Candida Zeylanoides Research Articles

Differentiation of yeasts growing on dry-cured Iberian ham by mitochondrial DNA restriction analysis, RAPD-PCR and their volatile compounds production

The efficiency of mitochondrial DNA (mtDNA) restriction analysis, RAPD-PCR and volatile compounds analysis to differentiate yeast biotypes involved in flavour development of dry-cured Iberian ham throughout the ripening process is evaluated. For this purpose, 86 yeasts isolated from Iberian hams in the main ripening stages at different industries of the four Protected Designations of Origin of this product, were used. The combination of mtDNA restriction analysis and RAPD-PCR using the primer (GACA) 4 showed a higher variability in the yeast species detected than obtained using only mtDNA restriction analysis. Only two species, Debaryomyces hansenii and Candida zeylanoides, were identified throughout the whole ripening process and a wide diversity of biotypes was found in these two species, with those of D. hansenii predominating. Clear differences between biotypes were detected in the generation of volatile compounds, with the biotype C2-2 of D. hansenii showing the highest concentrations of volatiles. The combined use of mtDNA restriction analysis and RAPD-PCR distinguishes yeast biotypes with different production of volatile compounds. In addition, analysis of the production profile of volatile compounds is needed to differentiate yeast strains of the same biotype recovered at different stages of ripening. Thus, the combination of these three methods could be very useful to select or monitor yeasts as starter cultures in dry-cured meat products.

Temporal trends, risk factors and outcomes in albicans and non- albicans candidaemia: an international epidemiological study in four multidisciplinary intensive care units

This multicentre study (i) evaluated geographic and temporal changes in candidaemia ecology in the critically ill, (ii) identified risk factors associated with non- albicans candidaemia and (iii) examined the association of Candida ecology with mortality. A retrospective cohort study of patients who developed candidaemia in four general Intensive Care Units located in Australia, Greece, Belgium and Brazil was performed. Two hundred Candida organisms were identified by positive blood culture in 189 patients, including 112 Candida albicans (56.0%), 38 Candida glabrata (19.0%), 21 Candida parapsilosis (10.5%), 18 Candida tropicalis (9.0%), 6 Candida krusei (3.0%), 1 Candida famata (0.5%), 1 Candida zeylanoides (0.5%) and 3 non-differentiated Candida spp. (1.5%). No trend towards increased non- albicans species over the study period ( P = 0.68) or by geographic area ( P = 0.35) was demonstrated. Independent risk factors for non- albicans candidaemia included: female gender [odds ratio (OR) 2.09, 95% confidence interval (CI) 1.13–3.86] and increased central venous catheter days (OR 1.16 per 5-day interval, 95% CI 1.05–1.28). Mortality in the non- albicans group was non-significantly higher than in the albicans group (65% vs. 53%; P = 0.10). This study is unique in that a large number of intensive care candidaemias in four geographically diverse units have been studied.

Occurrence and diversity of Candida genus in marine environments

A total of 317 yeast isolates from seawater, sediments, mud of salterns, guts of marine fishes and marine algae were obtained. The results of routine identification and molecular characterization showed that six isolates among these marine yeasts belonged to Candida genus as Candida intermedia for YA01a, Candida parapsilosis for 3eA2, Candida quercitrusa for JHSb, Candia rugosa for wl8, Candida zeylanoides for TJY13a, and Candida membranifaciens for W14-3. Isolates YA01a (Candida intermedia), wl8 (Candida rugosa), 3eA2 (Candida parapsilosis), and JHSb (Candida quercitrusa) were found producing cell-bound lipase, while isolate W14-3 (Candida membranifaciens) producing riboflavin. These marine yeast Candida spp. seem to have wide potential applications in biotechnology.

Evaluation and selection of yeasts isolated from dry-cured Iberian ham by their volatile compound production

One hundred and seventeen yeast strains isolated from dry-cured Iberian ham from the four different protected designations of origin of Spain were investigated for their volatile compound production. The yeast strains were grouped into the two main yeast species usually found in this product ( Debaryomyces hansenii and Candida zeylanoides) and 10 different biotypes by restriction mitochondrial DNA analysis. Yeast strains were grown in a designed model culture medium under conditions representative of dry-cured ham processing. Volatile compounds were extracted from this medium using solid-phase micro-extraction and were analysed by gas chromatography/mass spectrometry. Marked differences in volatile compound production were found between D. hansenii and C. zeylanoides and between the mitochondrial DNA patterns of these species . Two of the mitochondrial DNA patterns of D. hansenii exhibited the highest production of the volatile compounds involved in the dry-cured flavour. Consequently, these patterns of D. hansenii should be proposed as starter cultures for dry-cured ham.

Occurrence and growth of yeasts in processed meat products – Implications for potential spoilage

Spoilage of meat products is in general attributed to bacteria but new processing and storage techniques inhibiting growth of bacteria may provide opportunities for yeasts to dominate the microflora and cause spoilage of the product. With the aim of obtaining a deeper understanding of the potential role of yeast in spoilage of five different processed meat products (bacon, ham, salami and two different liver patés), yeasts were isolated, enumerated and identified during processing, in the final product and in the final product at the end of shelf life. Yeasts were isolated along the bacon production line in numbers up to 4.2 log (CFU/g). Smoking of the bacon reduced the yeast counts to lower than 1.0 log (CFU/g) or non-detectable levels. In general, yeasts were only isolated in low numbers during the production of salami, cooked ham and liver paté. In the final products yeasts were detected in low numbers in a few samples (3 out of 30) samples, 1.0–1.3 log (CFU/g). By the end of storage, yeasts were only detected in 1 out of 25 investigated samples 1.8 log (CFU/g). A combination of phenotypic and genotypic methods was used to identify the yeast microflora present during production of the processed meat products. The yeast microflora was complex with 4–12 different species isolated from the different production sites. In general, Candida zeylanoides, Debaryomyces hansenii and the newly described Candida alimentaria were found to be the dominant yeast species. In addition, three putatively previously undescribed yeast species were isolated. Fourteen isolates, representing seven different species isolated during the production of the processed meat products and one species isolated from spoiled, modified atmosphere packed, sliced ham, were screened for their ability to grow in a meat model substrate under a low oxygen/high carbon-dioxide atmosphere (0.5% O 2, 20% CO 2, 79.5% N 2) at two different temperatures (5 and 8 °C). Eleven out of the tested 14 strains were able to grow in the meat model substrate with C. zeylanoides, D. hansenii, Pichia guilliermondii and Candida sake reaching levels of 10 5–5 × 10 6 log (CFU/g), where sensoryical changes appear.

Studies on hydrazone derivatives as antifungal agents

The increasing clinical importance of drug-resistant fungal pathogens has urged additional need to fungal research and new antifungal compound development. For this purpose, some N-(1-benzyl-2-phenylethylidene)-N′-[4-(aryl)thiazol-2-yl]hydrazone (1a-e) and N-(1-phenylbutylidene)-N′-[4-(aryl)thiazol-2-yl]hydrazone (2a-e) derivatives were synthesised and evaluated for antifungal activity. Their antifungal activities against standard and clinical strands of Candida albicans, Candida glabrata, Candida utilis, Candida tropicalis, Candida krusei, Candida zeylanoides, and Candida parapsilosis were investigated. A significant level of activity was observed.

Synthesis and anticandidal activity of some imidazopyridine derivatives

New hydrazide derivatives of imidazo[1,2-a]pyridine have been synthesized and evaluated for anticandidal activity. The reaction of imidazo[1,2-a]pyridine-2-carboxylic acid hydrazides with various benzaldehydes gave N-(benzylidene)imidazo[1,2-a]pyridine-2-carboxylic acid hydrazide derivatives. Their anticandidal activities against Candida albicans and Candida glabrata (isolates obtained from Osmangazi University, Faculty of Medicine, Eskisehir, Turkey), Candida albicans (ATCC 90028), Candida utilis (NRLL Y-900), Candida tropicalis (NRLL Y-12968), Candida krusei (NRLL Y-7179), Candida zeylanoides (NRLL Y-1774), and Candida parapsilosis (NRLL Y-12696) were investigated.

CE separation of proteins and yeasts dynamically modified by PEG pyrenebutanoate with fluorescence detection

The optimized protocols of the bioanalytes separation, proteins and yeasts, dynamically modified by the nonionogenic tenside PEG pyrenebutanoate, were applied in CZE and CIEF with the acidic gradient in pH range 2-5.5, both with fluorescence detection. PEG pyrenebutanoate was used as a buffer additive for a dynamic modification of proteins and/or yeast samples. The narrow peaks of modified analytes were detected. The values of the pI's of the labeled proteins were calculated using new fluorescent pI markers in CIEF and they were found to be comparable with pI's of the native compounds. As an example of the possible use of the suggested CIEF technique, the mixed cultures of yeasts, Candida albicans, Candida glabrata, Candida kefyr, Candida krusei, Candida lusitaniae, Candida parapsilosis, Candida tropicalis, Candida zeylanoides, Geotrichum candidum, Saccharomyces cerevisiae, Trichosporon asahii and Yarrowia lipolytica, were reproducibly focused and separated with high sensitivity. Using UV excitation for the on-column fluorometric detection, the minimum detectable amounts of analytes, femtograms of proteins and down to ten cells injected on the separation capillary, were estimated.

Dynamics and characterization of yeasts during ripening of typical Italian dry-cured ham

The evolution of the yeast population during manufacturing and ripening of dry-cured Parma ham was investigated. Contamination levels ranged from 10 5 to 10 7 cfu/g on muscle surface, 10 4 to 10 6 cfu/g on covering fat and exceeded 10 7 cfu/g on spreadable fat mince (“sugna”). Two hundred and sixty one yeast isolates underwent identification test, showing that the predominant species of yeast population during the whole maturing process were Debaryomyces hansenii, Candida zeylanoides, Debaryomyces maramus, and to a lesser extent, Candida famata and Hyphopichia burtonii. The species Candida catenulata, Candida guilliermondii, Candida edax and other genera like Cryptococcus and Wingea were occasionally found. The yeast counts and species distribution changed according to the stage of processing and to the ham sampling location. At the end of the cold phase, the washing procedure was effective in lowering the yeast count in muscle and fat surface layers, but during the next ageing stages, yeast colonization of unskinned ham muscle increased again, though species distribution changed if compared to previous manufacturing phases. The ripening steps taken into account from the end of the cold phase to the final outcome, were always characterized by more than one yeast species, suggesting that yeasts other than Debaryomyces spp. could play a remarkable role on the sensory and safety properties of typical Italian dry-cured ham.

Dynamics and characterization of yeasts during natural fermentation of Italian sausages

A multiphasic approach was used to investigate the yeast ecology in Italian fermented sausages. Culture-dependent and -independent methods were applied to identify the yeast species during the maturation process and to characterize the numerically dominant species. Plating analysis and subsequent molecular identification of the isolates highlighted the dominance of Debaryomyces hansenii, but at least other three yeast species -Candida zeylanoides, Pichia triangularis and Metschnikowia pulcherrima - contributed to the fermentation as well. Direct denaturing gradient gel electrophoresis analysis confirmed that D. hansenii was the main yeast species present and its activity was also demonstrated. No other yeasts species were detected on the direct denaturing gradient gel electrophoresis gels, whereas DNA of Penicillium farinosum, Penicillium viridicatum and Mucor racemosus were present. Molecular characterization by RAPD-PCR analysis of the D. hansenii isolates demonstrated a shift in its population from the beginning to the end of the maturation of the sausages. Strains present during the early stages of the fermentation were grouped in clusters that differed from those isolated in the final phases of the maturation, underlining the genetic differences between these two populations of D. hansenii. However, all the isolates were able to grow in the presence of 3.5% sodium chloride and at 10 degrees C, evidence that these parameters did not select the species present at the end of the maturation period.

Phylogenetic analysis of pectin degrading yeasts from deep-sea environments

Deep-sea yeast strains were isolated from mud samples collected in Sagami Bay (1,100-1,400 m) and the Japan Trench (4,500-6,500 m). All of the 46 yeast isolates were capable of growth at 24°C and atmospheric pressure, suggesting that these strains might originate neritic regions or land but survived in deep sea. Based on sequencing of 26S rRNA gene, 15 strains were classified into basidiomycetous yeasts including Cryptococcus liquefaciens, Kondoa aeria, Rhodosporidium diobovatum, Rhodosporidium sphaerocarpum, Rhodotorula mucilaginosa, Rhodotorula dairenensis and Rhodotorula slooffiae, and 8 strains were classified into ascomycetous yeasts including Aureobasidium pullulans, Candida zeylanoides, Kluyveromyces nonfermentans, Metschnikowia bicuspidate and Williopsis saturnus. Screening of the 46 isolates appeared to yield a high frequency of polygalacturonase (PGase) producers capable of degrading pectin. We suggest that deep-sea yeasts are new sources of PGase producers.

DNA typing methods for differentiation of yeasts related to dry-cured meat products

RFLP analysis of the ITS and 18S rDNA, RAPD-PCR using mini- and microsatellite primers and RFLP analysis of mitochondrial DNA were examined to discriminate yeasts related to dry-cured meat products at species and strain level. Seven species and 35 strains of yeasts usually found in dry-cured meat products were tested. RFLP analysis of the ITS1-5.8S rDNA-ITS2 and 18S rDNA did not allow the separation at species level of all of the species tested. RAPD with a M13 primer was found to be useful for differentiation of Rhodotorula mucilaginosa, Candida zeylanoides, Yarrowia lipolytica, Debaryomyces hansenii and Saccharomyces cerevisiae. However, no differences were observed between Debaryomyces polymorphus and Pichia carsonii. RAPD analysis with microsatellite primers (GACA) 4, (GTG) 5 and (GAC) 5 enabled discrimination at species and strain level. However, the degree of discrimination by means of RAPD-PCR depends highly on the primers used. Thus, the PCR fingerprinting with primer (GACA) 4 enabled a higher level of discrimination than primers (GAC) 5 and (GTG) 5. The RFLP analysis of mtDNA allowed the discrimination at the species and strain level except for R. mucilaginosa, where no polymorphisms were observed in the strains tested. RAPD analysis with primer (GACA) 4 and the restriction analysis of mtDNA used in the present work are useful for the differentiation at species and strain level of yeasts related to dry-cured meat products.

Application of SSCP–PCR fingerprinting to profile the yeast community in raw milk Salers cheeses

Bacteria and yeasts are important sensory factors of raw-milk cheeses as they contribute to the sensory richness and diversity of these products. The diversity and succession of yeast populations in three traditional Registered Designation of Origin (R.D.O.) Salers cheeses have been determined by using phenotypic diagnoses and Single-Strand Conformation Polymorphism (SSCP) analysis. Isolates were identified by phenotypic tests and the sequencing of the D1–D2 domains of the 26S rRNA gene. Ninety-two percent of the isolates were identified as the same species in both tests. Yeast-specific primers were designed to amplify the V4 region of the 18S rRNA gene for SSCP analysis. The yeast species most frequently encountered in the three cheeses were Kluyveromyces lactis, Kluyveromyces marxianus, Saccharomyces cerevisiae, Candida zeylanoides and Debaryomyces hansenii. Detection of less common species, including Candida parapsilosis, Candida silvae, Candida intermedia, Candida rugosa, Saccharomyces unisporus, and Pichia guilliermondii was more efficient with the conventional method. SSCP analysis was accurate and could be used to rapidly assess the proportions and dynamics of the various species during cheese ripening. Each cheese was clearly distinguished by its own microbial community dynamics.

Malassezia and Candida colonisation on glans penis of circumcised men

The Malassezia yeast are members of the normal human cutaneous flora in adults. They also are reported as part of the microflora of the male genital region in mostly uncircumcised males. It has been reported that Malassezia sympodialis and Malassezia globosa are the most frequent yeast belonging to the resident microflora of the penis as in other human skin areas. The aim was to evaluate the prevalence of Malassezia and Candida yeast colonisation on the glans penis of circumcised males. Impression preparations were made on modified Dixon agar. The isolates were identified by morphological and physiological characteristics. A total of 245 circumcised males were included in the study. Of the 245 patients examined, 55 (22.4%) were found to have a mycologically proven yeast fungi on their glans penis. In 17 (30.9%) Malassezia, in 36 (65.5%) Candida, in one (1.8%) Malassezia and Candida, and in one (1.8%) Saccharomyces strains were detected. Malassezia furfur (66.7%) was the most common species among the lipophilic yeast, followed by Malassezia globosa (11.1%), Malassezia obtusa (11.1%) and Malassezia slooffiae (11.1%). Candida albicans was the most common non-lipophilic yeast (46.0%), that was isolated among the other yeast, followed by unidentified Candida strains (18.9%), Candida tropicalis (8.1%), Candida glabrata (8.1%), Candida parapsilosis (8.1%), Candida zeylanoides (5.4%), Candida guilliermondii (2.7%) and Saccharomyces cerevisiae (2.7%). The results of this study showed that Malassezia species were also colonised like Candida on the glans penis of circumcised males.

Identification of yeasts associated with milk products using traditional and molecular techniques

An integrated approach including phenotypic (morphological, biochemical and physiological characterization) and genotypic (RAPD-PCR, sequencing of D1/D2 domain of 26S rRNA encoding gene) methods was used for the identification of yeasts isolated from different milk products. There were 513 isolates in all, 460 ascomycetous and 53 basidiomycetous yeasts. The yeast isolates were characterized on the basis of their biochemical and physiological properties, and the D1/D2 domain of 26S rDNA was sequenced in selected strains. Relying on the obtained results from both the data-sets, corresponding type strains were selected and compared with the respective yeast isolates from milk products by RAPD fingerprinting. The strains showing a degree of similarity >80% were considered conspecific. By means of the applied techniques it was possible to identify 92% yeast isolates at species level. Debaryomyces hansenii, Geotrichum candidum, Kluyveromyces marxianus, Yarrowia lipolytica and Candida zeylanoides are the most frequently isolated species. The majority of the yeasts were isolated from fresh and sour curd cheese. A comparison of the results obtained by phenotypic and genotypic investigation revealed that the identification based on classical methods was supported by genotypic characterization in only 54% of examined isolates. The results described in this work show that the applied molecular identification is a reliable approach to the identification of yeasts associated with milk products in contrast to the conventional biochemical and physiological tests. The identification of new yeast species requires additional genetic markers such as sequencing of different genes or DNA:DNA hybridization.

Occurrence and characterization of yeasts isolated from artisanal Fiore Sardo cheese

The occurrence of yeast microflora in artisanal Fiore Sardo cheese during ripening was studied. Mean yeast counts ranged from 2.64±1 log 10 cfu ml −1 in milk to 0.65±1 log 10 cfu g −1 in 9 months cheese, with the higher counts observed in 48-h-old cheese. Strains belonging to the prevalent species Debaryomyces hansenii, Kluyveromyces lactis, Geotrichum candidum, Candida zeylanoides and Candida lambica were selected for technological and genotypic characterization. All D. hansenii strains fermented glucose and assimilated lactate, a high percentage assimilated citrate and only a few showed proteolytic and lipolytic activity. All K. lactis strains were able to both assimilate and ferment lactose, to assimilate lactate and to exhibit proteolytic activity on casein . G. candidum assimilated lactate and some strains showed proteolytic and lipolytic activity. C. zeylanoides showed lipolytic activity on tweens and the majority of strains assimilated citrate. C. lambica fermented glucose and assimilated lactate. Considering their diffusion and technological characteristics, an important role for K. lactis and G. candidum in the early stages of the ripening process and for D. hansenii after the first month of ripening can be suggested. RAPD-PCR analysis with M13 primer grouped the isolates in well-separated clusters with their type strains and confirmed the previous phenotypic identification. The high intraspecific homogeneity observed in tested strains could be explained by their isolation from a common substrate and from neighbouring geographical areas. This preliminary study allowed us to isolate autochthon yeast strains showing particular properties which can contribute to the production of typical cheese taste and flavour.

Influence of Antimicrobial Agents on the Spoilage of a Meat-Based Entomophage Diet

The microbial decomposition of a meat-based entomophage diet presented in Parafilm packets was investigated. Considerable bacteria but not fungi were associated with components used to prepare the diet (i.e., hens’ eggs, liver, and ground beef). At the initial sampling time, there were no differences among diet treatments in the size of bacterial or fungal populations. Bacterial populations in diets not containing antibacterial agents rapidly increased and reached an asymptote by 24 h (≈1010 colony-forming units per gram). Bacterial populations also increased in diets containing antibacterial agents, but they were significantly smaller than in diets not containing antibacterial agents. The most prevalent bacteria isolated were Carnobacterium piscicola, Carnobacterium divergens, Lactobacillus curvatus, Lactobacillus sakei, Leuconostoc mesenteroides, and Enterococcus spp., regardless of the antibacterial treatment used. The proliferation of fungi was delayed relative to bacteria, but significant differences were observed among the diet treatments. Fungi were most inhibited by sorbic acid and propionic acid in the absence of antibacterial agents. The most common fungi isolated were the yeasts Candida zeylanoides, Torulaspora globosa, and Yarrowia lipolytica. The pH of diets not containing antibacterial agents decreased rapidly and was highly correlated with increases in bacteria but not fungi. The results of this study demonstrate that antimicrobial agents significantly inhibit spoilage microorganisms in a meat-based diet and that alternative management strategies to delay the decomposition of such diets presented in Parafilm packets should target lactic acid spoilage bacteria, particularly Carnobacterium and Lactobacillus species.

Influence of Antimicrobial Agents on the Spoilage of a Meat-Based Entomophage Diet

The microbial decomposition of a meat-based entomophage diet presented in Parafilm packets was investigated. Considerable bacteria but not fungi were associated with components used to prepare the diet (i.e., hens' eggs, liver, and ground beef). At the initial sampling time, there were no differences among diet treatments in the size of bacterial or fungal populations. Bacterial populations in diets not containing antibacterial agents rapidly increased and reached an asymptote by 24 h (approximately 10(10) colony-forming units per gram). Bacterial populations also increased in diets containing antibacterial agents, but they were significantly smaller than in diets not containing antibacterial agents. The most prevalent bacteria isolated were Carnobacterium piscicola, Carnobacterium divergens, Lactobacillus curvatus, Lactobacillus sakei, Leuconostoc mesenteroides, and Enterococcus spp., regardless of the antibacterial treatment used. The proliferation of fungi was delayed relative to bacteria, but significant differences were observed among the diet treatments. Fungi were most inhibited by sorbic acid and propionic acid in the absence of antibacterial agents. The most common fungi isolated were the yeasts Candida zeylanoides, Torulaspora globosa, and Yarrowia lipolytica. The pH of diets not containing antibacterial agents decreased rapidly and was highly correlated with increases in bacteria but not fungi. The results of this study demonstrate that antimicrobial agents significantly inhibit spoilage microorganisms in a meat-based diet and that alternative management strategies to delay the decomposition of such diets presented in Parafilm packets should target lactic acid spoilage bacteria, particularly Carnobacterium and Lactobacillus species.

Monitoring yeast species in quarg, quarg products and their production environment during the manufacturing process

A total of 65 yeast cultures were isolated from quarg and its products, from the air of the production facilities and from rinses of equipment. The yeast isolates were identified and their proportion was determined. Of these isolates, 8 species were identified in the rinses of equipment, 2 from the air of the production facilities, and 9 from the final product, i.e., quarg and quarg products. Four species of the yeasts isolated from quarg, namely Debaryomyces hansenii, Trichosporon cutaneum, Kluyveromyces marxianus var. marxianus and Candida zeylanoides were also present in the rinses of equipment. Debaryomyces hansenii and Trichosporon cutaneum were the predominant species both in the rinses of equipment and in the final product.

Improved conditions for the aerobic reductive decolourisation of azo dyes by Candida zeylanoides

A number of anaerobic and aerobic bacterial species are known to decolourise azo dyes through the reduction of the azo bonds, forming the corresponding amines. In this work, we describe improved decolourisation conditions for model azo dyes by the ascomycete yeast Candida zeylanoides. The dyes were derived from the diazonium salts of metanilic and sulfanilic acids and N, N-dimethylaniline or 2-naphthol as coupling components. Total decolourisation times observed in culture media supplemented with 0.2 mM dye ranged from 40 to 60 h. The initial decolourisation rates were 14–52 μmol (g dry cell) −1 h −1, depending on dye structure. In the course of decolourisation either metanilic acid or sulfanilic acid were detected in the supernatant fluid, showing that decolourization by this yeast strain is due to azo bond reduction. None of those aminobenzenesulfonates supported microorganism growth as carbon and energy source but both could be used, to a limited extent, as nitrogen sources. The azo reductase activity is not significantly affected by pre-adaptation of the microorganism to the dyes.