Candida Deformans Research Articles

Presence, changes and technological properties of yeast species during processing of pastirma, a Turkish dry-cured meat product

Abstract In this study, alterations in yeast dynamics during pastirma processing, and technological (growth at different pH, temperature and NaCl conditions, lytic and enzymes activity) properties of yeast strains were investigated. The pH values of pastirma samples increased throughout pastirma processing, while moisture and water activity ( a w ) decreased. Initial yeast population decreased from 4.42 log cfu/g to 3.61 log cfu/g during the curing process. Considering the genotypic identification, a total of 100 isolates were obtained from pastirma samples. The dominant yeast species was Candida zeylanoides (58%) which was followed by Candida deformans (12%) and Candida galli (11%), respectively. These yeast species were found at all sampling points. Trichosporon japonicum (2%), Cryptococcus curvatus (1%) and Debaryomyces hansenii (1%) were the least frequent species isolated from the pastirma samples. C. zeylanoides and T. japonicum did not show any proteolytic activity, and their lipolytic activity was weak. Twenty-one of the yeast isolates had nitrate reductase activity.

Diversity and enzymatic profile of yeasts isolated from traditional llama meat sausages from north-western Andean region of Argentina

Llama meat fermented sausages are traditional products consumed in the Andean region of South America. The diversity and dynamics of yeasts present in these meat products were evaluated in two productions. The results demonstrated that yeast population increased during fermentation and it remained stable throughout the ripening period. A total of 414 yeasts isolated during different stages of production were identified and characterized by molecular methods. In both productions, Debaryomyces hansenii was found as the dominant species followed by Candida zeylanoides, although other species of the genera Candida, Cryptococcus, Metschnikowia, Rhodotorula, Rhodosporium, Trichosporon and Yarrowia also contributed to the fermentation. The fingerprinting analyses by RAPD-PCR of M13 minisatellite revealed the presence of different genotypes within D. hansenii and C. zeylanoides throughout the manufacturing process. Assay of proteolytic activity revealed that Yarrowia lipolytica from production A and Candida deformans, Cryptococcus curvatus, Rhodotorula mucilaginosa and Rhodosporium diobovatum from production B were able to hydrolyze meat proteins. All yeast species, excluding Torulaspora delbrueckii, exhibited lipolytic activity, whereas esterase activity was detected only in few species. A correspondence between enzymatic activity and RAPD M13 profiles was observed for yeasts of production A but this correspondence was not found in production B. An appropriate selection of yeast strains as starter cultures is fundamental for quality improvement of artisanal meat products.

The teleomorph state of Candida deformans Langeron & Guerra and description of Yarrowia yakushimensis comb. nov.

Yarrowia lipolytica is the only known species in the teleomorph (i.e. sexual) genus Yarrowia and has its anamorph (i.e. asexual state) classified in the genus Candida Berkhout as Candida lipolytica. This species can be found readily in nature, has significant industrial value and is important to the food and medical fields. Candida deformans was first considered a variety of C. lipolytica and was later listed as a synonym of this species. More recent studies based on sequence variation in the nuclear rRNA gene sequences suggested C. deformans to be a separate species with no known teleomorph linked to it. In this study we show that C. deformans strains, obtained from South Africa, can mate with strains present in the CBS yeast collection and produce a Yarrowia teleomorph, described here as Yarrowia deformans. Strains of Candida yakushimensis nom. inval., a species also belonging to the Yarrowia phylogenetic clade, were also studied and described as a novel species of Yarrowia.

The Influence of Debaryomyces hansenii, Candida deformans and Candida zeylanoides on the aroma formation of dry-cured “lacón”

The volatile profile of dry-cured “lacón” that has been inoculated with three different yeasts were determined and compared with a non-inoculated dry-cured “lacón”. Yeasts (Debaryomyces hansenii, Candida deformans and Candida zeylanoides) that were used as starter cultures in the present study were selected among yeasts that were isolated from native dry-cured “lacón” at different stages of ripening process. These starters were spread on dry-cured “lacón” surface in order to test their capacity to contribute on the generation of volatile compounds. A total of forty two volatile compounds were detected by dynamic headspace sampling followed by gas chromatography–mass spectrometry analysis. Significant differences (P<0.001) on the volatile profiles of different batches were found in comparison with non-inoculated samples, showing the highest total area values for the inoculated ones. Esters were the most abundant chemical family in all batches studied except for C. zeylanoides batch, which showed greater amount of hydrocarbons than esters. The second more abundant family was hydrocarbons for control and C. deformans batches (147.6 and 445.24×106 area units, respectively), alcohols for D. hansenii (363.77×106 area units) and esters for C. zeylanoides (248.33×106 area units). However, the aldehyde compound group in control batch samples was found to be significantly higher than in the inoculated ones (P<0.001). Among inoculated batches, D. hansenii batch showed the lowest hexanal content (14.42×106 area units) in comparison with non-inoculated batch (105.99×106 area units). Among all batches studied, D. hansenii batch presented the highest area values for esters, alcohols, linear hydrocarbons, ketones, acids and furans; control batch for aldehydes and C. zeylanoides batch for branched hydrocarbons. Therefore, the study showed that every yeast strain produced a specific volatile profile which was also different from that of the control dry-cured “lacón”.

Mitochondrial genomes of yeasts of the Yarrowia clade

Candida alimentaria, Candida deformans, Candida galli, and Candida phangngensis have been recently reported to be the close relatives of Yarrowia lipolytica. To explore this clade of yeasts, we sequenced the mitochondrial genome (mtDNA) of these four species and compared it with the mtDNA of Y.lipolytica. The five mtDNAs exhibit a similar architecture and a high level of similarity of protein coding sequences. Genome sizes are variable, ranging from 28017bp in C.phangngensis to 48508bp in C.galli, mainly because of the variations in intron size and number. All introns are of group I, except for a group II intron inserted in the cob gene of a single species, C.galli. Putative endonuclease coding sequences were present in most group I introns, but also twice as free-standing ORFs in C.galli. Phylogenetic relationships of the five species were explored using protein alignments. No close relative of the Yarrowia clade could be identified, but protein and rRNA gene orders were partially conserved in the mtDNA of Candida salmanticensis.

Polyphasic re-examination of Yarrowia lipolytica strains and the description of three novel Candida species: Candida oslonensis sp. nov., Candida alimentaria sp. nov. and Candida hollandica sp. nov.

The type strain of Yarrowia lipolytica and 38 strains identified as Yarrowia lipolytica, four strains of Candida deformans, including the type and two subcultures of the type, two strains of Candida galli and six unidentified strains that resembled Y. lipolytica were examined by PCR fingerprints using primers M13 and (GAC)5. The same strains, together with four strains of the recently introduced Candida yakushimensis nom. inval., were sequenced for the D1/D2 domain of the 26S rRNA gene and parts of the ITS domain and also studied for their physiological properties. Of the strains identified previously as Y. lipolytica, CBS 2076 had the same fingerprint as the type of C. deformans and strain CBS 4855 was distinct from all other strains. The six strains that resembled Y. lipolytica were separated into two groups distinct from any of the other clades. A total of six groups obtained by fingerprint and sequence data were evaluated by performing DNA reassociation reactions. Mating experiments among the 35 strains of Y. lipolytica sensu stricto showed that 15 strains represented one mating type and 16 strains represented the opposite mating type, while four strains were self-sporulating. Teleomorph states were not produced by C. deformans, C. galli or any of the unidentified isolates. However, positive mating reactions were rarely observed in crosses among C. galli and some strains of Y. lipolytica and C. deformans. Consequently, sharing the same mating type system, C. deformans and C. galli could be considered anamorphs of unnamed Yarrowia species. Results from PCR fingerprints, sequencing and mating studies support the grouping of the studied strains into Y. lipolytica, C. galli, C. deformans, C. yakushimensis nom. inval. and three novel species in the Yarrowia clade: Candida oslonensis sp. nov. (type strain CBS 10146(T) =NRRL Y-48252(T); Mycobank number MB 510769), Candida alimentaria sp. nov. (type strain CBS 10151(T) =NRRL Y-48253(T); Mycobank number MB 510770) and Candida hollandica sp. nov. (type strain CBS 4855(T) =NRRL Y-48254(T); Mycobank number MB 510771).

Purification and characterization of the extracellular lipase Lip2 from Yarrowia lipolytica

An extracellular lipase from Yarrowia lipolytica (YlLip2) has been purified by ion exchange chromatography on Q sepharose FF, followed by hydrophobic interaction chromatography on butyl sepharose FF. SDS-PAGE showed that the molecular weight of this lipase is about 38 kDa. N-terminal amino acid sequencing and MALDI-TOF mass spectral analysis showed that this lipase is encoded by gene LIP2 (GenBank accession no. AJ012632). Enzymatic deglycosylation showed that this lipase is a glycosylated protein which contains about 12% sugar. The corresponding deglycosylated lipase remained 88% specific activity of untreated lipase. There was a high amino acid sequence identity (91%) between YlLip2 and Candida deformans lipase CdLip1 (GenBank accession no. AJ428393). The optima temperature and pH for the purified lipase was 40 °C and 8.0, respectively. The lipase showed a preference for long chain fatty acid methyl esters (C12–C16), with the highest activity toward methyl myristate (C14). Lipase activity was stimulated by Ca 2+ and Mg 2+ and inhibited by Zn 2+, Ni 2+ and Cu 2+, whereas EDTA had no effect on its activity. A 0.1% of Tween 80 and Span 65 increased slightly the enzyme activity and SDS inhibited it.

Identification and characterisation of LIP7 and LIP8 genes encoding two extracellular triacylglycerol lipases in the yeast Yarrowia lipolytica

In the lipolytic yeast Yarrowia lipolytica, the LIP2 gene was previously reported to encode an extracellular lipase. The growth of a Δ lip2 strain on triglycerides as sole carbon source suggest an alternative pathway for triglycerides utilisation in this yeast. Here, we describe the isolation and the characterisation of the LIP7 and LIP8 genes which were found to encode a 366 and a 371-amino acid precursor protein, respectively. These proteins which belong to the triacylglycerol hydrolase family (EC 3.1.1.3) presented a high homology with the extracellular lipase Cd LIP2 and Cd LIP3 from Candida deformans. The physiological function of the lipase isoenzymes was investigated by creating single and multi-disrupted strains. Lip7p and Lip8p were found to correspond to active secreted lipases. The lack of lipase production in a Δ lip2 Δ lip7 Δ lip8 strain suggest that no additional extracellular lipase remains to be discovered in Y. lipolytica. The substrate specificity towards synthetic ester molecules indicates that Lip7p presented a maximum activity centred on caproate (C 6) while that of Lip8p is in caprate (C 10).

Identification of a triacylglycerol lipase gene family in Candida deformans: molecular cloning and functional expression.

The yeast Candida deformans CBS 2071 produces an extracellular lipase which was shown to catalyse the production of various esters by the esterification of free fatty acids, even in the presence of a large molar excess of water. To clone the gene encoding this extracellular lipase, Saccharomyces cerevisiae was transformed with C. deformans genomic libraries and screened for lipolytic activity on a medium containing rapeseed oil emulsion and rhodamine B. Three members of a lipase gene family (CdLIP1, CdLIP2 and CdLIP3) were cloned and characterized. Each deduced lipase sequence has a Gly-His-Ser-Leu-Gly-(Gly/Ala)-Ala conserved motif, eight cysteine residues and encodes an N-terminal signal sequence. MALDI-TOF mass spectrometry analysis of a proteolytic digest of the lipase produced was used to obtain experimental evidence that the CdLIP1 gene encoded the extracellular lipase. Recombinant expression studies confirmed that the cloned genes encoded functional lipases. The three lipases are very similar to lipases from the related species Yarrowia lipolytica. Significant homologies were also found with several yeast and fungal lipases. As C. deformans CBS 2071 was previously considered to be synonymous with Y. lipolytica, the strains were compared for the extent of nucleotide divergence in the variable regions (D1/D2) at the 5'-end of the large-subunit (26S) ribosomal DNA (rDNA) gene. This rDNA region has diverged sufficiently to suggest that C. deformans is a separate species. The nucleotide sequences of the CdLIP1, CdLIP2 and CdLIP3 genes will appear in the EMBL nucleotide sequence database under Accession Nos AJ428393, AJ428394 and AJ428395, respectively.

Chain-length selectivity of various lipases during hydrolysis, esterification and alcoholysis in biphasic aqueous medium

Chain-length selectivity profiles of seven lipases (EC 3.1.1.3) have been determined by multiple substrate competition for three reactions at 30 and 50 °C: hydrolysis and synthesis of fatty acid methyl esters and acyl transfer between fatty acid ethyl esters and methyl esters. All acyl chains were even-numbered and saturated. Chain-length specificity profiles depended on the enzyme and the reaction considered, due to the combined influence of enzyme and substrate properties. Changing temperature had a significant influence on substrate specificity for esterification only. The results led to the selection of Candida deformans lipase for the selective hydrolysis of short-chain (C 8, C 10) esters in an equimolar mixture of C 8–C 18 methyl esters in the presence of various methanol concentrations. The reaction resulted in a long-chain-enriched methyl ester and a short-chain-enriched fatty acid fractions, respectively, consisting of 96% C 12–C 18 and 68% C 8–C 10 in a given example.

Factors influencing ester synthesis catalysed in aqueous media by the lipase from candida deformans (zach) langeron and guerra

The purified lipase from Candida deformans was shown to catalyse ester production in aqueous media by esterification of free fatty acids but not by alcoholysis. As the enzyme also catalysed ester hydrolysis, the influence of various physico-chemical factors on ester hydrolysis and synthesis was studied and compared. Substrate specificities were also studied. Both activities had the same pH and temperature optima, and did not require a metal cofactor. Tyrosine appeared to be one of the amino acids of the enzyme required by both catalytic activities, whereas serine-reactive reagents inhibited synthesis only. The highest synthesis and hydrolysis activities were obtained with mono-, di-and tri-unsaturated fatty acids containing a [ cis] Δ-9 unsaturation and 16 to 18 carbon atoms. Only esters of primary alcohols were hydrolysed and synthesised. For alcohols ranging from methanol to butanol, synthesis activity increased with the length of the alcohol whereas hydrolysis activity of the corresponding esters decreased. The presence of alcohol inhibited hydrolysis. An optimum concentration was found for each primary alcohol at which ester synthesis was maximum and hydrolysis was low.

A new cerebroside of the n-Alkane-assimilating yeast Candida deformans

A glucocerebroside was isolated from the n-alkane-assimilating yeast Candida deformans MIL 4041 and named “Deformin”. The cerebroside contained only a single sugar, glucose, attached to ceramide in a β-configuration, and only a single fatty acid, 2-hydroxypalmitic acid. The long-chain base was identified as (5 E,9 Z)-C 19-sphinga-5,9-diene. Therefore, the structure of the cerebroside was determined to be (5 E,9 Z)- N-2′-hydroxypalmitoyl-1- O- β-glucopyranosyl-2-amino-5,9- nonadecadiene-1,3-diol. Deformin showed the fruiting-inducing activity of fungi.

The production of lipases by some Candida and Yarrowia yeasts

AbstractThe activities of the free and the cell‐bound lipases of Candida rugosa, Candida guilliermondii, Candida curvata, Candida deformans and Yarrowia lipolytica that had been grown on a complex medium with various carbon and nitrogen sources were examined. The highest activities were observed with citrate and succinate as the carbon sources. In some cases olive oil was used as the efficient inducer of the cell‐bound lipases.The cells grown in the presence of the organic nitrogen sources (peptone, urea, soy flour) exhibited higher lipase levels than those cultivated in the medium containing ammonium as the nitrogen source. The extracellular enzyme was detected only in cultures grown with urea, peptone or soy flour as the nitrogen source.