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To prevent microbial growth and its consequences, preservatives such as sorbic acid or its salts, commonly known as sorbates, are added to foods. However, some moulds and yeasts are capable of decarboxylating sorbates and producing 1,3-pentadiene. This is a volatile compound with an unpleasant &ldquo, petroleum-like &ldquo, odour, which causes consumer rejection of the contaminated products. In this work, we studied the production of 1,3-pentadiene in 91 strains of the yeast Debaryomyces hansenii, and we found that nearly 96% were able to produce this compound. The sequence of the FDC1Dh gene was analysed showing differences between 1,3-pentadiene producer (P) and non-producer (NP) strains. A specific PCR assay with degenerated primers based on the gene sequence was developed to discern NP and P strains. It was tested on D. hansenii strains and on some physiologically related species frequently isolated from foods, such as D. fabrii, D. subglobosus and Meyerozyma guillermondii. This method could be applied for the selection of NP D. hansenii strains, useful in biotechnological food production and as a biocontrol agent.

Despite previously published methods, there is still a lack of rapid and affordable methods for genotyping the Meyerozyma guilliermondii yeast species. The development of microsatellite markers is a useful genotyping method in several yeast species. Using the Tandem Repeat Finder Software, a total of 19 microsatellite motifs (di-, tri-, and tetra- repetition) were found in silico in seven of the nine scaffolds published so far. Primer pairs were designed for all of them, although only four were used in this work. All microsatellite amplifications showed size polymorphism, and the results were identical when repeated. The combination of three microsatellite markers (sc15F/R, sc32 F/R and sc72 F/R) produced a different pattern for each of the Type Culture Collection strains of M. guilliermondii used to optimize the method. The three primer pairs can be used in the same PCR reaction, which reduces costs, in tandem with the fluorescent labeling of only the forward primer in each primer pair. Microsatellite typing was applied on 40 more M. guilliermondii strains. The results showed that no pattern is repeated between the different environmental niches. Four M. guilliermondii strains were only amplified with primer pair sc32 F/R, and subsequently identified as Meyerozyma caribbica by Taq I-RFLP of the 5.8S ITS rDNA. Most out-group species gave negative results even for physiologically similarly species such as Debaryomyces hansenii. The microsatellite markers used in this work were stable over time, which enables their use as a traceability tool.

Four yeast strains were isolated from Spanish traditional fermented sausages (chorizo) spoiled by gas production. Using the classical identification procedures, they were identified as Debaryomyces hansenii. However, they fermented galactose and did not produce positive results in Debaryomyces differential medium (DDM), a growth medium highly specific for this species. Phylogenetic analysis showed identical sequences for the D1/D2 domain of the 26S rRNA gene and almost identical sequences for the 5.8S-ITS region with those of the recently described yeast species Candida cretensis. This result was confirmed by sequencing the gene encoding actin of the type and the new strains. Candida cretensis is a new species included in the so-called Candida kruisii clade that was described from a single strain, isolated from a decaying mushroom in Crete, Greece. The discovery of new strains of C. cretensis in fermented food expands its physiological and ecological diversity. With the description of these new strains isolated from food, three groups of strains can be distinguished within C. cretensis according to the restriction patterns of the intergenic spacer rRNA gene region and on the basis of some physiological properties that are of ecological relevance.

In this work, we developed a specific PCR assay for Debaryomyces hansenii strains that uses a putative homologous PAD1 region (729 bp) present in this yeast species as a target. The amplification of this sequence with the D. hansenii specific primer pair (DhPADF/DhPADR) was found to be a rapid, specific and an affordable method enabling identification of D. hansenii from other yeast strains. Primers were tested in almost 100 strains, 49 strains from Type Culture Collection belonging to the genus Debaryomyces and to other yeast species commonly found in foods or related genera. These primers were able to discriminate between closely related species of Debaryomyces, such as Debaryomyces fabryi and Debaryomyces subglobosus, with a 100% detection rate for D. hansenii. Also, the method was tested in 45 strains from different foods. Results confirmed the specificity of the PCR method and detected two earlier misidentifications of D. hansenii strains obtained by RFLP analysis of the 5.8S ITS rDNA region. Subsequently we confirmed by sequencing the D1/D2 domain of 26S rDNA that these strains belonged to D. fabryi. We call attention in this work to the fact that the RFLPs of the 5.8S ITS rDNA profiles of D. hansenii, D. fabryi and D. subglobosus are the same and this technique will thus lead to incorrect identifications.

The amplification by PCR of the intergenic spacer region (IGS) of rDNA followed by restriction fragment length polymorphism (RFLP) analysis was evaluated as a potential method for discriminating the 16 species belonging to the genus Debaryomyces. The digestion of this region with some or all the enzymes used in this study (HapII, HhaI and MboI) produced species-specific patterns that permitted differentiation of the species in the genus. With the exception of Debaryomyces vanrijiae, the technique was also efficient for distinguishing the varieties in the species Debaryomyces hansenii (var. hansenii, var. fabryi), Debaryomyces occidentalis (var. occidentalis, var. persoonii) and Debaryomyces polymorphus (var. africanus, var. polymorphus), respectively. PCR-RFLP analysis of the IGS region of rDNA is proposed as a clear and reproducible technique for the practical discrimination of species of the yeast genus Debaryomyces.

The amplification by PCR of the Intergenic Spacer region (IGS) of rDNA followed by Restriction Fragment Length Polymorphism (RFLP) analysis was evaluated as a potential method for the identification of Debaryomyces hansenii among other yeast species that frequently contaminate Intermediate-Moisture Foods (IMFs). For a first rapid differentiation at the species level, the determination of the IGS-PCR fragment size was found to be a useful approach. The digestion of this region with the enzymes HhaI, HapII and MboI resulted in specific patterns that permit the identification of D. hansenii among other yeast species. This method also permitted the discrimination between the D. hansenii varieties (var. hansenii and var. fabryi) as well as the differentiation of D. hansenii from other species of the genus, such as Debaryomyces pseudopolymorphus or Debaryomyces polymorphus var. polymorphus. The IGS-PCR RFLP method was assayed for the differential detection of D. hansenii in contaminated or spoiled IMF products and compared with traditional identification procedures, resulting in a 100% detection rate for D. hansenii.

A selective and differential solid medium, Debaryomyces differential medium (DDM), was used for the isolation of Debaryomyces hansenii. This medium is formulated to allow detection of the beta-glucuronidase enzyme using the chromogenic substrate magenta-glucuro.CHA (5Br-6Cl-3indolyl-beta-D-glucuronide, cyclohexylammonium salt). Of the more than 120 microorganisms tested, including yeasts, bacteria, and a filamentous fungus, only D. hansenii produced violet colonies, thus permitting its easy discrimination from other organisms. When quality assessment tests were performed, optimal productivity and selectivity were obtained for D. hansenii. The medium was also satisfactory when used to test naturally contaminated food products.