Analysis of composition and molecular characterization of mycobiota occurring on surface of cheese ripened in Dossena's mine.

Accurate identification of the fungal community spontaneously colonizing food products, aged in natural and not controlled environments, provides information about potential mycotoxin risk associated with its consumption. Autochthonous mycobiota colonizing cheese aging in Dossena mines, was investigated and characterized by two approaches: microbial isolations and metabarcoding. Microbial isolations and metabarcoding analysis were conducted on cheese samples, obtained by four batches, produced in four different seasons of the year, aged for 90 and 180 days, by five dairy farms. The two approaches, with different taxonomical resolution power, highlighted Penicillium biforme among filamentous fungi, collected from 58 out of 68 cheeses, and Debaryomyces hansenii among yeasts, as the most abundant species (31 ÷ 65%), none representing a health risk for human cheese consumption. Shannon index showed that the richness of mycobiota increases after 180 days of maturation. Beta diversity analysis highlighted significant differences in composition of mycobiota of cheese produced by different dairy farms and aged for different durations. Weak negative growth interaction between P. biforme and Aspergillus westerdijkiae by in vitro analysis was observed leading to hypothesize that a reciprocal control is possible, also affected by natural environmental conditions, possibly disadvantageous for the last species.

A PCR method to identify ochratoxin A-producing Aspergillus westerdijkiae strains on dried and aged foods.

Ochratoxins are a group of mycotoxins that frequently occur as contaminants in agricultural commodities and foods, including dry-cured meats and cheeses. The fungus Aspergillus westerdijkiae is frequently isolated from aged foods and can produce ochratoxin A (OTA). However, individual strains of the fungus can have one of two OTA production phenotypes (chemotypes): OTA production and OTA nonproduction. Monitoring and early detection of OTA-producing fungi in food are the most effective strategies to manage OTA contamination. Therefore, we examined genome sequence data from five A. westerdijkiae strains isolated from the surface of cheese from southern Italy to identify genetic markers indicative of the twoOTA chemotypes. This analysis revealed a naturally occurring deletion of the OTA regulatory gene, otaR, in an OTA-nonproducing isolate.We used this information to design a polymerase chain reaction (PCR) method that could identify A. westerdijkiae and distinguish between the two OTA chemotypes. In this method, the PCR primers were complementary to conserved sequences flanking otaR and yielded different-sized amplicons from strains with the different chemotypes. The primers did not yield ota-region-specific amplicons from other OTA-producing species. Because the method is specific to A. westerdijkiae and can distinguish between the two OTA chemotypes, it has potential to significantly improve OTA monitoring programs.

Fungal mycobiota and mycotoxin risk for traditional artisan Italian cave cheese.

Cave cheese is a surface mold-ripened variety of cheese produced also in South of Italy, exploiting fungal population naturally occurring on cave walls, as part of secondary microbiota for ripening. In this study, 148 fungal strains were isolated from 22 independent cave cheese samples, collected in 13 Italian geographical locations, mostly in Apulian area. DNA-based identification showed the presence of twenty-four fungal species in the outer part of the cheese ripened in caves. Aspergillus westerdijkiae and Penicillium biforme resulted the most frequently isolated species, followed by Penicillium roqueforti and Penicillium solitum. The 86% of cheese sample presented at least one toxigenic species and the 45% revealed the presence of ochratoxigenic species, A. westerdijkiae and A. steynii, suggesting possible mycotoxin risk during ripening stage in caves, confirmed by the presence of ochratoxin A (OTA) in the rind of 36% of samples. In conclusion, cave cheese is a susceptible product for toxigenic mold growth and in particular OTA contamination, therefore adeguate scientific tools for matching organolectic consumer expectations and complete safety of food should be developed, as well as spontaneously molded and not monitored cheeses should not be consumed to avoid mycotoxin risk.

Penicillium gravinicasei, a new species isolated from cave cheese in Apulia, Italy.

Several species of the genus Penicillium were isolated during a survey of the mycobiota of Apulian cave cheeses ripened in a cave in Gravina di Puglia, Italy. A novel species, Penicillium gravinicasei, is described in Penicillium section Cinnamopurpurea. Its taxonomic novelty was determined using a polyphasic approach, combining phenotypic, molecular (ß-tubulin, calmodulin, ITS and DNA dependent RNA polymerase) DNA sequences and mycotoxin production data. Phylogenetic analyses of the RPB2 data showed that isolates of the novel species form a clade most closely related to Penicillium cinnamopurpureum and P. parvulum with high bootstrap support. The fungus did not produce ochratoxin A, citrinin, patulin, sterigmatocystin or aflatoxin B1 on standard agar media. The novel species had a high growth rate on agar media supplemented with 5% NaCl, and could be distinguished from other Penicillium section Cinnamopurpurea species by phenotypic and molecular characteristics.