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PubMed2006

Use of whey protein beads as a new carrier system for recombinant yeasts in human digestive tract.

A new immobilizing protocol using whey protein isolates was developed to entrap recombinant Saccharomyces cerevisiae. The model yeast strain expresses the heterologous P45073A1 that converts trans-cinnamic acid into p-coumaric acid. Beads resulted from a cold-induced gelation of a whey protein solution (10%) containing yeasts (7.5 x 10(7)cells ml(-1)) into 0.1M CaCl(2). The viability and growth capability of yeasts were not altered by our entrapment process. The release and activity of immobilized yeasts were studied in simulated human gastric conditions. During the first 60 min of digestion, 2.2+/-0.9% (n=3) of initial entrapped yeasts were recovered in the gastric medium suggesting that beads should cross the gastric barrier in human. The P45073A1 activity of entrapped yeasts remained significantly higher (p<0.05) than that of free ones throughout digestion (trans-cinnamic acid conversion rate of 63.4+/-1.6% versus 51.5+/-1.8% (n=3) at 120 min). The protein matrix seemed to create a microenvironment favoring the activity of yeasts in the stringent gastric conditions. These results open up new opportunities for the development of drug delivery system using recombinant yeasts entrapped in whey protein beads. The main potential medical applications include biodetoxication or the correction of digestive enzyme deficiencies.

Artificial Organs↗
PubMed2001

The interaction between yeasts and bacteria in dairy environments.

The general environment from which raw dairy products originate and the microbiological quality of the products in its processed state inevitably admit yeast growth and spoilage. Only part of the primary microflora survives under the selective pressures exerted by the intrinsic and extrinsic biotic factors present, processing procedures and preservatives. Yeasts that possess the proper physiological attributes to counteract the specific ecological determinants will be favored. Eventually, a particular yeast community will develop, and if the environmental factors permit, this characteristic yeast community will result in a specific association contributing positively or negatively to the final product. The association that develops between yeasts and bacteria is governed by specific key properties selecting for a few predominant yeasts. These yeasts may either stimulate or inhibit normal bacterial growth. The extent to which interaction between yeasts and bacteria contribute to the final product is discussed.

Animals↗
PubMed1997

Optimisation of methodology for enumeration of xerophilic yeasts from foods.

Xerophilic yeasts grow in intermediate moisture foods (aw, 0.65-0.85) such as sugar syrups, fruit concentrates, jams and brines. Non-osmophilic yeasts are enumerated by diluting in 0.1% peptone and then plated onto media such as malt extract or glucose yeast extract agar. In the presence of moulds the yeasts are enumerated in dichloran rose bengal chloramphenicol agar (DRBC). These procedures were demonstrated to be unsatisfactory for the enumeration of xerophilic yeasts in low aw foods. Investigations using pure cultures of xerophilic yeasts as well as naturally contaminated apple juice concentrates and glacé cherries have shown that a reduced aw diluent, in particular 30% w/w glycerol in combination with tryptone 10% glucose yeast extract agar (TGY) optimises the recovery of the yeasts, especially sublethally injured cells. The inclusion of sodium chloride in either the diluents or the culture media was not necessary to optimise the recovery of D. hansenii growing in 20% sodium chloride broths.

Agar↗
PubMed1998

Yeast profile in Gouda cheese during processing and ripening.

The yeasts present in Gouda cheese during processing were monitored in a single cheese factory during a 32 day ripening period. Despite the predominance of lactic acid bacteria during Gouda making, yeasts played a significant role in the ripening process in reaching counts as high as 10(5) cfu g(-1) at the later stages of ripening. The increase in the number of yeasts corresponded with the depletion in lactose content and the simultaneous stabilization of lactic acid bacteria numbers. The sources of yeast contamination which may lead to contamination of the curd were also determined. The brine and equipment surfaces were responsible for the highest yield of contaminating yeasts. A diverse variety of 23 yeast species representing 13 genera were present in the factory environment, during processing and ripening. Samples were taken at critical control points in the manufacturing process and analysed after incubation at 25 degrees C for 96 h. Although a broad spectrum of yeasts were found in Gouda cheese, Debaryomyces hansenii was the most abundant yeast isolated. Other species encountered were Saccharomyces cerevisiae and Yarrowia lipolytica, Kluyveromyces marxianus, Torulaspora delbrueckii, Rhodotorula glutinis, Cryptococcus albidus and Candida catenulata.

Cheese↗
PubMed2002

Diversity of commensal yeasts within and among healthy hosts.

We sampled commensal yeasts from three body sites of 24 healthy individuals to examine the patterns of commensal yeast species distribution and strain relatedness within and among individuals. To examine the short-term dynamics, each individual was sampled three times every 35-40 days at each of three body sites: mouth, fingernail, and toenail. The hosts included six genealogically unrelated individuals and 18 that belonged to four families. A total of 63 morphologically distinct colonies were isolated, identified, and genotyped. Nine yeast species were recovered, including 28 isolates of Candida albicans; 26 of C. parapsilosis; 2 each of C. krusei and C. tropicalis; and 1 each of C. famata, C. glabrata, C. guilliermondii, C. lusitaniae and Trichosporon beigelii. A significant difference in total yeast recovery rate between families was observed. However, body sites did not differ in the rates of yeast recovery. The three body sites showed different species distributions with the fingernail sample containing the highest species diversity, followed by the toenail sample. The oral sample contained the lowest species diversity with all 23 oral isolates being C. albicans. Among the 63 strains, forty-six unique genotypes were identified by PCR fingerprinting. Eleven shared-genotypes were identified, seven of which were from the same body site of the same host. The other four were from different members of the same family. Several family-specific genotypes and genotype clusters were found but the results were inconsistent with strict familial transmission of human commensal yeasts. A single host can have multiple species or multiple genotypes of the same species at the same or different body sites. Changes of species and genotypes over the sampling period for the same body site of individual hosts were also observed, including one direct observation of familial yeast transmission between two members of the same family during our sampling period. Our results indicate dynamic processes of yeast colonization, maintenance and evolution in healthy human hosts.

Adolescent↗
PubMed2002

S-adenosylhomocysteine, but not homocysteine, is toxic to yeast lacking cystathionine beta-synthase.

Elevated plasma homocysteine is associated with a variety of diseases in humans including coronary heart disease, stroke, peripheral vascular disease, and birth defects. However, the mechanism by which plasma homocysteine affects cells is unknown. We have examined the growth of isogenic wild-type and cystathionine beta-synthase (CBS) deficient yeast in response to homocysteine and its immediate metabolic precursor, S-adenosylhomocysteine (SAH). CBS deficient yeast export significantly more homocysteine into the media than wild-type yeast and have elevated internal pools of homocysteine and SAH. We found that 5 mM homocysteine added to the media had very little effect on the growth of wild-type or CBS deficient yeast, although intracellular homocysteine concentrations increased five- to tenfold. In contrast, as little as 25 microM S-adenosylhomocysteine inhibited the growth of CBS deficient yeast, but had no effect on wild-type yeast. Measurements of the intracellular S-adenosylmethionine (SAM) and SAH indicate that CBS deficient yeast contain reduced SAM/SAH ratios relative to wild-type, and this ratio is further reduced by adding SAH to the media. Growth inhibition by SAH in CBS deficient yeast can be totally reversed by addition of SAM to the media, indicating that the ratio and not absolute level is critical for cell growth. These results suggest that CBS plays a key role in the regulation of the SAM/SAH ratio inside cells and that excessive perturbations of this ratio can inhibit growth. We hypothesize that elevated extracellular homocysteine present in humans may reflect an altered intracellular SAM/SAH ratio and that this may be related to disease pathogenesis.

Chromatography, High Pressure Liquid↗
PubMed2001

Systemic ketoconazole is an effective treatment of atopic dermatitis with IgE-mediated hypersensitivity to yeasts.

BACKGROUND: IgE-mediated hypersensitivity to yeasts is often seen in atopic dermatitis (AD) patients, especially when dermatitis is located in the head, neck, and shoulder regions. Two studies have shown the efficacy of ketoconazole in the treatment of this type of AD, in contrast to results of topical treatment. The objective was to assess the clinical efficacy of antifungal treatment in AD in a randomized, double-blind, placebo-controlled study with oral ketoconazole and yeast-specific IgE levels and saprophytic yeast growth monitored simultaneously. METHODS: Eighty patients with AD and positive P. ovale and/or C. albicans RAST/skin prick test results were randomized to receive ketoconazole or placebo for 30 days. The yeast growth of skin and pharynx; P. ovale, C. albicans, andS. cerevisiae RAST; serum total IgE; and the severity of the eczema (SCORAD) were assessed at day 0 and thereafter at 1 and 3 months. RESULTS: A significant improvement was seen in the SCORAD scale in the ketoconazole group at the second visit in comparison to the first visit (P<0.0005; n=36), but not in the placebo group (n=39). Of the individual determinants of the SCORAD, itching (P<0.005), the extent of dermatitis (area percentage), excoriation, lichenification (P<0.01), erythema, papulation, and dryness (P<0.05) improved significantly in the ketoconazole group. In the placebo group, only the extent of dermatitis (area percentage) decreased significantly (P<0.05). In the ketoconazole group, the number of positive P. ovale cultures decreased from 60% to 31% (n=35) compared to the placebo group (64% to 56%; n=39). The clinical response was most significant in female patients with positive yeast cultures. CONCLUSION: Saprophytic yeasts may be a source of allergens in AD. Thus, patients with AD, yeast growth, and elevated IgE levels to yeasts should be offered antifungal treatment.

Adolescent↗
PubMed1990

Effects of extracts of high- and low-chromium brewer's yeast on metabolism of glucose by hepatocytes from rats fed on high- or low-Cr diets.

Brewer's yeast was grown on a defined medium containing glucose, ammonia, salts and vitamins plus tracer 51Cr without (low-Cr) or with (high-Cr) carrier Cr. The two batches of yeast differed by more than 100-fold in Cr content, containing 80 ng and 10 micrograms Cr/g dry yeast respectively. Extraction and fractionation procedures were designed to isolate Cr complexes with properties similar to those reported for glucose tolerance factor. After weaning, rats were reared on rat cubes (normal diet) or on a diet containing less than 0.1 microgram Cr/kg (low-Cr diet), or on the low-Cr diet supplemented with Cr (1 mg Cr/kg). Hepatocytes from these rats were incubated with [U-14C]glucose and incorporation of 14C into glycogen was measured. Incorporation of glucose-C into glycogen was enhanced by some yeast fractions in the presence of insulin, but had less effect in the absence of insulin. No difference could be detected between the responses to fractions from high- or low-Cr yeast extracts, or between responses by hepatocytes from animals fed on normal or low-Cr diets with or without Cr supplementation. Glycogen synthetase (EC 2.4.1.11) activity (total and percentage in the a form) was similar in hepatocytes isolated from animals on the normal and low-Cr diets. Those yeast fractions which enhanced the response to insulin in the 14C-incorporation experiments also enhanced the percentage of the enzyme in the a form in the presence of insulin, but not in the absence of insulin. The presence in yeast extracts of material which enhances the response to insulin by hepatocytes may help to explain the reported beneficial effects of dietary yeast supplements on glucose tolerance.

Animals↗
PubMed2005

Yeast programmed cell death: an intricate puzzle.

Yeasts as eukaryotic microorganisms with simple, well known and tractable genetics, have long been powerful model systems for studying complex biological phenomena such as the cell cycle or vesicle fusion. Until recently, yeast has been assumed as a cellular 'clean room' to study the interactions and the mechanisms of action of mammalian apoptotic regulators. However, the finding of an endogenous programmed cell death (PCD) process in yeast with an apoptotic phenotype has turned yeast into an 'unclean' but even more powerful model for apoptosis research. Yeast cells appear to possess an endogenous apoptotic machinery including its own regulators and pathway(s). Such machinery may not exactly recapitulate that of mammalian systems but it represents a simple and valuable model which will assist in the future understanding of the complex connections between apoptotic and non-apoptotic mammalian PCD pathways. Following this line of thought and in order to validate and make the most of this promising cell death model, researchers must undoubtedly address the following issues: what are the crucial yeast PCD regulators? How do they play together? What are the cell death pathways shared by yeast and mammalian PCD? Solving these questions is currently the most pressing challenge for yeast cell death researchers.

Apoptosis↗
PubMed1989

[The prececal and total intestinal nutrient digestibility and amino acid absorption of food yeasts in swine].

In a difference experiment the apparent precaecal (pc) and total intestinal (tot) nutrient digestibility and amino acid (AA) absorption of 5 yeasts (n-alkane yeasts Fermosin and Paprin, molasses/molasses distillers residue yeast, two sulfite waste liquor yeasts) were studied. The two n-alkane yeasts were very highly digested pc, the difference to tot digestibility is only small in most cases. In contrast to this, the pc and tot digestibility values of molasses/molasses distillers residue yeast differed considerably more. The probable reason for this is too hot drying due to which pc digestibility, particularly of the crude protein, and pc AA absorption are considerably reduced. In the two sulfite waste liquor yeasts, sulfite waste liquor residues containing lignin impair especially the crude carbohydrate digestibility and the energetic feed value; the better processing conditions of origin A, however, reduced considerably the negative effects. The results confirm that each yeast origin has its specific particularities, which have to be taken into consideration when they are used in feeding.

Amino Acids↗
PubMed2000

Effect of probiotic bacteria on prevalence of yeasts in oropharyngeal biofilms on silicone rubber voice prostheses in vitro.

The proliferation of yeasts in the mixed bacterial and fungal biofilms colonising silicone rubber voice prostheses in laryngectomised patients is the main cause of malfunctioning of the valve mechanism on the oesophageal side of the prostheses. Indwelling voice prostheses usually have to be replaced every 3-4 months. The consumption of probiotic bacteria is largely motivated by health claims related to the urogenital and lower digestive tract, but not to the upper digestive tract. The present study examined the influence of probiotic bacteria on the prevalence of yeasts in oropharyngeal biofilms on silicone rubber voice prostheses, as formed in a modified Robbins device. Exposure of oropharyngeal biofilms on voice prostheses to suspensions of Bifidobacterium infantis 420 or Enterococcus faecium 603 did not significantly reduce the number of yeasts in the biofilm. However, suspensions of Lactobacillus fermentum B54, L. rhamnosus 744 or L. lactis cremoris SK11 led to a reduction in the number of yeasts harvested from the voice prostheses. Suspensions of L. casei Shirota and Streptococcus thermophilus B significantly reduced the number of yeasts in the biofilm to 39% and 33%, respectively. The reduction brought about in yeast prevalence in the mixed biofilm was greatest by exposure to a suspension of L. lactis 53, with yeast prevalence only 4% of the control. In conclusion, the study demonstrated that the prevalence of yeasts in oropharyngeal biofilms on silicone rubber voice prostheses might be controlled by consumption of probiotic bacteria.

Bifidobacterium↗
PubMed1993

Relationships amongst some bacterial and yeast lactate and mandelate dehydrogenases.

Five yeast strains were isolated by enrichment culture on the basis of their ability to grow on mandelate and two of these strains were identified as Rhodotorula glutinis. In addition, a range of yeasts from culture collections was screened for growth on mandelate. The results suggest that mandelate utilization is a widespread but not universal characteristic within the genus Rhodotorula. Several of the yeasts contained an inducible NAD-dependent D(-)-mandelate dehydrogenase and an inducible dye-linked (presumably flavoprotein) L(+)-mandelate dehydrogenase. All the D(-)-mandelate dehydrogenases from the yeasts showed immunological cross-reactivity with each other (as judged by both immunoinhibition and immunoblotting), as did all the yeast L(+)-mandelate dehydrogenases that were tested. Determination of N-terminal amino acid sequences of several bacterial and yeast lactate and mandelate dehydrogenases, together with the evidence from the immunological studies, confirmed and extended previous proposals that there are several major groups of such dehydrogenases: FMN-dependent, membrane-bound L(+)-lactate and L(+)-mandelate dehydrogenases (M(r) = approx. 44,000) in bacteria, mitochondrial flavocytochrome b2 L(+)-lactate and L(+)-mandelate dehydrogenases (M(r) = approx. 59,000) in yeasts, FAD-dependent, membrane-bound D(-)-lactate and D(-)-mandelate dehydrogenases in bacteria, and soluble NAD-dependent D(-)-mandelate dehydrogenases in both bacteria and yeasts.

Alcohol Oxidoreductases↗
PubMed2004

New PCR-based methods for yeast identification.

AIMS: To characterize reference yeast strains and identify indigenous strains isolated from wine fermentations by PCR methods. METHODS AND RESULTS: We compared several PCR techniques for yeast identification. We used oligonucleotide primers that are complementary to (i) intron splice sites, (ii) REP and (iii) ERIC elements to produce PCR fingerprints that display specific patterns between the different yeast species. These three techniques were used to characterize 41 reference yeast strains belonging to 15 different species and to identify 40 indigenous strains isolated from grape must and wine fermentations. Species-specific banding patterns were obtained with the three PCR-techniques with different degrees of intraspecific differentiation depending on the method. By comparing the PCR fingerprints of unknown isolates with those produced by reference strains, we identified yeast strains isolated from an industrial wine fermentation. CONCLUSIONS: All three PCR techniques are rapid, reliable and simple methods of yeast identification. As far as we know, this is the first time that the primers designed for amplifying repetitive elements in bacteria have been successfully used in yeast. SIGNIFICANCE AND IMPACT OF THE STUDY: Industry needs rapid, reliable and simple methods of yeast identification. The proposed PCR techniques will allow to achieve this objective.

DNA Fingerprinting↗
PubMed2004

A modified yeast assay used on archival samples of localized prostate cancer tissue improves the detection of p53 abnormalities and increases their predictive value.

OBJECTIVE: To determine the frequency and predictive value of p53 mutations in localized prostate cancer, comparing the accuracy of detection using immunohistochemistry (IHC) with a modified yeast assay, on archival tissue samples. MATERIALS AND METHODS: Prostate cancer tissue was obtained from 98 patients who had >/= 2 years of clinical follow-up after radical prostatectomy. DNA sequencing was used to verify the presence of p53 mutations in samples that were immunopositive or that gave evidence for p53 alterations using the yeast assay. The IHC and yeast findings were compared with patient outcome to determine the predictive value of these two test types. RESULTS: Fifty-five tumours (57%) were immunopositive, and 58 (59%) were positive using the yeast assay. Sequence-confirmed p53 mutations occurred in 44 (45%) cases. The IHC protocol generated 49% (27/55) false-positive and 36% (15/42) false-negative results, and was 65% sensitive and 50% specific, with an overall accuracy of 57%. The yeast assay resulted in 24% (14/58) false-positive results with a specificity of 74% and an accuracy of 86%. When the p53 status of these patients was correlated with their clinical outcome, patients who had sequence-confirmed p53 mutations had a 2.6-fold greater failure rate (P = 0.026) and a 2.5-fold greater risk of dying from prostate cancer (P = 0.05). Notably, mutations in exon 6 predicted a six-fold increase in treatment failure (P = 0.043) and a 5.3-fold increase in the chance of dying from prostate cancer (P = 0.009). Abnormal yeast-assay findings gave similar predictive results to those obtained for DNA sequencing, while immunopositivity did not correspond to patient outcome. CONCLUSIONS: Mutations of p53 occurred in 45% of localized prostate cancers. These alterations have important prognostic implications. The yeast assay was more accurate for detecting p53 mutations than the IHC protocol used and, unlike IHC, the results of the yeast assay were predictive of patient outcome.

Adult↗
PubMed2006

Inhibition of Listeria monocytogenes by food-borne yeasts.

Many bacteria are known to inhibit food pathogens, such as Listeria monocytogenes, by secreting a variety of bactericidal and bacteriostatic substances. In sharp contrast, it is unknown whether yeast has an inhibitory potential for the growth of pathogenic bacteria in food. A total of 404 yeasts were screened for inhibitory activity against five Listeria monocytogenes strains. Three hundred and four of these yeasts were isolated from smear-ripened cheeses. Most of the yeasts were identified by Fourier transform infrared spectroscopy. Using an agar-membrane screening assay, a fraction of approximately 4% of the 304 red smear cheese isolates clearly inhibited growth of L. monocytogenes. Furthermore, 14 out of these 304 cheese yeasts were cocultivated with L. monocytogenes WSLC 1364 on solid medium to test the antilisterial activity of yeast in direct cell contact with Listeria. All yeasts inhibited L. monocytogenes to a low degree, which is most probably due to competition for nutrients. However, one Candida intermedia strain was able to reduce the listerial cell count by 4 log units. Another four yeasts, assigned to C. intermedia (three strains) and Kluyveromyces marxianus (one strain), repressed growth of L. monocytogenes by 3 log units. Inhibition of L. monocytogenes was clearly pronounced in the cocultivation assay, which simulates the conditions and contamination rates present on smear cheese surfaces. We found no evidence that the unknown inhibitory molecule is able to diffuse through soft agar.

Agar↗
PubMed1995

New and emerging yeast pathogens.

The most common yeast species that act as agents of human disease are Candida albicans, Candida tropicalis, Candida glabrata, Candida parapsilosis, and Cryptococcus neoformans. The incidence of infections by other yeasts has increased during the past decade. The most evident emerging pathogens are Malassezia furfur, Trichosporon beigelii, Rhodotorula species, Hansenula anomala, Candida lusitaniae, and Candida krusei. Organisms once considered environmental contaminants or only industrially important, such as Candida utilis and Candida lipolytica, have now been implicated as agents of fungemia, onychomycosis, and systemic disease. The unusual yeasts primarily infect immunocompromised patients, newborns, and the elderly. The role of central venous catheter removal and antifungal therapy in patient management is controversial. The antibiograms of the unusual yeasts range from resistant to the most recent azoles and amphotericin B to highly susceptible to all antifungal agents. Current routine methods for yeast identification may be insufficient to identify the unusual yeasts within 2 days after isolation. The recognition of unusual yeasts as agents of sometimes life-threatening infection and their unpredictable antifungal susceptibilities increase the burden on the clinical mycology laboratory to pursue complete species identification and MIC determinations. Given the current and evolving medical practices for management of seriously ill patients, further evaluations of the clinically important data about these yeasts are needed.

Carrier State↗
PubMed1997

The incidence of killer activity and extracellular proteases in tropical yeast communities.

The presence of killer and proteolytic yeasts was studied among 944 isolates representing 105 species from tropical yeast communities. We found 13 killer toxin producing species, with Pichia kluyveri being the most frequent. Other killer yeast isolates were Candida apis, Candida bombicola, Candida fructus, Candida krusei, Candida sorbosa, Hanseniaspora uvarum, Issatchenkia occidentalis, Kloeckera apis, Kluyveromyces marxianus, Pichia membranaefaciens, Pichia ohmeri-like, and Sporobolomyces roseus. The communities from which killer yeasts were isolated had strains sensitive to them, and there were interspecific and intraspecific differences in the spectra of their killer activities. Pichia kluyveri had the broadest spectra of activity against sensitive isolates, and it apparently produced different toxins. The coexistence of sensitive and killer yeasts using the same substrate suggests that there is spatial separation in microhabitats or temporal separation in different stages of successions. Basidiomycetous yeasts were more frequently proteolytic than ascomycetous yeasts. Extracellular proteases could be important for the yeasts to have access to more nitrogen nutrients and obtain a better balance with available carbon sources.

Endopeptidases↗
PubMed1983

Yeast DNA plasmids.

The study of yeast DNA plasmids has been initiated with the discovery of the 2-micron DNA in Saccharomyces cerevisiae. This multiple copy plasmid, organized into chromatin structure in vivo, probably exists in the nucleus and provides a good system to obtain information on eukaryotic DNA replication. Yeast transformation with the 2-micron DNA or artificially constructed chimeric plasmids had contributed significantly to the study of the molecular biology of yeast and eukaryotes, allowing the isolation and characterization of various genes, ars, centromeres, and telomeres, and also serving as a tool to study the expression of various heterologous genes. Encouraged by these fruitful results, new yeast plasmids have been screened among phylogenetically distant yeasts. The linear DNA plasmids (pGKl1 and pGKl2) from Kluyveromyces lactis are the first case of yeast plasmids associated with biological function (killer phenotype). This plasmid system would be ideal as a model to study the structure and function of eukaryotic linear chromosomes. The extracellular secretion of protein toxin suggests the plasmids to be an excellent candidate for a secretion vector. The importance of yeasts as suitable materials for the study of eukaryotic cell biology would be much enhanced by the advent of new transformation systems with diverse host yeasts of genetically and phylogenetically distinct properties.

Chimera↗