Fungal communities on alpine cheese rinds in Southern Switzerland.

The biodiversity of the mycobiota of soft cheese rinds such as Brie or Camembert has been extensively studied, but scant information is available on the fungi colonizing the rinds of cheese produced in the Southern Switzerland Alps. This study aimed at exploring the fungal communities present on rinds of cheese matured in five cellars in Southern Switzerland and to evaluate their composition with regards to temperature, relative humidity, type of cheese, as well as microenvironmental and geographic factors. We used macro- and microscopical morphology, matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry, and sequencing to characterize the fungal communities of the cheeses, and compared them with metabarcoding targeting the ITS region. Isolation by serial dilution yielded 201 isolates (39 yeasts and 162 filamentous fungi) belonging to 9 fungal species. Mucor and Penicillium were dominant, with Mucor racemosus, M. lanceolatus, P. biforme, and P. chrysogenum/rubens being the most frequent species. All but two yeast isolates were identified as Debaryomyces hansenii. Metabarcoding detected 80 fungal species. Culture work and metabarcoding produced comparable results in terms of similarity of the fungal cheese rind communities in the five cellars. Our study has shown that the mycobiota on the rinds of the cheeses studied is a comparatively species-poor community influenced by temperature, relative humidity, type of cheese, and manufacturing steps, as well as microenvironmental and possibly geographic factors.

De Respinis S ，Caminada A ，Pianta E ，Buetti-Dinh A ，Riva Scettrini P ，Petrini L ，Tonolla M ，Petrini O ... -  《-》

Mycotoxigenic and phylogenetic perspective to the yeasts and filamentous moulds in mould-matured Turkish cheese.

This study was conducted to determine the diversity of yeasts and filamentous moulds in mould-matured cheese (MMC) consumed in Turkey. Overall, 120 samples were collected from 12 different geographical locations between March 2016 and April 2017. The morphological observation was applied in combination with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and molecular analyses to determine yeasts and filamentous moulds in the cheeses. High-performance liquid chromatography (HPLC) technique was used to evaluate the ability of mycotoxins production of fungal isolates and the presence of mycotoxins in cheese samples. A total of 241 fungi (81 filamentous moulds and 160 yeast) were recovered, and Penicillium roqueforti and Debaryomyces hansenii were the most frequently isolated species in all cheese samples. The rep-PCR results indicated a high level of genetic diversity among fungal isolates, regardless of isolation source or geographical origin. Filamentous mould strains isolated from MMC were found to synthesize at least one mycotoxin (Aflatoxin B1, B2, G1 and G2, citrinine, cyclopiazonic acid, mycophenolic acid, ochratoxin A, penicillic acid and roquefortine C). Although mycotoxin producing ability was observed from all isolates, none of the cheese samples were found positive for these mycotoxins. AFM1 was detected in 8 (6.6%) MMC samples from which 2 (1.6%) were above the legal limits (0.05 μg/kg) set by the Turkish Food Codex (TFC) and European Commission (EC). In conclusion, Turkish MMCs were found to be contaminated with toxigenic fungi, so a potential public health risk, while low, exists. Therefore, the selection of nontoxigenic filamentous mould strains for cheese manufacturing and control of the ripening conditions is a critical need to ensure the quality and safety of Turkish MMC.

Ertas Onmaz N ，Gungor C ，Al S ，Dishan A ，Hizlisoy H ，Yildirim Y ，Kasap Tekinsen F ，Disli HB ，Barel M ，Karadal F ... -  《-》

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.

Anelli P ，Dall'Asta C ，Cozzi G ，Epifani F ，Carella D ，Scarpetta D ，Brasca M ，Moretti A ，Susca A ... -  《-》

The wooden shelf surface and cheese rind mutually exchange microbiota during the traditional ripening process.

The rind acts as a protective barrier for internally-bacterial ripened cheeses. Unlike surface-inoculated smear cheeses, centripetal maturation is not assumed to occur in these cheeses. This research was aimed to evaluate the microbial diversity of the wooden shelves used for the ripening of Protected Denomination of Origin (PDO) Pecorino di Filiano and Protected Geographical Indication (PGI) Canestrato di Moliterno cheeses. The microorganisms associated with the rind of these cheeses were also investigated. Both wooden shelf surfaces and cheese rinds were sampled by brushing method to collect their biofilms. Wooden shelves showed levels of total mesophilic microorganisms (TMM) between 5.6 and 7.2 log CFU/cm2, while cheese rinds between 6.1 and 7.8 log CFU/cm2. The major dairy pathogens (Salmonella spp., Listeria monocytogenes, Escherichia coli, and Staphylococcus aureus) were never detected, while mesophilic and thermophilic bacteria dominated the surfaces of all wooden shelves and cheese rinds. LAB community was represented by Enterococcus spp., Leuconostoc spp., and Marinilactibacillus spp. Among yeasts, Debaryomyces spp., Candida spp., were identified, while Aspergillus spp., and Penicillium spp., dominated the community of filamentous fungi. MiSeq Illumina analysis identified 15 phyla, 13 classes, 28 orders, 54 families, and 56 genera among bacteria. Staphylococcus spp. was identified from all wooden surfaces, with a maximum abundance of 71 %. Brevibacterium, Corynebacterium and halophilic bacteria were detected in almost all samples. Regarding fungi, wooden shelves mainly hosted Aspergillus, Penicillium and Debaryomyces hansenii, while cheese rinds especially Penicillium and D. hansenii. Alpha diversity confirmed a strict correlation between the microbiota of wooden shelves and that of cheese rinds for the majority of factories. This study confirmed that the wooden shelves used for cheese ripening are microbiologically active and represent safe systems. Furthermore, the results of this work clarified the transfer flow between wooden shelves and PDO Pecorino di Filiano and PGI Canestrato di Moliterno cheese surfaces: smear-active microorganisms are mainly transferred from wooden shelves to cheese rind, which potentially contribute to the development of the final organoleptic characteristics; meanwhile, cheeses transfer LAB that are potentially involved in defining the safety aspects of the shelves.

Busetta G ，Garofalo G ，Claps S ，Sardina MT ，Franciosi E ，Alfonzo A ，Francesca N ，Moschetti G ，Settanni L ，Gaglio R ... -  《-》

Cultivation-independent analysis of microbial communities on Austrian raw milk hard cheese rinds.

"Vorarlberger Bergkäse" (VB) is an Austrian artisanal hard cheese produced from raw cow's milk. The composition of its rind microbiota and the changes in the microbial communities during ripening have not previously been investigated. This study used 16S and 18S rRNA gene cloning and sequencing to characterize the bacterial and fungal communities of seven pooled cheese rind samples taken in seven different ripening cellars of three Austrian dairy facilities. A total of 408 clones for 16S and 322 clones for 18S rRNA gene libraries were used for taxonomic classification, revealing 39 bacterial and seven fungal operational taxonomic units (OTUs). Bacterial OTUs belonged to four different phyla. Most OTUs were affiliated to genera often found in cheese, including high numbers of coryneforms. The most abundant OTU from 16S rRNA gene libraries showed highest similarity to Halomonas. Young cheese rinds were dominated by Actinobacteria or Proteobacteria, particularly by Halomonas and Brevibacterium aurantiacum, while Staphyloccocus equorum was most abundant in old cheeses. The most abundant 18S rRNA OTU had highest similarity to the filamentous fungus Scopulariopsis brevicaulis. Pairwise correlation analyses revealed putative co-occurrences between a number of OTUs. It was possible to discriminate the different cheese rind microbiota at the community-level by facility affiliation and ripening time. This work provides insights into the microbial composition of VB cheese rinds and might allow the processing- and ripening conditions to be improved to enhance the quality of the product.

Schornsteiner E ，Mann E ，Bereuter O ，Wagner M ，Schmitz-Esser S ... -  《-》