Mechanisms involved in reduction of ochratoxin A produced by Aspergillus westerdijkiae using Debaryomyces hansenii CYC 1244.

Aspergillus westerdijkiae is one of the most relevant ochratoxin A (OTA) producing species within the Section Circumdati contaminating a number of agroproducts. The yeast Debaryomyces hansenii CYC 1244 was previously reported to be able to reduce growth and extracellular OTA produced by A. westerdijkiae. In this work, we examined several mechanisms possibly involved in this OTA reduction in in vitro experiments. OTA biosynthesis was evaluated by quantitation of expression levels of pks (polyketide synthase) and p450-B03 (cytochrome p450 monooxygenase) genes using newly developed and specific real time RT-PCR protocols. Both genes showed significant lower levels in presence of D. hansenii CYC 1244 suggesting an effect on regulation of OTA biosynthesis at transcriptional level. High levels of removal of extracellular OTA were observed by adsorption to yeast cell walls, particularly at low pH (98% at pH 3). On the contrary, no evidences were obtained of absorption of OTA into yeast cells or the production of constitutively expressed enzymes that degrade OTA by D. hansenii CYC 1244. These results described the potential of this yeast strain as a safe and efficient biocontrol agent to decrease OTA in A. westerdijkiae and two important mechanisms involved which may permit its application at different points of the food chain.

Differential detection of Debaryomyces hansenii isolated from intermediate-moisture foods by PCR-RFLP of the IGS region of rDNA.

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.