAn antifungal substance, 2-phenylethanol (PEA), was isolated from K. apiculata and demonstrated to have antimicrobial activity against selected phytopathogenic fungi. Experiments on P. italicum cells identified the mitochondria and the nucleus as particularly sensitive to inhibition. Regulation of P. italicum gene expression was investigated using RNA-Seq. PEA up-regulated genes involved with the peroxisome, regulation of autophagy, phosphatidylinositol signaling system, protein processing in endoplasmic reticulum, fatty acid metabolism, and inhibited ribosome, RNA polymerase, DNA replication, amino acid biosynthesis, aminoacyl-tRNA biosynthesis and cell cycle. Inhibitory responses revealed by RNA-Seq suggest that PEA might compete for attachment on the active site of phenylalanyl-tRNA synthetase (PheRS).ConclusionThis study provided new insight on the mode of action of biocontrol yeast agents in controlling postharvest pathogenic fungi.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-014-0242-2) contains supplementary material, which is available to authorized users.

Effect of physicochemical factors on glycerol production by simultaneous cultures of wine micro-organisms using the response surface method.

To evaluate the effect of temperature, pH and SO2 on growth and glycerol production improvement by Saccharomyces cerevisiae mc2, Kloeckera apiculata mF and Oenococcus oeni X2L using the response surface method (RSM). Multifactorial design of cultures with physicochemical factors variations was performed. The micro-organisms grew in all cultures conditions. Overall, after 6 days yeasts prevailed, especially S. cerevisiae (10(9) CFU ml(-1)), while O. oeni reached 10(7) CFU ml(-1). At initial fixed pH 5·5, metabolic behaviour of cultures showed a temperature-dependent response. Total malate consumption occurred at 26°C, 50 mg l(-1) SO2. Glucose and pentoses utilization was highly modified when varying SO2. Ethanol showed negative interaction with temperature-SO2 relationship. At low SO2, glycerol and acetate production increased when temperature enhanced. Predictive results of RSM indicate that 26°C, 60·24 mg l(-1) SO2 and pH 5·5 were the optimal conditions for glycerol and organic acids synthesis compatible with wine quality. We propose a predictive condition to improve the performance of mixed cultures for must fermentations. To optimize the culture conditions to design mixed starters containing autochthonous yeasts and O. oeni strains for winemaking and to obtain products with high glycerol content, low acidity and maintenance of regional characteristics.

The effects of Kloeckera apiculata on the cuticular waxes of navel orange fruit

Kloeckera apiculata 34-9 was selected from the rhizosphere soil for its high efficacy in controlling citrus green and blue mold. In this study, the effect of the antagonistic yeast K. apiculata on citrus cuticular wax was investigated in ripening Newhall navel orange (Citrus sinensis L. Osbeck). Our results show that K. apiculata triggers the production of cuticular waxes and surface wax morphology changes in the fruit surface. 15 K. apiculata-responsive differentially expressed genes (DEGs) were identified for wax metabolism by using the Affymetrix citrus genome GeneChip. Using GC–MS, 46 wax compounds were found in the Newhall fruit surface. On one hand, esters including docosanoic acid, 1,2,3-propanetriyl ester and 9-hexadecenoic acid, 9-octadecenyl ester were up-regulated approximately twofold in the treatment condition compared with the control; and on the other hand, the fatty acids and fatty alcohols decreased by 74.4% and 72.3%, respectively, in the treatment condition. The new wax production and increased hydrophobicity of the Newhall surface resulting from the treatment may influence spore adhesion and germination. Furthermore, the response of the fruit waxes to the K. apiculata stimulus is likely to be regulated by intra-cellular H2O2 signaling. This study demonstrated the response fruit waxes to K. apiculata in Newhall navel oranges, thus providing new clues that aid our understanding of the mechanisms of action of antagonistic yeasts in postharvest fruits.

Phenylethanol promotes adhesion and biofilm formation of the antagonistic yeastKloeckera apiculatafor the control of blue mold on citrus

The yeast Kloeckera apiculata strain 34-9 is an antagonist with biological control activity against postharvest diseases of citrus fruit. In a previous study it was demonstrated that K.apiculata produced the aromatic alcohol phenylethanol. In the present study, we found that K.apiculata was able to form biofilm on citrus fruit and embed in an extracellular matrix, which created a mechanical barrier interposed between the wound surface and pathogen. As a quorum-sensing molecule, phenylethanol can promote the formation of filaments by K.apiculata in potato dextrose agar medium, whereas on the citrus fruit, the antagonist remains as yeast after being treated with the same concentration of phenylethanol. It only induced K.apiculata to adhere and form biofilm. Following genome-wide computational and experimental identification of the possible genes associated with K.apiculata adhesion, we identified nine genes possibly involved in triggering yeast adhesion. Six of these genes were significantly induced after phenylethanol stress treatment. This study provides a new model system of the biology of the antagonist-pathogen interactions that occur in the antagonistic yeast K.apiculata for the control of blue mold on citrus caused by Penicillium italicum.

Glycerol production by Oenococcus oeni during sequential and simultaneous cultures with wine yeast strains.

Growth and fermentation patterns of Saccharomyces cerevisiae, Kloeckera apiculata, and Oenococcus oeni strains cultured in grape juice medium were studied. In pure, sequential and simultaneous cultures, the strains reached the stationary growth phase between 2 and 3 days. Pure and mixed K. apiculata and S. cerevisiae cultures used mainly glucose, producing ethanol, organic acids, and 4.0 and 0.1 mM glycerol, respectively. In sequential cultures, O. oeni achieved about 1 log unit at 3 days using mainly fructose and L-malic acid. Highest sugars consumption was detected in K. apiculata supernatants, lactic acid being the major end-product. 8.0 mM glycerol was found in 6-day culture supernatants. In simultaneous cultures, total sugars and L-malic acid were used at 3 days and 98% of ethanol and glycerol were detected. This study represents the first report of the population dynamics and metabolic behavior of yeasts and O. oeni in sequential and simultaneous cultures and contributes to the selection of indigenous strains to design starter cultures for winemaking, also considering the inclusion of K. apiculata. The sequential inoculation of yeasts and O. oeni would enhance glycerol production, which confers desirable organoleptic characteristics to wines, while organic acids levels would not affect their sensory profile.

Characterization of competition for nutrients in the biocontrol of Penicillium italicum by Kloeckera apiculata

Kloeckera apiculata strain 34–9 was isolated for its biocontrol effectiveness against Penicillium italicum, the causal agent of blue mold of citrus. Competition for nutrients by strain 34–9 was investigated as a potential mechanism of antagonism. The results indicated that the antagonist inhibited conidial germination of the pathogen in diluted orange juice but did not kill the spores. Exposing these conidia to fresh orange juice increased conidial germination to the control level. In addition, the conidia were able to germinate in conditions of sufficient nutrient availability when co-cultured with K. apiculata. A total of 20 carbonaceous substances and 22 nitrogenous substances were tested for utilization by the antagonist and the pathogen. Twenty nitrogenous compounds were utilized by both K. apiculata and P. italicum, and one additional compound was utilized by P. italicum. P. italicum utilized 17 carbon sources, and K. apiculata utilized only four carbon sources. The addition of yeast nitrogen base without amino acids and ammonium sulfate, l-phenylalanine, l-tryptophan or l-tyrosine to the fruit wound reduced the biocontrol effectiveness of the antagonist. These results support the hypothesis that K. apiculata competes with the phytopathogen for nutrients and vitamins.

Polyphenol content and biological activities of Mesembryanthemum edule organs after fractionation

Mesembryanthemum edule (Aizoaceae) is an edible halophyte widely used as a traditional remedy against fungal and bacterial infections. This study investigates phenolic contents and biological activities of aqueous methanolic fractions (methanol/acidified water, v/v: 20/80, 40/60 and 60/40) of M. edule leaves, stems and roots. The most phenol-rich fractions were leaf 20%, stem 60%, and root 40% (from 671 to 989mg GAEg−1 DR). The highest ferric reducing power was found in leaf 40% and stem 40% (86 and 94μgml−1, respectively) whereas the highest total antioxidant activity was noted in root 40% (395mg GAEg−1 DR). Concerning the antimicrobial activity, organ extracts were assayed for their activity against food borne bacteria and fungi. The inhibitory percentages ranged from 0 to 94%. Stems showed the strongest antibacterial activity (inhibition of the growth in 6 of the 7 strains tested) especially against Micrococcus luteus (82%) followed by leaves and roots. Regarding fraction polarity, 60% and 20% aqueous methanolic fractions were the most and the less active fractions, respectively. All the organ fractions showed a high antifungal activity, notably against Kloeckera apiculata (85% for leaf 40%) and Candida albicans (77% for stem 40%). Even more, leaf, stem and root fractions were very potent in inhibiting growth of filamentous fungi, with inhibition percentages varying between 23% and 99%. Overall, the fractionation process enabled a better assessment of M. edule antioxidant and antimicrobial activities, which were attributed to various metabolites with different polarities. Our findings also indicate that M. edule organs could be used as a potent source of natural antioxidants and antibiotics.

PHENOLIC POTENTIAL DETERMINATION AND YEASTS IDENTIFICATION WITH SIGNIFICANT LEAVENS IN ISABELLA GRAPE (<I>Vitis labrusca</I>) FROM VILLA DEL ROSARIO (Norte de Santander) FOR WINE MAKING

The Isabella grape (Vitis labrusca) is a red variety that has been optimally adapted to the edafoclimatic conditions in the region of Villa del Rosario (Norte de Santander) and has been accepted also in local markets. Currently, the grape is eaten fresh and its potential for winemaking is unknown. Therefore, the purpose of this study was to establish the phenolic potential and identify important yeasts of this kind of grape for winemaking. To this end, berries taken from two consecutive vintages were analyzed. Parameters analyzed were density, soluble solids, pH, acidity and total and removable phenolic potential. The yeasts were identified morphologically and biochemically. The results of the physicochemical parameters of the two vintages were averaged and compared with results for grapes from Valle del Cauca. The results indicated that the berries of the two regions were similar in physical characteristics but significantly different in terms of chemical composition. The total phenolic potential was higher in grapes from Valle del Cauca, while removable was similar in the two regions. Five yeast species: Kloeckera apiculata, Criptococcus laurentii, Candida guillermondii, Candida novergensis y Rhodotorula glutinis were microbiologically isolated and identified.

Characterization of wines produced by mixed culture of autochthonous yeasts and Oenococcus oeni from the northwest region of Argentina

Two autochthonous yeasts from the northwest region of Argentina, Kloeckera apiculata mc1 and Saccharomyces cerevisiae mc2, were used as pure or mixed starter cultures in microvinification trials conducted in Malbec red must. Also, the effect of Oenococcus oeni X(2)L was evaluated. S. cerevisiae mc2 showed adequate growth and fermentative activity in single and composite fermentations, producing standard concentration of ethanol. The amount of esters was higher in fermentations conducted using mixed yeast starters. Independent of the timing of inoculation of O. oeni, this malolactic bacterium completely depleted malic acid. Sensory evaluation indicated that young wines fermented with mixed yeast cultures and sequential inoculation of O. oeni were preferred, achieving the highest scores for positive descriptors and they allowed better control of the sensory quality. Consequently, this study proposes inclusion of autochthonous K. apiculata mc1 as an adjunct culture to S. cerevisiae mc2 during Malbec must fermentation to improve the organoleptic properties of red wines. Furthermore, sequential inoculation of O. oeni X(2)L should be carried out after completion of the alcoholic fermentation to enhance sensory characteristics.

The inter-generic fungicidal activity of Xanthophyllomyces dendrorhous

In this study, the existence of intra-specific and inter-generic fungicidal activity in Xanthophyllomyces dendrorhous and Phaffia rhodozyma strains isolated from different regions of the earth was examined. Assays were performed under several culture conditions, showing that all the analyzed X. dendrorhous and P. rhodozyma strains have killing activity against Kloeckera apiculata, Rhodotorula sloffiae, and R. minuta. This activity was greater in rich media at a pH from 4.6 to 5.0. Extracellular protein extracts with fungicidal activity were obtained from cultures of all strains, and their characterization suggested that a protein of 33 kDa is the antifungal factor. According to peptide mass fingerprinting and an analysis of the results with the MASCOT search engine, this protein was identified as an aspartic protease. Additionally, extrachromosomal double-stranded DNA elements (dsDNAs) were observed in all X. dendrorhous and P. rhodozyma strains. Although there is a high variability, two dsDNAs of 5.4 and 6.8 kb are present in all strains.

Diversity and dynamics of the fungal community in Lake Ohrid

Fungi are universally present in freshwater systems and are of great significance in the structural and functional organization of these ecosystems. The aim of this study was to assess the diversity of fungal communities in the ancient, oligotrophic Lake Ohrid and to investigate their spa- tial and temporal dynamics. The fungal community in Lake Ohrid consists of 36 species identified from 213 isolates. The autochthonous community is made up of 10 species belonging to the orders Saprolegniales and Peronosporales: Achlya americana, A. racemosa, Aphanomyces leavis, Dic- tyuchus monosporus, D. sterile, Pythium ultimum, Saprolegnia ferax, S. hypogina, S. monoica, and Saprolegnia sp. The allochthonous community is composed of 26 species from 15 genera of fungi. The dominant genera are Aspergillus (5 species, 13.86% of determined species), Penicillium (6 species, 16.66%), and Cladosporium and Fusarium (3 species, 8.33%). The species Kloeckera apiculata, Didiostible sp., Gliocladium roseum, and Varicosporium delicatum are identified here for the first time in freshwater ecosystems of the Balkans. Quantitative analysis of the fungal community showed that the number of propagules varied in the range of 0 to 19.5 × 10 3 l -1 . The diversity and abundance of both the autochthonous and the allochthonous fungal communities are greater in the littoral than in the pelagial zone. Our results represent the first hydromycological data for Lake Ohrid the region of Macedonia.

Quantitative and qualitative analysis of non-Saccharomyces yeasts in spontaneous alcoholic fermentations

In this study, we looked at the yeast population present in four spontaneous alcoholic fermentations in the Rioja appellation (D.O.Ca. Rioja, Spain). The study was conducted through the identification of the yeasts via the PCR–RFLP technique of the ITS region of rDNA. In a first harvest, the qualitative diversity of the species present in spontaneous alcoholic fermentation was studied, and in a second harvest, their quantitative significance. In spontaneous fermentations, 17 different yeast species were found, although only two of them, Candida stellata and Kloeckera apiculata, as well as Saccharomyces cerevisiae, appeared in major proportions during fermentation. The significance of the non-Saccharomyces yeasts during the spontaneous alcoholic fermentation was conditioned by the presence of Saccharomyces cerevisiae in the medium. Species not cited in literature in connection with winemaking and yeasts associated with wines spoilage have been detected in all the alcoholic fermentations carried out in the qualitative study.

Influence of indigenous yeasts on the fermentation and volatile profile of plum brandies

The aim of this study was to determine the influence of different yeasts isolated from fresh blue plum fruits ( Aureobasidium sp.) and spontaneously fermenting plum musts ( Kloeckera apiculata and Saccharomyces cerevisiae), as well as commercial wine and distillery strains, on the fermentation and chemical composition of plum brandies. Gas chromatography methods were used to detect major volatile components. The most rapid fermentation occurred in musts inoculated with S. cerevisiae. However, the highest concentration of ethanol was detected in samples after spontaneous fermentation (8.40% v/v). Plum brandies obtained after distillation contained from 66.3 ( K. apiculata) up to 74.3% v/v ethanol (spontaneous fermentation). The samples after spontaneous fermentation were distinguished by a high content of acetoin, ethyl acetate and total esters, accompanied by a low level of methanol and fusel alcohols. Non- Saccharomyces yeasts were responsible for higher concentrations of esters and methanol, while S. cerevisiae strains resulted in increased levels of higher alcohols. It was also found that isolated indigenous strains of S. cerevisiae synthesized relatively low amounts of higher alcohols compared to commercial cultures. Samples obtained using the distillery strain of S. cerevisiae received the highest score (18.2) during sensory analysis and were characterized by a well-harmonised taste and aroma.

Wine-growing Areas in Oltenia (Romania) Major Natural Sources for the Isolation, Identification and Selection of Oenological Micoorganisms

The isolation and identification of the microbial agents responsible with the alcoholic fermentation process which cover all types of wine and the malolactic fermentation that is carried only by the red wines, begun almost when it was established the fact that the fermentations are biological processes done under the action of the enzymes that are produced by the microorganisms. As the passage of time, the searches of this kind have been boosted, so that almost no country is producing wines without having such concerns in the oenological microbiology field our researches also join on this line and have began with the isolation and identification of the yeasts present in different wine growing areas in Oltenia (Romania) then continuing with the isolation and identification of lactic bacteria present in these areas. During the study were isolated and identified yeasts strains of species belonging to Saccharomyces ellipsoideus, Saccharomyces oviformis, Saccharomyces rosei, Metschikowia reukaufii, Pichia membranaefaciens, Candida vinaria, Kloeckera apiculata, Rhodotorula glutinis and strains of the lactic bacteria that belong to the species: Lactobacillus plantarum, Leuconostoc mesenteroides, Oenococcus oeni. Knowing the physiologic and biochemical characteristics of these organisms, we can determine how they can influence positive or negative quality of the wine, the research will be continued to determine what types of relationships are established between these types of microorganisms both in the vineyard area and in wine.

Enhancement of survival of probiotic and non-probiotic lactic acid bacteria by yeasts in fermented milk under non-refrigerated conditions

The effects of yeasts on the survival of probiotic and non-probiotic lactic acid bacteria (LAB) were studied in fermented milk under non-refrigerated conditions (30 °C) with a view to develop ambient-stable fermented milk with live LAB. Five yeasts tested ( Saccharomyces bayanus, Williopsis saturnus var. saturnus, Yarrowia lipolytica, Candida kefyr and Kluyveromyces marxianus) enhanced the survival of Lactobacillus bulgaricus (but not Streptococcus thermophilus) in a mixed yoghurt culture in yoghurt by ~ 10 2 to 10 5-fold. Seven yeasts examined ( Candida krusei, Geotrichum candidum, Pichia subpelliculosa, Kloeckera apiculata, Pichia membranifaciens, Schizosaccharomyces pombe and Y. lipolytica) improved the survival of Lactobacillus rhamnosus in fermented milk by ~ 10 3 to 10 6-fold. W. saturnus var. saturnus enhanced the survival of Lactobacillus acidophilus, L. rhamnosus (probiotic) and Lactobacillus reuteri by up to 10 6-fold, but the same yeast failed to improve the survival of Lactobacillus johnsonii (probiotic), S. thermophilus and L. bulgaricus in fermented milk. These results provide definitive evidence that yeasts possess stability-enhancing effects on LAB and that the specific effects of yeasts on LAB stability vary with yeasts as well as with LAB. However, the molecular mechanism of such interaction of yeasts with LAB remains to be found.

Growth Inhibition of Pathogenic Bacteria and Some Yeasts by Selected Essential Oils and Survival ofL. monocytogenesandC. albicansin Apple–Carrot Juice

Food safety is a fundamental concern of both consumers and the food industry. The increasing incidence of foodborne diseases increases the demand of using antimicrobials in foods. Spices and plants are rich in essential oils and show inhibition activity against microorganisms, which are composed of many compounds. In this research, effects of garlic, bay, black pepper, origanum, orange, thyme, tea tree, mint, clove, and cumin essential oils on Listeria monocytogenes AUFE 39237, Escherichia coli ATCC 25922, Salmonella enteritidis ATCC 13076, Proteus mirabilis AUFE 43566, Bacillus cereus AUFE 81154, Saccharomyces uvarum UUFE 16732, Kloeckera apiculata UUFE 10628, Candida albicans ATCC 10231, Candida oleophila UUPP 94365, and Metschnikowia fructicola UUPP 23067 and effects of thyme oil at a concentration of 0.5% on L. monocytogenes and C. albicans in apple-carrot juice during +4 degrees C storage (first to fifth day) were investigated. Strong antibacterial and antifungal activities of some essential oils were found. Thyme, origanum, clove, and orange essential oils were the most inhibitory against bacteria and yeasts. Cumin, tea tree, and mint oils inhibited the yeasts actively. It is concluded that some essential oils could be used as potential biopreservatives capable of controlling foodborne pathogens and food spoilage yeasts.

Kloeckera apiculata strain (34-9) to controlBotrytis cinerea during the pre- and postharvest handling of strawberries

The efficacy ofKloeckera apiculata strain (34-9) in controlling gray mould (Botrytis cinerea) of strawberry fruit was evaluated in pre- and postharvest handling. Dynamic growth ofK. apiculata strain (34-9) was tested in the field on strawberry. Antagonist population was 2.2×106 CFU/ml in strawberry fruit after it was treated 2 h in the field, then decreased slightly, and then the population stabilized at the concentration 105 CFU/ml during the period of strawberry growth. The effect ofK. apiculata strain (34-9) (1.0×108 CFU/ml) onB. cinerea was evaluated. Preharvest (34-9) treatment was the most effective, while postharvest (34-9) and Sumilex treatment equally reduced the incidence of decay, caused by gray moulds (B. cinerea). Light microscopy revealed attachment of the yeast cells to the pathogen hyphae.Kloeckera apiculata strain (34-9) did not alter any quality parameters of fruit when assessed at the end of storage.

Optimising the inactivation of grape juice spoilage organisms by pulse electric fields

The effect of some pulsed electric field (PEF) processing parameters (electric field strength, pulse frequency and treatment time), on a mixture of microorganisms ( Kloeckera apiculata, Saccharomyces cerevisiae, Lactobacillus plantarum, Lactobacillus hilgardii and Gluconobacter oxydans) typically present in grape juice and wine were evaluated. An experimental design based on response surface methodology (RSM) was used and results were also compared with those of a factorially designed experiment. The relationship between the levels of inactivation of microorganisms and the energy applied to the grape juice was analysed. Yeast and bacteria were inactivated by the PEF treatments, with reductions that ranged from 2.24 to 3.94 log units. All PEF parameters affected microbial inactivation. Optimal inactivation of the mixture of spoilage microorganisms was predicted by the RSM models at 35.0 kV cm − 1 with 303 Hz pulse width for 1 ms. Inactivation was greater for yeasts than for bacteria, as was predicted by the RSM. The maximum efficacy of the PEF treatment for inactivation of microorganisms in grape juice was observed around 1500 MJ L − 1 for all the microorganisms investigated. The RSM could be used in the fruit juice industry to optimise the inactivation of spoilage microorganisms by PEF.

Evolution of Microbial Load of Grapes During the Ripening Process

In the process of growth of the grapes, as well as in the time of the alcoholic fermentation, the yeast’s structure abide many changes, that means that some kinds of yeast’s that were on the surface of the grapes can be found poorly in the first steps of the alcoholic fermentation and nearly none at the end of this process. Due to that, we decided to investigate the presence of the yeast’s during the maturation, fermentation of the grapes and also during different treatments given to the must. The research takes place at the Segarcea vineyard, on a sort of white grapes called Feteasca alba. 11 species have been found: Saccharomyces ellipsoideus, Saccharomyces oviformis, Saccharomyces rosei, Candida vini, Pichia membranaefaciens, Hansenula anomala, Kloeckera apiculata, Hanseniaspora uvarum, Metschnikowia pulcherima, Rhodotorula glutinis, Rhodotorula rubra. The smallest load of yeast’s has been found in the wine free running must, and the biggest one in the must made at the second presser. The yeast’s load depends also on the kind of wine clearance here have been used two kinds: the bentonite and sulphur dioxide and a second kind with cooling and sulphur dioxide. Due to the determination we found out that the reduction of the yeast’s emphasised by the first clarification with bentonite and sulphur dioxide. The researches will continue to analyse the influence of other clarification methods of the must on the number of yeast cells.