Diversity Of Yeast Species Research Articles

Changes of diversity and population of yeasts during the fermentations by pure and mixed inoculation of Saccharomyces cerevisiae strains

Mixed inoculation of Saccharomyces cerevisiae strains is used in winemaking for achieving high sensory quality of the wine. However, information on the diversity and population of yeasts during inoculated fermentation is very limited. In this study, we evaluated the effect of mixed inocula with different inoculation timing on the yeast community during fermentations of Cabernet Sauvignon. Grape must was inoculated with pure cultures of S. cerevisiae RC212 or S. cerevisiae R312, and simultaneous and sequential inoculation of both strains. Wallersterin Laboratory Nutrient (WLN) medium and sequence of the 26S rDNA D1/D2 domain were used to compare the diversity of yeast species. Five species, including Candida diversa, Hanseniaspora opuntiae, H. uvarum, Issatchenkia orientalis and I. terricola, were identified in the grape must, with Issatchenkia sp. being predominant (67.5 %). Three to four species were involved in each fermentation treatment. The fermentations by mixed inocula presented more yeast species than by pure inocula. Interdelta sequence typing was used to identify S. cerevisiae strains. Ten genotypes were identified among 322 isolated S. cerevisiae strains. Their distribution varied among different stages of fermentations and different inoculation treatments. The inoculated strains were not predominant, while indigenous genotypes I, III, and V showed strong competitiveness during fermentation. In general, this study provided information on the change of population structure and genetic diversity of yeasts in fermentations inoculated with pure and mixed S. cerevisiae strains.

In situ analysis of metabolic characteristics reveals the key yeast in the spontaneous and solid-state fermentation process of Chinese light-style liquor.

The in situ metabolic characteristics of the yeasts involved in spontaneous fermentation process of Chinese light-style liquor are poorly understood. The covariation between metabolic profiles and yeast communities in Chinese light-style liquor was modeled using the partial least square (PLS) regression method. The diversity of yeast species was evaluated by sequence analysis of the 26S ribosomal DNA (rDNA) D1/D2 domains of cultivable yeasts, and the volatile compounds in fermented grains were analyzed by gas chromatography (GC)-mass spectrometry (MS). Eight yeast species and 58 volatile compounds were identified, respectively. The modulation of 16 of these volatile compounds was associated with variations in the yeast population (goodness of prediction [Q(2)]>20%). The results showed that Pichia anomala was responsible for the characteristic aroma of Chinese liquor, through the regulation of several important volatile compounds, such as ethyl lactate, octanoic acid, and ethyl tetradecanoate. Correspondingly, almost all of the compounds associated with P. anomala were detected in a pure culture of this yeast. In contrast to the PLS regression results, however, ethyl lactate and ethyl isobutyrate were not detected in the same pure culture, which indicated that some metabolites could be generated by P. anomala only when it existed in a community with other yeast species. Furthermore, different yeast communities provided different volatile patterns in the fermented grains, which resulted in distinct flavor profiles in the resulting liquors. This study could help identify the key yeast species involved in spontaneous fermentation and provide a deeper understanding of the role of individual yeast species in the community.

Yeast diversity associated to sediments and water from two Colombian artificial lakes.

In Colombia, knowledge of the yeast and yeast-like fungi community is limited because most studies have focused on species with clinical importance. Sediments and water represent important habitats for the study of yeast diversity, especially for yeast species with industrial, biotechnological, and bioremediation potential. The main purpose of this study was to identify and compare the diversity of yeast species associated with sediment and water samples from two artificial lakes in Universidad del Valle (Cali-Colombia). Yeast samplings were performed from fifteen sediment samples and ten water samples. Grouping of similar isolates was initially based on colony and cell morphology, which was then complemented by micro/mini satellite primed PCR banding pattern analysis by using GTG5 as single primer. A representative isolate for each group established was chosen for D1/D2 domain sequencing and identification. In general, the following yeast species were identified: Candida albicans, Candida diversa, Candida glabrata, Candida pseudolambica, Cryptococcus podzolicus, Cryptococcus rajasthanensis, Cryptococcus laurentii, Williopsis saturnus, Hanseniaspora thailandica, Hanseniaspora uvarum, Rhodotorula mucilaginosa, Saccharomyces cerevisiae, Torulaspora delbrueckii, Torulaspora pretoriensis, Tricosporon jirovecii, Trichosporon laibachii and Yarrowia lypolitica. Two possible new species were also found, belonging to the Issatchenkia sp. and Bullera sp. genera. In conclusion, the lakes at the Universidad del Valle campus have significant differences in yeast diversity and species composition between them.

Evaluation of lactic acid bacteria and yeast diversity in traditional white pickled and fresh soft cheeses from the mountain regions of Serbia and lowland regions of Croatia

The goal of this study was the characterisation of indigenous lactic acid bacteria (LAB) and yeasts isolated from nine white pickled (BG) and nine fresh soft (ZG) artisanal cheeses collected in Serbia and Croatia. While LAB were present in all of the cheeses collected, yeasts were found in all BG cheeses but only in three ZG cheese samples. High LAB and yeast species diversity was determined (average H′L=0.4 and H′Y=0.8, respectively). The predominant LAB species in white pickled (BG) cheeses were Lactococcus lactis, Lactobacillus plantarum, and Leuconostoc mesenteroides, while in fresh soft (ZG) cheeses the most dominant LAB species were L. lactis, Enterococcus faecalis, and Leuconostoc pseudomesenteroides. Among the 20 yeast species found, Debaryomyces hansenii, Candida zeylanoides, and Torulaspora delbrueckii were found to be predominant in BG cheeses, while Yarrowia lipolytica was predominant in ZG cheeses. The characterisation of metabolic and technological potentials revealed that 53.4% of LAB isolates produced antimicrobial compounds, 44.3% of LAB strains showed proteolytic activity, while most of the yeast species possessed either lipolytic or proteolytic activity. In conclusion, the results obtained in this study showed that the composition of LAB and yeast populations in white pickled and fresh soft cheeses is region specific. The knowledge gained in this study could eventually be used to select region specific LAB and yeast strains for the production of white pickled and fresh soft artisanal cheeses with geographically specific origins under controlled conditions.

Grape berry yeast communities: Influence of fungicide treatments

The yeast communities colonising grape berry surfaces were evaluated for the influence of fungicide treatments in an organic vineyard (copper/sulphur-based products) and a conventional vineyard (commonly used fungicides). Analysis of yeast abundance and diversity was carried out on grape berries and juice during fermentation, using culture-dependent and -independent approaches. Yeast abundance was as generally reported for mature grapes and it was slight higher from grapes treated with conventional fungicides. Initial grape samples showed less yeast species diversity in the organic vineyard compared with the conventional one. In both vineyards, the dominant yeast were Candida zemplinina and Hanseniaspora uvarum (>50%), respectively, typical species that colonise surfaces of mature grape berries. Metschnikowia pulcherrima was widely found in the conventional samples while it was only occasionally found in organic ones.Saccharomyces cerevisiae was isolated only at the end of natural fermentation (conducted in sterile condition), with lower levels in the organic samples. S. cerevisiae strains showed less intraspecies diversity in the organic samples (two genotypes), in comparison with the conventional samples (six genotypes).

Mitochondrial Genome Evolution in a Single Protoploid Yeast Species

Mitochondria are organelles, which play a key role in some essential functions, including respiration, metabolite biosynthesis, ion homeostasis, and apoptosis. The vast numbers of mitochondrial DNA (mtDNA) sequences of various yeast species, which have recently been published, have also helped to elucidate the structural diversity of these genomes. Although a large corpus of data are now available on the diversity of yeast species, little is known so far about the mtDNA diversity in single yeast species. To study the genetic variations occurring in the mtDNA of wild yeast isolates, we performed a genome-wide polymorphism survey on the mtDNA of 18 Lachancea kluyveri (formerly Saccharomyces kluyveri) strains. We determined the complete mt genome sequences of strains isolated from various geographical locations (in North America, Asia, and Europe) and ecological niches (Drosophila, tree exudates, soil). The mt genome of the NCYC 543 reference strain is 51,525 bp long. It contains the same core of genes as Lachancea thermotolerans, the nearest relative to L. kluyveri. To explore the mt genome variations in a single yeast species, we compared the mtDNAs of the 18 isolates. The phylogeny and population structure of L. kluyveri provide clear-cut evidence for the existence of well-defined geographically isolated lineages. Although these genomes are completely syntenic, their size and the intron content were found to vary among the isolates studied. These genomes are highly polymorphic, showing an average diversity of 28.5 SNPs/kb and 6.6 indels/kb. Analysis of the SNP and indel patterns showed the existence of a particularly high overall level of polymorphism in the intergenic regions. The dN/dS ratios obtained are consistent with purifying selection in all these genes, with the noteworthy exception of the VAR1 gene, which gave a very high ratio. These data suggest that the intergenic regions have evolved very fast in yeast mitochondrial genomes.

Diversity of yeast species during fermentative process contributing to Chinese Maotai-flavour liquor making

The famous traditional Chinese Maotai-flavour liquor is produced by a unique spontaneous simultaneous saccharification and fermentation process, which contributes to a distinctive yeast community with specific physiological properties and performances. Therefore, it would be useful to investigate this yeast community and reveal the novelty of its characteristics. Nine yeast species were obtained from the fermentation period. A combination of physiological and functional analyses revealed a very high diversity of yeast populations. In particular, the extremely high temperature and low acidity of the fermentation conditions led to an accumulation of species with distinctive heat- and acid-resistant properties. Moreover, these yeast species were also significant flavour contributors, for various alcohols, acids and esters. The Chinese Maotai-flavour liquor-fermentation process is rich in yeast resources with distinctive fermentation properties, which were different from other beverages. This work is the first to study the complex yeast ecology of the Maotai-flavour liquor-making process. It allows a deeper insight into the mechanism of this process.

Biodiversity in Oscypek, a Traditional Polish Cheese, Determined by Culture-Dependent and -Independent Approaches

Oscypek is a traditional Polish scalded-smoked cheese, with a protected-designation-of-origin (PDO) status, manufactured from raw sheep's milk without starter cultures in the Tatra Mountains region of Poland. This study was undertaken in order to gain insight into the microbiota that develops and evolves during the manufacture and ripening stages of Oscypek. To this end, we made use of both culturing and the culture-independent methods of PCR followed by denaturing gradient gel electrophoresis (PCR-DGGE) and pyrosequencing of 16S rRNA gene amplicons. The culture-dependent technique and PCR-DGGE fingerprinting detected the predominant microorganisms in traditional Oscypek, whereas the next-generation sequencing technique (454 pyrosequencing) revealed greater bacterial diversity. Besides members of the most abundant bacterial genera in dairy products, e.g., Lactococcus, Lactobacillus, Leuconostoc, Streptococcus, and Enterococcus, identified by all three methods, other, subdominant bacteria belonging to the families Bifidobacteriaceae and Moraxellaceae (mostly Enhydrobacter), as well as various minor bacteria, were identified by pyrosequencing. The presence of bifidobacterial sequences in a cheese system is reported for the first time. In addition to bacteria, a great diversity of yeast species was demonstrated in Oscypek by the PCR-DGGE method. Culturing methods enabled the determination of a number of viable microorganisms from different microbial groups and their isolation for potential future applications in specific cheese starter cultures.

Survey of yeast diversity during wine bottling processes using restriction analysis of 26S ribosomal DNA (rDNA)

Background and Aims: Microbiological stability of bottled wine is largely dependent on the bottling process as the last technological operation. In this survey, yeast contamination was monitored and characterised during wine bottling. Methods and Results: Samples of wine, apparatus surfaces and bottles were collected. Yeast counts were recorded and colony morphotypes were differentiated. Isolates were characterised by restriction enzyme digests of polymerase chain reaction amplified regions of 26S ribosomal DNA. For unknown patterns, DNA sequencing was performed. The presence of Zygosaccharomyces bailii and Torulaspora delbrueckii in the filling tubes was also confirmed in the final product. Another important point of contamination was the corker jaws. Conclusions: A great diversity of yeast species was found, indicating that these habitats are prone to microbial development. Significance of the Study: Evaluation of the level and diversity of yeast contamination during wine bottling allowed to alert producers for the need to evaluate and control microbial contamination.

Yeast species associated with spontaneous wine fermentation of Cabernet Sauvignon from Ningxia, China

The eastern base of the Helan Mountains in Ningxia is a fast developing wine production area in China. Of urgent necessity to the Ningxia wine industry is to be able to produce wines with typical regional characteristics. It is well known that autochthonous yeast species and strains play an important role in introducing local character or terroir into the winemaking practice. The aim of this study was to investigate indigenous yeast species diversity and preselect desirable S. cerevisiae strains in the Ningxia region. Four hundred wine-related yeast colonies were isolated from Cabernet Sauvignon musts in three vineyards at the beginning, middle and final stages of spontaneous fermentations. Yeast species were first classified according to colony morphologies on Wallerstein Laboratory Nutrient Agar (WL) and confirmed by sequencing of the D1/D2 domain of the 26S rRNA gene. Nine unique colony morphology types were profiled on WL agar and three new types were found. After sequence analysis of representative colonies from each WL group, nine yeast species were identified, namely H. uvarum, H. occidentalis, M. pulcherrima, C. zemplinina, H. vineae, I. orientalis, Z. bailii, P. kluyveri and S. cerevisiae. The non-Saccharomyces yeasts appeared mainly in the early stage of the fermentation while H. uvarum, I. orientalis and P. kluyveri were able to remain in the final stage. Fourty-six S. cerevisiae isolates were preselected according to physiological characteristics and twelve strains with valuable fermentation properties were obtained.

Occurrence and dominance of yeast species in naturally fermented milk from the Tibetan Plateau of China

To determine which yeasts are present in the naturally fermented milks of China, 69 samples made by the nomads of Tibet were collected from the Tibetan Plateau in China. From these samples, 225 strains of yeast were isolated and identified using conventional microbiological analysis and gene sequencing analysis of the D1/D2 domain of the large subunit (26S) ribosomal DNA. The results showed that the total concentration of yeasts in these samples ranged from 5.01 to 8.97 log10 colony-forming units (CFU)/mL (6.91± 1.02 log10 CFU/mL; mean± SD). The number of cultivable yeasts was higher in the samples from Qinghai (7.55± 0.75 log10 CFU/mL) than those from Tibet (6.21± 0.79 log10 CFU/mL, P< 0.05). Moreover, there were 15 phylotypes in these 69 samples. Among these phylotypes, Kluyveromyces marxianus (49.3%, frequency percentage), Saccharomyces cerevisiae (62.3%), and Pichia fermentans (46.4%) appeared frequently and can be considered the most common culturable species in naturally fermented milk products. Traditional fermented Mongolian cow milk featured a wide diversity of yeast species, including Issatchenkia orientalis, Kazachstania unisporus, Rhodotorula mucilaginosa, Candida pararugosa, Torulaspora delbrueckii, Geotrichum sp., Kazachstania unisporus, Geotrichum fragrans, Debaryomyces hansenii, Yarrowia lipolytica, Trichosporon gracile, and Pichia membranifaciens. This study provides new data on yeast composition in naturally fermented milk and shows the yeast biodiversity of fermented milk products from the Tibetan Plateau of China.

Isolation and molecular identification of wine yeasts from a Brazilian vineyard

Traditional winemaking is carried out by spontaneous yeasts from the vineyard that persist throughout the alcoholic fermentation. This study examined the diversity of yeast species isolated from grape skin and musts of two varieties of Vitis labrusca from a vineyard in the southeast region of Brazil (Jales, Sao Paulo), which produces artisanal wines. Molecular identification was achieved by a combination of PCR-RFLP/sequencing of the internal transcribed spacers (ITS) and sequencing of the D1/D2 domain of ribosomal DNA. Eighty yeast samples were isolated from grapes and musts, and seven different species were identified. The diversity of species varied according to the grape variety. The most frequent species were Hanseniaspora uvarum with 28 isolates, followed by Issatchenkia occientalis with 19 isolates and Issatchenkia orientalis with 16 isolates. Other species with a lower number of isolates were: Issatchenkia terricola, Saccharomyces cerevisiae, Aureobasidium pullulans and Sporidiobolus pararoseus. Our results showed that molecular identification is a very powerful identification method in which natural isolates of ascomycetous or basidiomycetous yeasts can be rapidly and reliably identified with better reproducibility and higher throughput than conventional phenotypic methods. This is the first report of the diversity of indigenous yeast species from a vineyard in Brazil.

Yeast species associated with wine grapes in China

Having more information on the yeast ecology of grapes is important for wine-makers to produce wine with high quality and typical attributes. China is a significant wine-consuming country and is becoming a serious wine-producer, but little has been reported about the yeast ecology of local ecosystems. This study provides the first step towards the exploitation of the yeast wealth in China's vine-growing regions. The aim of this study was to investigate the yeast population density and diversity on three grape varieties cultivated in four representative vine-growing regions of China. Yeast species diversity was evaluated by using polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) and sequence analysis of the 5.8S internal transcribed spacer (ITS) ribosomal DNA (rDNA) region of cultivable yeasts. The grapes harbored yeast populations at 10 2–10 6 CFU/mL, consisting mostly of non- Saccharomyces species. Seventeen different yeast species belonging to eight genera were detected on the grape samples tested, including Hanseniaspora uvarum, Cryptococcus flavescens, Pichia fermentans, Candida zemplinina, Cryptococcus carnescens, Candida inconpicua, Zygosaccharomyces fermentati, Issatchenkia terricola, Candida quercitrusa, Hanseniaspora guilliermondii, Candida bombi, Zygosaccharomyces bailii, Sporidiobolus pararoseus, Cryptococcus magnus, Metschnikowia pulcherrima, Issatchenkia orientalis and Pichia guilliermondii. H. uvarum and C. flavescens were the dominant species present on the grapes. For the first time Sporidiobolus pararoseus was discovered as an inhabitant of the grape ecosystem. The yeast community on grape berries was influenced by the grape chemical composition, vine-variety and vine-growing region. This study is the first to identify the yeast communities associated with grapes in China using molecular methods. The results enrich our knowledge of wine-related microorganisms, and can be used to promote the development of the local wine industry.

Yeast heterogeneity during spontaneous fermentation of black Conservolea olives in different brine solutions

To assess the yeast community structure and dynamics during Greek-style processing of natural black Conservolea olives in different brine solutions. Black olives were subjected to spontaneous fermentation in 6% (w/v) NaCl brine solution or brine supplemented with (i) 0.5% (w/v) glucose, (ii) 0.2% (v/v) lactic acid and (iii) both glucose and lactic acid. Yeast species diversity was evaluated at the early (2 days), middle (17 days) and final (35 days) stages of fermentation by restriction fragment length polymorphism and sequence analyses of the 5.8S internal transcribed spacer and the D1/D2 ribosomal DNA (rDNA) regions of isolates. Analysis revealed a relatively broad range of biodiversity composed of 10 genera and 17 species. In all treatments, yeasts were the main micro-organisms involved in fermentation together with lactic acid bacteria that coexisted throughout the processes. Metschnikowia pulcherrima was the dominant yeast species at the onset of fermentation, followed by Debaryomyces hansenii and Aureobasidium pullulans. Species heterogeneity changed as fermentations proceeded and Pichia membranifaciens along with Pichia anomala evolved as the main yeasts of olive elaboration, prevailing at 17 and 35 days of the process. Molecular techniques allowed for the identification of five yeast species, namely A. pullulans, Candida sp., Candida silvae, Cystofilobasidium capitatum and M. pulcherrima, which have not been reported previously in black olive fermentation. By using molecular techniques, a rich yeast community was identified from Conservolea black olive fermentations. Metschnikowia pulcherrima was reported for the first time to dominate in different brines at the onset of fermentation, whereas Pichia anomala and P. membranifaciens evolved during the course. The addition of glucose and/or lactic acid perturbed yeast succession and dominance during fermentation. Yeasts have an important role in black olive fermentation and contribute to the development of the organoleptic characteristics of the final product. At the same time, certain species can cause significant spoilage. The present study adds to a better knowledge of yeast communities residing in olive fermentations towards a well-controlled process with minimization of product's losses.

Yeast diversity during tapping and fermentation of palm wine from Cameroon

In the present study, we have investigated the occurrence of yeast flora during tapping and fermentation of palm wine from Cameroon. The yeast diversity was investigated using both traditional culture-dependent and culture-independent methods. Moreover, to characterize the isolates of the predominant yeast species ( Saccharomyces cerevisiae) at the strain level, primers specific for δ sequences and minisatellites of genes encoding the cell wall were used. The results confirm the broad quantitative presence of yeast, lactic acid bacteria and acetic acid bacteria during the palm wine tapping process, and highlight a reduced diversity of yeast species using both dependent and independent methods. Together with the predominant species S. cerevisiae, during the tapping of the palm wine the other species found were Saccharomycodes ludwigii and Zygosaccharomyces bailii. In addition, denaturing gradient gel electrophoresis (DGGE) analysis detected Hanseniaspora uvarum, Candida parapsilopsis, Candida fermentati and Pichia fermentans. In contrast to the progressive simplification of yeast diversity at the species level, the molecular characterization of the S. cerevisiae isolates at the strain level showed a wide intraspecies biodiversity during the different steps of the tapping process. Indeed, 15 different biotypes were detected using a combination of three primer pairs, which were well distributed in all of the samples collected during the tapping process, indicating that a multistarter fermentation takes place in this particular natural, semi-continuous fermentation process.

Influence of grape treatment on the wine yeast populations isolated from spontaneous fermentations

To study the influence of different methods of grape treatment in wineries on the diversity of the yeast species in spontaneous fermentations. Grapes were crushed and pressed in three different ways followed by spontaneous fermentation. The same grape material picked and crushed aseptically directly in the vineyard served as control. Yeasts isolated at different stages of the fermentation were characterized by 5.8S-ITS-RFLP. Yeasts of the Saccharomyces sensu stricto complex were additionally analysed by microsatellite polymerase chain reaction fingerprinting. The diversity of yeast species isolated from winery fermentations was much greater than from the vineyard fermentation in respect to yeasts of the genus Saccharomyces as well as non-Saccharomyces. Oenonogical methods alter significantly the yeast diversity in spontaneous fermentations of grape juice. Managing spontaneous fermentations successfully depends not only on choosing the suitable grapes but also on the crushing and pressing techniques leading to different yeast populations.

Partial 26S rDNA restriction analysis as a tool to characterise non- Saccharomyces yeasts present during red wine fermentations

Restriction patterns of amplified regions of ribosomal large subunit RNA encoding genes (26S rDNA) were evaluated as a routine methodology to examine yeast species diversity during red wine fermentation. The results were confirmed by sequencing of D1/D2 region of 26S rDNA. Red wine production was carried out using a yeast starter culture together with different commercial products, namely enzymes, fermentation activators and tannins and their influence on the non- Saccharomyces yeast population was studied. Yeast strains were isolated using lysine agar as a selective medium for non- Saccharomyces yeasts, after morphological characterisation of colonies. Amplification of 26S rDNA followed by digestion with three restriction enzymes applied to the 121 isolates, generated 19 profiles and a very high correlation with sequencing results was achieved. Although a starter yeast culture was added, results showed that several yeast species were present during all stages of fermentation, independent of the conditions tested, emphasizing the diversity of microorganisms associated with winemaking. On the other hand commercial additives did not significantly influence the diversity of yeast population during the fermentation process. For non- Saccharomyces strains, restriction patterns of a PCR amplified 26S rDNA region proved to be an adequate tool for clustering strains at species level and enabled the monitoring of yeast population dynamics during red wine fermentation.

Yeast interslope divergence in soils and plants of "Evolution Canyon", Lower Nahal Oren, Mount Carmel, Israel

This is the first report on yeast species diversity recorded in Israel at the Evolution Canyon microsite in Mount Carmel, Israel, subdivided into a xeric, African;, savanna-like south-facing slope, and a mesic, forested, European north-facing slope. A total of 25 species belonging to 14 genera were recorded--19 species of soil yeasts from 11 genera and 14 species of plant yeasts from 10 genera, including 8 species and 7 genera common to plants and soil. The total number, as well as the total frequency of occurrence of yeast species, was higher on the European slope than on the African slope in samples from both plants and soil. The basidiomycetous yeast species, Cryptococcus magnus, the ascomycetous species, Debaryomyces hansenii var. hansenii, with its anamorph, Candida famata, and the black yeast, Aureobasidium pullulans, were dominant on both slopes and in both plant and soil samples.

Geographical differences in human oral yeast flora.

The oral yeast flora of healthy humans from eastern North America and China were sampled and compared. Chinese persons harbored a greater number and diversity of yeast species in the mouth. Furthermore, Candida albicans, which is the predominant commensal and etiologic species of candidiasis in Europe and the Western Hemisphere, was relatively rare in China.

Changes in the microflora of manouri, a traditional Greek whey cheese, during storage

Six batches of manouri cheese were manufactured, and selected microbial groups were counted throughout storage for 20 days at 4°C. The counts of all the microbial groups increased ( P &lt; 0.05) throughout storage and reached higher levels in cheeses made in summer than in those made in spring. Moreover, the micro‐organisms developed better on the cheese surfaces than in the interiors, especially in summer. The pH (6.78–7.33) and salt‐in‐moisture content (2.53–3.72) of the cheeses did not seem to affect the growth of the bacteria and yeasts present. The isolates of enterobacteriaceae were mainly Hafnia (68.75%), while the isolates from the Baird–Parker medium were mainly staphylococci. There was a great diversity of yeast species, but Debaryomyces hansenii and Pichia membranefasciens predominated. The isolates of enterobacteriaceae, staphylococci and—to a lesser extent—yeasts were proteolytic, but the free amino acid (N‐NH2) content of the cheese did not increase significantly during storage (136.4–225.2 mg/kg glycine equivalent). It also seemed that milk fat was not degraded to any great extent (acid degree value 0.09–0.19) by the lipolytic activity of the strains. The main enzymes detected in selected isolates of enterobacteriaceae were leucine aminopeptidase and phosphohydrolase.