The incidence of killer activity of non-Saccharomyces yeasts towards indigenous yeast species of grape must: potential application in wine fermentation

Fourteen killer yeasts were assayed for their ability to kill species of yeast that are commonly associated with fermenting grape must and wine. A total of 147 of a possible 364 killer-sensitive interactions were observed at pH 4.5. Of the killer yeasts studied, Pichia anomala NCYC 434 displayed the broadest killing range. At a pH value comparable with those of wine ferments, pH 3.5, the incidence of killer-sensitive interactions was reduced by 700% across all the yeasts. Williopsis saturnus var. mrakii CBS 1707 exhibited the broadest killing range at the lower pH, killing more than half of the tester strains. Intraspecific variation in sensitivity to killer yeasts was observed in all species where more than one strain was tested. Also, in strains of Pichia anomala, Kluyveromyces lactis and Pichia membranifaciens, the three species in which more than one killer yeast was analysed, intraspecific variation in killer activity was observed.     

Yeasts in Ensiled High-Moisture Corn

A total of 1,365 yeasts were selected from ensiled high-moisture corn at various stages in the ensiling process to determine the sequence and relative numbers of yeast species. The yeast species most frequently isolated from freshly harvested corn were Candida parapsilosis and C. intermedia; these two species were isolated infrequently after the third week of storage. Species of yeasts that predominate after the 12th day of storage were Hansenula anomala (66% of the isolates studied) and C. krusei (26% of the isolates studied). The preponderance of H. anomala and C. krusei in ensiled corn is believed to be associated with the ability of these two species to assimilate lactic acid.     

Pathogenicity of Hansenula anomala in a model of immunocompromised mice

Systemic infections caused by opportunistic fungi have shown an increased frequency in the past 10 years, particularly in immunocompromised patients. Hansenula anomala is an ascosporogenous yeast of the Ascomycetes class found in the skin, throat, and digestive tract transient normal flora. This study was conducted to compare the pathogenicity of H. anomala and Candida albicans in a model of immunocompromised mice. Thirty-eight Swiss mice were divided into two groups as follows: 30 animals received an intraperitoneal (i.p.) injection of cyclophosphamide (200 mg/kg) four days before the induction of infection with H. anomala (1 × 10⁶ yeasts/mL), and 8 animals received 100 mg/kg of cyclophosphamide at 3-day intervals during 3 weeks before inoculation of 1 × 10⁷ yeasts/mL. All animals were treated with amoxicillin/clavulanic acid (40 mg/kg) four days before induction of infection. A group of mice inoculatd with C. albicans (ATCC 64548) served as control. Tissue samples from the lung, spleen, liver, and kidney for histological and mycologic studies were obtained at necropsy. In each animal, the number of viable yeasts per gram of kidney was determined. The organs most frequently infected by H. anomala were the kidneys and the liver (20%), and the lung (10%). However, in conditions of sustained immunosuppression, H. anomala was found in 65.5% of the organs examined. It is concluded that in an experimental model of immunocompromised mice, the pathogenicity of H. anomala was low. This revised version was published online in August 2006 with corrections to the Cover Date.     

The Influence of pH and Temperature on Cellulolytic and Pectolytic Activity of Cylindrocarpon destructans (Zins.) Scholt.

In studies on the effect of pH and temperature on cellulolytic and pectolytic activity of C. destructans, it was found that the isolates used produced only endoglucanases. The temperature and pH affected the synthesis of these enzymes. Fungi cultured at 26°C produced more of these enzymes than those grown at the two other temperatures. At 10°C, only one isolate produced minute amounts of endoglucanases. None of fungi studied exhibited cellulolytic activity in cultures grown at 20°C. Cellulolytic activity was found only in acidic media (pH 5.0). The fungi studied exhibited higher pectolytic than cellulolytic activity. In the post culture liquids of these organisms, both types of pectolytic enzymes (exo- and endo-PMG) were detected. Different temperature and pH values affected the production of these enzymes differently in various isolates.     

Resistance of some wine spoilage yeasts to combinations of ethanol and acids present in wine

The most important factors affecting microbial growth in an alcoholic beverage are the ethanol content and the low pH. The effectiveness of different organic acids in conjunction with ethanol concentration in controlling growth of yeasts was determined for Saccharomyces cerevisiae, Schizosaccharomyces pombe, Brettanomyces lambicus, Pichia anomala, Zygosaccharomyces bailii, Saccharomycodes ludwigii and Kluyveromyces thermotolerans in malt extract broth (MEB). The results are summarized as undissociated concentrations of different acids required to inhibit growth of yeasts in MEB containing 10% (v/v) ethanol. About half the amount of undissociated malic or tartaric acid is necessary for inhibition of the yeasts, compared with acetic and lactic acid but the concentrations of acid necessary to inhibit growth were generally very high and unrealistic in wines for controlling growth of most of the yeasts tested. All the yeasts tested were able to grow in acidified non- or low alcoholic beverages but, at higher ethanol concentrations, Sacch. cerevisiae and Zygosacch. bailii have the greatest spoilage potential.     

Occurrence of killer yeast strains in industrial and clinical yeast isolates

The secretion of proteinaceous toxins is a widespread characteristic in environmental and laboratory yeast isolates, a phenomenon called "killer system". The killer phenotype (K+) can be encoded by extrachromosomal genetic elements (EGEs) as double stranded DNA or RNA molecules (dsDNA, dsRNA) or in nuclear genes. The spectrum of action and the activity of killer toxins are influenced by temperature, salinity and pH of media. In the present work we determined the existence of K+ in a collection of S. cerevisiae and P. anomala yeasts isolated from environmental, industrial and clinical sources. The assays were performed in strains belonging to three yeast genera used as sensitive cells and under a wide range of pH and temperatures. Approximately 51 % of isolates tested showed toxicity against at least one sensitive yeast strain under the conditions tested. The K+ P. anomala isolates showed a wide spectrum of action and two of them had toxic activity against strains of the three yeast genera assayed, including C. albicans strains. In all S. cerevisiae K+ isolates an extrachromosomal dsRNA molecule (4.2 Kb) was observed, contrary to P. anomala K+ isolates, which do not possess any EGEs. The K+ phenotype is produced by an exported protein factor and the kinetics of killer activity production was similar in all isolates with high activity in the log phase of growth, decaying in the stationary phase.     

Biotechnology, physiology and genetics of the yeast Pichia anomala

The ascomycetous yeast Pichia anomala is frequently associated with food and feed products, either as a production organism or as a spoilage yeast. It belongs to the nonSaccharomyces wine yeasts and contributes to the wine aroma by the production of volatile compounds. The ability to grow in preserved food and feed environments is due to its capacity to grow under low pH, high osmotic pressure and low oxygen tension. A new application of P. anomala is its use as a biocontrol agent, which is based on the potential to inhibit a variety of moulds in different environments. Although classified as a biosafety class-1 organism, cases of P. anomala infections have been reported in immunocompromised patients. On the other hand, P. anomala killer toxins have a potential as antimicrobial agents. The yeast can use a broad range of nitrogen and phosphor sources, which makes it a potential agent to decrease environmental pollution by organic residues from agriculture. However, present knowledge of the physiological basis of its performance is limited. Recently, the first studies have been published dealing with the global regulation of the metabolism of P. anomala under different conditions of oxygenation.     

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.     

The Fungal and Bacterial Flora of Stored White Cabbage

1. The predominant organisms isolated from the outer wrapper leaves of freshly harvested white cabbages were: bacteria, yeasts, Alternaria spp., Aureobasidium pullulans, Botrytis cinerea, Cladosporium spp. and Penicillium spp.
2. Few qualitative or quantitative changes were seen in the leaf surface flora during storage at 2°C for up to 33 weeks.
3. Numbers of bacteria, particularly fluorescent and pectolytic pseudomonads, were considerably higher on cabbages drenched with fungicide or water than on corresponding undrenched cabbages.     

Presence of pathogenic yeasts in the feces of the semi-domesticated pigeon (Columba livia, Gmelin 1789, urban type) from the city of Turin

The results of a survey on the presence of pathogenic yeasts in pigeon droppings collected in Turin, are shown. The study was carried out in 8 densely populated areas, where human-animal contact is highest. A total of 427 pigeon dropping samples, most of which fresh, were collected. 550 yeast colonies, clinically interesting or at least pathologically significant, were isolated by the identification routine methods. The yeasts belong to the following species: Candida albicans, C. humicola, C. krusei, C. guillermondii, C. lypolitica, C. lambica, C. parapsilosis, C. tropicalis, C. rugosa, C. zeylanoides, Cryptococcus albidus, C. laurentii, C. neoformans, Hansenula anomala, Geotrichum sp., Kloekera apiculata, Rhodotorula glutinis, R. rubra, Saccharomyces cerevisiae, Torulopsis candida, T. glabrata, Trichosporon beigelii, T. capitatum, T. cutaneum, T. pullulans.     

Influence of pesticides on yeasts colonizing leaves

The effect of nine different pesticides on the growth of yeasts isolated from the leaves of fruit and forest trees was investigated. Four insecticides (with the active ingredients: thiacloprid, deltamethrin, lambdacyhalothrin, and thiamethoxam) and five fungicides (with the effective substances: bitertanol, kresoxim-methyl, mancozeb, trifloxystrobin, and cupric oxychloride) were tested. The concentrations of chemicals were those recommended by the manufacturers for the spraying of trees. The yeast strains isolated from the leaves of fruit trees were not sensitive to any of the insecticides. The majority of yeast strains isolated from the leaves of forest trees were either not sensitive or only to a small extent. While Rhodotorula mucilaginosa and Pichia anomala were not affected by any insecticide, the strains of Cryptococcus laurentii and Rhodotorula glutinis showed the highest sensitivity. The effects of fungicides on the growth of isolated yeasts were more substantial. The fungicide Dithane DG (mancozeb) completely inhibited the growth of all yeasts. All strains isolated from fruit tree leaves were more resistant to the tested fungicides than those isolated from the leaves of forest trees. The most resistant strains from the leaves of fruit trees belonged to the species Metschnikowia pulcherrima, Pichia anomala, and Saccharomyces cerevisiae, whereas Cryptococcus albidus and C. laurentii, originating from the leaves of forest trees, showed the highest sensitivity to fungicides.     

Physiological characteristics of the biocontrol yeast Pichia anomala J121

The yeast Pichia anomala J121 prevents mold spoilage and enhances preservation of moist grain in malfunctioning storage systems. Development of P. anomala J121 as a biocontrol agent requires in-depth knowledge about its physiology. P. anomala J121 grew under strictly anaerobic conditions, at temperatures between 3 degrees C and 37 degrees C, at pH values between 2.0 and 12.4, and at a water activity of 0.92 (NaCl) and 0.85 (glycerol). It could assimilate a wide range of C- and N-sources and produce killer toxin. A selective medium containing starch, nitrate, acetic acid, and chloramphenicol was developed for P. anomala. P. anomala was equally sensitive as Candida albicans to common antifungal compounds. Growth ability at a range of environmental conditions contributes to the competitive ability of the biocontrol yeast P. anomala J121.     

Glycerol production by yeasts under osmotic and sulfite stress.

The yeasts Saccharomyces cerevisiae, Candida boidinii, Pichia augusta, and Pichia anomala were tested for glycerol production both under osmotic stress and by addition of a sulfite-steering agent. The osmotic pressure was increased by employing glucose concentrations from 50 to 200 g/L and by supplementing with NaCl (40 g/L). Of all the yeasts, S. cerevisiae exhibited the highest level of osmotolerance. The increased osmotic pressure affected glycerol formation the most in C. boidinii. In both Pichia species, glycerol formation was not sufficiently induced when exposed to sugar and salt stress. The addition of 40 g/L Na2SO3 to the medium containing 100 g/L glucose shifted the metabolism of all yeasts towards glycerol formation. Saccharomyces cerevisiae achieved 68.6%, while C. boidinii reached 25.5% of the theoretical glycerol yield, respectively. The highest glycerol yield, 82.3% of the theoretical, was produced by S. cerevisiae under microaerophilic conditions.     

Recovery of heat-stressed yeasts in media containing plant oleoresins

C onner , D.E. & B euchat , L.R. 1985. Recovery of heat-stressed yeasts in media containing plant oleoresins. Journal of Applied Bacteriology 59 , 49–55.
Oleoresins from seven plants (allspice, cinnamon, clove, garlic, onion, oregano, and thyme) were tested for their effects on eight genera of sublethally heated food-spoilage and industrially important yeasts ( Candida lipolytica, Debaryomyces han-senii, Hansenula anomala, Kloeckera apiculata, Lodderomyces elongisporus, Rhodotorula rubra, Saccharomyces cerevisiae, and Torulopsis glabrata ). All heat-stressed yeasts had increased sensitivity to oleoresins in recovery media. Heated cells were most sensitive to cinnamon oleoresin, a lethal effect being observed to concentrations as low as 5 μg/ml. Results indicate that all eight yeasts underwent metabolic and/or structural injury as a result of exposure to elevated temperature. Whilst changes in sensitivity to oleoresins were not the same for all yeasts, it is concluded that the oleoresins may have a pronounced synergistic or additive effect on thermal inactivation of yeasts in foods. Furthermore, carryover of oleoresins in low dilutions of food to enumeration media could adversely influence the recovery of heat-stressed cells, thus resulting in apparent low populations.     

Effect of osmotic stress on the derepression of invertase synthesis in nonconventional yeasts

AIMS: The aim of this study was to analyse the effect of osmotic stress on the biosynthesis of invertase enzyme in nonconventional yeasts. METHODS AND RESULTS: Invertase activities of the nonconventional yeast species belonging to Kluyveromyces, Schwanniomyces and Pichia genus were measured either in the presence or in the absence of various amounts of NaCl. The effect of hyperosmotic stress on the glucose consumption of Saccharomyces cerevisiae and Pichia anomala were also compared. Like S. cerevisiae, derepression of invertase synthesis in Kluyveromyces lactis, Schwanniomyces occidentalis and Pichia jadinii is inhibited by hyperosmotic stress. However, derepression of invertase synthesis in P. anomala is not affected by hyperosmotic stress. In addition, low levels of osmotic stress activated invertase synthesis three- to fourfold in P. anomala and K. lactis. CONCLUSIONS: This study shows that low levels of osmotic stress induces the invertase synthesis at very high levels in P. anomala and K. lactis. Glucose consumption was not influenced at significant levels by the hyperosmotic stress in P. anomala. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows the activation of invertase synthesis by low levels of osmotic stress in P. anomala and K. lactis.     

Pectolytic Enzymes in Three Populations of Ditylenchus dipsaci

Extracts of nematodes of the Raleigh, North Carolina (RNC), Waynesville, N. C. (WNC), and onion populations of Ditylenchus dipsaci were examined for pectolytic activity. RNC nematodes contained a NaCl-stimulated endo-polymethylgalacturonase with optimal pH for activity of 6.0, whereas nematodes of the WNC and onion populations possessed a NaCl-stimulated endo-polygalacturonase with pH optimum of 4.0. Nematodes of each population also contained a CaCl₂-activated endo-pectin methyl-trans-eliminase with optimal pH of 9.0. Nematode extracts containing 0.5 M NaCl macerated potato discs. RNC and onion nematodes induced gall formation in Wando pea seedlings, but WNC nematodes induced a resistant, hypersensitive response. Thus pectolytic activity was not correlated with pathogenicity of D. dipsaci on Wando pea.     

Lactobacillus coryniformis subsp. coryniformis strain Si3 produces a broad-spectrum proteinaceous antifungal compound

The antifungal activity spectrum of Lactobacillus coryniformis subsp. coryniformis strain Si3 was investigated. The strain had strong inhibitory activity in dual-culture agar plate assays against the molds Aspergillus fumigatus, A. nidulans, Penicillium roqueforti, Mucor hiemalis, Talaromyces flavus, Fusarium poae, F. graminearum, F. culmorum, and F. sporotrichoides. A weaker activity was observed against the yeasts Debaryomyces hansenii, Kluyveromyces marxianus, and Saccharomyces cerevisiae. The yeasts Rhodotorula glutinis, Sporobolomyces roseus, and Pichia anomala were not inhibited. In liquid culture the antifungal activity paralleled growth, with maximum mold inhibition early in the stationary growth phase, but with a rapid decline in antifungal activity after 48 h. The addition of ethanol to the growth medium prevented the decline and gave an increased antifungal activity. The activity was stable during heat treatment and was retained even after autoclaving at 121 degrees C for 15 min. Maximum activity was observed at pH values of between 3. 0 and 4.5, but it decreased rapidly when pH was adjusted to a level between 4.5 and 6.0 and was lost at higher pH values. The antifungal activity was fully regained after readjustment of the pH to the initial value (pH 3.6). The activity was irreversibly lost after treatment with proteolytic enzymes (proteinase K, trypsin, and pepsin). The antifungal activity was partially purified using ion-exchange chromatography and (NH(4))(2)SO(4) precipitation, followed by gel filtration chromatography. The active compound(s) was estimated to have a molecular mass of approximately 3 kDa. This is the first report of the production of a proteinaceous antifungal compound(s) from L. coryniformis subsp. coryniformis.     

Comparison between the yeast flora of Middle Eastern and Western European soft drinks

Samples of a carbonated orange drink, raw materials, and intermediate products originating from 6 Iraqi bottling plants were examined. 69 drinks, 4 flavoured syrups and 19 simple syrups contained yeasts, whereas all samples from one plant and all samples of beverage base were free from viable yeasts. From the orange drink 2 species were isolated viz. Saccharomyces montanus and Torulopsis stellata. The following species were present in simple syrup: Hansenula anomala, Sacch. bisporus var. mellis, T. candida and T. stellata. Sacch. bisporus var. mellis was also isolated from flavoured syrup. Representative strains were submitted to routine growth tests at reduced oxygen tension, at reduced water activity and on solid soft-drink media containing various amounts of anti-microbially active benzoic acid at pH 3.0. The results are discussed and compared to those obtained in European soft drinks.     

Hybridation ADN-ADN chez quelques espèces du genre Hansenula

The genus Hansenula was considered a long time ago as a good pattern for phylogenetic research. In 1969, Wickerham proposed an evolutive scheme based upon morphological, physiological and ecological criteria. Recently, relatedness among yeasts were analysed by DNA-DNA hybridization in liquid medium. H. anomala var. anomala (G + C content: 37,1%) was compared with H. anomala var. schneggii (37.6%), H. subpelliculosa (33,8%), H. ciferrii (33,1%), H. holstii (37%) belonging to the same line 2, and also with H. beckii (38,3%) line 3, H. sydowiorum (40,1%) and H. muscicola (37,1%).These results showed little relatedness between H. anomala var. anomala /H. ciferrii and H. anomala var. anomala/ H. subpelliculosa. On the other hand, H. anomala var. schneggii shared 89,5% of its nucleotide sequences with H. anomala var. anomala. These 2 strains were considered to represent the same species. H. holstii showed 67.1% complementarity with H. anomala var. anomala: this strain is considered to represent valid species, different from H. anomala var. anomala, but H. muscicola with 72.5% relatedness to H. anomala var. anomala could be considered as a limit species. An unexpected finding was that H. beckii was closely related to H. anomala var. anomala (84.8%). These data suggested the inadequacy of current criteria used to establish the phylogenetic lines in genus Hansenula.     

Interactions between killer yeasts and pathogenic fungi

Abstract A total of 17 presumptive killer yeast strains were tested in vitro for growth inhibitory and killing activity against a range of fungal pathogens of agronomic, environmental and clinical significance. Several yeasts were identified which displayed significant activity against important pathogenic fungi. For example, isolates of the opportunistic human pathogen, Candida albicans , were generally very sensitive to Williopsis mrakii killer yeast activity, whilst killer strains of Saccharomyces cerevisiae and Pichia anomala markedly inhibited the growth of certain wood decay basidiomycetes and plant pathogenic fungi. Results indicate that such yeasts, together with their killer toxins, may have potential as novel antimycotic biocontrol agents.