Josué Delgado

Presence of ochratoxin A on the surface of dry-cured Iberian ham after initial fungal growth in the drying stage

Accumulation of ochratoxin A (OTA) on the surface and to a 0.5 cm depth of dry-cured Iberian ham after initial fungal growth was investigated. For this, 20 dry-cured Iberian hams from the drying stage showing incipient fungal growth on the surface were analyzed. In addition, the presence of OTA-producing molds was examined on the surface of the hams by real-time quantitative PCR (qPCR) based on the otanpsPN gene. Quantification of specific OTA-producing molds, such as Penicillium nordicum and Penicillium verrucosum was also achieved on the hams by specific qPCR methods. Ten of 20 dry-cured hams showed OTA at higher levels than those established by legal regulation. OTA was even detected in the deep section of hams. OTA-producing molds ranged from 1.5 to 7.3 log cfu/cm². Accumulation of OTA on the hams seems to be related to the presence of OTA-producing molds and especially to P. nordicum.

Growth inhibition and stability of PgAFP from Penicillium chrysogenum against fungi common on dry-ripened meat products

Dry-ripened foods favor the development of a superficial fungal population that may include toxigenic molds. To combat unwanted molds, an antifungal protein from Penicillium chrysogenum (PgAFP) can be useful. The aim of the present work was to study the antimicrobial activity of PgAFP against microorganisms common in dry-ripened foods, and to evaluate its sensitivity to proteolytic enzymes and heat treatments that may be applied to foods, as well as to different pH values. The inhibitory effect of the purified protein on 38 microbial strains grown in culture medium was determined. PgAFP sensitivity to various proteases, heat treatments, and preincubation at different pH values was tested by means of the residual activity on selected reference strains. Inhibitory activity of PgAFP against unwanted molds was tested in a dry-fermented sausage. This protein exhibited potent inhibitory activity against unwanted molds, including the main mycotoxin-producing species of Aspergillus and Penicillium of concern for dry-ripened foods. PgAFP withstood most proteases, intense heat and a wide range of pH values. PgAFP efficiently reduced counts of A. flavus and P. restrictum inoculated on a dry-fermented sausage. This protein can be of interest to control hazardous molds in dry-ripened foods.

Effect of temperature and water activity on growth and aflatoxin production by Aspergillus flavus and Aspergillus parasiticus on cured meat model systems

Dry-cured hams may be colonised by aflatoxin-producing Aspergillus flavus and Aspergillus parasiticus during the ripening process. The objective of this study was to evaluate the interaction between non-ionic water stress and temperatures may have on lag phases prior to growth, growth rates and aflatoxin production by two strains of each A. parasiticus and A. flavus on meat matrices over a period of 12days. Results showed that A. flavus CBS 573.65 had shorter lag phases than A. parasiticus CECT 2688, however the growth rates were quite similar. For both species, no growth occurred at 10°C and all aw tested and optimum growth happened at 25°C and 0.95 aw. Similar aflatoxin B1 production profiles between both species were found, however A. flavus produced much higher concentration of such toxin than A. parasiticus. Both species produced aflatoxins when the temperature and the aw were ≥15°C and ≥0.90.

Increased chitin biosynthesis contributes to the resistance of Penicillium polonicum against the antifungal protein PgAFP

Antifungal proteins from molds have been proposed as a valuable tool against unwanted molds, but the resistance of some fungi limits their use. Resistance to antimicrobial peptides has been suggested to be due to lack of interaction with the mold or to a successful response. The antifungal protein PgAFP produced by Penicillium chrysogenum inhibits the growth of various ascomycetes, but not Penicillium polonicum. To study the basis for resistance to this antifungal protein, localization of PgAFP and metabolic, structural, and morphological changes were investigated in P. polonicum. PgAFP bound the outer layer of P. polonicum but not regenerated chitin, suggesting an interaction with specific molecules. Comparative two-dimensional gel electrophoresis (2D-PAGE) and comparative quantitative proteomics revealed changes in the relative abundance of several proteins from ribosome, spliceosome, metabolic, and biosynthesis of secondary metabolite pathways. The proteome changes and an altered permeability reveal an active reaction of P. polonicum to PgAFP. The successful response of the resistant mold seems to be based on the higher abundance of protein Rho GTPase Rho1 that would lead to the increased chitin deposition via cell wall integrity (CWI) signaling pathway. Thus, combined treatment with chitinases could provide a complementary means to combat resistance to antifungal proteins.

Manuscript title: antifungal proteins from moulds: analytical tools and potential application to dry-ripened foods

Moulds growing on the surface of dry-ripened foods contribute to their sensory qualities, but some of them are able to produce mycotoxins that pose a hazard to consumers. Small cysteine-rich antifungal proteins (AFPs) from moulds are highly stable to pH and proteolysis and exhibit a broad inhibition spectrum against filamentous fungi, providing new chances to control hazardous moulds in fermented foods. The analytical tools for characterizing the cellular targets and affected pathways are reviewed. Strategies currently employed to study these mechanisms of action include ‘omics’ approaches that have come to the forefront in recent years, developing in tandem with genome sequencing of relevant organisms. These techniques contribute to a better understanding of the response of moulds against AFPs, allowing the design of complementary strategies to maximize or overcome the limitations of using AFPs on foods. AFPs alter chitin biosynthesis, and some fungi react inducing cell wall integrity (CWI) pathway. However, moulds able to increase chitin content at the cell wall by increasing proteins in either CWI or calmodulin-calcineurin signalling pathways will resist AFPs. Similarly, AFPs increase the intracellular levels of reactive oxygen species (ROS), and moulds increasing G-protein complex β subunit CpcB and/or enzymes to efficiently produce glutathione may evade apoptosis. Unknown aspects that need to be addressed include the interaction with mycotoxin production by less sensitive toxigenic moulds. However, significant steps have been taken to encourage the use of AFPs in intermediate-moisture foods, particularly for mould-ripened cheese and meat products.

Detection of changes in mould cell wall stress-related gene expression by a novel reverse transcription real-time PCR method

The cell wall integrity (CWI) pathway is activated in response to cell wall stresses due to different food-related environments. Rho1 is one of the main regulators within such pathway. The objective of this work was to design an easy-to-use RT-qPCR technique for the evaluation of the Rho1 gene expression useful to measure responses to the presence of cell wall stressors such as the antifungal protein PgAFP. Two primer pairs were designed from published conserved regions. Their specificity initially was determined by in silico analysis for several fungal species. After optimising the qPCR, the primer pair Rho1-F1/R2 was selected due to the lowest Cq values obtained and its specificity. The qPCR method showed efficiencies between 97.5% and 100.5%. Applicability of the designed qPCR method was evaluated in the presence of the stressor PgAFP. The PgAFP-resistant Penicillium polonicum and the PgAFP-sensitive Aspergillus flavus showed Rho1 gene over- and under- expression, respectively, indicating that the CWI pathway is activated in the former species but not activated in the latter one in response to the stress caused by PgAFP. This novel qPCR methodology able to detect changes in CWI-related gene expression in filamentous fungi will be useful in future studies to evaluate physiological mould responses to different food environmental challenges.

Influence of ochratoxin A on adaptation of Penicillium nordicum on a NaCl-rich dry-cured ham-based medium

Iberian dry-cured ham is an important meat product with high consumption worldwide. The special ecological conditions occurring throughout its ripening favour surface colonisation of filamentous fungi. Normally, moulds contribute to the development of the sensory qualities of the ham; however, some toxigenic species, such as Penicillium nordicum, are able to successfully adapt to the NaCl-rich environment found in dry-cured ham and produce ochratoxin A (OTA) in this product. Moreover, it was suggested that the biosynthesis of OTA by P. nordicum itself may support the adaptation to this food environment. However, this mechanism has not been completely elucidated yet. The objective of this work was to evaluate the influence of different concentrations of commercial OTA (cOTA, at 0, 0.2, 1 and 5 ppb) on growth rate, biosynthetic- and stress-related gene expression and OTA production by two P. nordicum strains (Pn15 and Pn69) on dry-cured ham based-media. Two NaCl conditions (0% and 10%) were evaluated for each cOTA level. In general, no intra-strain and inter-strain differences in growth rates were found among the conditions tested. The stress-related Hog1 gene expression of the strain Pn15 was affected by cOTA and NaCl concentration whilst the strain Pn69 was not affected by these variables. The expression of OTA-related otapks and otanps genes of the strain Pn15 was affected by several NaCl and cOTA combinations. However, the strain Pn69 showed no differences in relative gene expression. Regarding to OTA production, different behaviours were displayed by the two strains. The strain Pn15, which produced high OTA amounts by itself, produced OTA without the necessity of the presence of NaCl or cOTA as stressors. However, the presence of cOTA triggers OTA production by the weak OTA producing Pn69 in the absence of NaCl. In addition, although a moderate correlation was found between the expression of the OTA-related genes and mycotoxin produced by P. nordicum in the absence of NaCl, none was obtained between Hog1 gene expression and mycotoxin production. This study is a step forward for a better understanding of the ability of P. nordicum producers of OTA to colonise NaCl-rich habitats such as Iberian ham for proposing actions to minimise OTA contamination in this meat product.

Inhibitory Effect of PgAFP and Protective Cultures on Aspergillus parasiticus Growth and Aflatoxins Production on Dry-Fermented Sausage and Cheese

Aflatoxigenic molds can grow and produce aflatoxins on dry-fermented meat and cheese. The small, basic, cysteine-rich antifungal protein PgAFP displays a time-limited inhibitory ability against unwanted molds by increasing reactive oxygen species (ROS), which can lead to increased aflatoxin production. However, calcium abolishes the inhibitory effect of PgAFP on certain Aspergillus spp. To maximize the antifungal effect, this protein may be combined with protective cultures. Yeasts and lactic acid bacteria may counteract the impact of calcium on PgAFP fungal inhibition. The objective of this work was to study the effect of PgAFP and different combined treatments with Debaryomyces hansenii and/or Pediococcus acidilactici against growth of and aflatoxin production by an aflatoxigenic strain of Aspergillus parasiticus in both culture media and dry-fermented foods with low or high calcium levels. Aflatoxins production was increased by PgAFP but dramatically reduced by P. acidilactici in low calcium culture medium, whereas in the Ca-enriched culture medium, all treatments tested led to low aflatoxins levels. To study whether PgAFP and the protective microorganisms interfere with ROS and aflatoxin production, the relative expression of genes foxA, which is involved in peroxisomal β-oxidation, and aflP, which is required for aflatoxin biosynthesis, were evaluated. The aflatoxin overproduction induced by PgAFP seems not to be linked to peroxisomal β-oxidation. The combination of PgAFP and D. hansenii provided a successful inhibitory effect on A. parasiticus growth as well as on aflatoxin production on sliced dry-fermented sausage and cheese ripened up to 15 days, whereas P. acidilactici did not further enhance the protective effect of the two former agents. Therefore, the combined treatment of PgAFP and D. hansenii seems to provide a promising protective mean against aflatoxin-producing A. parasiticus on dry-fermented foods.

Gene Expression Analysis as a Method to Predict OTA Accumulation in Dry-Cured Meat Products

Dry-cured meat products are frequently colonised by toxigenic Penicillium nordicum and Penicillium verrucosum throughout their ripening that can produce ochratoxin A (OTA). The predictive nature of the molecular analyses can be used to determine the risk of toxigenic species. For this reason, the objective of the present work was to analyse the temporal changes in the expression of the otapks and otanps genes by P. nordicum and P. verrucosum in relation to OTA production on slices of dry-fermented sausage salchichón and dry-cured ham. Gene expression was higher in P. nordicum than in P. verrucosum in both meat matrices. The otapks gene was overexpressed by both Penicillium species, especially in salchichón. OTA was only detected in inoculated salchichón regardless of the ochratoxigenic species used. The high significant correlation found between the early relative expression of the otapks gene and OTA production in salchichón leads to propose the relative gene expression of the otapks gene as a good indicator to predict OTA accumulation throughout the ripening of this product.