Randomly amplified polymorphic DNA (RAPD) PCR for the identification of yeasts isolated from dairy products

In the present work randomly amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) with primers M13 and RF2 was applied to the identification at species level of yeast strains isolated from cheeses. RAPD-PCR analysis of the type strains of different yeast species gave distinctive band profiles that allowed a clear differentiation of all the considered species. Forty-two of the 48 dairy associated yeasts were clearly assigned to the species Saccharomyces cerevisiae, Kluyveromyces marxianus (anamorph Candida kefyr), Kluyveromyces lactis (anamorph Candida sphaerica), Debaryomyces hansenii (anamorph Candida famata), Yarrowia lipolytica and Torulaspora delbrueckii (anamorph Candida colliculosa). The method, which is rapid and easy to perform, could be a useful tool for the identification of yeasts present in dairy products.     

Identification of the bacterial microflora in dairy products by temporal temperature gradient gel electrophoresis

Numerous microorganisms, including bacteria, yeasts, and molds, are present in cheeses, forming a complex ecosystem. Among these organisms, bacteria are responsible for most of the physicochemical and aromatic transformations that are intrinsic to the cheesemaking process. Identification of the bacteria that constitute the cheese ecosystem is essential for understanding their individual contributions to cheese production. We used temporal temperature gradient gel electrophoresis (TTGE) to identify different bacterial species present in several dairy products, including members of the genera Lactobacillus, Lactococcus, Leuconostoc, Enterococcus, Pediococcus, Streptococcus, and STAPHYLOCOCCUS: The TTGE technique is based on electrophoretic separation of 16S ribosomal DNA (rDNA) fragments by using a temperature gradient. It was optimized to reveal differences in the 16S rDNA V3 regions of bacteria with low-G+C-content genomes. Using multiple control strains, we first set up a species database in which each species (or group of species) was characterized by a specific TTGE fingerprint. TTGE was then applied to controlled dairy ecosystems with defined compositions, including liquid (starter), semisolid (home-made fermented milk), and solid (miniature cheese models) matrices. Finally, the potential of TTGE to describe the bacterial microflora of unknown ecosystems was tested with various commercial dairy products. Subspecies, species, or groups of species of lactic acid bacteria were distinguished in dairy samples. In conclusion, TTGE was shown to distinguish bacterial species in vitro, as well as in both liquid and solid dairy products.     

Identification of the Bacterial Microflora in Dairy Products by Temporal Temperature Gradient Gel Electrophoresis

Numerous microorganisms, including bacteria, yeasts, and molds, are present in cheeses, forming a complex ecosystem. Among these organisms, bacteria are responsible for most of the physicochemical and aromatic transformations that are intrinsic to the cheesemaking process. Identification of the bacteria that constitute the cheese ecosystem is essential for understanding their individual contributions to cheese production. We used temporal temperature gradient gel electrophoresis (TTGE) to identify different bacterial species present in several dairy products, including members of the genera Lactobacillus, Lactococcus, Leuconostoc, Enterococcus, Pediococcus, Streptococcus, and Staphylococcus. The TTGE technique is based on electrophoretic separation of 16S ribosomal DNA (rDNA) fragments by using a temperature gradient. It was optimized to reveal differences in the 16S rDNA V3 regions of bacteria with low-G+C-content genomes. Using multiple control strains, we first set up a species database in which each species (or group of species) was characterized by a specific TTGE fingerprint. TTGE was then applied to controlled dairy ecosystems with defined compositions, including liquid (starter), semisolid (home-made fermented milk), and solid (miniature cheese models) matrices. Finally, the potential of TTGE to describe the bacterial microflora of unknown ecosystems was tested with various commercial dairy products. Subspecies, species, or groups of species of lactic acid bacteria were distinguished in dairy samples. In conclusion, TTGE was shown to distinguish bacterial species in vitro, as well as in both liquid and solid dairy products.     

Molecular fingerprinting of dairy microbial ecosystems by use of temporal temperature and denaturing gradient gel electrophoresis

Numerous microorganisms, including bacteria, yeasts, and molds, constitute the complex ecosystem present in milk and fermented dairy products. Our aim was to describe the bacterial ecosystem of various cheeses that differ by production technology and therefore by their bacterial content. For this purpose, we developed a rapid, semisystematic approach based on genetic profiling by temporal temperature gradient electrophoresis (TTGE) for bacteria with low-G+C-content genomes and denaturing gradient gel electrophoresis (DGGE) for those with medium- and high-G+C-content genomes. Bacteria in the unknown ecosystems were assigned an identity by comparison with a comprehensive bacterial reference database of approximately 150 species that included useful dairy microorganisms (lactic acid bacteria), spoilage bacteria (e.g., Pseudomonas and Enterobacteriaceae), and pathogenic bacteria (e.g., Listeria monocytogenes and Staphylococcus aureus). Our analyses provide a high resolution of bacteria comprising the ecosystems of different commercial cheeses and identify species that could not be discerned by conventional methods; at least two species, belonging to the Halomonas and Pseudoalteromonas genera, are identified for the first time in a dairy ecosystem. Our analyses also reveal a surprising difference in ecosystems of the cheese surface versus those of the interior; the aerobic surface bacteria are generally G+C rich and represent diverse species, while the cheese interior comprises fewer species that are generally low in G+C content. TTGE and DGGE have proven here to be powerful methods to rapidly identify a broad range of bacterial species within dairy products.     

Molecular Polymorphism of β-Galactosidase LAC4 Genes in Dairy and Natural Strains of Kluyveromyces Yeasts

Molecular Biology - The ability to ferment lactose is a characteristic peculiarity of dairy Kluyveromyces lactis yeasts; the vast majority of other yeast species are not able to assimilate this...     

Molecular Fingerprinting of Dairy Microbial Ecosystems by Use of Temporal Temperature and Denaturing Gradient Gel Electrophoresis

Numerous microorganisms, including bacteria, yeasts, and molds, constitute the complex ecosystem present in milk and fermented dairy products. Our aim was to describe the bacterial ecosystem of various cheeses that differ by production technology and therefore by their bacterial content. For this purpose, we developed a rapid, semisystematic approach based on genetic profiling by temporal temperature gradient electrophoresis (TTGE) for bacteria with low-G+C-content genomes and denaturing gradient gel electrophoresis (DGGE) for those with medium- and high-G+C-content genomes. Bacteria in the unknown ecosystems were assigned an identity by comparison with a comprehensive bacterial reference database of ~150 species that included useful dairy microorganisms (lactic acid bacteria), spoilage bacteria (e.g., Pseudomonas and Enterobacteriaceae), and pathogenic bacteria (e.g., Listeria monocytogenes and Staphylococcus aureus). Our analyses provide a high resolution of bacteria comprising the ecosystems of different commercial cheeses and identify species that could not be discerned by conventional methods; at least two species, belonging to the Halomonas and Pseudoalteromonas genera, are identified for the first time in a dairy ecosystem. Our analyses also reveal a surprising difference in ecosystems of the cheese surface versus those of the interior; the aerobic surface bacteria are generally G+C rich and represent diverse species, while the cheese interior comprises fewer species that are generally low in G+C content. TTGE and DGGE have proven here to be powerful methods to rapidly identify a broad range of bacterial species within dairy products.     

Intraspecific Polymorphism of the Yeast Kluyveromyces lactis: Genetic Populations

Microbiology - According to the modern classification of yeasts, the species Kluyveromyces lactis includes two taxonomic varieties: cultural dairy yeast K. lactis var. lactis and...     

Identification of clinically relevant yeasts by PCR/RFLP

For molecular diagnosis of fungal disease using DNA amplification procedures in the routine laboratory, choice of appropriate target structures and rapid and inexpensive identification of amplification products are important prerequisites. Most diagnostic procedures described thus far are characterized by limited applicability, considerable cost for laboratory equipment or low power of discrimination between species. This study aimed at identification of a PCR target appropriate for diagnosis of clinically relevant yeasts and an affordable procedure for characterization of the PCR products to the species level. Here, we describe a PCR-based system using amplification of intergenic spacers ITS1 and ITS2 and restriction length polymorphism of PCR products after sequence-specific enzymatic cleavage. We show the evaluation of the system for clinically relevant Candida species. The simple and inexpensive procedure should be instrumental for rapid identification of medically important yeasts.     

Safety and regulation of yeasts used for biocontrol or biopreservation in the food or feed chain

Yeasts have been important components of spontaneous fermentations in food and beverage processing for millennia. More recently, the potential of utilising antagonistic yeasts, e.g. Pichia anomala and Candida spp., for post-harvest biological control of spoilage fungi during storage of plant-derived produce (‘biopreservation’) has been clearly demonstrated. Although some yeast species are among the safest microorganisms known, several have been reported in opportunistic infections in humans, including P. anomala and bakers’ yeast, Saccharomyces cerevisiae. More research is needed about the dominant pathogenicity and virulence factors in opportunistic yeasts, and whether increased utilisation of biopreservative yeasts in general could contribute to an increased prevalence of yeast infections. The regulatory situation for yeasts used in post-harvest biocontrol is complex and the few products that have reached the market are mainly registered as biological pesticides. The qualified presumption of safety (QPS) approach to safety assessments of microorganisms intentionally added to food or feed, recently launched by the European Food Safety Authority, can lead to more efficient evaluations of new products containing microbial species with a sufficient body of knowledge or long-term experience on their safety. P. anomala is one of several yeast species that have been given QPS status, although the status is restricted to use of this yeast for enzyme and metabolite production purposes. With regard to authorisation of new biopreservative yeasts, we recommend that the possibility to regulate microorganisms for food biopreservation as food additives be considered.     

A mixed culture of Propionibacterium jensenii and Lactobacillus paracasei subsp. paracasei inhibits food spoilage yeasts

Screening for antimicrobial features of 197 propionibacteria and tests with several antifungal lactobacilli led to the development of three protective cultures containing Propionibacterium jensenii SM11 and Lactobacillus paracasei subsp. paracasei strain SM20, SM29 or SM63. These cultures showed inhibitory activities (up to 5 orders of magnitude) against yeasts in dairy products such as yoghurt or cheese surface at refrigerator temperatures (6 degrees C) without an influence on the quality properties of the food. Initial cell numbers of 5 x 10(7) cells/g of propionibacteria and 1 x 10(8) cells/g of lactobacilli were the optimal concentrations to yield a total inhibition of the spoilage yeasts (Candida pulcherrima, Candida magnoliae, Candida parapsilosis and Zygosaccharomyces bailii).     

Formation of Dulcitol in Aerobic Dissimilation of d-Galactose by Yeasts

During the study of aerobic dissimilation of galactose by yeasts, polyhydric products were isolated in crystalline form from the fermented broths and identified. Yeast species may be divided into two groups on basis of sugar alcohol production: type I yeasts form the same end products from galactose as from glucose; type II yeasts produce dulcitol from galactose with or without other sugar alcohols but they produce no dulcitol from glucose. Isolation of dulcitol from microorganism has not been previously described.     

Biotechnology of non-Saccharomyces yeasts—the ascomycetes

Saccharomyces cerevisiae and several other yeast species are among the most important groups of biotechnological organisms. S. cerevisiae and closely related ascomycetous yeasts are the major producer of biotechnology products worldwide, exceeding other groups of industrial microorganisms in productivity and economic revenues. Traditional industrial attributes of the S. cerevisiae group include their primary roles in food fermentations such as beers, cider, wines, sake, distilled spirits, bakery products, cheese, sausages, and other fermented foods. Other long-standing industrial processes involving S. cerevisae yeasts are production of fuel ethanol, single-cell protein (SCP), feeds and fodder, industrial enzymes, and small molecular weight metabolites. More recently, non-Saccharomyces yeasts (non-conventional yeasts) have been utilized as industrial organisms for a variety of biotechnological roles. Non-Saccharomyces yeasts are increasingly being used as hosts for expression of proteins, biocatalysts and multi-enzyme pathways for the synthesis of fine chemicals and small molecular weight compounds of medicinal and nutritional importance. Non-Saccharomyces yeasts also have important roles in agriculture as agents of biocontrol, bioremediation, and as indicators of environmental quality. Several of these products and processes have reached commercial utility, while others are in advanced development. The objective of this mini-review is to describe processes currently used by industry and those in developmental stages and close to commercialization primarily from non-Saccharomyces yeasts with an emphasis on new opportunities. The utility of S. cerevisiae in heterologous production of selected products is also described.     

Yeasts in foods and beverages: impact on product quality and safety

The role of yeasts in food and beverage production extends beyond the well-known bread, beer and wine fermentations. Molecular analytical technologies have led to a major revision of yeast taxonomy, and have facilitated the ecological study of yeasts in many other products. The mechanisms by which yeasts grow in these ecosystems and impact on product quality can now be studied at the level of gene expression. Their growth and metabolic activities are moderated by a network of strain and species interactions, including interactions with bacteria and other fungi. Some yeasts have been developed as agents for the biocontrol of food spoilage fungi, and others are being considered as novel probiotic organisms. The association of yeasts with opportunistic infections and other adverse responses in humans raises new issues in the field of food safety.     

新疆传统发酵乳品中乳酸菌与 酵母菌的益生特性

目的观察新疆传统发酵乳品中分离的14种菌株的生长特点及产酸能力,筛选出具有较强耐胆盐能力,并能在人工胃肠液中存活的菌株。方法对10株乳酸菌和4株酵母菌进行生长曲线、pH、耐胆盐能力和耐人工胃肠液检测。结果 10株乳酸菌和4株酵母菌具有良好的生长曲线和产酸能力;马乳酒样乳杆菌具有较强的耐胆盐能力;希氏乳杆菌、马乳酒样乳杆菌、乙醇假丝酵母和东方伊萨酵母具有较强的耐人工胃液能力;乳酸乳球菌、哈尔滨乳杆菌、瑞士乳杆菌、马乳酒样乳杆菌、乙醇假丝酵母和东方伊萨酵母具有较强的耐人工肠液能力。结论 10株乳酸菌和4株酵母菌具有优良的益生特性,有望成为益生菌制剂的备用菌株。     

西藏曲拉和云南乳饼中酵母菌的鉴定及其生物多样性

【目的】探讨西藏曲拉和云南乳饼中酵母菌的生物多样性及其分布特征,为我国传统乳制品中酵母菌资源的利用提供基础数据。【方法】从西藏和云南分别采集的5份曲拉样品和8份乳饼样品中分离出41株酵母菌,利用26SrDNAD1/D2区域序列分析对这些菌株进行了分类鉴定。【结果】曲拉和乳饼样品中酵母菌的总数分别在106-107cfu/g和102-106cfu/g之间,曲拉样品的酵母菌平均数比乳饼样品中的高34倍。共鉴定出10属12种,其中西藏曲拉的优势菌株为发酵毕赤氏酵母(Pichia fermentans)和酿酒酵母(Saccharomyces cerevisiae);云南乳饼的优势菌株为类筒假丝酵母(Candida zeylanoides)和喜仙人掌毕赤氏酵母(Pichia cactophila)。毕赤氏酵母属(Pichia)是曲拉和乳饼的共同优势属。【结论】西藏曲拉和云南乳饼中的酵母菌都具有丰富的生物多样性,但其差异性很大。     

A new method for quantitative determination of members of the genusSaccharomyces in mixtures with other yeast genera

A new method for the quantitative determination of members of the genusSaccharomyces in mixtures with other yeasts is described. It is based on the higher resistance of theSaccharomyces species toward phenylhydrazine. The method is not applicable only toSaccharomyces fragilis and to some of the species grouped sometimes under the genus Zygosaccharomyces. The method described can be used for detecting Saccharomyces individuals in cannery or wine-making raw materials and products, for purity control of production and collection strains of yeasts and for detecting contamination withSaccharomyces species during fodder yeast production.     

Purification, characterization, and amino terminal sequence of xylose reductase from Candida shehatae.

D-Xylose is a major component of the carbohydrates derived from agricultural residues and forest products. Among more than two hundred known xylose-utilizing yeasts, only a few species are known to be able to ferment xylose anaerobically. Candida shehatae is one of such xylose-fermenting yeasts. Xylose reductase (E.C. 1.1.1.21) is a key enzyme responsible for xylose metabolism in xylose-utilizing as well as xylose-fermenting yeasts. In this paper, we report the development of a convenient and reliable procedure for the purification of xylose reductase from C. shehatae to near homogeneity. The amino acid composition and N-terminal sequence of the enzyme have also been analyzed. C. shehatae seems to contain only a single xylose reductase, but the enzyme has a dual coenzyme specificity for both NADPH and NADH. The enzyme is remarkably stable at room temperature and 4 degrees C.     

Lactic acid bacteria and yeasts associated with gowé production from sorghum in Bénin

AIMS: To identify the dominant micro-organisms involved in the production of gowé, a fermented beverage, and to select the most appropriate species for starter culture development. METHODS AND RESULTS: Samples of sorghum gowé produced twice at three different production sites were taken at different fermentation times. DNA amplification by internal transcribed spacer-polymerase chain reaction of 288 lactic acid bacteria (LAB) isolates and 16S rRNA gene sequencing of selected strains revealed that the dominant LAB responsible for gowé fermentation were Lactobacillus fermentum, Weissella confusa, Lactobacillus mucosae, Pediococcus acidilactici, Pediococcus pentosaceus and Weissella kimchii. DNA from 200 strains of yeasts was amplified and the D1/D2 domain of the 26S rRNA gene was sequenced for selected isolates, revealing that the yeasts species were Kluyveromyces marxianus, Pichia anomala, Candida krusei and Candida tropicalis. CONCLUSIONS: Gowé processing is characterized by a mixed fermentation dominated by Lact. fermentum, W. confusa and Ped. acidilactici for the LAB and by K. marxianus, P. anomala and C. krusei for the yeasts. SIGNIFICANCE AND IMPACT OF THE STUDY: The diversity of the LAB and yeasts identified offers new opportunities for technology upgrading and products development in gowé production. The identified species can be used as possible starter for a controlled fermentation of gowé.     

Seasonal dynamics of the structure of epiphytic yeast communities

The seasonal dynamics of the species structure of epiphytic yeasts on the leaves and in the flowers of 25 plant species was studied throughout the period of their vegetation. It was shown that, on average for the vegetation period, the composition of epiphytic yeast communities was nonspecific. The same species of epiphytic yeasts dominated on different plant species, irrespective of their taxonomic identity and ecological peculiarities. However, different species of yeasts exhibited different types of seasonal dynamics of relative abundance. Therefore, a combination of the dynamics of yeast species and the ontogenetic cycles of plants creates a pattern of the dynamics of the epiphytic yeast population, which is unique for each plant species. The species diversity of yeasts on the leaves of a plant is determined by the duration of its ontogenetic cycle: the longer the vegetation of a plant, the higher the diversity of the epiphytic yeasts population. The greatest diversity of epiphytic yeasts was revealed on the leaves of perennial hygrophytes and mesophytes; the minimal diversity, on ephemeroids and annuals with a short ontogenetic cycle.     

Identification and tracing of Bifidobacterium species by use of enterobacterial repetitive intergenic consensus sequences

Eighty-nine Bifidobacterium strains from 26 species were identified and classified to the species level with an enterobacterial repetitive intergenic consensus (ERIC)-PCR approach. We demonstrated that ERIC-PCR is useful for a phylogenetic and taxonomical analysis but as well as for a species composition analysis of mixed bifidobacterial cultures isolated from dairy products and other environments.