Valérie Vasseur

Filamentous Fungi and Mycotoxins in Cheese: A Review

Important fungi growing on cheese include Penicillium, Aspergillus, Cladosporium, Geotrichum, Mucor, and Trichoderma. For some cheeses, such as Camembert, Roquefort, molds are intentionally added. However, some contaminating or technological fungal species have the potential to produce undesirable metabolites such as mycotoxins. The most hazardous mycotoxins found in cheese, ochratoxin A and aflatoxin M1, are produced by unwanted fungal species either via direct cheese contamination or indirect milk contamination (animal feed contamination), respectively. To date, no human food poisoning cases have been associated with contaminated cheese consumption. However, although some studies state that cheese is an unfavorable matrix for mycotoxin production; these metabolites are actually detected in cheeses at various concentrations. In this context, questions can be raised concerning mycotoxin production in cheese, the biotic and abiotic factors influencing their production, mycotoxin relative toxicity as well as the methods used for detection and quantification. This review emphasizes future challenges that need to be addressed by the scientific community, fungal culture manufacturers, and artisanal and industrial cheese producers.

Quantification of Penicillium camemberti and P. roqueforti mycelium by real-time PCR to assess their growth dynamics during ripening cheese.

Real-time PCR has been applied to quantify mycelium of Penicillium camemberti and Penicillium roqueforti during ripening of model cheese curd and surface mould-ripened cheeses. Total fungal DNA was first validated as an indicator of mycelial biomass in pure liquid culture and then in model curds at different stages of ripening. To imitate cheese matrix effects, DNA was extracted from curd mixed with known amounts of fresh mycelium of P. camemberti or P. roqueforti and was used as biomass standards for further quantitative real-time PCR. Mycelial mass per cheese (mg/g) was then directly obtained from fluorescence data. In model cheese curd, mycelial mass of P. camemberti increased from 2.8 at d4 to 596 mg/g at d11 whereas P. roqueforti increased from 0.3 to 6.3 mg/g during the same period. P. camemberti showed a fast development in Coulommiers from d2 to d9 (66 to 119 mg/g) and a 100-fold increase in Carré (0.85 to 85 mg/g). While mycelial biomass reached a maximum at d9 in Coulommiers, it still developed in Carré until d45. For the first time, cheese manufacturers have a powerful technique to monitor mycelial growth dynamics of their fungal cultures, which represents an important step for controlling cheese making.

Physiological traits of Penicillium glabrum strain LCP 08.5568, a filamentous fungus isolated from bottled aromatised mineral water

Penicillium glabrum is a ubiquitous fungus distributed world wide. This fungus is a frequent contaminant in the food manufacturing industry. Environmental factors such as temperature, water activity and pH have a great influence on fungal development. In this study, a strain of P. glabrum referenced to as LCP 08.5568, has been isolated from a bottle of aromatized mineral water. The effects of temperature, a(w) and pH on radial growth rate were assessed on Czapeck Yeast Agar (CYA) medium. Models derived from the cardinal model with inflection [Rosso et al., 1993 An unexpected correlation between cardinal temperatures of microbial growth highlighted by a new model. J. Theor. Bio. 162, 447-463.] were used to fit the experimental data and determine for each factor, the cardinal parameters (minimum, optimum and maximum). Precise characterisation of the growth conditions for such a fungal contaminant, has an evident interest to understand and to prevent spoilage of food products.

Temperature, water activity and pH during conidia production affect the physiological state and germination time of Penicillium species

Conidial germination and mycelial growth are generally studied with conidia produced under optimal conditions to increase conidial yield. Nonetheless, the physiological state of such conidia most likely differs from those involved in spoilage of naturally contaminated food. The present study aimed at investigating the impact of temperature, pH and water activity (aw) during production of conidia on the germination parameters and compatible solutes of conidia of Penicillium roqueforti and Penicillium expansum. Low temperature (5°C) and reduced aw (0.900 aw) during sporulation significantly reduced conidial germination times whereas the pH of the sporulation medium only had a slight effect at the tested values (2.5, 8.0). Conidia of P. roqueforti produced at 5°C germinated up to 45h earlier than those produced at 20°C. Conidia of P. roqueforti and P. expansum produced at 0.900 aw germinated respectively up to 8h and 3h earlier than conidia produced at 0.980 aw. Furthermore, trehalose and mannitol assessments suggested that earlier germination might be related to delayed conidial maturation even though no ultra-structural modifications were observed by transmission electron microscopy. Taken together, these results highlight the importance of considering environmental conditions during sporulation in mycological studies. The physiological state of fungal conidia should be taken into account to design challenge tests or predictive mycology studies. This knowledge may also be of interest to improve the germination capacity of fungal cultures commonly used in fermented foods.

Modelling the effect of temperature, water activity and pH on the growth of Serpula lacrymans

Aims:  To predict the risk factors for building infestation by Serpula lacrymans, which is one of the most destructive fungi causing timber decay in buildings.

Application of capillary electrophoresis single-stranded conformation polymorphism (CE-SSCP) analysis for identification of fungal communities in cheese.

As major contributors of the ripening process, yeasts and filamentous fungi play a fundamental role in cheese-making. Still, there is no rapid and affordable identification method available for both yeasts and filamentous fungi encountered in cheeses. In the present study, we developed a method based on CE-SSCP analysis of nuclear ribosomal DNA ITS amplicons, along with a species pattern database comprising 37 fungal species. By combining analyses of the ITS1 and ITS2 conformers, 25 out of 37 species were discriminated using CE-SSCP analysis. This reproducible and sensitive method was applied to determine the fungal community composition of 36 cheeses including blue-veined, pressed-cooked, pressed-uncooked, red-smear and surface-mould ripened cheeses. Overall, each cheese contained between 1 and 6 fungal species and 23 different species of fungi were detected including 8 yeast species, 9 filamentous species and 6 unidentified species. Comparison of the fungal diversity obtained after cloning and sequencing (rDNA ITS) versus CE-SSCP for 8 cheeses showed that CE-SSCP was at least as exhaustive as cloning and sequencing of thirty clones per cheese. In conclusion, this CE-SSCP method was an effective tool to identify the fungi present in various cheese varieties and may be of interest for the cheese industry to rapidly describe the composition of cheese fungal communities.

Application of denaturing high-performance liquid chromatography (DHPLC) for yeasts identification in red smear cheese surfaces

Aims:  To evaluate and optimize the use of denaturing high‐performance liquid chromatography (DHPLC) for yeasts identification in red smear cheese surfaces.

Cladosporium lebrasiae, a new fungal species isolated from milk bread rolls in France.

The fungal genus Cladosporium (Cladosporiaceae, Dothideomycetes) is composed of a large number of species, which can roughly be divided into three main species complexes: Cladosporium cladosporioides, Cladosporium herbarum, and Cladosporium sphaerospermum. The aim of this study was to characterize strains isolated from contaminated milk bread rolls by phenotypic and genotypic analyses. Using multilocus data from the internal transcribed spacer ribosomal DNA (rDNA), partial translation elongation factor 1-α, actin, and beta-tubulin gene sequences along with Fourier-transform infrared (FTIR) spectroscopy and morphological observations, three isolates were identified as a new species in the C. sphaerospermum species complex. This novel species, described here as Cladosporium lebrasiae, is phylogenetically and morphologically distinct from other species in this complex.

Modeling the Effect of Modified Atmospheres on Conidial Germination of Fungi from Dairy Foods

Modified atmosphere packaging (MAP) is commonly applied to extend food shelf-life. Despite growth of a wide variety of fungal contaminants has been previously studied in relation to modified-atmospheres, few studies aimed at quantifying the effects of dioxygen (O2) and carbon dioxide (CO2) partial pressures on conidial germination in solid agar medium. In the present study, an original culture method was developed, allowing microscopic monitoring of conidial germination under modified-atmospheres in static conditions. An asymmetric model was utilized to describe germination kinetics of Paecilomyces niveus, Mucor lanceolatus, Penicillium brevicompactum, Penicillium expansum, and Penicillium roquefoti, using two main parameters, i.e., median germination time (τ) and maximum germination percentage (Pmax). These two parameters were subsequently modeled as a function of O2 partial pressure ranging from 0 to 21% and CO2 partial pressure ranging from 0.03 to 70% (8 tested levels for both O2 and CO2). Modified atmospheres with residual O2 or CO2 partial pressures below 1% and up to 70%, respectively, were not sufficient to totally inhibit conidial germination,. However, O2 levels < 1% or CO2 levels > 20% significantly increased τ and/or reduced Pmax, depending on the fungal species. Overall, the present method and results are of interest for predictive mycology applied to fungal spoilage of MAP food products.

Development and application of MALDI-TOF MS for identification of food spoilage fungi

Filamentous fungi are frequently involved in food spoilage and cause important food losses and substantial economic damage. Their rapid and accurate identification is a key step to better manage food safety and quality. In recent years, MALDI-TOF MS has emerged as a powerful tool to identify microorganisms and has successfully been applied to the identification of filamentous fungi especially in the clinical context. The aim of this study was to implement a spectral database representative of food spoilage molds. To this end, after application of a standardized extraction protocol, 6477 spectra were acquired from 618 fungal strains belonging to 136 species and integrated in the VITEK MS database. The performances of this database were then evaluated by cross-validation and ∼95% of correct identification to the species level was achieved, independently of the cultivation medium and incubation time. The database was also challenged with external isolates belonging to 52 species claimed in the database and 90% were correctly identified to the species level. To our best knowledge, this is the most comprehensive database of food-relevant filamentous fungi developed to date. This study demonstrates that MALDI-TOF MS could be an alternative to conventional techniques for the rapid and reliable identification of spoilage fungi in food and industrial environments.