Safety evaluation of the food enzyme triacylglycerol lipase from the non-genetically modified Penicillium caseifulvum strain AE-LRF.

The food enzyme triacylglycerol lipase (triacylglycerol acylhydrolase; EC 3.1.1.3) is produced with the non-genetically modified Penicillium caseifulvum strain AE-LRF by Amano Enzyme Inc. The food enzyme was free from viable cells of the production organism. It is intended to be used in four food manufacturing processes. Dietary exposure to the food enzyme-total organic solids (TOS) was estimated to be up to 0.013 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 69 mg TOS/kg bw per day, the highest dose tested, which when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 5308. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and no match was found. However, the Panel noted that traces of ■■■■■, used in the manufacture of the triacylglycerol lipase, may be found in the food enzyme. The Panel considered that the risk of allergic reactions upon dietary exposure could not be excluded, particularly in individuals sensitised to fish. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns, under the intended conditions of use.

PrlaeA Affects the Production of Roquefortine C, Mycophenolic Acid, and Andrastin A in Penicillium roqueforti, but It Has Little Impact on Asexual Development.

The regulation of fungal specialized metabolism is a complex process involving various regulators. Among these regulators, LaeA, a methyltransferase protein originally discovered in Aspergillus spp., plays a crucial role. Although the role of LaeA in specialized metabolism has been studied in different fungi, its function in Penicillium roqueforti remains unknown. In this study, we employed CRISPR-Cas9 technology to disrupt the laeA gene in P. roqueforti (PrlaeA) aiming to investigate its impact on the production of the specialized metabolites roquefortine C, mycophenolic acid, and andrastin A, as well as on asexual development, because they are processes that occur in the same temporal stages within the physiology of the fungus. Our results demonstrate a substantial reduction in the production of the three metabolites upon disruption of PrlaeA, suggesting a positive regulatory role of LaeA in their biosynthesis. These findings were further supported by qRT-PCR analysis, which revealed significant downregulation in the expression of genes associated with the biosynthetic gene clusters (BGCs) responsible for producing roquefortine C, mycophenolic acid, and andrastin A in the ΔPrlaeA strains compared with the wild-type P. roqueforti. Regarding asexual development, the disruption of PrlaeA led to a slight decrease in colony growth rate, while conidiation and conidial germination remained unaffected. Taken together, our results suggest that LaeA positively regulates the expression of the analyzed BGCs and the production of their corresponding metabolites in P. roqueforti, but it has little impact on asexual development.

Antifungal activity of star anise extract against Penicillium roqueforti and Aspergillus niger for bread shelf life.

Because star anise is underutilized in the baking sector and the antifungal targets are unclear, this study aimed to investigate the antifungal effect and mechanism of star anise extract (SAE) on spoilage fungi in bread. SAE was prepared by ethanol extraction and 31 substances were identified by GC-MS, among which trans-anethole (62.62%), estragole (7.82%) and linalool (4.66%) were the major components. The antifungal activity of SAE and the three main components against Penicillium roqueforti and Aspergillus niger were determined by using the Oxford cup method and the sesqui-dilution method. The inhibition zones were 9.88 mm and 15.09 mm, while the minimum inhibitory concentrations were 125.00 µL/mL and 31.25 µL/mL. Trans-anethole and estragole both showed antifungal activity against Penicillium roqueforti and Aspergillus niger, while linalool only showed antifungal activity against Aspergillus niger. Propidium iodide and fluorescein diacetate staining analysis, leakage of cellular components (nucleic acids and proteins) and rise in ergosterol content indicated that SAE disrupted the integrity and permeability of the cell membrane. Malondialdehyde was increased after SAE treatment, indicating that SAE caused lipid peroxidation in the cell membrane, further confirming that it disrupted the cell membrane. At the same time, SAE interacted with membrane proteins and altered their conformation, resulting in cell membrane dysfunction. Finally, the shelf life test showed that SAE extended the shelf life of the bread by up to 6 days. In general, this study highlights the antifungal effect of SAE against Penicillium roqueforti and Aspergillus niger, which indicated that SAE can be used as an antifungal agent to extend the shelf life of bread.

New secondary metabolites with cytotoxicity from fungus Penicillium roqueforti.

Two novel compounds including a cyclohelminthol type polyketide (namely oxaleimide K, 1) and a maleimide derivative (namely peniroquefortine A, 2), and a new natural product (namely 2-(acetylamino)-N-[(1E)-2-phenylethenyl]-acetamide, 3), together with four known compounds (4-7), were isolated and identified from fungus Penicillium roqueforti, which was separated from the root soil of Hypericum beanii N. Robson collected from the Shennongjia Forestry District, Hubei Province. Their structures including absolute configurations were mainly established by the NMR spectroscopy analyses and single-crystal X-ray diffraction experiment. Compound 1 represents the second example of a cyclohelminthol type polyketide, which features a rare 6/6/5/5 tetracyclic system and a branched aliphatic chain containing a terminal olefin (oct-1-en-3-yl) moiety, and compound 2 possesses an unprecedented carbon skeleton that is uniquely defined by a maleimide moiety linked to the respective 4-methylene-2-(3-methylbut-2-en-1-yl)-phenol and para-substituted aromatic moieties via the carbon-carbon bonds. Remarkably, the absolute configuration of a cyclohelminthol type polyketide as exemplified by compound 1 is determined by the single-crystal diffraction analysis for the first time, highlighting an E-configuration for the linkage of a succinimide moiety and a tetrahydrofuran moiety for 1 rather than a Z-configuration as previously reported in the biosynthesis study, which gives a new insight into the structural elucidation of this category of polyketides. Additionally, compound 1 exhibited significant cytotoxic activity against multiple tumor cells, especially against the Farage and SU-DHL-2 cells (IC50 < 20 µM, 48 h). Further mechanism study revealed that compound 1 significantly induced cell cycle arrest in Farage and SU-DHL-2 cells by causing abnormal ROS level and triggering oxidative stress.

Ten undescribed eremophilane and guaiane sesquiterpenes from Penicillium roqueforti.

Nine undescribed eremophilane sesquiterpenes, one undescribed guaiane sesquiterpene, along with ten known analogues were isolated and identified from fungus Penicillium roqueforti, which was separated from the root soil of Hypericum beanii N. Robson collected from the Shennongjia Forestry District, Hubei Province. Their structures were elucidated on the basis of various spectroscopic analyses, mainly including NMR and HRESIMS data, 13C NMR calculations with DP4+ probability analyses, ECD calculations, and single-crystal X-ray diffraction experiments. Furthermore, all twenty compounds were evaluated for the in vitro cytotoxic activities against seven human tumor cell lines, and the result suggested that 14-hydroxymethylene-1(10)-ene-epi-guaidiol A exhibited considerable cytotoxic activity against the Farage (IC50 < 10 µM, 48 h), SU-DHL-2, and HL-60 cells. Further mechanism study demonstrated that 14-hydroxymethylene-1(10)-ene-epi-guaidiol A could significantly promote apoptosis by inhibiting tumor cell respiration and decreasing intracellular ROS levels, thereby inducing S-phase blockade in tumor cells.

Secondary Metabolites Produced by the Blue-Cheese Ripening Mold Penicillium roqueforti; Biosynthesis and Regulation Mechanisms.

Filamentous fungi are an important source of natural products. The mold Penicillium roqueforti, which is well-known for being responsible for the characteristic texture, blue-green spots, and aroma of the so-called blue-veined cheeses (French Bleu, Roquefort, Gorgonzola, Stilton, Cabrales, and Valdeón, among others), is able to synthesize different secondary metabolites, including andrastins and mycophenolic acid, as well as several mycotoxins, such as Roquefortines C and D, PR-toxin and eremofortins, Isofumigaclavines A and B, festuclavine, and Annullatins D and F. This review provides a detailed description of the biosynthetic gene clusters and pathways of the main secondary metabolites produced by P. roqueforti, as well as an overview of the regulatory mechanisms controlling secondary metabolism in this filamentous fungus.

The potential of UVC decontamination to prolong shelf-life of par-baked bread.

The effect of UVC (254 nm) treatment on the mould-free shelf-life of par-baked wholemeal, rye and six-grain bread was examined. Currently, these breads are par-baked, wrapped in high-density polyethylene (HDPE)-foil and transported or stored at room temperature for a couple of days before being full-baked and sold/consumed. Generally, after five days, these breads show signs of mould spoilage. A shelf-life increase in one or more days would already offer immense economical and logistic benefits for the baker or retailer. In this study, the parameters fluence rate (irradiation intensity), fluence (UV dose), distance to the UV-lamp (DTL) and number of layers of a common wrapping HDPE-foil (20 µm) were diversified. The breads were subjected to a UVC treatment (0-2502 mJ/cm²), packed and stored at room temperature for a period of 15 days (21.5 ± 0.8°C). Similar as for the breads, agar plates with mould spores of Aspergillus niger, Aspergillus montevidensis and Penicillium roqueforti were UVC treated (0-1664 mJ/cm²) and checked daily for visible mould growth during 15 days (25°C). Aspergillus niger showed the strongest resistance towards UVC, a fluence of 800 mJ/cm² was needed to inhibit growth during 15 days of storage, whereas for P. roqueforti and A. montevidensis, respectively, UV levels of 291 and 133 mJ/cm² were found sufficient. Furthermore, the shelf-life of wholemeal, rye and six-grain bread can be prolonged from 5 to 6, 8 and 9 days, respectively, using 2502 mJ/cm². The effect of higher UVC dosage on shelf-life reached a maximal level and was strongly impacted by the wide spread on data of mould-free shelf-life. The main factors influencing the potential of UV decontamination were the rough bread surface, differences in DTL, the possibility of post-contamination and UV permeability of packaging materials.

Unlocking the biosynthetic potential of Penicillium roqueforti for hyperproduction of the immunosuppressant mycophenolic acid: Gamma radiation mutagenesis and response surface optimization of fermentation medium.

Based on the broad clinical utility of the immunosuppressant mycophenolic acid (MPA), this article aims to intensify the biosynthetic potential of Penicillium roqueforti for more effective hyperproduction of the drug. Several mutants were generated from irradiation mutagenesis and screened. Two strains (GM1013 and GM1093) presented an elevated MPA productivity with significant yield constancy over 10 subsequent generations. By investigating the effect of some phosphorous sources and mineral salts on MPA production by the two mutants, KH2 PO4 and FeSO4 ·7H2 O were most preferred by the two mutants for higher MPA production rates. Statistics-dependent experimental designs were also employed for optimizing medium components for maximum MPA production. Medium components were primarily screened using the Plackett-Burman model to demonstrate the most important components that most significantly affect MPA production. The concentrations of these significant components were then optimized through a central composite rotatable model. In conclusion, gamma-radiation mutation and response surface optimization resulted in a promising MPA productivity by P. roqueforti GM1013. To our knowledge, the MPA-yield achieved in this study (2933.32 mg L-1 ) is the highest reported by academic laboratories from P. roqueforti cultures, which could be of economic value for a prospective large industrialized application.

Cyclopiazonic acid in soft-ripened and blue cheeses marketed in the USA.

Strains of Penicillium camemberti and P. roqueforti are used in the production of soft-ripened and blue-veined cheeses. However, some strains can produce toxic secondary metabolites (mycotoxins), including α-cyclopiazonic acid (CPA), a neurotoxin. Data on the levels of CPA in cheeses marketed in the USA are extremely limited. An enzyme-linked immunosorbent assay was adapted for measuring CPA in soft-ripened and blue-veined cheeses. Recoveries from cheese curds were 103 ± 27% (n = 30). A total of 254 samples of soft-ripened, blue and miscellaneous cheeses were examined. CPA was detected in 36/79 (45.6%) of soft-ripened cheeses and in 41/168 (24.4%) of blue-veined cheeses. Median levels in positive samples were 48.5 µg/kg and 30 µg/kg, respectively. The highest levels found were 3,820 µg/kg (in a Brie), 1,250 µg/kg (in a blue) and 7,900 µg/kg (in a Monte Enebro). The implication of such exposures is unknown, as a consensus on acceptable intake remains to be established.

Intraspecific variability in heat resistance of fungal conidia.

Microbial species are inherently variable, which is reflected in intraspecies genotypic and phenotypic differences. Strain-to-strain variation gives rise to variability in stress resistance and plays a crucial role in food safety and food quality. Here, strain variability in heat resistance of asexual spores (conidia) of the fungal species Aspergillus niger, Penicillium roqueforti and Paecilomyces variotii was quantified and compared to bacterial variability found in the literature. After heat treatment, a 5.4- to 8.6-fold difference in inactivation rate was found between individual strains within each species, while the strain variability of the three fungal species was not statistically different. We evaluated whether the degree of intraspecies variability is uniform, not only within the fungal kingdom, but also amongst different bacterial species. Comparison with three spore-forming bacteria and two non-spore-forming bacteria revealed that the variability of the different species was indeed in the same order of magnitude, which hints to a microbial signature of variation that exceeds kingdom boundaries.

Lacticaseibacillus rhamnosus C1 effectively inhibits Penicillium roqueforti: Effects of antimycotic culture supernatant on toxin synthesis and corresponding gene expression.

Recently, consumers are increasingly concerned about the contamination of food by molds and the addition of chemical preservatives. As natural and beneficial bacteria, probiotics are a prospective alternative in food conservation because of their antimycotic activities, although the mechanism has not been explained fully at the level of metabolites. This study aimed at investigating the antifungal activities and their mechanisms of five potential probiotic strains (Lacticaseibacillus rhamnosus C1, Lacticaseibacillus casei M8, Lactobacillus amylolyticus L6, Schleiferilactobacillus harbinensis M1, and Limosilactobacillus fermentum M4) against Penicillium roqueforti, the common type of mold growth on the bread. Results showed that C1 emerged the strongest effectiveness at blocking mycelium growth, damaging the morphology of hyphae and microconidia, decreasing DNA content and interfering in the synthesis of the fungal toxins patulin, roquefortine C and PR-toxin, as well as downregulating the expression of key genes associated with the toxin biosynthesis pathways. Further metabonomic investigation revealed that protocatechuic acid with the minimum inhibitory concentration of 0.40 mg/mL, may be most likely responsible for positively correlated with the antimycotic effects of C1. Thus, C1 is expected to be both a potentially greatly efficient and environmental antimycotic for controlling P. roqueforti contamination in foods.

Intraspecific variability in cardinal growth temperatures and water activities within a large diversity of Penicillium roqueforti strains.

Different strains of a given fungal species may display heterogeneous growth behavior in response to environmental factors. In predictive mycology, the consideration of such variability during data collection could improve the robustness of predictive models. Among food-borne fungi, Penicillium roqueforti is a major food spoiler species which is also used as a ripening culture for blue cheese manufacturing. In the present study, we investigated the intraspecific variability of cardinal temperatures and water activities (aw), namely, minimal (Tmin and awmin), optimal (Topt and awopt) and maximal (Tmax) temperatures and/or aw estimated with the cardinal model for radial growth, of 29 Penicillium roqueforti strains belonging to 3 genetically distinct populations. The mean values of cardinal temperatures and aw for radial growth varied significantly across the tested strains, except for Tmax which was constant. In addition, the relationship between the intraspecific variability of the biological response to temperature and aw and putative genetic populations (based on microsatellite markers) within the selected P. roqueforti strains was investigated. Even though no clear relationship was identified between growth parameters and ecological characteristics, PCA confirmed that certain strains had marginal growth response to temperature or aw. Overall, the present data support the idea that a better knowledge of the response to abiotic factors such as temperature and aw at an intraspecific level would be useful to model fungal growth in predictive mycology approaches.

Preservation stress resistance of melanin deficient conidia from Paecilomyces variotii and Penicillium roqueforti mutants generated via CRISPR/Cas9 genome editing.

BACKGROUND: The filamentous fungi Paecilomyces variotii and Penicillium roqueforti are prevalent food spoilers and are of interest as potential future cell factories. A functional CRISPR/Cas9 genome editing system would be beneficial for biotechnological advances as well as future (genetic) research in P. variotii and P. roqueforti. RESULTS: Here we describe the successful implementation of an efficient AMA1-based CRISPR/Cas9 genome editing system developed for Aspergillus niger in P. variotii and P. roqueforti in order to create melanin deficient strains. Additionally, kusA- mutant strains with a disrupted non-homologous end-joining repair mechanism were created to further optimize and facilitate efficient genome editing in these species. The effect of melanin on the resistance of conidia against the food preservation stressors heat and UV-C radiation was assessed by comparing wild-type and melanin deficient mutant conidia. CONCLUSIONS: Our findings show the successful use of CRISPR/Cas9 genome editing and its high efficiency in P. variotii and P. roqueforti in both wild-type strains as well as kusA- mutant background strains. Additionally, we observed that melanin deficient conidia of three food spoiling fungi were not altered in their heat resistance. However, melanin deficient conidia had increased sensitivity towards UV-C radiation.

Thermoplastic starch and bioactive chitosan sub-microparticle biocomposites: Antifungal and chemico-physical properties of the films.

In this study, chitosan (C) tripolyphosphate (T) sub-micro particles containing ungeremine (CTUn), an alkaloid particularly active against Penicilliumroqueforti, a fungus responsible of the bakery products deterioration, were prepared through external gelation crosslinking process. The particles were included in a thermoplastic starch based polymer Mater-Bi (MBi), and MBi/CTUn bioactive biocomposites were obtained. The films showed bioactivity against P. roqueforti. In particular, the bioassays were performed on films with different concentration of CTUn and at different pH values. CTUn particles influenced MBi crystallization (DSC analysis) and promoted thermal degradation of MBi starch component (TGA). Morphological analysis confirmed even distribution of sub-micro particles into the polymeric matrix. Water permeability slightly increased, as expected, whereas oxygen permeability decreased. Tensile tests showed CTUN sub-microparticles improved rigidity and tensile strength of the films at the expense of ductility. Finally, MBi/CTUn biocomposites evidenced interesting performances potentially exploitablein bioactive bakery based food packaging materials.

Functional Metabolome Analysis of Penicillium roqueforti by Means of Differential Off-Line LC-NMR.

UPLC-TOF/MS profiling, followed by the recently reported differential off-line LC-NMR (DOLC-NMR) and quantitative 1H NMR spectroscopy (qHNMR), led to the differential qualitative analysis and accurate quantitation of l-tryptophan-induced metabolome alterations of Penicillium roqueforti, which is typically used in making blue-mold cheese. Among the 24 metabolites identified, two tetrapeptides, namely, d-Phe-l-Val-d-Val-l-Tyr and d-Phe-l-Val-d-Val-l-Phe, as well as cis-bis(methylthio)silvatin, are reported for the first time as metabolites of P. roqueforti. Antimicrobial activity tests showed strong effects of the catabolic l-tryptophan metabolites 3-hydroxyanthranilic acid, anthranilic acid, and 3-indolacetic acid against Saccharomyces cerevisiae, with IC50 values between 15.6 and 24.0 µg/mL, while roquefortine C and cis-bis(methylthio)silvatin inhibited the growth of Gram-negative Escherichia coli and Gram-positive Bacillus subtilis with IC50 values between 30.0 and 62.5 µg/mL.

Effect of the solid state fermentation of cocoa shell on the secondary metabolites, antioxidant activity, and fatty acids.

During cocoa (Theobroma cacao L.) processing, the accumulated cocoa shell can be used for bioconversion to obtain valuable compounds. Here, we evaluate the effect of solid-state fermentation of cacao flour with Penicillium roqueforti on secondary metabolite composition, phenol, carotenoid, anthocyanin, flavonol, and fatty acids contents, and antioxidant activity. We found that the total concentrations of anthocyanins and flavonols did not change significantly after fermentation and the phenolic compound and total carotenoid concentrations were higher. The fermentation process produced an increase in saponin concentration and antioxidant activity, as well as significant changes in the levels of oleic, linoleic, gamma-linolenic, and saturated fatty acids. Based on our findings, we propose that the reuse of food residues through solid state fermentation is viable and useful.

The developmental regulator Pcz1 affects the production of secondary metabolites in the filamentous fungus Penicillium roqueforti.

Penicillium roqueforti is used in the production of several kinds of ripened blue-veined cheeses. In addition, this fungus produces interesting secondary metabolites such as roquefortine C, andrastin A and mycophenolic acid. To date, there is scarce information concerning the regulation of the production of these secondary metabolites. Recently, the gene named pcz1 (Penicillium C6 zinc domain protein 1) was described in P. roqueforti, which encodes for a Zn(II)2Cys6 protein that controls growth and developmental processes in this fungus. However, its effect on secondary metabolism is currently unknown. In this work, we have analyzed how the overexpression and down-regulation of pcz1 affect the production of roquefortine C, andrastin A and mycophenolic acid in P. roqueforti. The three metabolites were drastically reduced in the pcz1 down-regulated strains. However, when pcz1 was overexpressed, only mycophenolic acid was overproduced while, on the contrary, levels of roquefortine C and andrastin A were diminished. Importantly, these results match the expression pattern of key genes involved in the biosynthesis of these metabolites. Taken together, our results suggest that Pcz1 plays a key role in regulating secondary metabolism in the fungus Penicillium roqueforti.

PR Toxin - Biosynthesis, Genetic Regulation, Toxicological Potential, Prevention and Control Measures: Overview and Challenges.

Out of the various mycotoxigenic food and feed contaminant, the fungal species belonging to Penicillium genera, particularly Penicillium roqueforti is of great economic importance, and well known for its crucial role in the manufacturing of Roquefort and Gorgonzola cheese. The mycotoxicosis effect of this mold is due to secretion of several metabolites, of which PR toxin is of considerable importance, with regard to food quality and safety challenges issues. The food products and silages enriched with PR toxin could lead into damage to vital internal organs, gastrointestinal perturbations, carcinogenicity, immunotoxicity, necrosis, and enzyme inhibition. Moreover, it also has the significant mutagenic potential to disrupt/alter the crucial processes like DNA replication, transcription, and translation at the molecular level. The high genetic diversities in between the various strains of P. roqueforti persuaded their nominations with Protected Geographical Indication (PGI), accordingly to the cheese type, they have been employed. Recently, the biosynthetic mechanism and toxicogenetic studies unraveled the role of ari1 and prx gene clusters that cross-talk with the synthesis of other metabolites or involve other cross-regulatory pathways to negatively regulate/inhibit the other biosynthetic route targeted for production of a strain-specific metabolites. Interestingly, the chemical conversion that imparts toxic properties to PR toxin is the substitution/oxidation of functional hydroxyl group (-OH) to aldehyde group (-CHO). The rapid conversion of PR toxin to the other derivatives such as PR imine, PR amide, and PR acid, based on conditions available reflects their unstability and degradative aspects. Since the PR toxin-induced toxicity could not be eliminated safely, the assessment of dose-response and other pharmacological aspects for its safe consumption is indispensable. The present review describes the natural occurrences, diversity, biosynthesis, genetics, toxicological aspects, control and prevention strategies, and other management aspects of PR toxin with paying special attention on economic impacts with intended legislations for avoiding PR toxin contamination with respect to food security and other biosafety purposes.

Antimould microbial and plant metabolites with potential use in intelligent food packaging.

Moulds food infestation is a heavy dangerous problem for human health and also could generate heavy economic losses. The intelligent packaging using eco-friendly biodegradable biofilm incorporating bioactive natural safe compounds represents a new frontier. This manuscript reports the inhibitory activity of 12 bacterial, fungal and plant metabolites against Penicillium roqueforti and Aspergillus niger. Among them α-costic acid and ungeremine (3 and 12) are the most promising as potential biofungicide against both fungal strains. They inhibited fungal growth by more than 60% respect to the control at 72 h and this activity persisted also at 96 h. Ungeremine showed MIC90 lower than 0.003 mg/mL after 48 h of incubation and of 0.025 mg/mL at 72 h against P. roqueforti. The MIC90 value for A. niger was 0.2 mg/mL at 48 h for both compounds. The α-costic acid showed generally MIC values at 48 and 72 h higher than ungeremine.

A Case of Fungus Ball-Type Maxillary Sinusitis Due to Penicillium Roqueforti.

The Penicillium genera, encompassing about 225 different species of fungi, are naturally present in the environment. These genera are poorly linked to human disease, except for Penicillium marneffei causing septicemia in immunocompromised hosts. Thus, Penicillium species recovered from respiratory tract samples are often considered as inhaled contaminants in the clinical laboratory. However, we report here a case of fungal maxillary sinusitis due to Penicillium roqueforti diagnosed in a 40-year-old female, a teacher, complaining of moderate pain for months in the maxillary sinus and chronic posterior rhinorrhea. CT scanner and MRI enabled a preliminary diagnosis of left maxillary fungus ball-type sinusitis with calcified material seen on CT and marked very low signal in T2 weighted images seen on MRI. Anatomopathological and mycological examination of sinusal content showed septate hyphae. Direct sequencing of the sinusal content revealed P. roqueforti. P. roqueforti has been traditionally used in France for more than 200 years for cheese ripening. However, to our knowledge, this ascomycetous fungus has very rarely been associated in the literature with human disease. P. roqueforti is associated only with cheese worker's lung, a hypersensitivity pneumonitis affecting employees in blue cheese factories. Other species in the Penicillium genus are reported to cause various disorders such as invasive infection, superficial infection or allergic diseases. P. roqueforti has never previously been reported as a cause of human infection. Thus, we report the first case of fungus ball due to P. roqueforti in an immunocompetent patient.