Molecular identification of yeasts associated with traditional Egyptian dairy products.

This study aimed to examine the diversity and ecology of yeasts associated with traditional Egyptian dairy products employing molecular techniques in yeast identification. A total of 120 samples of fresh and stored Domiati cheese, kariesh cheese, and "Matared" cream were collected from local markets and examined. Forty yeast isolates were cultured from these samples and identified using the restriction-fragment length polymorphism (RFLPs) of 5.8S-ITS rDNA region and sequencing of the domains D1 and D2 of the 26S rRNA gene. Yeasts were identified as Issatchenkia orientalis (13 isolates), Candida albicans (4 isolates), Clavispora lusitaniae (Candida lusitaniae) (9 isolates), Kodamaea ohmeri (Pichia ohmeri) (1 isolate), Kluyveromyces marxianus (6 isolates), and Candida catenulata (7 isolates). With the exception of C. lusitaniae, the D1/D2 26S rRNA gene sequences were 100% identical for the yeast isolates within the same species. Phylogenetic reconstruction of C. lusitaniae isolates grouped them into 3 distinguished clusters. Kariesh cheese was found to be the most diverse in its yeast floras and contained the highest total yeast count compared with other examined dairy products. This was linked to the acidic pH and lower salt content of this cheese, which favor the growth and survival of yeasts in foodstuffs. Stored Domiati cheese also contained diverse yeast species involving isolates of the pathogenic yeast C. albicans. This raises the possibility of dairy products being vehicles of transmission of pathogenic yeasts.

El-Sharoud WM ，Belloch C ，Peris D ，Querol A ... -  《-》

Identification of yeasts associated with milk products using traditional and molecular techniques.

An integrated approach including phenotypic (morphological, biochemical and physiological characterization) and genotypic (RAPD-PCR, sequencing of D1/D2 domain of 26S rRNA encoding gene) methods was used for the identification of yeasts isolated from different milk products. There were 513 isolates in all, 460 ascomycetous and 53 basidiomycetous yeasts. The yeast isolates were characterized on the basis of their biochemical and physiological properties, and the D1/D2 domain of 26S rDNA was sequenced in selected strains. Relying on the obtained results from both the data-sets, corresponding type strains were selected and compared with the respective yeast isolates from milk products by RAPD fingerprinting. The strains showing a degree of similarity >80% were considered conspecific. By means of the applied techniques it was possible to identify 92% yeast isolates at species level. Debaryomyces hansenii, Geotrichum candidum, Kluyveromyces marxianus, Yarrowia lipolytica and Candida zeylanoides are the most frequently isolated species. The majority of the yeasts were isolated from fresh and sour curd cheese. A comparison of the results obtained by phenotypic and genotypic investigation revealed that the identification based on classical methods was supported by genotypic characterization in only 54% of examined isolates. The results described in this work show that the applied molecular identification is a reliable approach to the identification of yeasts associated with milk products in contrast to the conventional biochemical and physiological tests. The identification of new yeast species requires additional genetic markers such as sequencing of different genes or DNA:DNA hybridization.

Lopandic K ，Zelger S ，Bánszky LK ，Eliskases-Lechner F ，Prillinger H ... -  《FOOD MICROBIOLOGY》

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.

Bai M ，Qing M ，Guo Z ，Zhang Y ，Chen X ，Bao Q ，Zhang H ，Sun TS ... -  《-》

The diversity of eukaryotic microbiota in the traditional Slovak sheep cheese--bryndza.

We investigated the occurrence and diversity of yeasts and filamentous fungi in bryndza an artisanal Slovak soft spreadable cheese prepared from raw sheep milk or from a mixture of sheep and cow milk. Samples collected during four months of the summer production period from two locations (northern and southern parts of central Slovakia) contained 10(5)-10(7) (cfu) yeasts and about 10(2) (cfu) of mold per gram of wet weight. Further characterization by conventional taxonomy and sequence comparison of D1/D2 region from 26S rRNA gene revealed Mucor circinelloides v. Tieghem as the predominant filamentous fungus. A novel Geotrichum sp. together with Kluyveromyces (K. lactis/K. marxianus) was identified as the most abundant yeast species. Occasionally other yeasts, such as Candida inconspicua, Candida silvae, Pichia fermentans and Trichosporon domesticum were found. Conventional taxonomy readily identified isolates to the genus level, but DNA sequence comparison was capable of discriminating them at the species level.

Laurencík M ，Sulo P ，Sláviková E ，Piecková E ，Seman M ，Ebringer L ... -  《INTERNATIONAL JOURNAL OF FOOD MICROBIOLOGY》

Development and evaluation of a real-time quantitative PCR assay for detection and enumeration of yeasts of public health interest in dairy products.

Yeast contamination is a problem in the food industry as a cause of spoilage. Moreover, various species of yeasts are known to be capable of causing opportunistic infections in humans. We have developed a real-time quantitative PCR (qPCR) assay to directly detect and quantify nine emerging opportunistic yeast species (Candida albicans, Candida glabrata, Candida parapsilosis, Candida tropicalis, Clavispora lusitaniae, Filobasidiella neoformans, Issatchenkia orientalis, Trichosporon asahii, and Trichosporon jirovecii) in dairy product samples. We designed six primer pairs, conserved sequences of the variable D1/D2 domains of the 26S rRNA gene, to detect the yeasts and demonstrated their specificity. The qPCR assay could accurately quantify emerging opportunistic yeasts in an artificially contaminated dairy product. qPCR with the primer pairs we designed, was very sensitive and will allow producers to enumerate contaminating yeasts and identify whether they are opportunistic pathogens, in only 4 to 5h. This assay can easily be extended to other food items and to a variety of food-monitoring initiatives.

Makino H ，Fujimoto J ，Watanabe K 《-》