Penicillium Roqueforti Research Articles

Yellow mombin and jackfruit seeds residues applied in the production of reducing sugars by a crude multi‐enzymatic extract produced by Penicillium roqueforti ATCC 101110

As an alternative to the use of widely investigated agro-industrial residues, the present study aimed to promote the valorization of two selected residues, yellow mombin seed (YS) and jackfruit seed (JS), as a result of their enhanced performance. YS was applied as a solid state substrate for Penicillium roqueforti ATCC 101110 cultivation (25 °C, Aw = 0.963, 107 spores g-1 and 142 h) to produce a crude multi-enzymatic extract (CE-YS) containing activities of CMCase = 31.95 U g-1 , xylanase = 56.85 U g-1 , exoglucanase = 5.55 U g-1 and FPase = 24.60 U g-1 . CE-YS was then applied to six different residues saccharification and the best performance was obtained with jackfruit seed residue (JS), which was selected for enzymatic saccharification. The highest productivity of reducing sugars expressed as glucose (6.26 mg g-1 h-1 ) was obtained under the conditions: 40.7 g L-1 JS, 5 mmol L-1 MgCl2 , 65 °C, 120 rpm, pH 3.0 (citrate buffer 50 mmol L-1 ) and 18 h. The residues, YS and JS, can be used satisfactorily for the production of bioproducts of great industrial applicability, such as crude extracts (containing cellulolytic enzymes) and RS (which can be converted, for example, into bioethanol). © 2020 Society of Chemical Industry.

Major components in Lilac and Litsea cubeba essential oils kill Penicillium roqueforti through mitochondrial apoptosis pathway

Agriculture products spoilage caused by mold not only causes huge economic losses, but also poses a safety hazard to human health. Although essential oils (EOs) are widely used for agriculture products antisepsis, little is known about the synergistic antifungal mechanisms of their major components. In this paper, the synergistic mechanism of eugenol and citral (SEC) on Penicillium roqueforti was studied. The results showed that SEC caused irreversible damage to the cell membrane of P. roqueforti, resulting in significant changes in membrane protein conformation and fatty acid content, among which citral had a greater effect on fatty acid content than eugenol. Through the analysis of mitochondrial morphology and membrane potential, it was found that SEC treatment resulted in serious collapse of mitochondria and significant increase of membrane potential. In this process, the contribution rate of eugenol was higher than that of citral. Further analysis of the tricarboxylic acid cycle (TCA) pathway showed that SEC could inhibit the activity of key enzymes and the production of energy charge in the TCA pathway, resulting in significant down-regulation of gene expression of three key molecules (succinate dehydrogenase, isocitrate dehydrogenase and fumarate hydratase). In conclusion, these results indicated that SEC could kill P. roqueforti through the tricarboxylic acid cycle.

Analysis of the synergistic antifungal mechanism of eugenol and citral

The synergistic effects of different essential oils can not only reduce their dosage and cost, but also improve their biological activity. The aim of this study was to evaluate the synergistic antifungal effect of eugenol and citral (SEC) on Penicillium roqueforti. Determination of reactive oxygen species (ROS) and malondialdehyde content indicated that SEC could induce membrane lipid peroxidation, and the contribution of citral was greater than that of eugenol. In addition, downregulation of the gene encoding an NADPH oxidase subunit indicated that the ROS burst induced by SEC was mediated by NADPH oxidase. Scanning electron microscope (SEM) and transmission electron microscope (TEM), propidium iodide treatment, DNA gel electrophoresis and cell leakage experiments showed that SEC destroyed the integrity of the cell membrane and internal structures, and degraded the cell contents, and the contribution of eugenol was greater than that of citral. These results showed that SEC inhibited the activity of P. roqueforti more effectively than eugenol or citral alone, by inducing membrane lipid peroxidation and promoting the ability of eugenol to destroy the cell membrane. The combined agents eventually caused leakage of the cell contents, and ultimately cell death.

Independent domestication events in the blue-cheese fungus Penicillium roqueforti.

Domestication provides an excellent framework for studying adaptive divergence. Using population genomics and phenotypic assays, we reconstructed the domestication history of the blue cheese mould Penicillium roqueforti. We showed that this fungus was domesticated twice independently. The population used in Roquefort originated from an old domestication event associated with weak bottlenecks and exhibited traits beneficial for pre‐industrial cheese production (slower growth in cheese and greater spore production on bread, the traditional multiplication medium). The other cheese population originated more recently from the selection of a single clonal lineage, was associated with all types of blue cheese worldwide except Roquefort, and displayed phenotypes more suited for industrial cheese production (high lipolytic activity, efficient cheese cavity colonization ability and salt tolerance). We detected genomic regions affected by recent positive selection and putative horizontal gene transfers. This study sheds light on the processes of rapid adaptation and raises questions about genetic resource conservation.

Synergistic properties of citral and eugenol for the inactivation of foodborne molds in vitro and on bread

To control the spoilage of bread products and reduce the costs associated with preventing their spoilage, the synergistic antifungal effects of natural agents against the main bread spoilage fungi Penicillium roqueforti and Aspergillus niger were studied. After screening 66 groups antifungals using the checkerboard method, only one combination, i.e., that of eugenol and citral (SEC), showed synergistic effects against both the fungi. Their average minimal inhibitory concentrations (MICs) decreased significantly when they were present as a combination, i.e., by 3.40-fold, compared to the case when each agent was used separately. SEC inhibited the spore germination of both the fungi by more than 95% at 2 × MIC. At the same time, their mycelial growth was almost completely inhibited. The morphology of the mycelia was further observed by SEM. Serious wrinkles and deformations were found on the surface of the mycelia at 2 × MIC. Finally, the shelf life tests showed that treatment with SEC significantly prolonged the molding time of bread. These results showed that SEC may serve as a spoilage control measure and reduce the costs associated with preventing bread spoilage.

Antimicrobial and antioxidant activities of natural and fermented bee pollen

Fermentation of various food products leads to better nutritional value, improved biological activities, enhanced extraction properties, etc. The impact of artificial solid-state lactic acid fermentation on bee pollen properties has not been fully studied. The aim of this study was to evaluate antibacterial, antifungal and antioxidant activities of natural and pasteurized polyfloral bee pollen after spontaneous fermentation and fermentation with added Lactococcus lactis and Lactobacillus rhamnosus bacteria. Total phenolic content, total flavonoid content and radical scavenging activity increased by 1.27–2.40 times, antibacterial activity against Micrococcus luteus, Staphylococcus aureus and Escherichia coli increased by 1.08–16.9 times and antifungal activity against Penicillium roqueforti increased by 1.96–5.52 times after bee pollen fermentation. Chemometric analysis, including a similarity measure (based on Euclidean distance) estimation and self-organizing maps, were used to determine the impact of different fermentations and pasteurization on bee pollen's antioxidant and antimicrobial properties. The analysis showed that the natural microflora of the bee pollen contributes to the fermentation process together with the added bacteria, therefore pasteurized bee pollen showed significantly lower antimicrobial and antioxidant activities after fermentation than fermented natural bee pollen. Overall, the fermentation had a positive effect on bee pollen biological activities, the improvements being dependent on the botanical origin of the pollen and fermentation type (spontaneous or with added bacteria).

Occurrence of filamentous fungi isolated from matured blue cheese

Abstract Matured blue cheese can be made from cow's milk associated with the inoculated fungus Penicillium roqueforti, which guarantees specific sensorial characteristics. Recently, Brazil’s fine cheese production and consumption have increased by more than 200%, highlighting the relevance of microbiological quality control of these products. Fungal contaminations are responsible for significant losses in cheese production and provide a suitable environment for mycotoxins production, constituting a hazard to public health. In this work, we evaluated the mycological contamination profile of matured blue cheeses commercialized in Brazil. Samples of ten different brands were analyzed by serial dilution method, by plating in the Dicloran Rose of Bengal Chloramphenicol (DRBC) culture medium and Dicloran Glycerol Medium Base (DG18). Subsequently, different fungi morphotypes were isolated and morphologically identified. As a result, 461 fungi were isolated and identified, notably Aspergillus aculeatus, Penicillium roqueforti and Penicillium solitum. All samples were contaminated by filamentous fungi, amongst those, many already reported as mycotoxin producers, which underlines the relevance of microbiological monitoring.

Roquefornine A, a sesterterpenoid with a 5/6/5/5/6-fused ring system from the fungus Penicillium roqueforti YJ-14

Roquefornine A, a sesterterpenoid with an unprecedented 5/6/5/5/6-membered pentacyclic system, was characterized from Penicillium roqueforti YJ-14.

Occurrence of filamentous fungi and mycotoxins in wrapped forages in Sweden and Norway and their relation to chemical composition and management

AbstractA study was conducted on 124 farms in Sweden and Norway where fungal (all farms) and mycotoxin (100 of the 124 farms) presence was examined in wrapped forage bales. Samples were also analysed for chemical composition, and data on the bale production and storage system on each farm were collected. Fungi, analysed by three common sampling methods, were found in bales from 89% of the visited farms (110 farms). The most frequently isolated fungal species from the bale surface was Penicillium roqueforti. Mycotoxins were present in 39% of the 100 samples analysed. The most common mycotoxins present were enniatin B (14 farms) followed by deoxynivalenol (12 farms). The risk of finding fungi in baled forage samples was higher with odds ratio (OR) of 5.1 when less than eight layers of stretch film were applied, low seal integrity of wrapping (OR 172 at <10 s of seal integrity), higher dry‐matter content (OR 1.17–1.56 per % unit DM) and higher concentrations of acetic acid (OR 47.5 per g DM) and ethanol (OR 3.4 per g DM). Mycotoxin presence was not found to be correlated with any of the chemical characteristics of the baled forage, or with any forage production and management factors. A positive correlation was present between total fungal counts and general mycotoxin presence, but not between specific mycotoxins and the toxin‐producing fungal species.

Novel antimicrobial and antioxidative activity by endophytic Penicillium roqueforti and Trichoderma reesei isolated from Solanum surattense

Crop protection against phyto-pathogens has become a global challenge that can be tackled efficiently through natural resources, including endophytic fungi. Endophytes serve as a reservoir for the vast array of potent bioactive metabolites. We investigated the antioxidant and antibacterial potency of endophytes from the roots of Solanum surattense. The non-polar fraction of the cultural filtrate from the isolated strains was tried for antibacterial potency through agar plate diffusion assay. Among the isolated strains, Penicillium roqueforti (CGF-1) and Trichoderma reesei (CGF-11) had broad-spectrum antibacterial activity against phyto-pathogenic bacteria (Xanthomonas oryzae, Pseudomonas syringae, Agrobacterium tumefaciens, and Ralstonia solanacearum). The extracts of CGF-1 and CGF-11 achieved the best result against A. tumefaciens. Similarly, qualitative analysis of the ethyl acetate extracts P. roqueforti and T. reesei exposed the occurrence of alkaloids, flavonoids, phenols, steroids, and tannins. HPLC analysis also confirmed the presence ferulic acid, cinnamic acid, quercetin, and rutin in the non-polar fraction of the cultural filtrate from the isolated strains. The results conclude that P. roqueforti and T. reesei can play an active role against the plant pathogens by secreting the bioactive compounds to protect host plant. Furthermore, the antibacterial and antioxidant potential of the P. roqueforti and T. reesei suggests its use in agriculture and pharmaceutical industry.

Application of MALDI-TOF MS to species complex differentiation and strain typing of food related fungi: Case studies with Aspergillus section Flavi species and Penicillium roqueforti isolates

Filamentous fungi are one of the main causes of food losses worldwide and their ability to produce mycotoxins represents a hazard for human health. Their correct and rapid identification is thus crucial to manage food safety. In recent years, MALDI-TOF emerged as a rapid and reliable tool for fungi identification and was applied to typing of bacteria and yeasts, but few studies focused on filamentous fungal species complex differentiation and typing. Therefore, the aim of this study was to evaluate the use of MALDI-TOF to identify species of the Aspergillus section Flavi, and to differentiate Penicillium roqueforti isolates from three distinct genetic populations. Spectra were acquired from 23 Aspergillus species and integrated into a database for which cross-validation led to more than 99% of correctly attributed spectra. For P. roqueforti, spectra were acquired from 63 strains and a two-step calibration procedure was applied before database construction. Cross-validation and external validation respectively led to 94% and 95% of spectra attributed to the right population. Results obtained here suggested very good agreement between spectral and genetic data analysis for both Aspergillus species and P. roqueforti, demonstrating MALDI-TOF applicability as a fast and easy alternative to molecular techniques for species complex differentiation and strain typing of filamentous fungi.

Simple microencapsulation of plant essential oil in porous starch granules: Adsorption kinetics and antibacterial activity evaluation

In this work, corn porous starch was used as a food grade polymer to microencapsulate essential oil (EO), the effects of temperature on adsorption of EO were investigated, and adsorption kinetics curves were plotted. Using this approach, the sustained-release antibacterial effects of porous starch microcapsules containing EOs (PSM-EOs) against Penicillium roqueforti and Aspergillus niger were explored under accelerated volatilization under high temperature conditions, and microencapsulation was successfully applied to bread preservation. The results showed that temperature had a significant effect on the adsorption capacity, and adsorption rate was controlled by both liquid film diffusion and intraparticle diffusion. The activation energy (Ea) of the adsorption process was 7.96 K J/mol as calculated using the Arrhenius equation. The results of antibacterial experiments showed that PSM-EOs exhibited superior sustained-release antibacterial effects and significantly prolonged the shelf life of bread, compared with controls. Practical applications The spoilage caused by mold contamination is a serious challenge for the bread industry. At present, the main preservative used in baking products is calcium propionate, a chemical preservative. However, calcium propionate can be decomposed to produce propionic acid under the action of intestinal microorganisms. Propionic acid is an important factor in inducing diabetes mellitus. Therefore, it is necessary to explore new preservation techniques rather than bread with calcium propionate. In this paper, porous starch microcapsules containing essential oil were prepared, and the adsorption kinetics curve of porous starch to essential oil was plotted. On this basis, the inhibition effect of porous starch microcapsules containing essential oil in high temperature accelerated volatilization on the main mold (P. roqueforti and A. niger) causing bread corruption was investigated. The aim is to provide new ideas for bread preservation.

Spoilage fungi in a bread factory in Brazil: Diversity and incidence through the bread-making process.

This study aimed to verify the main fungal species involved in the deterioration of different types of bread and to identify the possible sources of contamination of these products. Samples of raw materials (n = 127), environmental air (n = 50) and moldy bread (n = 90) were analyzed. Aspergillus candidus, Wallemia sebi, and Penicillium roqueforti were the predominant species in the raw materials and were isolated in samples of wheat flour, in two-thirds of the samples of rye and 62.5% of the wheat flour. Penicillium roqueforti was isolated from all types of moldy bread analyzed and Hyphopichia burtoni was also present in samples of moldy wheat and rye bread. These two species were also recovered during air sampling from baking industry facilities (cooling and slice and package areas), which may be crucial for product contamination after baking. Hygienic measures to reduce airborne contamination during the cooling and packaging of food should be taken to prevent the early deterioration of bread.

Tyrosine Induced Metabolome Alterations of Penicillium roqueforti and Quantitation of Secondary Key Metabolites in Blue-Mold Cheese.

To map qualitative and quantitative metabolome alterations when Penicillium roqueforti is grown in an environment where l-tyrosine levels are perturbed, the recently established differential off-line LC-NMR (DOLC-NMR) approach was successfully applied in connection with an absolute metabolite quantitation using a quantitative 1H NMR protocol following the ERETIC 2 (Electronic REference To access In vivo Concentrations) methodology. Among the 23 influenced metabolites, amino acid degradation products like 2-(4-hydroxyphenyl)acetic acid and 2-(3,4-dihydroxyphenyl)acetic acid underwent a tremendous upregulation in the amino acid perturbed approach. Moreover, the output of secondary metabolites like andrastin A, eremofortin B, and the tetrapeptide d-Phe-l-Val-d-Val-l-Tyr was affected in the case of the presence or absence of the added aromatic amino acid. Furthermore, the isolated secondary metabolites of P. roqueforti have been quantified for the first time in five divergent Penicillium isolates by means of a validated LC-ECHO-MS/MS method. This technique is used to compensate the effect of co-extracted matrix compounds during the analysis and to utilize quasi-internal standards to quantify all metabolites of interest accurately. This screening outlined the great variety between the different fungi of the same species. The metabolite spectra of wild-type fungi included more toxic intermediates compared to a selected fungi used as a starter culture for blue-mold cheese production. In addition, these secondary metabolites were quantified in commercially available white- and blue-mold cheese samples. The main differences between the analyte profiles of white and blue cheeses were linked to the impact of the used starter culture. Specific metabolites detected from P. roqueforti like andrastin A and B or roquefortine C could not be detected in white cheese. Among the blue cheese samples, different metabolite pattern could be observed regarding various P. roqueforti starter cultures.

Proteolysis in Danish blue cheese during ripening

Proteolysis in Danish blue cheese was studied during 9 weeks' ripening. Levels of pH 4.6-soluble N as a percentage of total N increased from 7.2% to 25%, indicating extensive proteolysis. Urea-polyacrylamide gel electrophoretograms confirmed the extent of proteolysis through chymosin and plasmin action early in ripening, but later the action of Penicillium roqueforti proteinases became apparent. The proteolytic specificity of Penicillium roqueforti PR-R proteinases on αS1- and β-casein was determined in a model system. Regions most susceptible to proteinase action in αS1-casein were 6–40, 69–99, 124–147 and 155–199, with a total of 91 cleavage sites identified; regions in β-casein susceptible to proteolysis were 43–87, 101–119, 161–185 and 192–209 with a total of 118 cleavage sites identified. A large number of peptides was identified cheese extracts during 9 weeks ripening, principally from αS1-casein regions 1–40, 105–136 and 150–176 and β-casein regions 6–14, 46–68, 101–140 and 193–209.

‘Lipase and protease production of dairy Penicillium sp. on milk‐protein‐based solid substrates’

The lipolytic and proteolytic activity of Penicillium camemberti PC TT033 and Penicillium roqueforti PR G3, cultured on the whey solids or simulated cheese media, were compared under several pH reaction conditions. Lipolytic activity was higher when both strains had been cultured on the whey medium than on the simulated cheese medium, whereas proteolytic activity was less influenced by the culture medium. The relationship between the reaction pH and these enzyme activities was dependent on the culture medium, which suggested that the expression level and balance of isozyme rely on the culture substrate.

Antifungal activity of commercial sanitizers against strains of Penicillium roqueforti, Penicillium paneum, Hyphopichia burtonii, and Aspergillus pseudoglaucus: Bakery spoilage fungi

Information on the sensitivity of spoilage fungi of bakery products to sanitizing agents is scarce in the literature. Thus, the aim of this study was to evaluate the antifungal activity of different classes of commercial sanitizers, which have permitted use in the food industry, on the main fungi involved in spoiling bakery products. The tests were carried out according to the protocol for testing the antifungal effect of chemical sanitizers of the European Committee for Standardization (CEN), with adaptations. Different strains of six isolated fungal species responsible for spoiling bakery products (Penicillium roqueforti, Penicillium paneum, Hyphopichia burtonii, and Aspergillus pseudoglaucus) were tested against five sanitizers at three concentrations: benzalkonium chloride (0.3%, 2.5%, 5%), biguanide (2%, 3.5%, 5%), peracetic acid (0.15%, 1.5%, 3%), quaternary ammonium (0.3%, 2.5%, 5%), and sodium hypochlorite (0.01%, 0.1%, 0.2%). Peracetic acid was the most effective sanitizes considering the genera, species, and concentrations evaluated, generally being capable of reductions between 2 and 4 logs of initial control tested. Biguanide should not be the compound of choice when the main goal of the bakery industry is fungal control.

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.

Pesticides in agriculture

Pesticides in agriculture

Lemna gibba and Eichhornia crassipes extracts: Clean alternatives for deacidification, antioxidation and fungicidal treatment of historical paper

In this research Lemna gibba (L) and Eichhornia crassipes (E) ethanolic extracts were added to pure cellulose substrate to produce impregnated interleaving papers (LIP) and (EIP). These products are proposed to cure historical paper undergoing acidification, oxidation and fungal infection. The extracts were analysed using Gas Chromatography/Mass Spectrometer (GC/MS) and polyphenolic compounds were assessed. The products were tested on model decayed paper showing identical characteristics of historical paper, on which pH values were measured, transition metals concentrations were evaluated using Inductively Coupled Plasma- Optical Emission Spectrometry (ICP-OES) and samples were investigated using Scanning Electron Microscope combined with Energy Dispersive Spectroscopy (SEM-EDX) respectively. Radical scavenging and antioxidation activities of the extracts were also assessed using 1,1-diphenyl-2-picryl-hydrazyl free radical DPPH·. Results demonstrated a significant increase (P < 0.01) in the DPPH· free radical scavenging with increasing concentration in the following order vitamin C > BHT ≈ (L) extract > (E) extract. Results showed also that both (LIP) and (EIP) products effectively neutralized the acidity of decayed paper after 7 day treatment and evidently chelated transition metals. Zn2+ and Fe2+ were better chelated by (LIP) treatment in the order; Zn2+ > Fe2+ > Pb2+ > Cu2+, while Pb2+ and Cu2+ were greater removed by (EIP) treatment in the order Pb2+ > Cu2+ > Zn2+ > Fe2+. Up to 90% radical scavenging was achieved at 320.00 μg/mL concentration of (L) extract and 667.296 μg/mL concentration of (E) extracts. Aspergillus niger, Penicillium roqueforti and Eurotium chevalieri growth are inhibited at 1000 μg/mL concentration of LIP with considerable fungal spots elimination. Our findings established LIP and EIP products as novel natural origin alternative for effective synergic deacidification, antioxidation, antiradical, metal chelation and fungicidal treatment of historical paper.