Immunosuppressive effects of the mycotoxin patulin in macrophages.

Patulin (PAT) is a fungi-derived secondary metabolite produced by numerous fungal species, especially within Aspergillus, Byssochlamys, and Penicillium genera, amongst which P. expansum is the foremost producer. Similar to other fungi-derived metabolites, PAT has been shown to have diverse biological features. Initially, PAT was used as an effective antimicrobial agent against Gram-negative and Gram-positive bacteria. Then, PAT has been shown to possess immunosuppressive properties encompassing humoral and cellular immune response, immune cell function and activation, phagocytosis, nitric oxide and reactive oxygen species production, cytokine release, and nuclear factor-κB and mitogen-activated protein kinases activation. Macrophages are a heterogeneous population of immune cells widely distributed throughout organs and connective tissue. The chief function of macrophages is to engulf and destroy foreign bodies through phagocytosis; this ability was fundamental to his discovery. However, macrophages play other well-established roles in immunity. Thus, considering the central role of macrophages in the immune response, we review the immunosuppressive effects of PAT in macrophages and provide the possible mechanisms of action.

Brazilian Table Olives: A Source of Lactic Acid Bacteria with Antimycotoxigenic and Antifungal Activity.

Food and feed contamination by fungi, especially by toxigenic ones, is a global concern because it can pose serious health problems when the production of mycotoxins is involved. Lactic acid bacteria (LAB), well-known for fermenting foods, have been gaining attention for their antifungal and anti-mycotoxin properties. This work tested 14 LAB strains isolated from naturally fermented Brazilian table olives for growth inhibition of Aspergillus flavus, Aspergillus carbonarius, Penicillium nordicum, and Penicillium expansum. The strains Lacticaseibacillus paracasei subsp. paracasei CCMA 1764, Levilactobacillus brevis CCMA 1762, and Lactiplantibacillus pentosus CCMA 1768 showed the strongest antifungal activity, being more active against P. expansum. Aflatoxin B1 (AFB1), ochratoxin A (OTA), and patulin (PAT) production was reduced essentially by mycelia growth inhibition. The main organic acids detected in the cell free supernatant (CFS) were lactic and acetic acids. Tested LAB exhibited adsorption capacity against AFB1 (48-51%), OTA (28-33%), and PAT (23-24%). AFB1 was converted into aflatoxin B2a (AFB2a) by lactic and acetic acids produced by the strain CCMA 1764. A similar conversion was observed in solutions of these organic acids (0.1 M). These findings demonstrate the potential of isolated LAB strains as natural agents to control toxigenic fungi and their mycotoxins in fermented products, such as table olives.

Patulin in tomatoes and the inhibitory capacity of their phenolic extracts against Penicillium expansum.

This study aimed to determine Patulin (PAT) in samples of organically and conventionally grown tomato varieties, to correlate it with their phenolic profile and at evaluating effects of phenolic extracts of tomato samples against the Penicillium expansum CCT 7549 strain. Four varieties of tomatoes (Cherry, Khaki, Italian and Long Life) subject to conventional and organic management were collected in markets in the south of Brazil. PAT was determined in samples by validated method Matrix Solid Phase Dispersion (MSPD) with detection in HPLC-PDA. Titratable acidity, phenolic profile and antifungal activity (minimum inhibitory concentration - MIC and minimum fungicidal concentration - MFC) against P. expansum were determined. In most tomatoes PAT was found at levels that were below the Maximum Recommended Level (MRL = 50 µg/kg). Samples of conventionally grown Cherry tomato showed high acidity and PAT contamination, whose correlation was confirmed by the Principal Component Analysis (PCA). Phenolic extracts of tomatoes subject to conventional management were more efficient to inhibit P. expansum, a fact that may be explained by the content of caffeic acid. However, since PAT production was not inhibited in fruit, it is recommended that PAT should be monitored and a new management strategy should be investigated.

Diversity and metabolomic characterization of Penicillium expansum isolated from apples grown in Argentina and Spain.

Apples (Malus domestica) are one of the most consumed fruits globally. It is a relevant crop in Argentina and Spain, and one of the main fruits for export and industrialization in these countries. Quality control of apples, fundamentally in the postharvest stage, is critical to prevent fungal diseases. The blue mould, caused by Penicillium expansum, is responsible for great economic losses due to the deterioration of the fruit and mycotoxin production. Many studies have characterized this pathogen; however, little is known about the differences between populations from distant geographical origins. The objective of the present study was to characterize two P. expansum populations, from Argentina and Spain, through morphological, metabolomic and molecular approaches, and to evaluate the existence of differences related to their geographical source. A total of 103 isolates, 53 from Argentina and 50 from Spain were studied. Their morphological features were consistent with the species description. The secondary metabolite profiles revealed low chemical diversity. All 103 isolates shared the production of 13 compounds, namely andrastins, aurantioclavine, chaetoglobosins, communesins, expansolides, roquefortine C and patulin. Penostatins and citrinin were produced by 102 and 101 isolates, respectively. A region of the ß-tubulin gene was selected to analyse the diversity of the P. expansum isolates. No substantial differences were observed between isolates of different geographical origins through morphology, patulin accumulation, secondary metabolite profiles and phylogenetic analysis. However, the analysis of polymorphisms revealed 29 haplotypes with a relative separation between isolates of both populations; 13 haplotypes contained Argentinean isolates, while Spanish isolates were separated into 16 haplotypes. The diversity indices of Shannon (H'=2.075; H'=2.402) and Simpson (SiD = 0.850; SiD = 0.895) for isolates from Argentina and Spain, respectively, indicated that the diversity of P. expansum is greater in Spain than in Argentina. This distribution could be explained both by the existence of haplotype exchange between both countries, with the ancestral haplotypes originating in Spain, and the subsequent adaptation to the environmental conditions or apples varieties grown in each region.

Fungal growth, patulin accumulation and volatile profile in 'Fuji Mishima' apples under controlled atmosphere and dynamic controlled atmosphere.

The objective of this study was to evaluate fungal and patulin contamination, together with its correlation with the volatile compounds (VCs), in 'Fuji Mishima' apples (up to 25% decayed) under controlled atmosphere (CA) and dynamic controlled atmosphere with respiratory quotient (DCA-RQ) of 1.3 combined with different partial pressures of carbon dioxide (0.8, 1.2, 1.6 and 2.0 pCO2). Fruits were stored under the above conditions for 8 months at 0.5 °C plus 7 days shelf life at 20 °C. Toxigenic fungi and patulin accumulation were found in apples from all treatments. Penicillium expansum was the most prevalent species. For all storage conditions, patulin concentrations were above the maximum level allowed in Brazil (50  µg  kg-1) with an exception of DCA-RQ1.3 + 0.8 kPa CO2. This condition, with lower pCO2, showed the lowest patulin accumulation, below the legal limit. The CA provided the highest patulin concentration (166 µg  kg-1). It was observed that fungal growth could also contribute to changes in the volatile composition. Styrene and 3-methyl-1-butanol are considered P. expansum markers in some apple cultivars and were detected in the samples. However, it was not possible to identify volatile organic compounds (VOCs) that are biomarkers from P. expansum, because there were other fungi species present in all samples. In this study, styrene, n-decanoic acid, toluene, phenol and alpha-farnesene were the compounds that showed the most positive correlation with patulin accumulation. On the other hand, a negative correlation of patulin with acids has been shown, indicating that in treatments with a higher patulin concentration there were less acidic compounds.

Laser ablation electrospray ionization mass spectrometry imaging as a new tool for accessing patulin diffusion in mold-infected fruits.

This work describes the use of laser ablation electrospray ionization mass spectrometry imaging (LAESI imaging) to investigate the diffusion of the mycotoxin patulin from rotten to healthy areas of fruits. Slices of mold-infected and uninfected (control) apples and strawberries were prepared, and this was the only sample preparation step used. An infrared laser beam (2.94 µm) was used to irradiate the slices, resulting in the ablation of sample compounds directly ionized by electrospray and analyzed by mass spectrometry. Multivariate curve resolution - alternating least squares was applied in unfolded LAESI images to obtain relative quantity information. Patulin was not detected in the control samples but was seen in all mold-infected fruits. LAESI images showed the diffusion of patulin from the rotten area to unaffected parts of the fruits. This study points out the advantage of LAESI imaging over traditional analytical methods used to study the diffusion of mycotoxins in fruits.

Patulin inhibits LPS-induced nitric oxide production by suppressing MAPKs signaling pathway.

Patulin (PAT) is a natural product isolated from several species of fungi. Here, we evaluated the effect of PAT (62.5-4,000 ng/ml) in lipopolysaccharide (LPS)-activated murine peritoneal macrophages. Cell viability assay showed that PAT at concentrations up to 250 ng/ml did not affect macrophage viability. PAT (250 ng/ml) significantly reduced LPS-induced nitric oxide production (by 98.4%), inducible nitric oxide synthase (iNOS) expression (by 83.5%), and iNOS messenger ribonucleic acid expression (by 100.0%). Moreover, PAT significantly reduced LPS-induced interleukin-1ß (by 80.6%), cluster of differentiation (CD) 69 (by 63.1%), and Toll-like receptor (TLR) 4 (by 91.9%) protein expression. Finally, PAT significantly reduced LPS-triggered phosphorylation of all mitogen-activated protein kinases (MAPK) assessed: extracellular signal-regulated kinase (ERK; by 89.5%), c-Jun N-terminal kinase (JNK; by 77.5%), and p38 (by 72.3%). Taken together, these data suggest that PAT downregulates acute inflammatory response, inhibiting nitric oxide production by suppressing CD69-TLR4/ERK-JNK-p38 MAPKs/Nos2/iNOS signaling pathway.

Development and validation of an extraction method using liquid chromatography-tandem mass spectrometry to determine patulin in apple juice.

Patulin (PAT) is a potent mycotoxin commonly found in apples and apple-based products such as juice, thus affecting global food safety. Therefore, development of fast and simple analytical methods to effectively control its contamination is of great importance. This study developed and validated a technique for the analysis of PAT in samples of industrialized apple juice based on liquid-liquid extraction and using acetonitrile as the extraction solvent. Detection via mass spectrometry was performed after Atmospheric Pressure Chemical Ionization (APCI). Mean recoveries of 97.5, 92.49 and 96.92% were reached for 4, 8 and 20 µg/L of PAT, respectively. The analyte was monitored with an APCI source in negative ion mode to identify its fragments. The 24 analyzed samples presented PAT levels below the limit of quantification. It may be concluded that the method fulfilled all of the validation criteria, thus being appropriate for routine surveillance of PAT in apple juice.

Kosakonia radicincitans and Cryptococcus laurentii controlled Penicillium expansum rot and decreased patulin production at 4 and 25 °C.

In the present work, we evaluated the effects of a mixture of biocontrol agents against two toxigenic strains of Penicillium expansum isolated in Argentine Patagonia from pome fruits. The two strains, INTA-5 and INTA-10, were previusly selected among ten strains coming from the Alto Valle (Rio Negro-Argentina) for their high production of patulin. For the biocontrol, Kosakonia radicincitans, Cryptococcus laurentii, and Rhodosporidium fluviale were tested in vitro experiments on Potato Dextrose Agar (PDA) dishes against the INTA-5 and INTA-10 strains. The bacterium K. radicincitans and the yeast C. laurentii were selected to be used in a mixture due to their capacity to control the fungus and reduce the mycotoxin severely. In vitro assays with the mixture showed a high antagonism against P. expansum INTA-5 and INTA-10, at 21 d of incubation at 25 °C and a patulin reduction of 98%. The mixture of microorganisms was also effective in apples stored at 25 °C for 10 d and 4 °C for 30 d. At cold storage, the mixture controlled moderately the development of rot and decreased patulin concentration. At 25 °C, the pathogen's optimal growth temperature, the mixture of Biological Control Agent (BCAs) assured both the control of rot and decrease of patulin concentration. The combination of two microorganisms, with different requirements and abilities, resulted in a mix with a strong antagonism against P. expansum with the capability to decrease the patulin concentration. Treatment with the selected mixture could be a good option for controlling strains with different behaviours and in different environmental conditions.

Distinct Transcriptional Changes in Response to Patulin Underlie Toxin Biosorption Differences in Saccharomyces Cerevisiae.

Patulin (4-hydroxy-4H-furo[3,2c]pyran-2[6H]-one) is a mycotoxin produced by a suite of fungi species. Patulin is toxic to humans and is a sporadic contaminant in products that were made from fungi-infected fruits. The baker yeast Saccharomyces cerevisiae (S. cerevisiae) has been shown to decrease patulin levels likely by converting it to the less harmful E-ascladiol, yet this capacity is dependent on the strain utilized. In this study we show that four representative strains of different S. cerevisiae lineages differ in their ability to tolerate and decrease patulin levels in solution, demonstrating that some strains are better suitable for patulin biocontrol. Indeed, we tested the biocontrol capacities of the best patulin-reducer strain (WE) in contaminated apple juice and demonstrated their potential role as an efficient natural biocontrol solution. To investigate the mechanisms behind the differences between strains, we explored transcriptomic changes of the top (WE strain) and worst (WA strain) patulin-biocontroller strains after being exposed to this toxin. Large and significant gene expression differences were found between these two strains, the majority of which represented genes associated with protein biosynthesis, cell wall composition and redox homeostasis. Interestingly, the WE isolate exhibited an overrepresentation of up-regulated genes involved in membrane components, suggesting an active role of the membrane towards patulin detoxification. In contrast, WA upregulated genes were associated with RNA metabolism and ribosome biogenesis, suggesting a patulin impact upon transcription and translation activity. These results suggest that different genotypes of S. cerevisiae encounter different stresses from patulin toxicity and that different rates of detoxification of this toxin might be related with the plasma membrane composition. Altogether, our data demonstrates the different molecular mechanisms in S. cerevisiae strains withstanding patulin exposure and opens new avenues for the selection of new patulin biocontroller strains.

Patulin accumulation in apples under dynamic controlled atmosphere storage.

The goal of this study was to evaluate patulin contamination in 'Galaxy' and 'Fuji Kiku' apples subjected to controlled atmosphere (CA) and dynamic controlled atmosphere (DCA) conditions. Experiments were performed and fruit were stored for nine months under refrigeration plus 7 days shelf life at 20 °C. CA and DCA were not effective in preventing patulin production in either 'Galaxy' or 'Fuji Kiku' apples. Healthy fruit were not contaminated with patulin, even when stored together with decayed apples. For 'Galaxy' apples, application of 1-methylcyclopropene increased the percentage of fruit with decay and patulin contamination. Patulin concentrations were above the maximum limit (50 µg kg-1) established in the Brazilian legislation, meaning the use of CA and DCA conditions were not advantageous in preventing patulin accumulation. In 'Fuji Kiku' apples, there was no significant difference in patulin concentration among CA, DCA-CF and DCA-RQ 1.3 treatments, and all were below the maximum.

Effects of patulin and ascladiol on porcine intestinal mucosa: An ex vivo approach.

Patulin (PAT) is a secondary metabolite mainly produced by Aspergillus and Penicillium that is frequently found contaminating apples and rotten fruits. Patulin can be transformed in potencially less toxic compounds such as ascladiol (ASC). Toxic effects of patulin were described in rats and in in vitro models, however concerning ascladiol, data are restricted to metabolic pathways. The aim of the present study was to evaluate the effects of different concentrations of PAT (10 µM, 30 µM, 100 µM) and ASC (30 µM, 100 µM) on intestinal tissue using the jejunal explant model. Explants from pigs were exposed for 4 h to PAT and ASC and after this period were processed for histological, morphometrical and immunohistochemical analysis. Mild histological changes were observed in jejunal explants exposed to PAT and ASC, however no significant difference in the lesional score or villi height was observed between the PAT/ASC-groups and the control. Also, explants exposed to 100 µM of PAT showed a significant decrease in goblet cells density and a significant increase in cell apoptosis. These results indicate that high levels of patulin can induce mild toxic effects on intestinal mucosa whereas ascladiol apparently is non-toxic to intestinal tissue.

Effect of the combination of crude extracts of Penicillium griseofulvum and Fusarium graminearum containing patulin and zearalenone on rumen microbial fermentation and on their metabolism in continuous culture fermenters.

Six single-flow continuous cultures were used to study the effects of the mycotoxins patulin (PAT) and zearalenone (ZEN) alone or in combination on rumen microbial fermentation. In each of the four 7-d periods, the fermenters were supplemented in a 2 × 3 factorial arrangement with two levels of PAT (0 and 20 mg/l) and three levels of ZEN (0, 5 and 10 mg/l). The treatments did not affect the apparent and true digestibility of organic matter. PAT alone decreased the digestibility of neutral detergent fibre (NDF) and acid detergent fibre (ADF) (p < 0.01), but in the presence of 5 or 10 mg/l of ZEN, there were no effects of PAT. In contrast, the digestibility of NDF and ADF was decreased at 10 mg/l of ZEN in the absence of PAT (p < 0.05). The pH of the fermenters increased after 2 and 3 d of PAT treatment (p < 0.01). PAT decreased the concentration of total volatile acids (VFA), the molar proportion of acetate and the acetate:proportionate ratio (p < 0.01). The molar concentrations of other VFA were unchanged. Ammonia N (NH3-N) flow increased (p < 0.05) and there was a tendency to a higher NH3-N concentration (p < 0.1) in fermenters with PAT. Total N, non-ammonia N and bacterial N as well as efficiency of microbial protein synthesis and efficiency of N utilisation were not affected by treatments. PAT was nearly completely degraded during incubation. The mean recovery of ZEN, α-zearalenol and ß-zearalenol expressed as a proportion of administered ZEN was less than 50% in effluents from fermenters receiving only ZEN and ZEN plus PAT, respectively. With exception of fibre digestion, the co-administration of PAT and ZEN did not elicit interaction effects on most measured parameters of rumen metabolism.

Eletrochemical reduction of patulin and 5-hydroxymethylfurfural in both neutral and acid non-aqueous media. Their electroanalytical determination in apple juices.

The electro-reduction of patulin mycotoxin and 5-hydroxymethylfurfural at glassy carbon electrodes in acetonitrile +0.1 mol L(-1) tetrabutylammonium perchlorate, in both the absence and the presence of different aliquots of trifluoroacetic acid is reported. 5-hydroxymethylfurfural is the most common interference in the determination of patulin in products derived from apples. The electrochemical techniques were cyclic and square wave voltammetries, and controlled potential bulk electrolysis. The number of electrons exchanged in the patulin electro-reduction of n=1 could be inferred from controlled potential bulk electrolysis measurements. Ultraviolet-visible and infrared spectroscopies were used to identify patulin electro-reduction product/s. A value of (2.1±0.1)×10(-5) cm(2) s(-1) for the patulin diffusion coefficient was calculated from convoluted cyclic voltammograms. A method based on square wave voltammetry was developed for the quantitative determination of patulin in both fresh, and commercial apple juices in the presence of 5-hydroxymethylfurfural. Calibration curves obtained from solutions of the commercial reagent, and commercial apple juices were linear in the range from 3.0×10(-7) to 2.2×10(-5) mol L(-1). The lowest concentration measured experimentally for a signal to noise ratio of 3:1 was 3×10(-7) mol L(-1) (45 ppb) and a recovery percent of 84% was determined for commercial apple juices. This electroanalytical methodology appears as a good screening method for the determination of patulin in apple juices.

Penicillium species present in Uruguayan salami.

The surface coverage of certain dry fermented sausages such as Italian salami by some species of Penicillium provides their characteristic flavor and other beneficial properties. One of them is the protective effect by means of a uniform film of white mold against undesirable microorganisms. The aim of this work was to identify and to isolate the fungal species present in mature Italian type of salami and to evaluate if it is possible to obtain some of them as starters. In addition, the effects of temperature (14 °C and 25 °C), water activity (a w) (0.90, 0.95 and 0.995) and 2.5 % sodium chloride (NaCl) on fungal growth were determined. Similarly, the proteolytic and lipolytic activity and the ability to produce toxic secondary metabolites were evaluated in order to characterize some possible starter strain. All species found belong to the genus Penicillium, including a performing starter as Penicillium nalgiovense and some potentially toxicogenic species. All the strains showed a higher growth rate at 25 °C. The production of extracellular proteases and lipases was significantly higher at 25 °C than at 14 °C with and without sodium chloride. Only Penicillium expansum produced patulin. On the other hand, Penicillium griseofulvum was the only species that produced ciclopiazonic acid but none of the strains produced penicillin. The species present on salami, Penicillium nalgiovense, Penicillium minioluteum, Penicillium brevicompactum and Penicillium puberulum were unable to produce any of the evaluated toxins. These findings suggest that some fungal isolates from the surface of salami such as P. nalgiovense are potentially useful as starters in sausage manufacture.

Penicillium species present in Uruguayan salami / Especies de Penicillium presentes en salamines uruguayos

The surface coverage of certain dry fermented sausages such as Italian salami by some species of Penicillium provides their characteristic flavor and other beneficial properties. One of them is the protective effect by means of a uniform film of white mold against undesirable microorganisms. The aim of this work was to identify and to isolate the fungal species present in mature Italian type of salami and to evaluate if it is possible to obtain some of them as starters. In addition, the effects of temperature (14 °C and 25 °C), water activity (a w) (0.90, 0.95 and 0.995) and 2.5 % sodium chloride (NaCl) on fungal growth were determined. Similarly, the proteolytic and lipolytic activity and the ability to produce toxic secondary metabolites were evaluated in order to characterize some possible starter strain. All species found belong to the genus Penicillium, including a performing starter as Penicillium nalgiovense and some potentially toxicogenic species. All the strains showed a higher growth rate at 25 °C. The production of extracellular proteases and lipases was significantly higher at 25 °C than at 14 °C with and without sodium chloride. Only Penicillium expansum produced patulin. On the other hand, Penicillium griseofulvum was the only species that produced ciclopiazonic acid but none of the strains produced penicillin. The species present on salami, Penicillium nalgiovense, Penicillium minioluteum, Penicillium brevicompactum and Penicillium puberulum were unable to produce any of the evaluated toxins. These findings suggest that some fungal isolates from the surface of salami such as P. nalgiovense are potentially useful as starters in sausage manufacture.

La cobertura de la superficie de los embutidos fermentados secos -como el salamín tipo italiano- por algunas especies de Penicillium les proporciona un sabor característico y otras propiedades beneficiosas. Una de ellas es el efecto de protección contra microorganismos indeseables, al formarse una película blanca uniforme de mohos. El objetivo de este trabajo fue aislar e identificar los hongos filamentosos encontrados en la superficie de salamines tipo italiano y evaluar la posibilidad de obtener especies para utilizarse como cultivos iniciadores. Se determinó el efecto de la temperatura, la actividad de agua y del cloruro de sodio sobre el crecimiento fúngico. La actividad proteolítica y lipolítica y la capacidad de producir metabolitos secundarios tóxicos fueron evaluadas con el fin de caracterizar algunos posibles cultivos iniciadores. Todas las cepas fúngicas aisladas e identificadas correspondieron a especies del género Penicillium, algunas benéficas, como Penicillium nalgiovense, y otras potencialmente toxicogénicas. Estas cepas tuvieron diferentes tasas de crecimiento en respuesta a las diferentes condiciones de cultivo. Todas las cepas mostraron mayor crecimiento a 25 °C. La producción de proteasas y lipasas extracelulares fue significativamente mayor a 25 °C que a 14 °C. Penicillium expansum fue la única especie que produjo patulina y Penicillium griseofulvum fue la única que produjo ácido ciclopiazónico. Ninguna de las especies produjo penicilina. Penicillium nalgiovense, Penicillium minioluteum, Penicillium brevicompactum y Penicillium puberulum no produjeron ninguna de las toxinas evaluadas. Estos resultados sugieren que algunos aislamientos fúngicos, como P. nalgiovense, son potencialmente útiles como cultivos iniciadores en la fabricación de estos productos.

Penicillium species present in Uruguayan salami / Especies de Penicillium presentes en salamines uruguayos

The surface coverage of certain dry fermented sausages such as Italian salami by some species of Penicillium provides their characteristic flavor and other beneficial properties. One of them is the protective effect by means of a uniform film of white mold against undesirable microorganisms. The aim of this work was to identify and to isolate the fungal species present in mature Italian type of salami and to evaluate if it is possible to obtain some of them as starters. In addition, the effects of temperature (14 °C and 25 °C), water activity (a w) (0.90, 0.95 and 0.995) and 2.5 % sodium chloride (NaCl) on fungal growth were determined. Similarly, the proteolytic and lipolytic activity and the ability to produce toxic secondary metabolites were evaluated in order to characterize some possible starter strain. All species found belong to the genus Penicillium, including a performing starter as Penicillium nalgiovense and some potentially toxicogenic species. All the strains showed a higher growth rate at 25 °C. The production of extracellular proteases and lipases was significantly higher at 25 °C than at 14 °C with and without sodium chloride. Only Penicillium expansum produced patulin. On the other hand, Penicillium griseofulvum was the only species that produced ciclopiazonic acid but none of the strains produced penicillin. The species present on salami, Penicillium nalgiovense, Penicillium minioluteum, Penicillium brevicompactum and Penicillium puberulum were unable to produce any of the evaluated toxins. These findings suggest that some fungal isolates from the surface of salami such as P. nalgiovense are potentially useful as starters in sausage manufacture.(AU)

La cobertura de la superficie de los embutidos fermentados secos -como el salamín tipo italiano- por algunas especies de Penicillium les proporciona un sabor característico y otras propiedades beneficiosas. Una de ellas es el efecto de protección contra microorganismos indeseables, al formarse una película blanca uniforme de mohos. El objetivo de este trabajo fue aislar e identificar los hongos filamentosos encontrados en la superficie de salamines tipo italiano y evaluar la posibilidad de obtener especies para utilizarse como cultivos iniciadores. Se determinó el efecto de la temperatura, la actividad de agua y del cloruro de sodio sobre el crecimiento fúngico. La actividad proteolítica y lipolítica y la capacidad de producir metabolitos secundarios tóxicos fueron evaluadas con el fin de caracterizar algunos posibles cultivos iniciadores. Todas las cepas fúngicas aisladas e identificadas correspondieron a especies del género Penicillium, algunas benéficas, como Penicillium nalgiovense, y otras potencialmente toxicogénicas. Estas cepas tuvieron diferentes tasas de crecimiento en respuesta a las diferentes condiciones de cultivo. Todas las cepas mostraron mayor crecimiento a 25 °C. La producción de proteasas y lipasas extracelulares fue significativamente mayor a 25 °C que a 14 °C. Penicillium expansum fue la única especie que produjo patulina y Penicillium griseofulvum fue la única que produjo ácido ciclopiazónico. Ninguna de las especies produjo penicilina. Penicillium nalgiovense, Penicillium minioluteum, Penicillium brevicompactum y Penicillium puberulum no produjeron ninguna de las toxinas evaluadas. Estos resultados sugieren que algunos aislamientos fúngicos, como P. nalgiovense, son potencialmente útiles como cultivos iniciadores en la fabricación de estos productos.(AU)

Penicillium species present in Uruguayan salami / Especies de Penicillium presentes en salamines uruguayos

The surface coverage of certain dry fermented sausages such as Italian salami by some species of Penicillium provides their characteristic flavor and other beneficial properties. One of them is the protective effect by means of a uniform film of white mold against undesirable microorganisms. The aim of this work was to identify and to isolate the fungal species present in mature Italian type of salami and to evaluate if it is possible to obtain some of them as starters. In addition, the effects of temperature (14 °C and 25 °C), water activity (a w) (0.90, 0.95 and 0.995) and 2.5 % sodium chloride (NaCl) on fungal growth were determined. Similarly, the proteolytic and lipolytic activity and the ability to produce toxic secondary metabolites were evaluated in order to characterize some possible starter strain. All species found belong to the genus Penicillium, including a performing starter as Penicillium nalgiovense and some potentially toxicogenic species. All the strains showed a higher growth rate at 25 °C. The production of extracellular proteases and lipases was significantly higher at 25 °C than at 14 °C with and without sodium chloride. Only Penicillium expansum produced patulin. On the other hand, Penicillium griseofulvum was the only species that produced ciclopiazonic acid but none of the strains produced penicillin. The species present on salami, Penicillium nalgiovense, Penicillium minioluteum, Penicillium brevicompactum and Penicillium puberulum were unable to produce any of the evaluated toxins. These findings suggest that some fungal isolates from the surface of salami such as P. nalgiovense are potentially useful as starters in sausage manufacture.(AU)

La cobertura de la superficie de los embutidos fermentados secos -como el salamín tipo italiano- por algunas especies de Penicillium les proporciona un sabor característico y otras propiedades beneficiosas. Una de ellas es el efecto de protección contra microorganismos indeseables, al formarse una película blanca uniforme de mohos. El objetivo de este trabajo fue aislar e identificar los hongos filamentosos encontrados en la superficie de salamines tipo italiano y evaluar la posibilidad de obtener especies para utilizarse como cultivos iniciadores. Se determinó el efecto de la temperatura, la actividad de agua y del cloruro de sodio sobre el crecimiento fúngico. La actividad proteolítica y lipolítica y la capacidad de producir metabolitos secundarios tóxicos fueron evaluadas con el fin de caracterizar algunos posibles cultivos iniciadores. Todas las cepas fúngicas aisladas e identificadas correspondieron a especies del género Penicillium, algunas benéficas, como Penicillium nalgiovense, y otras potencialmente toxicogénicas. Estas cepas tuvieron diferentes tasas de crecimiento en respuesta a las diferentes condiciones de cultivo. Todas las cepas mostraron mayor crecimiento a 25 °C. La producción de proteasas y lipasas extracelulares fue significativamente mayor a 25 °C que a 14 °C. Penicillium expansum fue la única especie que produjo patulina y Penicillium griseofulvum fue la única que produjo ácido ciclopiazónico. Ninguna de las especies produjo penicilina. Penicillium nalgiovense, Penicillium minioluteum, Penicillium brevicompactum y Penicillium puberulum no produjeron ninguna de las toxinas evaluadas. Estos resultados sugieren que algunos aislamientos fúngicos, como P. nalgiovense, son potencialmente útiles como cultivos iniciadores en la fabricación de estos productos.(AU)

DNA damage in organs of mice treated acutely with patulin, a known mycotoxin.

Patulin, a known mycotoxin, is considered a significant contaminant in apples, apple-derived products and feeds. This study investigated the genotoxic effects of patulin in multiple organs (brain, kidney, liver and urinary bladder) of mice using an in vivo comet assay. We assessed the mechanism underlying this genotoxicity by measuring the GSH content and the thiobarbituric acid-reactive species (TBARS) level. Male CF-1 mice were given 1.0-3.75 mg/kg patulin intraperitoneally. The effect of patulin was dose-dependent and the highest patulin dose induced DNA strand breaks in the brain (damage index, DI, in hippocampus increased from 36.2 in control animals to 127.5), liver (44.3-138.4) and kidneys (31.5-99); decreased levels of GSH (hippocampus--from 46.9 to 18.4 nmol/mg protein); and an increase in lipid peroxidation (hippocampus--from 5.8 to 20.3 MDA equivalents/mg protein). This finding establishes an interrelationship between the pro-oxidant and genotoxic effects of patulin. Pre-treatment administration of N-acetyl-cysteine reduced patulin-induced DNA damage (hippocampus--DI from 127.5 to 39.8) and lipid peroxidation (hippocampus--20.3 to 12.8 MDA equivalents/mg protein) by restoring cellular GSH levels, reinforcing the positive relationship between patulin-induced GSH depletion and DNA damage caused by systemic administration of this mycotoxin.

Patulin accumulation in apples during storage by Penicillium expansum and Penicillium griseofulvum strains

A part of apples destined to juice production is generally of poor quality. Apples from cold storage or recently harvest (ground harvested or low quality apples) are stored under ambient conditions until they are processed. Since Penicillium expansum and P. griseofulvum are the principal fungal species isolated from stored apples in Brazil, the objective of this study was to investigate the ability of these strains to produce patulin in apples and report the consequences of this type of storage in loss of quality. The toxin was quantified using thin layer chromatography and charge-coupled device camera (TLC-CCD). The rate and quantities that P. expansum and P. griseofulvum can grow and produce patulin are highly dependent on the fungal strain and time. Lesion diameter resulted to be independent of the strain considered. The maximum period of time which apples were kept at cold storage (4 ºC) without patulin accumulation was 27 days. When these apples were kept at 25 ºC during 3 days, both factors lesion diameter and patulin production increased significantly. These results confirm that time in which apples are taken out from cold storage room before juice production is critical in order to prevent patulin accumulation.