Activity of different ''killer'' yeasts on strains of yeast species undesirable in the food industry

Killer strains of the genera Saccharomyces, Hansenula and Kluyveromyces were tested for killing activity against yeasts that cause trouble in the food industry (in the genera Zygosaccharomyces, Kloeckera, Saccharomycodes and Schizosaccharomyces). Saccharomyces strains killed only Zygosaccharomyces rouxii strains, while non-Saccharomyces strains showed a wider anti-yeast spectrum. The Kluyveromyces phaffii killer strain was of particular interest because of its killer action against Kloeckera apiculata, Saccharomycodes ludwigii and Zygosaccharomyces rouxii.     

Molecular assessment of indigenous yeast population from traditional balsamic vinegar

AIMS: To identify and describe the indigenous yeast population involved in traditional balsamic vinegar (TBV) fermentation. METHODS AND RESULTS: Using the restriction analysis of the ribosomal region 5.8S (5.8S rRNA) and the internal transcribed spacers 1 and 2 (5.8S-ITS region) we were able to group 133 strains isolated from 17 cooked grape must samples into 10 different yeast species, included into 4 genera. Moreover, we sequenced the D1/D2 domains of the 26S rRNA and confirmed the reliability of each identification at species level. Most strains belonged to the genus Zygosaccharomyces. In particular, Zygosaccharomyces bailii was found in 41% of the samples, followed by Saccharomyces cerevisiae, Zygosaccharomyces pseudorouxii and Candida stellata. Strains belonging respectively to Zygosaccharomyces mellis, Zygosaccharomyces bisporus, Zygosaccharomyces rouxii, Hanseniaspora valbyensis, Hanseniaspora osmophila and Candida lactis-condensi species were also detected. Despite the great number of species recovered, the mtDNA restriction profiles showed low variability at strain level. Saccharomyces cerevisiae isolates with an higher degree of intraspecific variance were considered an exception. CONCLUSIONS: Many different indigenous yeast species were recovered and TBV yeasts population seems to be far more complex than what was reported in previous literature. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has allowed us to gain a better understanding of the indigenous yeast species of TBV cooked must.     

Natural wine yeasts as biocontrol agents

A total of 586 natural wine yeasts, belonging to different genera, were tested for their antagonistic effect on fungal pathogens. A low percentage of yeast strains completely inhibited the pathogens and the biocontrol activity was found to be a strain characteristic and did not solely depend on species or genus. Among the antagonists, two strains of Saccharomyces cerevisiae and one of Zygosaccharomyces showed a broad spectrum of antagonistic activity against 10 fungal pathogens.     

Diversity and evolution of non-Saccharomyces yeast populations during wine fermentation: effect of grape ripeness and cold maceration

We have evaluated the effect of grape maturity and cold maceration prior to fermentation on the yeast ecology during wine fermentation. Non-Saccharomyces strains were selectively isolated and identified using two rapid PCR techniques, namely enterobacterial repetitve intergenic consensus-PCR and PCR-intron splice sites, in various wine fermentation conditions. These identifications were further complemented and confirmed by restriction fragment length poymorphism and sequencing analysis of the 5.8S-ITS and D1/D2 ribosomal regions, respectively. Eleven species belonging to five genera were identified. Candida stellata, Hanseniaspora uvarum and Hanseniaspora osmophila were the dominant species, representing almost 90% of the isolates. Minor strains presented different species of the genera Candida, Issatchenkia, Zygoascus and Zygosaccharomyces. Selective isolation made it possible to isolate some species that were hardly related to the wine-making process, such as Issatchenkia hanoiensis, a new species that has only been described recently.     

Characterization of the yeast flora present in some Turkish high-sugar products

In this study, 129 Turkish high-sugar products were examined in terms of their yeast flora and 73 representative strains were isolated. Yeast isolates were identified at species level by using Apilab Plus (bioMérieux, France), a specific computer program developed for ID 32C strips (bioMérieux, France). While one of the isolates could be identified at genus level as Aureobasidium, 66 of them were identified as 21 species belonging to 8 different genera. The distribution of these isolates were as follows: Candida (38), Rhodotorula (8), Zygosaccharomyces (7), Cryptococcus (6), Saccharomyces (3), Debaryomyces (2), Pichia (1) and Torulaspora (1). Approximately 70% of the isolates were found to have the ability to grow on media with 50% (w/w) glucose. Hence, they were characterized as xerotolerant strains. Although Zygosaccharomyces rouxii is known as the most xerotolerant yeast species, only two strains of Z. rouxii could be isolated from Turkish high-sugar foods. During identification studies, it was observed that ID 32C test strips should certainly be supported by morphological and physiological tests for obtaining more reliable identification results. If not, closely related yeast species such as anamorph and telemorph forms can not be distinguished.     

Strain differentiation of pathogenic yeasts by the killer system

High sensitivity rates to the activity of killer toxins produced by 25 species of yeasts belonging to the genera Candida, Hansenula, Pichia, Rhodotorula, Saccharomyces and Trichosporon have been observed among 112 yeast isolates (25 Cryptococcus neoformans, 29 C. glabrata, 16 C. parapsilosis, 20 C. pseudotropicalis and 22 C. tropicalis). The highest sensitivity has been observed among the C. parapsilosis isolates, the lowest in C. glabrata strains. Genera Pichia and Hansenula proved to have the greatest killer activity. A killer system, formerly used for differentiating C. albicans isolates within the species, proved to be valid as epidemiological marker when applied to 112 strains of pathogenic yeasts.     

Comparative evaluation of various bacterial growth inhibitors as selective agents for isolation of soil Actinomyces]

Tobramycin and dioxidine sensitivity of 57 strains belonging to 14 actinomycetes genera was studied. The cultures of Streptomyces were much more sensitive to tobramycin than the cultures of rare genera. The majority of the Streptomyces cultures showed a high resistance to dioxidine (MIC greater than or equal to 50 micrograms/ml). At the same time the majority of the cultures of rare genera were inhibited by low concentrations of dioxidine (no more than 50 micrograms/ml). For isolation of actinomycetes from soil samples, tobramycin, dioxidine, ceftriaxone and novobiocin were used. Tobramycin added in a concentration of 10 micrograms/ml to the Gauze agar organic medium No. 2 promoted a 2-fold increase in detection of actinomycetes of the rare genera as compared to the control. It was especially favourable for detection of cultures belonging to Micromonospora, Amycolatopsis, Streptosporangium and Nocardiopsis. Dioxidine in concentrations of 10 and 50 micrograms/ml inhibited the growth of the cultures belonging to rare genera. Ceftriaxone in the same concentrations inhibited the growth of the cultures of both Streptomyces and the rare genera. Novobiocin favoured detection of the cultures belonging to Amicolatopsis and Micromonospora. Therefore, among the tested compounds tobramycin and novobiocin appeared to be the most useful selective agents for isolation of actinomycetes of rare genera.     

Differential killer sensitivity as a tool for fingerprinting wine-yeast strains ofSaccharomyces cerevisiae

The extreme variability of the killer phenomenon in nature, expressed differently in different strains of the same yeast species, embodies an exceptional potential for the discrimination of yeasts at the strain level. Killer-sensitive relationships between a killer reference panel of 24 yeasts belonging to 13 species of six genera, and different industrial wine-starters ofSaccharomyces cerevisiae can be used profitably for a rapid and simple fingerprinting procedure.     

Killer yeasts of Kluyveromyces and Hansenula genera with potential application in fermentation and therapy

The killing/immunity interactions among killer strains of the genera Kluyveromyces, Hansenula and Saccharomyces from the Czechoslovak Collection of Yeasts were studied with the aim to find the strains with broad specificity and killer activity targeted against a range of undesirable wild yeasts causing stuck fermentations. Among 49 tested Kluyveromyces strains, five strains were found, and among 55 Hansenula strains, ten yeast strains were found with activity against a sensitive strain of Saccharomyces. Hansenula mrakii CCY 38-7-1 and Hansenula saturnus var. subsufficiens CCY 38-4-2 showed exceptional activity against the wine contaminants, Zygosaccharomyces bailii, as well as against pathogenic Candida species within a broad range of pH 2.9–5.1. Their potential biotechnological application is discussed.     

红富士苹果园酵母多样性研究

从北京顺义和山东泰安红富士苹果园采集果实、叶片、树皮和土壤等不同基物,分离酵母菌,利用26S rDNA的D1/D2区域序列分析并结合形态学特征和SSCP分析对这些菌株进行了分类学研究,探讨了苹果园酵母的物种多样性及其分布。北京苹果园共分离酵母菌129株,鉴定为13属21种,优势属为Pichia（4个种）,Cryptococcus（3个种）,Pseudozyma（3个种）,子囊菌占较大优势,分布于8属12种,占总种数的57.1%。山东苹果园共分离酵母291株,鉴定为13属26种,优势属为假丝酵母Candida（6个种）,毕赤酵母Pichia（4个种）和隐球酵母Cryptococcus（3个种）,并且子囊菌占较大优势,分布于7属17种,占总种数的65.4%。     

The use of killer biotyping in an ecological survey of yeast in an old patagonian winery

An ecological study of Saccharomyces cerevisiae strains in spontaneous alcoholic fermentation has been made in the same winery on two consecutive years (1993 and 1994) with Merlot type musts, and with Malbec type must on a third year (1998). Saccharomyces cerevisiae strains associated with winery surfaces were also analysed. Differential killer sensitivity patterns related to a killer reference panel of 10 killer yeasts belonging to nine species of four genera were used as a quick and simple procedure to discriminate between indigenous S. cerevisiae isolates at the strain level. Although a great diversity of wild strains was observed, two main indigenous S. cerevisiae strains, designated as S. cerevisiae 9 and S. cerevisiae 13, took over the Merlot type fermentation in both years. These strains also appeared in Malbec must fermentation during the year 1998 and they were again found on the winery surface the next year. These results show that some few and stable indigenous S. cerevisiae strains remained in the environmental winery over the considered period of time (1993–1999) and they represent an additional evidence of the taking over of musts by local strains of S. cerevisiae.     

Lecithinase activity of various yeast strains]

The activity of lecithinase was studied among 50 yeast strains belonging to the genera Rhodotorula Harrison, Cryptococcus Kütz, and Lipomyces Lodder et Kreger van Rij. The maximum activity of lecithinase is typical of epiphyte yeast strains belonging to the genus Rhodotorula and is not manifested by species of the henus Lipomyces which inhabit soil. Strains of the genus Cryptococcus cannot be distinctly differentiated into soil and epiphyte cultures, and occupy an intermediate position by the activity of lecithinase.     

Spoilage yeasts from Patagonian cellars: characterization and potential biocontrol based on killer interactions

Indigenous yeasts associated with surfaces in three North Patagonian cellars were isolated by means of selective media developed for the isolation of Dekkera/Brettanomyces yeasts; 81 isolates were identified as belonging to Candida boidinii (16%), Hanseniaspora uvarum (38%), Pichia guilliermondii (3%), Saccharomyces cerevisiae (1%), Geotrichum silvicola (16%) and the new yeast species Candida patagonica (26%). No Dekkera/Brettanomyces isolate was obtained, however, 41 isolates (51% of the total isolates) produced some enologically undesirable features under laboratory conditions including the production of 4-ethylphenol and 4-vinylphenol, observed in the Candida boidinii and Pichia guilliermondii isolates. The sensitivity of the 41 spoilage isolates and seven Brettanomyces bruxellensis collection strains was evaluated against a panel of 55 indigenous and ten reference killer yeasts. Killer cultures belonging to Pichia anomala and Kluyveromyces lactis species showed the broadest killer spectrum against spoilage yeasts, including Dekkera bruxellensis collection strains. These killer isolates could be good candidates for use in biocontrol of regionally relevant spoilage yeasts.     

OBSERVATIONS SUR LES BESOINS EN VITAMINES DES DESMIDIÉES (CHLOROPHYCÉES-ZYGNEMATALES)1

The minimal nutrient requirements of 61 strains of desmids belonging to 18 different genera were investigated within 16 axenic cultures and 45 contaminated cultures. Thirty-nine strains were autotrophic: they included all 30 strains belonging to the genera Cosmarium, Staurastrum, and Micrasterias. Twenty-two strains were auxotrophic; they included all 14 strains belonging to the genus Closterium. Of the 22 auxotrophic strains, 13 required only vitamin B₁₂; the specific requirements of the other 9 strains were not determined.     

A comparison of the killer character in different yeasts and its classification

The interactions between 20 killer yeasts of various genera and species were examined. Ten distinct groups were recognised with respect to killer activity and 10 distinct groups with respect to resistance to killer action. Using both killing and resistance phenotypes, 13 classes of killer yeast were found. With the exception of Torulopsis glabrata NCYC 388, non-Saccharomyces strains of yeast were not killed by a member of the genus Saccharomyces.The killer character of the 3 killing groups of Saccharomyces identified could be cured by treatment with cycloheximide or incubation at elevated temperature and the effectiveness of these procedures was indicative of the category of killer yeast examined. Killer yeasts not belonging to the genus Saccharomyces could not be cured of their activity. Double-stranded ribonucleic acids were extracted only from Saccharomyces spp. and the molecular weights of the species present were a function of the killer class to which a strain belonged.By an analysis of the effects of proteolytic enzymes, temperature and pH on killer activity and by gel chromatography of crude preparations of killer factors, the toxins of different killer classes were shown to be biochemically distinct. However all toxins had certain properties in common consistent with there being a protein component essential to killer action.     

Nucleotide composition and homologies in the DNA of new extreme halophilic soil archaebacteria

DNA nucleotide composition was studied in extreme halophilic bacteria belonging to the genera Halobacterium, Halococcus, Natronobacterium and Natronococcus. The cultures were shown to be a monolithic group of microorganisms with the content of GC pairs typical of extreme halophilic archebacteria. The difference between the content of DNA major and minor components was twice as high in Halobacterium distributus strains isolated from sulfate saline soils as compared to cultures of this species isolated from natural waters with a high salinity. DNA minor components were not found in haloalkalophilic microorganisms from soda saline soils in contrast to those from soda lakes. The results of DNA-DNA hybridization indicate that the Halobacterium genus is highly heterogeneous. The newly isolated strains of extremely halophilic H. distributus are characterized by the low homology of their DNAs both among themselves and with other species of the genus. However, the hybridization data for the collection strains H. vallismortis 1398 and H. halobium 996 from the National Collection of Microorganisms are indicative of a high homology (80-100%) which is not characteristic of cultures belonging to different species. These results as well as some phenotypical properties of H. vallismortis 1398 different from those of this species type strain support the data reported in the literature about the genetic instability of extreme halophilic archebacteria. The analysis of homologies in DNA nucleotide sequences may be used to study the taxonomy of extreme halophilic archebacteria.     

A Circular Plasmid from the YeastTorulaspora delbrueckii

A new member of the 2-μm family of plasmids, named pTD1, was found in the yeastTorulaspora delbrueckii,a widespread yeast associated with food. Nucleotide sequences revealed the presence of a pair of inverted repeats and three open reading frames, one of which is a homologue of the FLP recombinase gene of 2-μm plasmid. An ARS region was identified, by replication inSaccharomyces cerevisiaeandT. delbrueckii,near one of the inverted repeats. By the use of pTD1 derivatives and auxotrophic mutant hosts, an efficient host–vector system was established forT. delbrueckii.So far, the 2-μm family of plasmids is restricted to four closely related genera (Q6 group):Saccharomyces, Zygosaccharomyces, Kluyveromyces,andTorulaspora.After a survey of 2500 strains belonging to about 500 species (80 genera) of yeast, no circular plasmids were found in other genera.     

Zygosaccharomyces kombuchaensis: the physiology of a new species related to the spoilage yeasts Zygosaccharomyces lentus and Zygosaccharomyces bailii

Zygosaccharomyces kombuchaensis was recently discovered in the 'tea fungus' used to make fermented tea. Z. kombuchaensis was shown by ribosomal DNA sequencing to be a novel species, and a close relative of Zygosaccharomyces lentus, from which it could not be distinguished by conventional physiological tests. Z. lentus was originally established as a new taxon by growth at 4 degrees C, sensitivity for heat and oxidative stress, and lack of growth in aerobic shaken culture at temperatures above 25 degrees C. Subsequent analysis of Z. kombuchaensis reveals that this species shares these unusual characteristics, confirming its close genealogical relationship to Z. lentus. Detailed physiological data from a number of Z. kombuchaensis and Z. lentus strains clearly demonstrate that these two species can in fact be distinguished from one another based on their differing resistance/sensitivity to the food preservatives benzoic acid and sorbic acid. The spoilage yeasts Zygosaccharomyces bailii and Z. lentus are resistant to both acetic acid and sorbic acid, whereas Z. kombuchaensis is resistant to acetic acid but sensitive to sorbic acid. This would indicate that Z. kombuchaensis strains lack the mechanism for resistance to sorbic acid, but possess the means of resistance to acetic acid. This observation would therefore suggest that these two resistance mechanisms are different, and that in all probability acetic and sorbic acids inhibit yeast growth by different modes of action. Z. kombuchaensis strains were also sensitive to benzoic acid, again suggesting inhibition dissimilar from that to acetic acid.     

A mitochondrial molecular marker, ori-rep-tra, for differentiation of yeast species.

Yeasts exhibit various mechanisms for the inheritance of their mitochondrial genomes. Differences among these mechanisms are based on variations within nuclear as well as mitochondrial genetic elements. Here we report diagnostic differences in the presence of biologically active mitochondrial intergenic sequences, ori-reptra, among related yeasts in the genera Saccharomyces, Arxiozyma, Debaryomyces, Kluyveromyces, Pachytichospora, Torulaspora, and Zygosaccharomyces. A molecular probe containing ori-rep-tra can be employed specifically for the differentiation and identification of isolates belonging to the species complex Saccharomyces sensu stricto.     

Comparative study of the fatty acid composition of some groups of purple nonsulfur bacteria

The fatty acid composition (FAC) of 43 strains of purple nonsulfur bacteria belonging to six genera—Rubrivivax, Rhodopseudomonas, Rhodoplanes, Blastochloris, Rhodobium, and Rhodomicrobium—was studied by capillary gas chromatography. The cultures were grown on standard medium under standard conditions. Automatic identification of the fatty acid methyl esters and statistical processing of the results were performed by the computerized Microbial Identification System (MIS). Significant differences between the FACs of different genera, species, and, sometimes, strains were revealed. 16S rRNA genes of some of the new isolates, primarily those having a specific FAC, were sequenced. The taxonomic status of a number of the strains in question was determined using the FAC characteristics as one of the criteria. It was shown that the FAC characteristics may be used both for affiliating isolates to known species and for revealing new taxa.