Penicillium roqueforti: an overview of its genetics,physiology, metabolism and biotechnological applications

Besides research on the model fungal genera Saccharomyces, Neurospora and Aspergillus, that has provided important biological knowledge in the areas of genetics, cell biology and physiology, recent investigations on non-model fungi used for food production offer insight into the mechanisms involved in food production but also adaptation and domestication processes. In this context, Penicillium roqueforti has been the most extensively studied species. This species is best known worldwide for its technological use for blue-veined cheese production and ripening. Recently, several advances related to taxonomy, population genetics, physiology and metabolism have been documented and provided deeper knowledge about this species. The methodological approaches used to study this species can be applied to other still largely understudied fungi associated with food production worldwide (e.g. P. camemberti, P. nalgiovense, P. salamii, Bisifusarium domesticum, Mucor spp.).     

Chemical characterisation of cheese associated fungi

Recent work in our laboratory has demonstrated that the most common contaminating fungi on different types of cheese are;Penicillium commune, P. nalgiovense, P. solitum, P. discolor, P. roqueforti, P. crustosum, P. nordicum andAspergillus versicolor. On blue cheese a new speciesP. caseifulvum has been discovered as a surface contaminant. A large number of known and unknown metabolites have been described from the above mentioned cheese associated fungi from both synthetic media and real samples. Based on chemotaxonomy our laboratory has discovered thatP. roqueforti should be divided into three species:P. roqueforti (from cheese),P. carneum (from meat) andP. paneum (from bread). SimilarlyP. verrucosum should be divided intoP. verrucosum (from cereals) andP. nordicum (from cheese and meat products). Both species produce ochratoxins, however, only the former species produce citrinin.     

Comparison of growth characteristics and roquefortin C production of<Emphasis Type="Italic">Penicillium roqueforti</Emphasis> from blue-veined cheese

The properties of 21 isolates ofPenicillium roqueforti from just as many commercial blue-veined cheeses, purchased from the Argentinean market (domestic and imported products) were comparatively examined. Isolates were investigated for their ability to grow at different temperatures, pH values and concentration of NaCl, as well as for their proteolytic and lipolytic activities, respectively. The potential of these strains to produce roquefortin in vitro, and the actual levels of roquefortin in 10 of these cheeses were analysed by TLC. All strains showed similar growth properties in aspects of salt concentration and pH-value of the medium, and all grew well at 10 °C. Only four strains showed proteolytic activity on casein agar, while all strains were lipolytic on trybutirin agar. After incubation at 25 °C for 16 days, all strains produced roquefortin in Yeast Extract Sucrose (25.6–426.7 μg/g) and in reconstituted (10%) sterile skim milk (26.9–488 μg/g). Roquefortin at >0.1 μg/g was also found in 9 out of 10 analysed samples of blue-veined cheeses (8 from Argentine, 1 from Spain), with a maximum value 3.6 μg/g. During the ripening process of blueveined cheese, production of roquefortin seems to be unavoidable. Care should be taken to select strains with low toxin production characteristics, to minimize potential health risks. Roquefortin C production byP. roqueforti in vitro was not correlated with roquefortin C levels found in cheese. Financial support: Research grants from the National University of Quilmes, Argentina     

Strain-Specific Synthesis of Mycophenolic Acid by Penicillium roqueforti in Blue-Veined Cheese

Twenty of 80 strains of Penicillium roqueforti were able to produce up to 600 mg of mycophenolic acid (MPA) liter⁻¹ in 2% yeast extract-5% sucrose broth. Sixty-two of these strains had been isolated from the main blue-veined cheese varieties of western Europe or from starter cultures. Of these 62 dairy strains, only 7 had MPA-producing potential in vitro. These seven strains had all been isolated during the period 1975 to 1981 from the blue cheese of one individual factory. In cheese from the market, MPA (up to 5 mg kg⁻¹) was only found in samples of this same factory. With MPA-producing and -nonproducing strains for the experimental manufacture of blue cheese, MPA synthesis in cheese was only detected with strains which form MPA in yeast extract-sucrose broth. The maximum MPA level at 4 mg kg⁻¹ was similar to that in commercial cheese. Toxicity of MPA was tested with two established human cell lines (Detroit 98 and Girardi Heart) and one established pig kidney cell line (AmII).     

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°C. At 400 MPa, the viability of P. roqueforti in cheese slurry decreased by >2-log-unit cycles at 10°C and by 6-log-unit cycles at temperatures of 20 and 30°C. S. aureus and E. coli were not detected after HP treatments in cheese slurry of >600 MPa at 20°C and >400 MPa at 30°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°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.     

Toxigenic fungi isolated from roquefort cheese

To evaluate the potential for mycotoxin production by fungi contaminating blue-veined cheese, as well as by the ripening fungus,Penicillium roqueforti, the fungal flora of six of local and imported brands was determined. A total of 19 fungi were isolated from the six brands tested. Fourteen of the isolates were toxic to chicken embryos. The toxigenic fungi produced the following mycotoxins:Aspergillus fumigatus, kojic acid;A. versicolor, sterigmatocystin;Penicillium roqueforti, penicillic acid and unidentified toxic metabolites.     

Fertility depression among cheese‐making Penicillium roqueforti strains suggests degeneration during domestication

Genetic differentiation occurs when gene flow is prevented, due to reproductive barriers or asexuality. Investigating the early barriers to gene flow is important for understanding the process of speciation. Here, we therefore investigated reproductive isolation between different genetic clusters of the fungus Penicillium roqueforti, used for maturing blue cheeses, and also occurring as food spoiler or in silage. We investigated premating and postmating fertility between and within three genetic clusters (two from cheese and one from other substrates), and we observed sexual structures under scanning electron microscopy. All intercluster types of crosses showed some fertility, suggesting that no intersterility has evolved between domesticated and wild populations despite adaptation to different environments and lack of gene flow. However, much lower fertility was found in crosses within the cheese clusters than within the noncheese cluster, suggesting reduced fertility of cheese strains, which may constitute a barrier to gene flow. Such degeneration may be due to bottlenecks during domestication and/or to the exclusive clonal replication of the strains in industry. This study shows that degeneration has occurred rapidly and independently in two lineages of a domesticated species. Altogether, these results inform on the processes and tempo of degeneration and speciation.     

Isolation and Identification of the Microbiota of Danish Farmhouse and Industrially Produced Surface-Ripened Cheeses

For studying the microbiota of four Danish surface-ripened cheeses produced at three farmhouses and one industrial dairy, both a culture-dependent and culture-independent approach were used. After dereplication of the initial set of 433 isolates by (GTG)5-PCR fingerprinting, 217 bacterial and 25 yeast isolates were identified by sequencing of the 16S rRNA gene or the D1/D2 domain of the 26S rRNA gene, respectively. At the end of ripening, the cheese core microbiota of the farmhouse cheeses consisted of the mesophilic lactic acid bacteria (LAB) starter cultures Lactococcus lactis subsp. lactis and Leuconostoc mesenteorides as well as non-starter LAB including different Lactobacillus spp. The cheese from the industrial dairy was almost exclusively dominated by Lb. paracasei. The surface bacterial microbiota of all four cheeses were dominated by Corynebacterium spp. and/or Brachybacterium spp. Brevibacterium spp. was found to be subdominant compared to other bacteria on the farmhouse cheeses, and no Brevibacterium spp. was found on the cheese from the industrial dairy, even though B. linens was used as surface-ripening culture. Moreover, Gram-negative bacteria identified as Alcalignes faecalis and Proteus vulgaris were found on one of the farmhouse cheeses. The surface yeast microbiota consisted primarily of one dominating species for each cheese. For the farmhouse cheeses, the dominant yeast species were Yarrowia lipolytica, Geotrichum spp. and Debaryomyces hansenii, respectively, and for the cheese from the industrial dairy, D. hansenii was the dominant yeast species. Additionally, denaturing gradient gel electrophoresis (DGGE) analysis revealed that Streptococcus thermophilus was present in the farmhouse raw milk cheese analysed in this study. Furthermore, DGGE bands corresponding to Vagococcus carniphilus, Psychrobacter spp. and Lb. curvatus on the cheese surfaces indicated that these bacterial species may play a role in cheese ripening.     

Construction of a dairy microbial genome catalog opens new perspectives for the metagenomic analysis of dairy fermented products

Background

Microbial communities of traditional cheeses are complex and insufficiently characterized. The origin, safety and functional role in cheese making of these microbial communities are still not well understood. Metagenomic analysis of these communities by high throughput shotgun sequencing is a promising approach to characterize their genomic and functional profiles. Such analyses, however, critically depend on the availability of appropriate reference genome databases against which the sequencing reads can be aligned.

Results

We built a reference genome catalog suitable for short read metagenomic analysis using a low-cost sequencing strategy. We selected 142 bacteria isolated from dairy products belonging to 137 different species and 67 genera, and succeeded to reconstruct the draft genome of 117 of them at a standard or high quality level, including isolates from the genera Kluyvera, Luteococcus and Marinilactibacillus, still missing from public database. To demonstrate the potential of this catalog, we analysed the microbial composition of the surface of two smear cheeses and one blue-veined cheese, and showed that a significant part of the microbiota of these traditional cheeses was composed of microorganisms newly sequenced in our study.

Conclusions

Our study provides data, which combined with publicly available genome references, represents the most expansive catalog to date of cheese-associated bacteria. Using this extended dairy catalog, we revealed the presence in traditional cheese of dominant microorganisms not deliberately inoculated, mainly Gram-negative genera such as Pseudoalteromonas haloplanktis or Psychrobacter immobilis, that may contribute to the characteristics of cheese produced through traditional methods.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1101) contains supplementary material, which is available to authorized users.     

Predictive formulae for goat cheese yield based on milk composition

Prediction of the yield and quality of different types of cheeses that could be produced from a given type and/or amount of goat milk is of great economic benefit to goat milk producers and goat cheese manufacturers. Bulk tank goat milk was used for manufacturing hard, semi-hard and soft cheeses (N = 25, 25 and 24, respectively) to develop predictive formulae of cheese yield based on milk composition. Fat, total solids, total protein and casein contents in milk and moisture-adjusted cheese yield were determined to establish relationships between milk composition and cheese yield. Soft, semi-hard and hard cheeses in this study had moisture contents of 66, 46 and 38%, respectively, which could be used as reference standards. In soft cheese, individual components of goat milk or a combination of two or three components predicted cheese yield with a reasonably high correlation coefficient (R² = 0.73–0.81). However, correlation coefficients of predictions were lower for both semi-hard and hard cheeses. Overall, total solids of goat milk was the strongest indicator of yield in all three types of cheeses, followed by fat and total protein, while casein was not a good predictor for both semi-hard and hard cheeses. When compared with moisture-adjusted cheese yield, there was no difference (P > 0.05) in predicting yield of semi-hard and hard goat milk cheeses between the developed yield formulae in this study and a standard formula (the Van Slyke formula) commonly used for cow cheese. Future research will include further validation of the yield predictive formulae for hard and semi-hard cheeses of goat milk using larger data sets over several lactations, because of variation in relationships between milk components due to breed, stage of lactation, season, feeding regime, somatic cell count and differences in casein variants.     

Sex in Cheese: Evidence for Sexuality in the Fungus Penicillium roqueforti

Although most eukaryotes reproduce sexually at some moment of their life cycle, as much as a fifth of fungal species were thought to reproduce exclusively asexually. Nevertheless, recent studies have revealed the occurrence of sex in some of these supposedly asexual species. For industrially relevant fungi, for which inoculums are produced by clonal-subcultures since decades, the potentiality for sex is of great interest for strain improvement strategies. Here, we investigated the sexual capability of the fungus Penicillium roqueforti, used as starter for blue cheese production. We present indirect evidence suggesting that recombination could be occurring in this species. The screening of a large sample of strains isolated from diverse substrates throughout the world revealed the existence of individuals of both mating types, even in the very same cheese. The MAT genes, involved in fungal sexual compatibility, appeared to evolve under purifying selection, suggesting that they are still functional. The examination of the recently sequenced genome of the FM 164 cheese strain enabled the identification of the most important genes known to be involved in meiosis, which were found to be highly conserved. Linkage disequilibria were not significant among three of the six marker pairs and 11 out of the 16 possible allelic combinations were found in the dataset. Finally, the detection of signatures of repeat induced point mutations (RIP) in repeated sequences and transposable elements reinforces the conclusion that P. roqueforti underwent more or less recent sex events. In this species of high industrial importance, the induction of a sexual cycle would open the possibility of generating new genotypes that would be extremely useful to diversify cheese products.     

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.     

Biosynthesis of flavors by Penicillium roqueforti

The development of the unique flavor of blue type cheese depends on the concerted action of numerous enzymes of Penicillium roqueforti involved in protein and lipid metabolism. Protease(s) by degrading casein modify the texture and background flavor of the ripening cheese. Lipase by hydrolyzing milk triglycerides provides flavorful fatty acids and precursors of methyl ketones. The enzyme complex involved in the partial oxidation of free fatty acids and the properties of β-ketoacyl decarboxylase which generates the major flavor components of blue cheese are discussed. Fermentation of P. roqueforti for the rapid production of methyl ketones is briefly reviewed.     

Surface Microflora of Four Smear-Ripened Cheeses

The microbial composition of smear-ripened cheeses is not very clear. A total of 194 bacterial isolates and 187 yeast isolates from the surfaces of four Irish farmhouse smear-ripened cheeses were identified at the midpoint of ripening using pulsed-field gel electrophoresis (PFGE), repetitive sequence-based PCR, and 16S rRNA gene sequencing for identifying and typing the bacteria and Fourier transform infrared spectroscopy and mitochondrial DNA restriction fragment length polymorphism (mtDNA RFLP) analysis for identifying and typing the yeast. The yeast microflora was very uniform, and Debaryomyces hansenii was the dominant species in the four cheeses. Yarrowia lipolytica was also isolated in low numbers from one cheese. The bacteria were highly diverse, and 14 different species, Corynebacterium casei, Corynebacterium variabile, Arthrobacter arilaitensis, Arthrobacter sp., Microbacterium gubbeenense, Agrococcus sp. nov., Brevibacterium linens, Staphylococcus epidermidis, Staphylococcus equorum, Staphylococcus saprophyticus, Micrococcus luteus, Halomonas venusta, Vibrio sp., and Bacillus sp., were identified on the four cheeses. Each cheese had a more or less unique microflora with four to nine species on its surface. However, two bacteria, C. casei and A. arilaitensis, were found on each cheese. Diversity at the strain level was also observed, based on the different PFGE patterns and mtDNA RFLP profiles of the dominant bacterial and yeast species. None of the ripening cultures deliberately inoculated onto the surface were reisolated from the cheeses. This study confirms the importance of the adventitious, resident microflora in the ripening of smear cheeses.     

Penicillium roqueforti conidia induced by L-amino acids can germinate without detectable swelling

Penicillium roqueforti is used for the production of blue-veined cheeses but is a spoilage fungus as well. It reproduces asexually by forming conidia. Germination of these spores can start the spoilage process of food. Germination is typically characterized by the processes of activation, swelling and germ tube formation. Here, we studied nutrient requirements for germination of P. roqueforti conidia. To this end,?>?300 conidia per condition were monitored in time using an oCelloScope imager and an asymmetric model was used to describe the germination process. Spores were incubated for 72 h in NaNO₃, Na₂HPO₄/NaH₂PO₄, MgSO₄ and KCl with 10 mM glucose or 10 mM of 1 out of the 20 proteogenic amino acids. In the case of glucose, the maximum number of spores (P_max) that had formed germ tubes was 12.7%, while time needed to reach 0.5 P_max (τ) was about 14 h. Arginine and alanine were the most inducing amino acids with a P_max of germ tube formation of 21% and 13%, respectively, and a τ of up to 33.5 h. Contrary to the typical stages of germination of fungal conidia, data show that P. roqueforti conidia can start forming germ tubes without a detectable swelling stage.

     

Characterization of Non-Starter Lactic Acid Bacteria from Italian Ewe Cheeses Based on Phenotypic, Genotypic, and Cell Wall Protein Analyses

Non-starter lactic acid bacteria (NSLAB) were isolated from 12 Italian ewe cheeses representing six different types of cheese, which in several cases were produced by different manufacturers. A total of 400 presumptive Lactobacillus isolates were obtained, and 123 isolates and 10 type strains were subjected to phenotypic, genetic, and cell wall protein characterization analyses. Phenotypically, the cheese isolates included 32% Lactobacillus plantarum isolates, 15% L. brevis isolates, 12% L. paracasei subsp. paracasei isolates, 9% L. curvatus isolates, 6% L. fermentum isolates, 6% L. casei subsp. casei isolates, 5% L. pentosus isolates, 3% L. casei subsp. pseudoplantarum isolates, and 1% L. rhamnosus isolates. Eleven percent of the isolates were not phenotypically identified. Although a randomly amplified polymorphic DNA (RAPD) analysis based on three primers and clustering by the unweighted pair group method with arithmetic average (UPGMA) was useful for partially differentiating the 10 type strains, it did not provide a species-specific DNA band or a combination of bands which permitted complete separation of all the species considered. In contrast, sodium dodecyl sulfate-polyacrylamide gel electrophoresis cell wall protein profiles clustered by UPGMA were species specific and resolved the NSLAB. The only exceptions were isolates phenotypically identified as L. plantarum and L. pentosus or as L. casei subsp. casei and L. paracasei subsp. paracasei, which were grouped together. Based on protein profiles, Italian ewe cheeses frequently contained four different species and 3 to 16 strains. In general, the cheeses produced from raw ewe milk contained a larger number of more diverse strains than the cheeses produced from pasteurized milk. The same cheese produced in different factories contained different species, as well as strains that belonged to the same species but grouped in different RAPD clusters.     

Biodiversity of Bacterial Ecosystems in Traditional Egyptian Domiati Cheese

Bacterial biodiversity occurring in traditional Egyptian soft Domiati cheese was studied by PCR-temporal temperature gel electrophoresis (TTGE) and PCR-denaturing gradient gel electrophoresis (DGGE). Bands were identified using a reference species database (J.-C. Ogier et al., Appl. Environ. Microbiol. 70:5628-5643, 2004); de novo bands having nonidentified migration patterns were identified by DNA sequencing. Results reveal a novel bacterial profile and extensive bacterial biodiversity in Domiati cheeses, as reflected by the numerous bands present in TTGE and DGGE patterns. The dominant lactic acid bacteria (LAB) identified were as follows: Leuconostoc mesenteroides, Lactococcus garvieae, Aerococcus viridans, Lactobacillus versmoldensis, Pediococcus inopinatus, and Lactococcus lactis. Frequent non-LAB species included numerous coagulase-negative staphylococci, Vibrio spp., Kocuria rhizophila, Kocuria kristinae, Kocuria halotolerans, Arthrobacter spp./Brachybacterium tyrofermentans. This is the first time that the majority of these species has been identified in Domiati cheese. Nearly all the dominant and frequent bacterial species are salt tolerant, and several correspond to known marine bacteria. As Domiati cheese contains 5.4 to 9.5% NaCl, we suggest that these bacteria are likely to have an important role in the ripening process. This first systematic study of the microbial composition of Domiati cheeses reveals great biodiversity and evokes a role for marine bacteria in determining cheese type.