Geotrichum candidum dominates in yeast population dynamics in Livarot, a French red-smear cheese

The diversity and dynamics of yeast populations in four batches of Livarot cheese at three points of ripening were determined. Nine different species were identified by Fourier transform infrared spectroscopy and/or sequencing, and each batch had its own unique yeast community. A real-time PCR method was developed to quantify the four main yeast species: Debaryomyces hansenii, Geotrichum candidum, Kluyveromyces sp. and Yarrowia lipolytica. Culture and molecular approaches showed that G. candidum was the dominant yeast in Livarot cheese. When D. hansenii was added as a commercial strain, it codominated with G. candidum. Kluyveromyces lactis was present only at the start of ripening. Yarrowia lipolytica appeared primarily at the end of ripening. We propose a scheme for the roles and dynamics of the principal Livarot yeasts.     

Production of volatile compounds by cheese-ripening yeasts: requirement for a methanethiol donor for S-methyl thioacetate synthesis by Kluyveromyces lactis

Five cheese-ripening yeasts (Geotrichum candidum, Saccharomyces cerevisiae, Kluyveromyces lactis, Yarrowia lipolytica and Debaryomyces hansenii) were compared with respect to their ability to generate volatile aroma compounds. K. lactis produced a variety of esters - ethylacetate (EA) being the major one - and relatively limited amounts of volatile sulphur compounds (VSCs). Conversely, G. candidum produced significant amounts of VSCs [with the thioester S-methyl thioacetate (MTA) being the most prevalent] and lower quantities of non-sulphur volatile compounds than K. lactis. We suspect that K. lactis is able to produce and/or accumulate acetyl CoA - a common precursor of MTA and EA - but that it produces limited amounts of methanethiol (MTL); both acetyl CoA and MTL are precursors for MTA synthesis. When supplemented with exogenous MTL, MTA production greatly increased in K. lactis cultures whereas it was unchanged in G. candidum cultures, suggesting that MTL is a limiting factor for MTA synthesis in K. lactis but not in G. candidum. Our results are discussed with respect to L-methionine catabolism.     

Malt-yeast extract-sucrose agar, a suitable medium for enumeration and isolation of fungi from silage.

A general medium named malt-yeast extract-sucrose agar (MYSA) containing oxgall was designed. The medium was intended for the enumeration and isolation of molds and yeasts in routine examinations of animal feed stuffs. In this study MYSA was tested as a general medium for mycological examination of silage. The medium was compared with dichloran-rose bengal medium (DRBC) in an examination of more than 500 specimens of big bale grass silage. Selected characteristics of known fungal species commonly isolated from feeds were examined after growth on MYSA and DRBC and on malt extract agar, used as a noninhibitory control medium. MYSA suppressed bacterial growth, without affecting the growth of fungi common in feeds. The fungi growing on MYSA were easily recognized, and the medium seemed to slow radial growth of fungal colonies, which permitted, easy counting. The number of species found was higher on MYSA than on DRBC. When we compared MYSA with DRBC for mycological examination of grass silage samples, MYSA was found to be the medium of choice.     

Antifungal Activity of Sodium Bicarbonate Against Fungal Agents Causing Superficial Infections

Although sodium bicarbonate—NaHCO₃ (SB) has many domestic and medical, traditional and empirical uses, only little scientific documentation of its activity is available. The aims of this study were to investigate the antifungal activity of SB on the three fungal groups (yeasts, dermatophytes and molds) responsible for human skin and nail infections. We first evaluated the in vitro antifungal activity of SB on 70 fungal strains isolated from skin and nail infections: 40 dermatophytes, 18 yeasts and 12 molds. A concentration of 10 g/L SB inhibited the growth of 80 % of all the fungal isolates tested on Sabouraud dextrose agar. The minimal inhibitory concentration 90 (MIC90) of SB measured on Sabouraud dextrose agar, Sabouraud dextrose broth and potato dextrose broth was 5 g/L for the yeasts, 20 g/L for the dermatophytes and 40 g/L for the molds. In a second step, we prospectively evaluated the ex vivo antifungal activity of SB on 24 infected (15 dermatophytes, 7 yeasts and 2 molds) clinical specimens (15 nails and 9 skin scrapings). The fungal growth was completely inhibited for 19 (79 %) specimens and reduced for 4 (17 %) specimens after 7 days of incubation on Sabouraud dextrose–chloramphenicol agar supplemented with 10 g/L of SB as compared to Sabouraud dextrose–chloramphenicol agar without SB. In conclusion, we documented the antifungal activity of SB on the most common agents of cutaneous fungal infection and onychomycosis, and we specified the effective concentrations for the different groups of pathogenic fungi. The mechanism of action of SB has yet to be explored.     

Quantitative and qualitative studies of the microflora of barley malt production

Populations of aerobic heterotrophic bacteria, mycelial fungi and yeasts occurring in malting barley were estimated by a plate technique and scanning electron microscopy. There was an increase in the total number of micro-organisms during germination, although populations declined after kilning. Bacteria dominated on all samples, with progressively lower populations of yeasts and filamentous fungi. There was no obvious spatial distribution of micro-organisms on the samples although there appeared to be high populations of bacteria and fungal hyphae on the inner surface of the kernels. The dominant groups of aerobic heterotrophic bacteria were presumptively identified as Alcaligenes sp., Arthrobacter globiformis, Clavibacter iranicum, Erwinia herbicola, Lactobacillus spp. and Pseudomonas fluorescens. The principal filamentous fungi were identified as Aiternaria alternata, Aspergillus glaucus (group), Cladosporium macrocarpum, Epicoccum purpurascens, Fusarium avenaceum, Geotrichum candidum and Penicillium spp. The yeasts isolated most frequently were Candida catenulata, C. vini, Debaryomyces hansenii, Hansenula polymorpha, Kloeckera apiculata, Rhodotorula mucilaginosa, Sporobolomyces roseus and Trichosporon beigelii.     

Kinetics of growth and medium de-acidification for Geotrichum candidum and Penicillium camemberti cultivated on complex liquid media

Growth kinetics of Geotrichum candidum and Penicillium camemberti in submerged cultures under conditions of low aeration rate and uncontrolled pH were continuously recorded turbidimetrically. In these conditions the exponential growth phase was short, and ceased at a total biomass concentration of about 0.5 gl^–1 for G. candidum and 0.8 gl^–1 for P. candidum. The succeeding linear growth phase was also short, and its end corresponded to a biomass of 1.5 and 2.5 gl^–1 for G. candidum and P. camemberti respectively. A fair growth was recorded for P. camemberti on peptone-lactate medium, but G. candidum required addition of trace elements. For neither of these species growth was stimulated by growth factors of yeast extract. In peptone-lactate medium, the final pH did not depend on supplementation: 8–8.4 was recorded for G. candidum and 8.7–8.8 for P. camemberti.     

SENSORY AND INSTRUMENTAL FLAVOR ANALYSES OF CHEESE CURD COCULTURED WITH SELECTED YEAST AND BACTERIA

Relationships between odor properties and volatile chemical composition were explored on 39 cocultures of three different yeasts, three Geotrichum candidum and five bacteria, commonly used in bacteria and mold surface ripened cheese. Sensory profiling was performed by ten trained judges by scoring the intensity of 14 odor attributes. At the same time, the volatile compounds of the cocultures were extracted and analyzed by dynamic headspace gas chromatography and mass spectrometry. Sensory and instrumental data were compared and correlated using correlation analysis and partial least squares regression analysis. The sample plot including the whole set of samples evidenced a clustering of the associations containing the yeast strain Kluyveromyces lactis and any bacteria. They developed strong fruity olfactory notes related to their high content of ethyl esters and various alcohols. The sample plot on a restricted set of samples evidenced the fruity characteristics of Debaryomyces hansenii and bacteria associations and the cheesy odors of Yarrowia lipolytica and Geotrichum candidum cocultures that produced sulfur compounds.     

Fungal diversity in activated sludge from membrane bioreactors in Berlin

The objective of this study was to evaluate the occurrence of fungi in aerobic and anoxic activated sludge from membrane bioreactors. Thirty-six samples from each aerobic and anoxic activated sludge were taken from two membrane bioreactors treating domestic wastewater. Over a period of 9?months, four samples from each plant were taken per month. The samples were prepared for count and identification of fungi. Sixty species belonging to 30 genera were collected from activated sludge samples under aerobic and anoxic conditions. In terms of fungal identification, under aerobic conditions Geotrichum candidum was found at 94.4% followed by Penicillium species at 80.6%, yeasts at 75.0%, and Trichoderma species at 50.0%; under anoxic conditions G. candidum at 86.1%, yeasts at 66.6%, and Penicillium species at 61.1% were the most prevalent. The results indicate that activated sludge is a habitat for growth and sporulation of different groups of fungi, both saprophytic and pathogenic.     

Scanning electron and light microscopic study of microbial succession on bethlehem st. Nectaire cheese

St. Nectaire cheese is a semisoft cheese of French origin that, along with Brie and Camembert cheeses, belongs to the class of surface mold-ripened cheese. The surface microorganisms that develop on the cheese rind during ripening impart a distinctive aroma and flavor to this class of cheese. We have documented the sequential appearance of microorganisms on the cheese rind and in the curd over a 60-day ripening period. Scanning electron microscopy was used to visualize the development of surface fungi and bacteria. Light microscopy of stained paraffin sections was used to study cross sections through the rind. We also monitored the development of bacterial and yeast populations in and the pH of the curd and rind. The earliest stage of ripening (0 to 2 days) is dominated by the lactic acid bacterium Streptococcus cremoris and multilateral budding yeasts, primarily Debaryomyces and Torulopsis species. Geotrichum candidum follows closely, and then zygomycetes of the genus Mucor develop at day 4 of ripening. At day 20, the deuteromycete Trichothecium roseum appears. From day 20 until the end of the ripening process, coryneforms of the genera Brevibacterium and Arthrobacter can be seen near the surface of the cheese rind among fungal hyphae and yeast cells.     

Why do some yeast species require niacin for growth? Different modes of NAD synthesis

NAD holds a key position in metabolism and cellular regulatory events as a major redox carrier and a signalling molecule. NAD biosynthesis pathways have been reconstructed and compared in seven yeast species with completely sequenced genomes, including Saccharomyces cerevisiae, Kluyveromyces lactis, Candida glabrata, Debaryomyces hansenii, Candida albicans, Yarrowia lipolytica and Schizosaccharomyces pombe. Both amino acid and nucleotide sequence similarity analysis in silico indicated that de novo NAD biosynthesis might not exist in K. lactis, C. glabrata and Schiz. pombe, while other species have the kynurenine pathway. It also showed that the NAD salvage pathway via nicotinic acid and nicotinic acid mononucleotide is conserved in all of these yeasts. Deletion of KlNPT1 (the gene for nicotinate phosphoribosyl-transferase) is lethal, which demonstrates that this salvage pathway, utilizing exogenous nicotinic acid, is the unique route to synthesize NAD in K. lactis. The results suggested that the basis of the variation of niacin requirements in yeasts lies in their different combinations of NAD biosynthesis pathways. The de novo pathway is absent but the salvage pathway is conserved in niacin-negative yeasts, while both pathways coexist in niacin-positive yeasts.     

Antimicrobial activity of food-related Penicillium sp. against pathogenic bacteria in laboratory media and a cheese model system

Food-related Penicillium species ( n ep fy1 = rs 34) and Geotrichum candidum ( n = 11) grown on Czapek Dox and brie agar were tested for their ability to suppress growth of pathogenic bacteria. Ten out of 13 P. camemberti showed antagonistic activity while the other species did not interact significantly with the bacterial growth. The order of inhibition was: Gram-negative bacteria and Bacillus cereus > Listeria monocytogenes , Lactococcus sp. > Micrococcus sp. whereas Lactobacillus sp., Staphylococcus aureus and some Micrococcus sp. were unaffected. When Salmonella typhimurium was inoculated together with P. camemberti P25 in brie agar, bacterial growth was inhibited during the first 6 d of incubation whereafter growth started. The inhibition of L. monocytogenes was similar but less pronounced. The antimicrobial activity produced by P. camemberti P25 and L84 was enhanced with increasing amount of sucrose in the medium. The activity was increased at low pH and destroyed at pH above 8. It was detectable at 15°C but not at 37°C indicating that volatile metabolites might be involved. No significant accumulation of organic acids and no secondary metabolites such as mycotoxins were detected. HSGC-MS analysis indicated that acetaldehyde, benzaldehyde, 3-methylbutanal and 1-octen-3-ol were produced by P. camemberti during the period when inhibitory activity was observed. Pure acetaldehyde and benzaldehyde were shown to be inhibitory to L. monocytogenes and Salm. typhimurium when grown at 15°C and pH 5·5 and 7·0.     

低温大曲酵母菌分离和计数培养方法优化

低温大曲是清香型白酒酿造的核心因素之一,为酿造过程提供了物系、酶系和菌系。酵母菌是白酒发酵过程中最重要的功能微生物类群,研究大曲中的酵母菌种类和含量具有重要意义,对大曲质量评价也具有重要参考价值。但用常规酵母菌分离技术和培养基从大曲中分离酵母菌时易受优势丝状真菌（霉菌）的干扰,霉菌常常很快长满培养皿,将酵母菌覆盖,难以对酵母菌进行定量计数、观察和分离纯化。本研究根据酒醅发酵过程中随乙酸和乳酸含量的升高,霉菌含量急剧降低而酵母菌含量逐渐升高的现象,用常规培养基YPD为基础培养基,测试了添加不同量的乙酸、乳酸和丙酸（后者为常用食品防腐和防霉剂）,以及不同比例的乙酸乳酸组合物,对低温大曲中霉菌的抑制效果和对酵母菌生长的影响进行探究,发现在灭菌后的YPD中添加3.3mL/L乙酸、2.0mL/L丙酸或在乙酸乳酸1:3的情况下添加2.0mL/L乙酸,30℃培养3-5d之内可有效抑制低温大曲中的霉菌,实现对酵母菌的有效分离、计数和纯化培养,而单加乳酸对霉菌,特别是黄曲霉的抑制效果差。随后测试了以前我们从清香型白酒大曲和酒醅发酵过程中分离的13属18种酵母菌在这些培养基上的生长情况,发现这些酵母菌中的绝大多数,尤其是大曲和酒醅中的优势酵母菌种,均可以在这些加酸培养基上良好生长。综合考虑培养基的成本、配制的简便性及分离效果等因素,本研究推荐将灭菌后添加3.3mL/L乙酸的YPD固体培养基作为低温大曲酵母菌分离和定量计数的优化培养基。     

Comparison of methods for estimating the biomass of three food-borne fungi with different growth patterns.

To evaluate the effectiveness of steps taken to reduce the growth of molds in food and feed, methods that can accurately quantify the degree of fungal contamination of solid substrates are needed. In this study, the ergosterol assay has been evaluated by comparing the results of this assay with spore counts and hyphal length measurements made with a microscope and with CFU counts. Three fungi with different growth patterns during cultivation on a synthetic agar substrate were used in these experiments. For the nonsporulating Fusarium culmorum, there was good agreement between changes in hyphal length, CFU, and ergosterol content. Penicillium rugulosum and Rhizopus stolonifer produced many spores, and the production of spores coincided with large increases in CFU but not with increases in hyphal length or ergosterol content. Spores constituted between 3 and 5% of the total fungal mass. Changes in ergosterol level were closely related to changes in hyphal length. It was concluded that ergosterol level is a suitable marker for use in quantitatively monitoring fungal growth in solid substrates.     

Phenotypic expression of Kluyveromyces lactis killer toxin against Saccharomyces spp.

The secretion of killer toxins by some strains of yeasts is a phenomenon of significant industrial importance. The activity of a recently discovered Kluyveromyces lactis killer strain against a sensitive Saccharomyces cerevisiae strain was determined on peptone-yeast extract-nutrient agar plates containing as the carbon source glucose, fructose, galactose, maltose, or glycerol at pH 4.5 or 6.5. Enhanced activity (50 to 90% increase) was found at pH 6.5, particularly on the plates containing galactose, maltose, or glycerol, although production of the toxin in liquid medium was not significantly different with either glucose or galactose as the carbon source. Results indicated that the action of the K. lactis toxin was not mediated by catabolite repression in the sensitive strain. Sensitivities of different haploid and polyploid Saccharomyces yeasts to the two different killer yeasts S. cerevisiae (RNA-plasmid-coded toxin) and K. lactis (DNA-plasmid-coded toxin) were tested. Three industrial polyploid yeasts sensitive to the S. cerevisiae killer yeast were resistant to the K. lactis killer yeast. The S. cerevisiae killer strain itself, however, was sensitive to the K. lactis killer yeast.     

Phenotypic expression of Kluyveromyces lactis killer toxin against Saccharomyces spp

The secretion of killer toxins by some strains of yeasts is a phenomenon of significant industrial importance. The activity of a recently discovered Kluyveromyces lactis killer strain against a sensitive Saccharomyces cerevisiae strain was determined on peptone-yeast extract-nutrient agar plates containing as the carbon source glucose, fructose, galactose, maltose, or glycerol at pH 4.5 or 6.5. Enhanced activity (50 to 90% increase) was found at pH 6.5, particularly on the plates containing galactose, maltose, or glycerol, although production of the toxin in liquid medium was not significantly different with either glucose or galactose as the carbon source. Results indicated that the action of the K. lactis toxin was not mediated by catabolite repression in the sensitive strain. Sensitivities of different haploid and polyploid Saccharomyces yeasts to the two different killer yeasts S. cerevisiae (RNA-plasmid-coded toxin) and K. lactis (DNA-plasmid-coded toxin) were tested. Three industrial polyploid yeasts sensitive to the S. cerevisiae killer yeast were resistant to the K. lactis killer yeast. The S. cerevisiae killer strain itself, however, was sensitive to the K. lactis killer yeast.     

Le Paraquat, un Outil pour la Révélation Rapide d'Infections Fongiques Latentes et de Champignons Endophytes

Paraquat: a tool for the quick development of latent fungal infections and endophytic fungi.
Detached winter wheat leaves without any symptom of fungal infection were thoroughly washed, surface desinfected, treated with paraquat (0.03–0.32% a.i.) and incubated on nutrient agar or in moist chambers under sterile conditions. Microscopic examinations showed that hypae and yeast cells emerged from several stomata within 48–72 h after treatment. Fungal colonies developed on agar within the same period of time. More than 27 filamentous fungal genera and yeasts were identified during a wheat cropin healthy, green leaves. Non-treated leaves showed very few early emergences of fungi from stomata, but senescent leaves eventually yielded several fungal taxa observed after paraquat treatment. Similar observations were made on non-detached wheat leaves, and on detached sugarbeet leaves. Paraquat also induced development of Fusarium spp. from detached healthy winter wheat ears before or after heading. In grape berries, paraquat induced the development of latent infections of Botrytis cinerea , a few days after treatment.
The usefulness of paraquat for the detection of endophytes and for the diagnosis of early and latent fungal infections in plants, and the possible involvement of endophytes in tissue senescence are discussed.     

Comparison of Media for Detection of Fungi on Spacecraft

Five media, including Trypticase soy agar (TSA; BBL) pour plates, spread plates of TSA, Mycophil agar with chloromycetin, Mycophil agar with chloromycetin and Actidione, and cornmeal agar with chloromycetin were quantitatively and qualitatively compared for the detection of fungi on spacecraft. Cornmeal agar with chloromycetin yielded the highest number of fungal colonies, although not always significantly higher than Mycophil agar with chloromycetin or TSA spread plates. Cornmeal agar with chloromycetin also gave the best qualitative representation of fungi on the spacecraft, recovering 68% of the genera found from all media. This medium yielded 10 times the number of fungal colonies and 3 times the number of genera found on TSA pour plates as currently used for spacecraft assay.     

A visual method for rapid screening of xylose-fermenting ethanolic strains of Klebsiella pneumoniae.

A simple and rapid screening method for selecting hyper-ethanolic strains of Klebsiella pneumoniae is described. The method involves a novel biological screening marker, namely, the yeast Candida ethanothermophilum. The screening marker was seeded on an agar plate to the surface of which agar blocks, each containing a colony of K. pneumoniae, were subsequently fixed. This seeded plate lacked sources of carbon and energy. Ethanol formed in the agar blocks by the K. pneumoniae colonies diffused into the seeded medium and served as a carbon and energy source for the ethanotrophic yeasts. Colonies of yeasts appeared around the agar blocks in regions of ethanolic diffusion. Hyper-ethanolic strains of K. pneumoniae were thus selected on the basis of the number of colonies of the screening marker that appeared around the blocks containing the ethanolic colonies of K. pneumoniae.     

Evaluation of Media for Selective Isolation of Yeasts from Oral, Rectal, and Burn Wound Specimens

Six media were evaluated to determine their ability to isolate yeasts and inhibit bacteria. The media included the following: Snyder, Snyder tellurite, Sabouraud tellurite, Littman-gentamicin, molybdate, and Mycosel (BBL). Doses of mixed intestinal gram-negative bacilli and enterococci were most effectively inhibited by Snyder tellurite agar. Klebsiella pneumoniae was the most common bacterial contaminant of the other media. All six media were comparable in isolating yeasts while preventing the growth of the oral bacterial flora. The selection of a basal fungal growth medium for tellurite incorporation to inhibit bacteria but permit growth of yeasts was affected by pH. The bacteriostatic effect of tellurite was decreased with increasing pH of media while fungistatic action was increased. The arbitrary selection of Snyder and Littman agars to isolate yeast from burn wound cultures demonstrated the need to include a selective medium for these specimens. Blood, phenylethyl alcohol blood agar, and Columbia CN blood agar were all inadequate for isolating yeasts from burns. Growth of a variety of filamentous saprophytic and pathogenic dimorphic fungi grew adequately on four of five selective media tested.