Antagonistic activity of the food-related filamentous fungus Penicillium nalgiovense by the production of penicillin.

Defined strains of the genus Penicillium used as starter cultures for food and strains isolated from mold-fermented foods were analyzed for their ability to inhibit the growth of Micrococcus luteus DSM 348 used as an indicator organism. Most of the strains belonging to the species Penicillium nalgiovense showed antagonistic activity in agar diffusion assays. Penicillium camemberti and Penicillium roqueforti strains proved to be inactive in these tests. The inhibitory substance excreted by P. nalgiovense strains was totally inactivated when treated with beta-lactamase (penicillinase), indicating that a beta-lactam antibiotic is produced by these strains. This observation was verified by PCRs with primer sets specific to the [delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine] synthetase gene (pcbAB), the isopenicillin-N-synthase gene (pcbC), and the acyl coenzyme A:6-aminopenicillanic acid acyltransferase gene (penDE) from Penicillium chrysogenum using chromosomal DNA of the fungal strains as a template. These results indicate that penicillin biosynthesis is a characteristic often found in strains of P. nalgiovense. No specific PCR signal could be identified with DNA from P. camemberti and P. roqueforti.     

Antigenic characterization of Penicillium camemberti and related common cheese contaminants

Twenty-four isolates of Penicillium (including a green-spored mutant from a French Brie cheese, Penicillium camemberti) with a proposed relationship to the white cheese mold P. camemberti were investigated by immunological procedures. These penicillia, which are representative of species that have caused considerable taxonomic confusion, had common micromorphology (terverticillate penicilli with rough and smooth stipes and smooth ellipsoidal to subglobose [(3 to 5) X 2 1/2 to 4 1/2 microns] conidia); growth rates; good growth on creatine sucrose agar, cheese, and other products with a high amount of protein and lipid as a primary habitat; production (with the exception of Penicillium solitum) of cyclopiazonic acid; and the ability to grow at low temperatures and water activities. The isolates that were investigated proved to be strictly antigenically related. Absorbed antiserum of the green-spored mutant of P. camemberti showed a specific precipitin band when tested by immunodiffusion either with its homologous reference antigen or with the exoantigens obtained from different isolates. The precipitin band was not present in any P. camemberti starter culture but in many unwanted cheese contaminants. The precipitin band can be used in the purity control of P. camemberti starter culture spore preparations. Analysis of the exoantigens of all the cultures by reversed phase high-performance liquid chromatography allowed us to subdivide these penicillia into nine groups below the species level. The results indicate that P. commune Thom is the wild-type ancestor of P. camemberti.     

Antigenic characterization of Penicillium camemberti and related common cheese contaminants.

Twenty-four isolates of Penicillium (including a green-spored mutant from a French Brie cheese, Penicillium camemberti) with a proposed relationship to the white cheese mold P. camemberti were investigated by immunological procedures. These penicillia, which are representative of species that have caused considerable taxonomic confusion, had common micromorphology (terverticillate penicilli with rough and smooth stipes and smooth ellipsoidal to subglobose [(3 to 5) X 2 1/2 to 4 1/2 microns] conidia); growth rates; good growth on creatine sucrose agar, cheese, and other products with a high amount of protein and lipid as a primary habitat; production (with the exception of Penicillium solitum) of cyclopiazonic acid; and the ability to grow at low temperatures and water activities. The isolates that were investigated proved to be strictly antigenically related. Absorbed antiserum of the green-spored mutant of P. camemberti showed a specific precipitin band when tested by immunodiffusion either with its homologous reference antigen or with the exoantigens obtained from different isolates. The precipitin band was not present in any P. camemberti starter culture but in many unwanted cheese contaminants. The precipitin band can be used in the purity control of P. camemberti starter culture spore preparations. Analysis of the exoantigens of all the cultures by reversed phase high-performance liquid chromatography allowed us to subdivide these penicillia into nine groups below the species level. The results indicate that P. commune Thom is the wild-type ancestor of P. camemberti.     

Differentiating Penicillium species by detection of indole metabolites using a filter paper method

The indole secondary metabolites chaetoglobosin C, cyclopiazonic acid, isofumigaclavine A and rugulovasine A and B produced by several Penicillium species growing on Czapek yeast autolysate agar were detected directly in the culture using filter paper wetted with Ehrlich reagent dissolved in ethanol. The filter paper was placed on the mycelial side of an agar plug and the metabolites were visualized as a violet zone on the paper within 10 min. It was shown that the combined characters of the violet reaction on filter paper and the ability to grow on creatine sucrose agar occurred in 5 out of 16 species of Penicillium examined. A few additional simple morphological and physiological criteria were then sufficient for identification of P. camemberti, P. commune, P. discolor, P. expansum and P. roqueforti var. roqueforti.     

The role of fungi in cheese ripening

Fungi are important in the manufacture of two types of cheese—blue-veined cheeses, and Camembert and Brie. Among the former are Roquefort, Gorgonzola and Stilton, dependent on the mold Penicillium roqueforti and the bacterium Streptococcus lactis. Camembert and Brie require Penicillium camemberti and lactic acid- producing streptococci; the mold Oospora lactis and the organism Bacterium linens may also play roles in their manufacture.     

Study of conditions of production of roquefortine and other metabolites of Penicillin roqueforti.

Experiments to determine optimum yields of roquefortine, isofumigaclavine A, and PR toxin, metabolites from Penicillum roqueforti Thom, were performed. Four strains, isolated from blue cheese, and five liquid media were evaluated, although not all permutations were studied. Sucrose (15%)-yeast extract (2%) was the medium chosen for time-course studies at 25 and 15 degrees C using one favorable strain. At 25 degrees C, maximum estimated yields of roquefortine were about 100 mg/liter in the mycelium by 16 days, and no subsequent degradation of this alkaloid was observed. On the other hand, production of PR toxin in the medium peaked at 770 mg/liter at 21 days. At 15 degrees C, yields of roquefortine and PR toxin after 49 days were 60 to 70% of the maximum yields obtained at 25 degrees C. However, about three times more isofumigaclavine A (up to 11 mg/liter) was formed in the mycelium at 15 degrees C than at 25 degrees C. All four strains of P. roqueforti procedure both roquefortine and PR toxin on the sucrose-yeast extract medium at 25 degrees C; isofumigaclavine A was detected in all but one strain grown on this medium.     

Efficiency of several micro-fiber glass filters for recovery of poliovirus from tape water.

Sixteen strains of Penicillium roqueforti Thom, isolated from blue-molded cheeses, were studied. In vitro, all of these strains produced mycophenolic acid, some on the order of 0.8 to 4 mg/g od dry culture. The greatest yields were obtained after 10 days of incubation of cultures at 15 degrees C. However, under some experimental conditions, mycophenolic acid was not alone responsible for the toxicity of culture extracts to chicken embryos.     

Headspace sorptive extraction and GC-TOFMS for the identification of volatile fungal metabolites

Volatiles from fungi cultivated in Petri dishes were collected by a simple headspace polydimethylsiloxane (PDMS) sorptive extraction technique (HSSE), thermally desorbed into a gas chromatographic capillary column and detected and identified by gas chromatography-time-of-flight mass spectrometry (GC-TOFMS). The method was used to compare metabolite profiles of seven species of fungi grown on two types of sterile agars - potato dextrose and Sabouraud dextrose. Three species from the genus Penicillium (P. italicum, P. camemberti, and P. roqueforti) and four outgroups, each from a different phylum (Saprolegnia sp.; Sordaria fimicola, wild-type; Coprinus cinereus; and Rhizopus stolonifer) were grown on the two types of agars and analyzed. Multivariate analysis (PCA) was used to determine whether separate classes of fungi can be distinguished from one another based on their metabolite profiles. PCA showed clear class separation between the three Penicillium samples and the outgroups. Slight differences were observed in metabolite profiles as a function of growth medium. HSSE/GC-TOFMS appears to be a relatively simple and accurate technique for classification of fungi based on their volatile metabolite profiles. The volatiles sampling technique reported here is non-destructive, so it can be applied with traditional methods for studying fungal growth and metabolism.     

Molecular identification of species from the Penicillium roqueforti group associated with spoiled animal feed

The Penicillium roqueforti group has recently been split into three species, P. roqueforti, Penicillium carneum, and Penicillium paneum, on the basis of differences in ribosomal DNA sequences and secondary metabolite profiles. We reevaluated the taxonomic identity of 52 livestock feed isolates from Sweden, previously identified by morphology as P. roqueforti, by comparing the sequences of the ribosomal internal transcribed spacer region. Identities were confirmed with random amplified polymorphic DNA analysis and secondary metabolite profiles. Of these isolates, 48 were P. roqueforti, 2 were P. paneum, and 2 were Penicillium expansum. No P. carneum isolates were found. The three species produce different mycotoxins, but no obvious relationship between mold and animal disease was detected, based on medical records. P. roqueforti appears to dominate in silage, but the ecological and toxicological importance of P. carneum and P. paneum as feed spoilage fungi is not clear. This is the first report of P. expansum in silage.     

Kinetics of growth and medium de-acidification for Geotrichum candidum and Penicillium camemberti cultivated on complex liquid media

Growth kinetics of Geotrichum candidum and Penicillium camemberti in submerged cultures under conditions of low aeration rate and uncontrolled pH were continuously recorded turbidimetrically. In these conditions the exponential growth phase was short, and ceased at a total biomass concentration of about 0.5 gl^–1 for G. candidum and 0.8 gl^–1 for P. candidum. The succeeding linear growth phase was also short, and its end corresponded to a biomass of 1.5 and 2.5 gl^–1 for G. candidum and P. camemberti respectively. A fair growth was recorded for P. camemberti on peptone-lactate medium, but G. candidum required addition of trace elements. For neither of these species growth was stimulated by growth factors of yeast extract. In peptone-lactate medium, the final pH did not depend on supplementation: 8–8.4 was recorded for G. candidum and 8.7–8.8 for P. camemberti.     

Methods for integrated air sampling and dna analysis for detection of airborne fungal spores

Integrated air sampling and PCR-based methods for detecting airborne fungal spores, using Penicillium roqueforti as a model fungus, are described. P. roqueforti spores were collected directly into Eppendorf tubes using a miniature cyclone-type air sampler. They were then suspended in 0.1% Nonidet P-40, and counted using microscopy. Serial dilutions of the spores were made. Three methods were used to produce DNA for PCR tests: adding untreated spores to PCRs, disrupting spores (fracturing of spore walls to release the contents) using Ballotini beads, and disrupting spores followed by DNA purification. Three P. roqueforti-specific assays were tested: single-step PCR, nested PCR, and PCR followed by Southern blotting and probing. Disrupting the spores was found to be essential for achieving maximum sensitivity of the assay. Adding untreated spores to the PCR did allow the detection of P. roqueforti, but this was never achieved when fewer than 1,000 spores were added to the PCR. By disrupting the spores, with or without subsequent DNA purification, it was possible to detect DNA from a single spore. When known quantities of P. roqueforti spores were added to air samples consisting of high concentrations of unidentified fungal spores, pollen, and dust, detection sensitivity was reduced. P. roqueforti DNA could not be detected using untreated or disrupted spore suspensions added to the PCRs. However, using purified DNA, it was possible to detect 10 P. roqueforti spores in a background of 4,500 other spores. For all DNA extraction methods, nested PCR was more sensitive than single-step PCR or PCR followed by Southern blotting.     

Measurement of mercury methylation in lake water and sediment samples

The production of various eremophilane-type sesquiterpenes by Penicillium roqueforti strains has allowed us to propose a biochemical pathway for PR toxin synthesis. A time-course study of P. roqueforti metabolite production by high-performance liquid chromatography was performed to check this hypothetical pathway. The results obtained suggested that eremofortin C was the direct precursor of PR toxin in the P. roqueforti cell. Attempts to determine the amount of PR toxin in the mycelium failed. It was shown that the absence of PR toxin in mycelium was due to its instability during the extraction procedure.     

Antimicrobial activity of food-related Penicillium sp. against pathogenic bacteria in laboratory media and a cheese model system

Food-related Penicillium species ( n ep fy1 = rs 34) and Geotrichum candidum ( n = 11) grown on Czapek Dox and brie agar were tested for their ability to suppress growth of pathogenic bacteria. Ten out of 13 P. camemberti showed antagonistic activity while the other species did not interact significantly with the bacterial growth. The order of inhibition was: Gram-negative bacteria and Bacillus cereus > Listeria monocytogenes , Lactococcus sp. > Micrococcus sp. whereas Lactobacillus sp., Staphylococcus aureus and some Micrococcus sp. were unaffected. When Salmonella typhimurium was inoculated together with P. camemberti P25 in brie agar, bacterial growth was inhibited during the first 6 d of incubation whereafter growth started. The inhibition of L. monocytogenes was similar but less pronounced. The antimicrobial activity produced by P. camemberti P25 and L84 was enhanced with increasing amount of sucrose in the medium. The activity was increased at low pH and destroyed at pH above 8. It was detectable at 15°C but not at 37°C indicating that volatile metabolites might be involved. No significant accumulation of organic acids and no secondary metabolites such as mycotoxins were detected. HSGC-MS analysis indicated that acetaldehyde, benzaldehyde, 3-methylbutanal and 1-octen-3-ol were produced by P. camemberti during the period when inhibitory activity was observed. Pure acetaldehyde and benzaldehyde were shown to be inhibitory to L. monocytogenes and Salm. typhimurium when grown at 15°C and pH 5·5 and 7·0.     

Potentiation effect of corn extract on the production of eremofortin C, EC oxidase, and PR toxin by Penicillium roqueforti.

Eremofortin C (EC) and PR toxin are secondary metabolites of Penicillium roqueforti. Their structures are similar and differ only by an alcohol and an aldehyde group at the C-12 position. EC has been demonstrated to be the precursor of PR toxin, and EC is transformed to PR toxin by EC oxidase. These two compounds and EC oxidase are secreted by P. roqueforti in the culture medium, which is usually composed of 15% sucrose and 2% yeast extract. Recently, we discovered that the addition of corn extract to this medium increased the production of EC and PR toxin and the activity of EC oxidase in a coordinative manner. In a time-course study, we found that the peak yield of EC and PR toxin and the maximum activity of EC oxidase in the culture medium containing 7.5% sucrose, 1% yeast extract, and 20% corn extract were increased 6.2, 4.6, and 4.7-fold, respectively, as compared with those obtained in the medium without corn extract. Moreover, corn extract increased the production of EC and PR toxin and the activity of EC oxidase by P. roqueforti in a dose-dependent manner. On the other hand, when the concentrations of sucrose and yeast extract were increased while fixing the ratio of corn extract, we found that the levels of EC and PR toxin and the enzyme activity were decreased concomitantly. We thus conclude that corn extract can enhance the production of EC, PR toxin and EC oxidase by P. roqueforti when grown in a minimal medium and that the potentiation effect of corn extract is suppressed when the fungi are grown in a rich medium.     

Calcium-ion-binding activity in human small-intestinal mucosal cytosol. Purification of two proteins and interrelationship of calcium-binding fractions.

Butane-2,3-dione inactivates the aspartyl proteinases from Penicillium roqueforti and Penicillium caseicolum, as well as pig pepsin, penicillopepsin and Rhizopus pepsin, at pH 6.0 in the presence of light but not in the dark. The inactivation is due to a photosensitized modification of tryptophan and tyrosine residues. In the dark none of the amino acid residues, not even arginine residues, is modified even after several days. In the light one arginine residue in pig pepsin is lost at a rate that is comparable with the rate of inactivation; however, the loss of the single arginine residue in the aspartyl proteinase of P. roqueforti and the second arginine residue of pig pepsin is slower than the loss of activity; penicillopepsin is devoid of arginine. Loss of most of the activity is accompanied by the following amino acid losses: P. roqueforti aspartyl proteinase, about two tryptophan and six tyrosine residues; penicillopepsin, about two tryptophan and three tyrosine residues; pig pepsin, about four tryptophan and most of the tyrosine residues. Modification of histidine residues was too slow to contribute to inactivation. None of the other residues, including half-cystine and methionine residues (when present), was modified even after prolonged incubation. The inactivation of P. roqueforti aspartyl proteinase and pig pepsin appears due to non-specific modification of several residues. With penicillopepsin, however, the reaction is more limited and initially affects only those tryptophan and tyrosine residues that lie in the active-site groove. In the presence of pepstatin the rate of inactivation is considerably diminished. After prolonged reaction a general structural breakdown occurs.     

Control of Penicillium roqueforti (Thom) infection in cultures of Drosophila melanogaster (Meigen) (Diptera: Drosophilidae)

Abstract  Microbial contamination of artificial insect food media can jeopardise the viability, productivity and survival of many insect cultures, including Drosophila melanogaster . Here we investigated and improved upon control methods for one common contaminant, Penicillium roqueforti . We found that the combined effect of methyl p -hydroxybenzoate (23.7 mM), propionic acid (67.5 mM) and sorbic acid (8.9 mM) (PS^NPS treatment) was the most effective of the four candidate treatments, at inhibiting the growth of P. roqueforti. PS^NPS treatment inhibited 100% of visible P. roqueforti growth for 21 days (a complete D. melanogaster life cycle) and thus reduced the risk of transmitting infection to the next generation. Although the PS^NPS treatment negatively affected the two D. melanogaster fitness components, survivorship (number of adults) and biomass (live weight), it did not prevent successful reproduction and is suitable for short-term treatment of P. roqueforti infections.     

The composition of Camembert cheese-ripening cultures modulates both mycelial growth and appearance

The fungal microbiota of bloomy-rind cheeses, such as Camembert, forms a complex ecosystem that has not been well studied, and its monitoring during the ripening period remains a challenge. One limitation of enumerating yeasts and molds on traditional agar media is that hyphae are multicellular structures, and colonies on a petri dish rarely develop from single cells. In addition, fungi tend to rapidly invade agar surfaces, covering small yeast colonies and resulting in an underestimation of their number. In this study, we developed a real-time quantitative PCR (qPCR) method using TaqMan probes to quantify a mixed fungal community containing the most common dairy yeasts and molds: Penicillium camemberti, Geotrichum candidum, Debaryomyces hansenii, and Kluyveromyces lactis on soft-cheese model curds (SCMC). The qPCR method was optimized and validated on pure cultures and used to evaluate the growth dynamics of a ripening culture containing P. camemberti, G. candidum, and K. lactis on the surface of the SCMC during a 31-day ripening period. The results showed that P. camemberti and G. candidum quickly dominated the ecosystem, while K. lactis remained less abundant. When added to this ecosystem, D. hansenii completely inhibited the growth of K. lactis in addition to reducing the growth of the other fungi. This result was confirmed by the decrease in the mycelium biomass on SCMC. This study compares culture-dependent and qPCR methods to successfully quantify complex fungal microbiota on a model curd simulating Camembert-type cheese.     

Identification of cheese-associated fungi using selected ion monitoring of volatile terpenes

The cheese-associated fungi Penicillium commune , P. roqueforti , P. solitum , P. discolor and Aspergillus versicolor have been investigated for production of volatile terpenes for chemical identification, when grown on yeast extract agar. Volatiles were collected by headspace solid-phase microextraction. Selected ion monitoring of four to seven of the most characteristic ions of mainly sesquiterpenes made it possible to identify the fungi to species level within 2 d. In a mixed culture of P. roqueforti and P. commune , inoculated in a ratio of 1000 : 1, volatiles from both fungi could be detected within 3 d, making identification possible.     

Mold-inhibitory activity of different yeast species during airtight storage of wheat grain

The yeast Pichia anomala J121 inhibits spoilage by Penicillium roqueforti in laboratory and pilot studies with high-moisture wheat in malfunctioning airtight storage. We tested the biocontrol ability of an additional 57 yeast species in a grain mini silo system. Most yeast species grew to CFU levels comparable to that of P. anomala J121 after 14 days of incubation (>10(6) CFU g(-1)). Of the 58 species, 38 (63 strains) had no mold-inhibitory effects (Pen. roqueforti levels >10(5) CFU g(-1)). Among these were 11 species (18 strains) that did not grow on the wheat grain. Several of the non-inhibiting yeast species have previously been reported as biocontrol agents in other postharvest environments. Weak inhibitory activity, reducing Pen. roqueforti levels to between 10(4) and 10(5) CFU g(-1), was observed with 11 species (12 strains). Candida silvicola and Pichia guillermondii reduced Pen. roqueforti to <10(4) CFU g(-1). Candida fennica, Candida pelliculosa, Candida silvicultrix, P. anomala (29 strains), Pichia burtonii, Pichia farinosa and Pichia membranifaciens strongly inhibited Pen. roqueforti (<10(3) CFU g(-1)) in the mini silos, but none had higher biocontrol activity than P. anomala strain J121. This report is the first of biocontrol activity of C. fennica and C. silvicultrix. The ability of 27 yeast species to grow to high CFU values without inhibiting mold growth suggests that nutrient competition may not be the main mode of action of P. anomala J121.     

Use of hydrostatic pressure for inactivation of microbial contaminants in cheese

The objective of this study was to determine the effect of high pressure (HP) on the inactivation of microbial contaminants in Cheddar cheese (Escherichia coli K-12, Staphylococcus aureus ATCC 6538, and Penicillium roqueforti IMI 297987). Initially, cheese slurries inoculated with E. coli, S. aureus, and P. roqueforti were used as a convenient means to define the effects of a range of pressures and temperatures on the viability of these microorganisms. Cheese slurries were subjected to pressures of 50 to 800 MPa for 20 min at temperatures of 10, 20, and 30 degrees C. At 400 MPa, the viability of P. roqueforti in cheese slurry decreased by >2-log-unit cycles at 10 degrees C and by 6-log-unit cycles at temperatures of 20 and 30 degrees C. S. aureus and E. coli were not detected after HP treatments in cheese slurry of >600 MPa at 20 degrees C and >400 MPa at 30 degrees C, respectively. In addition to cell death, the presence of sublethally injured cells in HP-treated slurries was demonstrated by differential plating using nonselective agar incorporating salt or glucose. Kinetic experiments of HP inactivation demonstrated that increasing the pressure from 300 to 400 MPa resulted in a higher degree of inactivation than increasing the pressurization time from 0 to 60 min, indicating a greater antimicrobial impact of pressure. Selected conditions were subsequently tested on Cheddar cheese by adding the isolates to cheese milk and pressure treating the resultant cheeses at 100 to 500 MPa for 20 min at 20 degrees C. The relative sensitivities of the isolates to HP in Cheddar cheese were similar to those observed in the cheese slurry, i.e., P. roqueforti was more sensitive than E. coli, which was more sensitive than S. aureus. The organisms were more sensitive to pressure in cheese than slurry, especially with E. coli. On comparison of the sensitivities of the microorganisms in a pH 5.3 phosphate buffer, cheese slurry, and Cheddar cheese, greatest sensitivity to HP was shown in the pH 5.3 phosphate buffer by S. aureus and P. roqueforti while greatest sensitivity to HP by E. coli was exhibited in Cheddar cheese. Therefore, the medium in which the microorganisms are treated is an important determinant of the level of inactivation observed.