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  • influence of an industrial dry fermented sausage processing on ochratoxin a production by Penicillium nordicum
    International Journal of Food Microbiology, 2021
    Co-Authors: Josue Delgado, Felix Nunez, Juan J Rondan, Alicia Rodríguez
    Abstract:

    Abstract Dry-fermented sausages are prone to be colonised by Penicillium nordicum, which is one of the main ochratoxin A (OTA)-producing species. Its ability to produce this mycotoxin on dry-fermented sausages has been reported. However, the influence of the conditions of a traditional processing of a Spanish dry-fermented sausage and the intrinsic physicochemical parameters of this product such as water activity (aw) and pH on OTA production has not been studied yet. Thus, the aim of this study was to evaluate the influence of traditional processing (interaction of relative humidity (RH) x temperature x ripening days) on the evolution of pH and aw during maturation of dry-fermented sausage “salchichon” and its relationship with OTA synthesis by P. nordicum. The expression of otapks and otanps genes, both involved in the biosynthesis of the mycotoxin, was also assessed. For this, 27 raw sausages were inoculated with P. nordicum and ripened for 26 days in a drying chamber (3 days at 5 °C and 84% RH, 17 days at 12 °C and 84% RH, and 6 days at 12 °C and 80% RH). From results, although it seems that the pH slightly influenced on OTA biosynthesis, the aw had a great impact on this mycotoxin production. In fact, the two highest OTA concentrations found coincided with a dramatic rise of the aw value (0.92 aw) by day 18 of incubation when the RH of the drying chamber was still 84% and at the end of the incubation time when the aw decreased noticeably (0.87 aw). The expression of the otapks and otanps genes correlated with the OTA produced by P. nordicum. Results from this work confirm that the traditional processing of Spanish dry-fermented sausages favours itself OTA synthesis by P. nordicum. Our findings may help in informed decision-making in relation to RH/temperature of drying chambers and shortening of the ripening process. This may be then effectively incorporated into the hygienic production system in the framework of HACCP together with other measures including the use of Penicillium nalgiovense as protective culture or the monitoring of otapks gene expression, and aw during the processing of dry-fermented sausages. All these strategies together may put ochratoxigenic Penicillia at a disadvantage and minimise OTA contamination risks in dry-fermented sausages.

  • competitiveness of three biocontrol candidates against ochratoxigenic Penicillium nordicum under dry cured meat environmental and nutritional conditions
    Fungal Biology, 2021
    Co-Authors: Micaela Alvarez, Felix Nunez, Mar Rodriguez, Josue Delgado, María J. Andrade, Alicia Rodríguez
    Abstract:

    Abstract The environmental conditions during the ripening of dry-cured meats and their nutritional composition promote the colonisation of their surface by Penicillium spp., including Penicillium nordicum producer of ochratoxin A (OTA). The objective of this work was to study the competitiveness of three potential biocontrol candidates (Debaryomyces hansenii FHSCC 253H, Enterococcus faecium SE920 and Penicillium chrysogenum CECT 20922) against the ochratoxigenic P. nordicum FHSCC4 under environmental and nutritional conditions simulating the ripening of dry-cured meat products. For this, the nutritional utilisation pattern, niche overlap index (NOI), interactions by dual-culture assays and OTA production were determined. The number of carbon sources (CSs) metabolised depended on the microorganism and the interacting water activity (aw) x temperature conditions. The number of CSs utilised by both filamentous fungi was quite similar and higher than those utilised by D. hansenii and E. faecium. The yeast isolate metabolised a number of CSs much larger than the bacterium. The NOI values showed that, in general, P. nordicum nutritionally dominated E. faecium and D. hansenii regardless of the environmental conditions evaluated. The relationship between the toxigenic and non-toxigenic fungal isolates depended on the aw x temperature combinations, although in none of the conditions a dominance of P. nordicum was observed. According to the interaction assays, both D. hansenii and P. chrysogenum decreased the growth of P. nordicum. The effect of D. hansenii could be attributed to the production of some extra-cellular compounds, while the action of P. chrysogenum is likely related to nutritional competition. In addition, both P. chrysogenum and D. hansenii reduced the OTA levels produced by P. nordicum. The effect of the yeast was more pronounced decreasing the concentration of OTA at quantities lower than the limit established by the Italian legislation. Therefore, P. chrysogenum and D. hansenii can be suggested as biocontrol candidates in the manufacture of dry-cured meat products.

  • competitiveness of three biocontrol candidates against ochratoxigenic Penicillium nordicum under dry cured meat environmental and nutritional conditions
    Fungal Biology, 2021
    Co-Authors: Micaela Alvarez, Felix Nunez, Mar Rodriguez, Josue Delgado, María J. Andrade, Alicia Rodríguez
    Abstract:

    Abstract The environmental conditions during the ripening of dry-cured meats and their nutritional composition promote the colonisation of their surface by Penicillium spp., including P. nordicum producer of ochratoxin A (OTA). The objective of this work was to study the competitiveness of three potential biocontrol candidates (Debaryomyces hansenii FHSCC 253H, Enterococcus faecium SE920 and Penicillium chrysogenum CECT, 20922) against the ochratoxigenic P. nordicum FHSCC4 under environmental and nutritional conditions simulating the ripening of dry-cured meat products. For this, the nutritional utilisation pattern, niche overlap index (NOI), interactions by dual-culture assays and OTA production were determined. The number of carbon sources (CSs) metabolised depended on the microorganism and the interacting water activity (aw) x temperature conditions. The number of CSs utilised by both filamentous fungi was quite similar and higher than those utilised by D. hansenii and E. faecium. The yeast isolate metabolised a number of CSs much larger than the bacterium. The NOI values showed that, in general, P. nordicum nutritionally dominated E. faecium and D. hansenii regardless of the environmental conditions evaluated. The relationship between the toxigenic and non-toxigenic fungal isolates depended on the aw x temperature combinations, although in none of the conditions a dominance of P. nordicum was observed. According to the interaction assays, both D. hansenii and P. chrysogenum decreased the growth of P. nordicum. The effect of D. hansenii could be attributed to the production of some extra-cellular compounds, while the action of P. chrysogenum is likely related to nutritional competition. In addition, both P. chrysogenum and D. hansenii reduced the OTA levels produced by P. nordicum. The effect of the yeast was more pronounced decreasing the concentration of OTA at quantities lower than the limit established by the Italian legislation. Therefore, P. chrysogenum and D. hansenii can be suggested as biocontrol candidates in the manufacture of dry-cured meat products.

  • in vitro antifungal effects of spices on ochratoxin a production and related gene expression in Penicillium nordicum on a dry cured fermented sausage medium
    Food Control, 2020
    Co-Authors: Micaela Alvarez, Felix Nunez, Alicia Rodríguez, Antonio Silva, María J. Andrade
    Abstract:

    Abstract Penicillium nordicum, an ochratoxin A (OTA) producer, widely contaminates the surface of dry-cured fermented sausages. Although the meat industry uses antifungal synthetic compounds during ripening, consumers' preferences currently stand up for natural preservatives. The aim of this study was to evaluate the antifungal effect of spices commonly added to dry-cured fermented sausages during their processing on P. nordicum for establishing their value as an alternative strategy to synthetic compounds. The mould strain was grown on a dry-cured fermented sausage-based medium containing oregano, rosemary or thyme. Moreover, an antifungal commercial preparation containing potassium sorbate and natamycin was tested alone and in combination with the spices. The growth rate, OTA accumulation and relative expression of genes involved in OTA biosynthesis (otapks and otanps) and some stress pathways (Hog1 and Rho1) were evaluated. In the growth assessment, the oregano alone or in combination with the antifungal preparation significantly reduced the growth rate. Different patterns were observed at both sampling times for the OTA production and gene expression analyses, and even differences between treatments at the same incubation period were observed. Regarding OTA accumulation, significant reductions were found when adding oregano or rosemary in the presence and absence of the antifungal compounds. While a stimulation of the expression of most of the tested genes was obtained at the initial stages, a repression was generally found at the end of the incubation. This makes sense since the mycotoxin accumulation was usually higher at the initial stages than at the end of the incubation. Accordingly, such spices would allow satisfying the current consumers’ demand for natural preservatives minimising the hazard associated with the OTA presence in cured meats at the same time.

  • diffusion of mycotoxins and secondary metabolites in dry cured meat products
    Food Control, 2019
    Co-Authors: Belen Peromingo, Marc Lemmens, Alicia Rodríguez, Michael Sulyok, Mar Rodriguez
    Abstract:

    Abstract The aim of this study was to analyse the pattern and diffusion capacity of secondary metabolites produced by ochratoxin A (OTA)- and cyclopiazonic acid (CPA)-producing mould species commonly growing in dry-cured meat products. Dry-fermented sausage and dry-cured ham pieces were inoculated with Penicillium nordicum, Penicillium verrucosum and Penillium griseofulvum, and incubated at 20 °C for 15 days at 94% relative humidity. After incubation, the samples were divided into 3 subsamples: A (0–1 cm; including the fungal colony), B (1–2 cm) and C (2–3 cm). The subsamples were analysed with a UHPLC-Q trap-MS method capable of detecting and quantifying 33 mycotoxins at values around 1 μg/kg. Mould strains produced from 5 to 12 secondary metabolites on the surface of the meat products. Besides, a higher number of metabolites was encountered in dry-fermented sausage than in dry-cured ham. Some of the fungal secondary metabolites diffused from the surface (layer A) into the inner core of meat products (layers B and C) contaminating the products up to 3 cm depth. In general, the concentration of fungal metabolites able to diffuse generally decreased as they diffused inward; while other compounds remained on the surface. This supposes a problem for the industry given that the removal of mouldy surface of dry-cured meats seems not to protect consumers’ health.

Brenda D Wingfield - One of the best experts on this subject based on the ideXlab platform.

Rolf Geisen - One of the best experts on this subject based on the ideXlab platform.

  • the application of transcriptomics to understand the ecological reasons of ochratoxin a biosynthesis by Penicillium nordicum on sodium chloride rich dry cured foods
    Trends in Food Science and Technology, 2011
    Co-Authors: Markus Schmidtheydt, Eva Graf, Julia Batzler, Rolf Geisen
    Abstract:

    Penicillium nordicum and to some extent also P. verrucosum can be found as contaminants on NaCl rich fermented foods like ham, cheeses or vegetables. Both fungal species are able to produce ochratoxin A. Ochratoxin A is a chloride containing mycotoxin which has toxic activities especially against the kidney. Further putative ochratoxin A producing species can be found in this environment. Recent results show that the production of ochratoxin A increases the competitiveness of the producing fungi under salt stress conditions which occur in these types of foods. This increased competitiveness may explain the frequent occurrence of these fungi in salt rich commodities.

  • influence of light on food relevant fungi with emphasis on ochratoxin producing species
    International Journal of Food Microbiology, 2011
    Co-Authors: Markus Schmidtheydt, Giancarlo Perrone, Corinna E Rufer, Frank Raupp, Anja Bruchmann, Rolf Geisen
    Abstract:

    Abstract The influence of light of varying wavelength on growth and ochratoxin A biosynthesis of Aspergillus carbonarius , A. niger , A. steynii and on Penicillium nordicum and P. verrucosum was analysed. For comparison the influence of light on various other food relevant fungi, including citrinin producers, was also tested. Generally the Aspergilli seem to be more resistant to light treatment than the Penicillia. Interestingly wavelengths from both sides of the spectrum, e. g. red (long wavelength, 627 nm) and blue (short wavelength 470–455 nm) had the strongest inhibitory effects on growth and ochratoxin A biosynthesis. Blue light generally had a stronger effect. Light of moderate wavelength, 590 to 530 nm, (yellow to green) had more a positive than a negative influence on growth or ochratoxin A biosynthesis compared to the control (dark incubation). The light effect on growth and ochratoxin A biosynthesis was dependent on the growth medium. In contrast to malt extract medium (MEA), YES medium, as an especially nutrient rich medium, had an attenuating effect on the reactivity against light. However the tendency of the response in both media was the same. Moreover, the light intensity strongly influences how the fungus reacts. Depending on the intensity and the resistance of the species a complete cessation of growth and/or inhibition of ochratoxin A biosynthesis could be achieved. Light irradiation has the opposite effect on ochratoxin A than citrinin, two mycotoxins which can be produced simultaneously in P. verrucosum. Citrinin was produced essentially under light conditions which inhibited ochratoxin A biosynthesis. The same was true for a derivative of ochratoxin, in particular a derivative of ochratoxin β in A. carbonarius . A. carbonarius produced high amounts of the ochratoxin β derivative under blue light when the production of ochratoxin A was ceased at the most inhibiting conditions used (MEA, royal blue light, 455 nm, 1700 lx). Light has a growth stalling but not inactivating effect on aerial mycelia. If a non-growing colony under light is shifted to the dark it immediately grows normally. However on spores blue light has a deactivating effect. After incubation of spores of P. verrucosum for 24 h under blue light up to 97% of the spores were no longer able to germinate. Again the spores of the Aspergilli were much more resistant.

  • expression of a gfp gene in Penicillium nordicum under control of the promoter of the ochratoxin a polyketide synthase gene
    International Journal of Food Microbiology, 2009
    Co-Authors: Markus Schmidtheydt, Tobias Schunck, Rolf Geisen
    Abstract:

    A gfp reporter gene strain of Penicillium nordicum was constructed in order to be able to study the influence of environmental parameters on ochratoxin A biosynthesis. To introduce the gfp gene an Agrobacterium tumefaciens mediated transformation system (ATMT) for P. nordicum was established resulting in a transformation efficiency of about 60 transformants per µg of DNA. The selection principle was based on the hygromycin B resistance gene located on the TI-DNA fragment of the binary vector system. PCR and Southern blot hybridization revealed that the TI-DNA was integrated into the chromosome of P. nordicum. To show that the GFP protein can be used as a reporter gene in P. nordicum, this species was subsequently transformed with a vector, carrying a gfp gene under the control of the strong constitutive gpd promoter of A. nidulans. Moreover in this vector construction the gfp gene contained a stuA nuclear localization domain. Successful transformed strains showed a strong GFP activity located in the nuclei after light stimulation in contrast to the wild type which showed only very weak unspecific auto fluorescence under these conditions. Based on this proof of principle a vector was constructed in which the promoter of the otapksPN gene, the gene of the ochratoxin A polyketide synthase of P. nordicum was placed in front of the gfp gene. This construct was transformed into P. nordicum by ATMT and the resulting transformants were analysed by fluorescence microscopy. In these transformants the whole mycelial cells showed GFP activity after light stimulation, whereas the wild type strain did not. When the transformed strains were grown on medium which suppressed ochratoxin A biosynthesis, a very low level of fluorescence could be detected, whereas a high level of fluorescence was seen after growth on medium supportive for ochratoxin A biosynthesis.

  • gene expression as an indication for ochratoxin a biosynthesis in Penicillium nordicum
    Mycotoxin Research, 2007
    Co-Authors: Markus Schmidtheydt, Rolf Geisen
    Abstract:

    The expression level of theotapksPN gene ofPenicillium nordicum was determined by Reverse Transcription Real Time PCR in relation to several growth parameters known to have an influence on ochratoxin A biosynthesis, like pH, medium composition and temperature. Generally there was a good correlation between the expression level of this key gene and ochratoxin A biosynthesis. The results, which were obtained by Real Time PCR for a single gene, could generally be confirmed by microarray analysis with a whole set of genes related to ochratoxin A biosynthesis. Growth conditions which favour the expression of the ochratoxin A biosynthetic genes also resulted in the biosynthesis of ochratoxin A by the fungal culture, indicating that the influence of abiotic parameters is mediated via the regulation of transcription of the ochratoxin A biosynthetic genes. The influence of several parameters, like pH, salt concentration and temperature on the expression of the ochratoxin A biosynthetic genes has been analysed. Optimum conditions for the activation of the ochratoxin A biosynthesis genes were: pH 6.0–8.0, 25 °C and a sodium chloride concentration of 20 g/l. This was paralleled by the capacity ofP. nordicum to produce ochratoxin A under the same conditions. After combining the most inhibitory conditions (15 °C, sodium chloride 10 g/l, pH 5.0) nearly a complete deactivation of the ochratoxin A biosynthetic genes could be observed resulting in a complete abolishment of ochratoxin A biosynthesis. In contrast, a high expression and a high production of ochratoxin A could be observed after growth ofP. nordicum under more optimal conditions (25 °C, 20 g/1 sodium chloride, pH 8.0).

  • development of a molecular detection and differentiation system for ochratoxin a producing Penicillium species and its application to analyse the occurrence of Penicillium nordicum in cured meats
    International Journal of Food Microbiology, 2006
    Co-Authors: Caroline Bogs, Paola Battilani, Rolf Geisen
    Abstract:

    A PCR method for differentiation and detection of the two known ochratoxin A producing Penicillium species, Penicillium verrucosum and Penicillium nordicum has been developed. It is based upon two genes of the ochratoxin A biosynthetic pathway, namely the ochratoxin A polyketide synthase gene (otapksPN) and a non-ribosomal peptide syntethase gene (otanpsPN) from P. nordicum. Both ochratoxin A producing Penicillia differ characteristically in the PCR result, making a taxonomic differentiation possible. P. verrucosum gives consistently only a positive reaction with the primers for the otanpsPN gene, whereas P. nordicum is positive for both genes. The PCR reaction is negative with all of other food related fungal species tested. This PCR system has been used to analyse 62 Penicillium strains isolated from cured meat products or ripening rooms, the natural habitat of P. nordicum. Among the 62 analysed strains 11 (18%) were positive with all specific PCR reactions. All 11 strains were able to produce ochratoxin A. In a RAPD analysis performed in parallel all 11 strains showed a pattern characteristic of P. nordicum, indicating the congruence of all data. None of the other strains isolated from cured meat produced ochratoxin A; most of them (30 out of 62) had a RAPD pattern characteristic for Penicillium nalgiovense. Interestingly some of the P. nalgiovense strains showed weak PCR product bands with varying length after electrophoresis. This was true for both primer pairs. None of these P. nalgiovense strains however produced detectable amounts of ochratoxin A. A more detailed analysis revealed that P. nalgiovense carries similar but non-transcribed sequences to the ochratoxin A biosynthetic genes of P. nordicum.

Wilhelm Z De Beer - One of the best experts on this subject based on the ideXlab platform.