Search Results (22)

Sixteen long-ripened, high-quality Cabrales cheeses from independent producers underwent a comprehensive biochemical and microbiological characterisation. Significant variations in total microbial counts and specific microbial groups were observed among the cheeses. A metataxonomic analysis identified 249 prokaryotic amplicon sequence variants (ASVs) and 99 eukaryotic ASVs, respectively, which were classified into 52 prokaryotic and 43 eukaryotic species. The predominant species included bacteria of the genera Tetragenococcus, Lactococcus (of which Lactococcus lactis was used as a starter), and Staphylococcus, followed by Brevibacterium and Corynebacterium species. The starter mould Penicillium roqueforti was highly abundant in all cheeses; Debaryomyces hansenii, Geotrichum candidum, and Kluyveromyces spp. constituted the subdominant fungal populations. Glutamic acid (≈20 mg g⁻¹) was the most abundant free amino acid in all samples, followed by lysine, leucine, and valine (≈10–13 mg g⁻¹). Moderate-to-high amounts of the biogenic amines tyramine and ornithine were detected. A large variation between cheeses of the main organic acids (lactic, acetic, or butyric) was detected. Differences between samples were also observed for the majority volatile compounds, which included organic acids, alcohols, esters, and ketones. Positive and negative correlations between bacterial and fungal species were detected, as well as between microbial populations and key biochemical markers. Among the latter, Tetragenococcus halophilus correlated positively with ethyl caprylate and hexanoic acid, and Loigolactobacillus rennini correlated positively with γ-aminobutyric acid. Conversely, Staphylococcus equorum showed a strong negative correlation with ethyl caprylate and capric acid. These microbial and biochemical insights enabled us to propose a microbiota-based starter culture comprising prokaryotic and eukaryotic components to enhance Cabrales cheese quality. Full article

Difenoconazole (DIF), a demethylation inhibitor fungicide, was registered in 2016 for the control of postharvest diseases of pome fruits. In this study, 162 isolates from P. expansum (n = 31) and 13 other “non-expansum” Penicillium spp., i.e., P. solitum (n = 52), P. roqueforti (n = 32), P. commune (n = 15), P. paneum (n = 9), P. psychrosexuale (n = 8), P. crustosum (n = 5), P. carneum (n = 3), P. palitans (n = 2), along with one isolate each of P. citrinum, P. griseofulvum, P. raistrickii, P. ribium, and P. viridicatum, were collected from multiple packinghouses in the U.S. Pacific Northwest. In vitro sensitivity assays showed similar sensitivities of spores and mycelia across species with the mean EC₅₀ values ranging from 0.01 for P. psychrosexuale (n = 8) to 1.33 μg mL⁻¹ for P. palitans (n = 2), whereas the mean EC_50s were 0.03, 0.12, 0.19, and 0.51 μg mL⁻¹ for P. expansum (n = 31), P. paneum (n = 9), P. solitum (n = 52), and P. crustosum (n = 5), respectively. The recommended rate of DIF controlled P. expansum and P. roqueforti isolates but not all isolates of four other Penicillium spp. on Fuji apples after five months at 1.5 °C. The mixture of DIF + fludioxonil (FDL) (Academy^TM) controlled all the dual-sensitive isolates (DIF^SFDL^S) and DIF single-resistant (DIF^R) isolates among the six species tested but not the FDL^R and dual DIF^RFDL^R isolates. Notable polymorphism was detected in the CYP51 gene of the “non-expansum” species with four mutations located at four residues. Although the isolates analyzed in this study had not previously been exposed to DIF, the findings indicate variable sensitivity levels among the Penicillium spp. Full article

The Penicillium genus exhibits a broad global distribution and holds substantial economic value in sectors including agriculture, industry, and medicine. Particularly in agriculture, Penicillium species significantly impact plants, causing diseases and contamination that adversely affect crop yields and quality. Timely detection of Penicillium species is crucial for controlling disease and preventing mycotoxins from entering the food chain. To tackle this issue, we implement a novel species identification approach called Analysis of whole GEnome (AGE). Here, we initially applied bioinformatics analysis to construct specific target sequence libraries from the whole genomes of seven Penicillium species with significant economic impact: P. canescens, P. citrinum, P. oxalicum, P. polonicum, P. paneum, P. rubens, and P. roqueforti. We successfully identified seven Penicillium species using the target we screened combined with Sanger sequencing and CRISPR-Cas12a technologies. Notably, based on CRISPR-Cas12a technology, AGE can achieve rapid and accurate identification of genomic DNA samples at a concentration as low as 0.01 ng/µL within 30 min. This method features high sensitivity and portability, making it suitable for on-site detection. This robust molecular approach provides precise fungal species identification with broad implications for agricultural control, industrial production, clinical diagnostics, and food safety. Full article

Gorgonzola is an Italian “erborinato” blue cheese from cow’s milk, bearing blue-green “parsley-like” spots due to the spread of Penicillium roqueforti mycelium. Due to its pH, water activity, and high nutrient content, as well as the environmental conditions required for its maturation, Gorgonzola constitutes an optimal ecological niche supporting the growth of both yeasts and filamentous fungi. Therefore, exploring the abundant mycobiota present in this peculiar habitat is of great interest regarding the search for new probiotic strains. The present investigation aimed to characterize the Gorgonzola mycobiota using both phenotypic (macroscopic and microscopic morphological analyses) and genotypic (DNA barcoding) analyses to find possible putative probiotic strains to be used in veterinary medicine in feed supplements. Among the different isolated filamentous fungi (Mucor and Penicillium) and yeasts (Yarrowia, Debaryomyces, Saccharomyces, and Sporobolomyces), we selected a strain of Saccharomyces cerevisiae var. boulardii. We tested its adaptation to thermal stress and its stability in feed matrices. The overall results highlight that the selected strain is stable for three months and can be considered as a possible candidate for use as a probiotic in veterinary feed supplements. Full article

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. Full article

Penicillium roqueforti is a fungal starter culture used for the production of blue-veined cheeses, such as Roquefort, Gorgonzola, Stilton, Cabrales, and Danablue. During ripening, this species grows in the veins of the cheese, forming the emblematic blue-green color and establishing the characteristic flavor owin to its biochemical activities. P. roqueforti synthesizes a diverse array of secondary metabolites, including the well-known compounds roquefortine C, clavine alkaloids, such as isofumigaclavine A and B, mycophenolic acid, andrastin A, and PR-toxin. This review provides an in-depth exploration of P. roqueforti’s secondary metabolites, focusing on their biosynthetic pathways, the gene clusters responsible for their production, and their bioactivities. The presence of these compounds in blue cheeses is also reviewed. Furthermore, the silent clusters and the potential of P. roqueforti for producing secondary metabolites were discussed. The review highlights recently identified metabolites, including sesterterpenoids; tetrapeptides, D-Phe-L-Val-D-Val-L-Tyr, and D-Phe-L-Val-D-Val-L-Phe; cis-bis(methylthio)silvatin; and the 1,8-dihydroxynaphthalene (DHN)-melanin precursor, scytalone. Additionally, a gene cluster for DHN–melanin biosynthesis is presented. Finally, a revised cluster for roquefortine C biosynthesis comprising three rather than four genes is proposed. Full article

Blue cheeses are those whose matrix is veined with a blue, blue-grey, or blue-green colour due to the development of Penicillium roqueforti. There are more than 45 varieties of blue cheese produced worldwide, with some distinct features, although the manufacture process is similar. In addition to P. roqueforti, complex microbial populations interact and succeed throughout the manufacturing and ripening at the cheese’s surface (the rind) and interior (matrix). The microbiota of blue cheeses is made up of a vast array of both prokaryotic and eukaryotic microorganisms. Acidification of the curd relies on the action of lactococci and other lactic acid bacteria (LAB) species. The ripened cheeses’ final quality and shelf-life properties largely depend on the enzymatic systems of the components of the microbiota, particularly on those of LAB, P. roqueforti, and yeast species. Proteolysis is the most complex and important primary biochemical process involved in blue-veined cheeses during ripening, with P. roqueforti being considered the main proteolytic agent. Lipolysis is also strong, originating, among other compounds, ketones, which are the main aroma compounds in blue-veined cheeses. In addition, several bioactive compounds are produced during ripening. The biochemical activities, mainly of microbial origin, are responsible for the sensory characteristics of these very appreciated cheese varieties worldwide. Full article

Technological defects in the organoleptic characteristics of cheese (odour, colour, texture, and flavour) reduce quality and consumer acceptance. A red colour defect in Cabrales cheese (a traditional, blue-veined, Spanish cheese made from raw milk) occurs infrequently but can have a notable economic impact on family-owned, artisanal cheesemaking businesses. This work reports the culture-based determination of Serratia marcescens as the microbe involved in the appearance of red spots on the surface and nearby inner areas of such cheese. Sequencing and analysis of the genome of one S. marcescens isolate, RO1, revealed a cluster of 16 genes involved in the production of prodigiosin, a tripyrrole red pigment. HPLC analysis confirmed the presence of prodigiosin in methanol extracts of S. marcescens RO1 cultures. The same was also observed in extracts from red areas of affected cheeses. The strain showed low survival rates under acidic conditions but was not affected by concentrations of up to 5% NaCl (the usual value for blue cheese). The optimal conditions for prodigiosin production by S. marscescens RO1 on agar plates were 32 °C and aerobic conditions. Prodigiosin has been reported to possess antimicrobial activity, which agrees with the here-observed inhibitory effect of RO1 supernatants on different bacteria, the inhibition of Enterobacteriaceae, and the delayed development of Penicillium roqueforti during cheesemaking. The association between S. marcescens and the red colour defect was strengthened by recreating the fault in experimental cheeses inoculated with RO1. The data gathered in this study point towards the starting milk as the origin of this bacterium in cheese. These findings should help in the development of strategies that minimize the incidence of pigmenting S. marcescens in milk, the red defect the bacterium causes in cheese, and its associated economic losses. Full article

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. Full article

Pit mud, a specific fermented soil, is an essential material for the fermentation of Chinese strong-flavour liquor. However, few studies to date have sought to characterize the spatial profiles of pit mud fungal communities in fermentation cellars from Chinese strong-flavour liquor distilleries. In this analysis, differences in fungal community structures and physicochemical properties in pit mud samples from different spatial positions within fermentation cellars were analyzed, revealing unique characteristic multidimensional pit mud fungal community profiles. Penicillium roqueforti, Pichia kudriavzevii, Aotearoamyces nothofagi, Penicillium robsamsonii, Alternaria arborescens, Trichosporon insectorum, Seltsamia ulmi, Trichosporon coremiiforme, Malassezia restricta were dominant in the pit mud samples form the upper cellar wall, whereas Metarhizium frigidum, Calonectria pseudoreteaudii, Penicillium clavigerum, Fusarium equiseti, Simplicillium chinense, Aspergillus intermedius, Trichosporon coremiiforme, Fusarium circinatum, Alternaria radicina, Aspergillus heterocaryoticus were predominant in the middle cellar wall. Alternaria radicina, Cladosporium chasmanthicola, Alternaria helianthiinficiens, Penicillium argentinense, Antarctomyces psychrotrophicus, and Trichosporon inkin are majorly present in the down cellar wall layer. Bipolaris axonopicola, Ramgea ozimecii, Penicillium argentinense, Calonectria queenslandica, Metarhizium robertsii, and Penicillium roqueforti were identified as the dominant fungi in pit mud samples from the cellar bottom. Additionally, Alternaria destruens and Alternaria doliconidium are present at notably high levels in all layers of pit mud samples. Moisture, pH, PO₄³⁻, acetic acid, humus, K⁺, Mg²⁺, Ca²⁺, butyric acid, and caproic acid levels in these different pit mud positions exhibited a rising incremental pattern from the upper wall layer to the bottom layer, whereas lactic acid levels were significantly lower in the bottom pit mud layer relative to these other layers. Moisture, pH, and NH₄⁺-N were identified as the three most significant factors associated with fungal community composition through a redundancy analysis. Overall, these findings may offer a theoretical foundation for future efforts to improve or standardize artificial pit mud. Full article

Food processes use different microorganisms, from bacteria to fungi. Yeast strains have been extensively studied, especially Saccharomyces cerevisiae. However, to date, very little is known about the potential beneficial effects of molds on gut health as part of gut microbiota. We undertook a comprehensive characterization of five mold strains, Penicillium camemberti, P. nalgiovense, P. roqueforti, Fusarium domesticum, and Geotrichum candidum used in food processes, on their ability to trigger or protect intestinal inflammation using in vitro human cell models and in vivo susceptibility to sodium dextran sulfate-induced colitis. Comparison of spore adhesion to epithelial cells showed a very wide disparity in results, with F. domesticum and P. roqueforti being the two extremes, with almost no adhesion and 20% adhesion, respectively. Interaction with human immune cells showed mild pro-inflammatory properties of all Penicillium strains and no effect of the others. However, the potential anti-inflammatory abilities detected for G. candidum in vitro were not confirmed in vivo after oral gavage to mice before and during induced colitis. According to the different series of experiments carried out in this study, the impact of the spores of these molds used in food production is limited, with no specific beneficial or harmful effect on the gut. Full article

Currently, Penicillium roqueforti and the closely related P. carneum and P. paneum are identified based on their macromorphology, micromorphology, and molecular properties, the determination of which involves time-consuming procedures. Culture collections focused on dairy isolates of P. roqueforti require quick and efficient tools for routine applications to identify the (a) taxonomy affiliation and (b) morphological properties of strains that influence the sensory properties of blue-veined cheeses. Here, we assessed the morphological variability of P. roqueforti, P. carneum, P. paneum, and P.crustosum on artificial, Edam-like, and Roquefort-like media. Molecular tools were used to test P. roqueforti strains and clones effectively. A novel primer, PrsF, was tested for specificity within strains and isolates of P. roqueforti compared to P. carneum, P. paneum, and P. crustosum. The results reveal that PrsF was specific to the P. roqueforti samples and did not amplify the other tested Penicillium species. Identification based simultaneously on the specificity of the PrsF primer pair and cultivation of P. roqueforti strains on Roquefort-like medium represents an effective method for expanding the collections and practical use of P. roqueforti in the dairy industry. Full article

Yeasts can have additional genetic information in the form of cytoplasmic linear dsDNA molecules called virus-like elements (VLEs). Some of them encode killer toxins. The aim of this work was to investigate the prevalence of such elements in D. hansenii killer yeast deposited in culture collections as well as in strains freshly isolated from blue cheeses. Possible benefits to the host from harboring such VLEs were analyzed. VLEs occurred frequently among fresh D. hansenii isolates (15/60 strains), as opposed to strains obtained from culture collections (0/75 strains). Eight new different systems were identified: four composed of two elements and four of three elements. Full sequences of three new VLE systems obtained by NGS revealed extremely high conservation among the largest molecules in these systems except for one ORF, probably encoding a protein resembling immunity determinant to killer toxins of VLE origin in other yeast species. ORFs that could be potentially involved in killer activity due to similarity to genes encoding proteins with domains of chitin-binding/digesting and deoxyribonuclease NucA/NucB activity, could be distinguished in smaller molecules. However, the discovered VLEs were not involved in the biocontrol of Yarrowia lipolytica and Penicillium roqueforti present in blue cheeses. Full article

Four taxa of the Lamiaceae family growing in Apulia (Clinopodium suaveolens, Satureja montana subsp. montana, Thymbra capitata, and Salvia fruticosa subsp. thomasii) that had not been previously studied for their potential use in the food sector, were analyzed for their essential oils (EOs) composition and antioxidant and antimicrobial properties against some microorganisms, isolated from bread and bakery products, including molds (Aspergillus niger, Penicillium roqueforti) and spore-forming bacteria (Bacillus amyloliquefaciens and Bacillus subtilis). Two different sites were considered for each plant species, and the strongest antimicrobial EOs, which were active against all of the microorganisms tested, were those from one S. montana subsp. montana sample (Sm2) and both T. capitata EOs (Tc1 and Tc2) with Minimal Inhibitory Concentration (MIC) values ranging between 0.093% and 0.375% (v/v) against molds, while higher values were registered for bacteria (0.75–1%). In particular, the biological activity of EOs from T. capitata and S. montana subsp. montana was maybe due to the high amount of thymol and carvacrol, which were also responsible for the highest antioxidant activity. S. fruticosa subsp. thomasii EOs had different chemical profiles but showed only a slight antibacterial effect and no antifungal activity. C. suaveolens showed no significant changes between EOs with an interesting antifungal activity (MIC 0.093%÷0.187% v/v), which may be due to the presence of pulegone. These plant species can be considered as promising sources of bioactive compounds to be exploited as biopreservatives in bread and bakery products mainly considering the low concentration needed to inhibit microorganism’s growth. Full article

Preventing food spoilage without the addition of chemical food additives, while increasing functional properties of wheat-based bakery products, is an increasing demand by the consumers and a challenge for the food industry. Within this study, lactic acid bacteria (LAB) isolated from sourdough were screened in vitro for the ability to utilize the typical wheat carbohydrates, for their antimicrobial and functional properties. The dual culture overlay assay revealed varying levels of inhibition against the examined fungi, with Lactiplantibacillus plantarum S4.2 and Lentilactobacillusparabuchneri S2.9 exhibiting the highest suppression against the indicator strains Fusarium graminearum MUCL43764, Aspergillus fumigatus, A. flavus MUCL11945, A. brasiliensis DSM1988, and Penicillium roqueforti DSM1079. Furthermore, the antifungal activity was shown to be attributed mainly to the activity of acids produced by LAB. The antibacillus activity was evaluated by the spot-on-the-lawn method revealing a high inhibition potential of the majority of LAB isolated from sourdough against Bacillus cereus DSM31, B. licheniformis DSM13, B. subtilis LMG7135, and B. subtilis S15.20. Furthermore, evaluating the presence of the glutamate decarboxylase gen in LAB isolates by means of PCR showed a strain dependency of a potential GABA production. Finally, due to improved functional activities, LAB isolated from sourdoughs exhibit promising characteristics for the application as natural preservatives in wheat-based bakery products. Full article