Antifungal Effect of Autochthonous Aromatic Plant Extracts on Two Mycotoxigenic Strains of Aspergillus flavus.

This study identified the compounds obtained from four native Dehesa plants, which were holm oak, elm, blackberry and white rockrose, and evaluated their ability to inhibit the growth and production of aflatoxins B1 and B2 of two strains of mycotoxigenic Aspergillus flavus. For this purpose, phenolic compounds present in the leaves and flowers of the plants were extracted and identified, and subsequently, the effect on the growth of A. flavus, aflatoxin production and the expression of a gene related to its synthesis were studied. Cistus albidus was the plant with the highest concentration of phenolic compounds, followed by Quercus ilex. Phenolic acids and flavonoids were mainly identified, and there was great variability among plant extracts in terms of the type and quantity of compounds. Concentrated and diluted extracts were used for each individual plant. The influence on mold growth was not very significant for any of the extracts. However, those obtained from plants of the genus Quercus ilex, followed by Ulmus sp., were very useful for inhibiting the production of aflatoxin B1 and B2 produced by the two strains of A. flavus. Expression studies of the gene involved in the aflatoxin synthesis pathway did not prove to be effective. The results indicated that using these new natural antifungal compounds from the Dehesa for aflatoxin production inhibition would be desirable, promoting respect for the environment by avoiding the use of chemical fungicides. However, further studies are needed to determine whether the specific phenolic compounds responsible for the antifungal activity of Quercus ilex and Ulmus sp. produce the antifungal activity in pure form, as well as to verify the action mechanism of these compounds.

Enterococcus faecium: a promising protective culture to control growth of ochratoxigenic moulds and mycotoxin production in dry-fermented sausages.

Moulds positively contribute to the development of typical characteristic flavour and aroma of dry-fermented sausages. However, some mould species, such as Penicillium nordicum and Penicillium verrucosum, may contaminate this product with ochratoxin A (OTA). For this reason, the control of toxigenic moulds is needed. Strategies based on the use of antifungal microorganisms present in the native microbial population in the dry-fermented sausage processing could be a promising strategy. The aim of this work was to study the effect of Enterococcus faecium strains on P. nordicum and P. verrucosum growth and OTA production in a dry-fermented sausage-based medium at conditions of temperature and water activity similar to those occurring during the ripening of these meat products. Six strains were screened to evaluate their growth capacity and antifungal activity against P. nordicum and P. verrucosum at three fixed temperatures related to the sausage ripening. The two E. faecium strains that decreased growth of both species were chosen to further evaluate their effect on growth of P. verrucosum and P. nordicum and their mycotoxin production under conditions simulating the dry-fermented sausage ripening. The presence of E. faecium SE920 significantly reduced OTA production of P. nordicum although it did not affect P. verrucosum. E. faecium SE920, isolated from dry-fermented sausages, could be a good candidate to reduce OTA production by P. nordicum in dry-fermented sausages.

Efficacy of the Combined Protective Cultures of Penicillium chrysogenum and Debaryomyces hansenii for the Control of Ochratoxin A Hazards in Dry-Cured Ham.

The ecological conditions during the ripening of dry-cured ham favour the development of moulds on its surface, being frequently the presence of Penicillium nordicum, a producer of ochratoxin A (OTA). Biocontrol using moulds and yeasts usually found in dry-cured ham is a promising strategy to minimize this hazard. The aim of this work is to evaluate the effect of previously selected Debaryomyces hansenii and Penicillium chrysogenum strains on growth, OTA production, and relative expression of genes involved in the OTA biosynthesis by P. nordicum. P. nordicum was inoculated against the protective cultures individually and combined on dry-cured ham for 21 days at 20 °C. None of the treatments reduced the growth of P. nordicum, but all of them decreased OTA concentration. The lower production of OTA could be related to significant repression of the relative expression of otapksPN and otanpsPN genes of P. nordicum. The efficacy of the combined protective cultures was tested in 24 dry-cured hams in industrial ripening (an 8 month-long production). OTA was detected in nine of the 12 dry-cured hams in the batch inoculated only with P. nordicum. However, in the batch inoculated with both P. nordicum and the combined protective culture, a considerable reduction of OTA contamination was observed. In conclusion, although the efficacy of individual use P. chrysogenum is great, the combination with D. hansenii enhances its antifungal activity and could be proposed as a mixed protective culture to control the hazard of the presence of OTA in dry-cured ham.

Effect of Spanish smoked paprika "Pimentón de La Vera" on control of ochratoxin A and aflatoxins production on a dry-cured meat model system.

Environmental conditions during ripening of dry-cured meat products favour growth of fungal population on their surface. Some of these moulds can produce mycotoxins. Paprika is one of the ingredients usually used in the formulation of raw-cured sausages, and its addition could influence the growth and production of mycotoxins of the moulds present in these products. In this work the effect of Spanish smoked paprika "Pimentón de la Vera" on growth of Aspergillus parasiticus and Penicillium nordicum and production of aflatoxins B1 (AFB1), G1 (AFG1) and ochratoxin A (OTA) respectively, was evaluated. Moulds were grown in a culture medium made from lyophilized fresh pork meat added with 4% salt and different concentrations of Spanish smoked paprika (1, 2 and 3%) at several water activity values (0.98, 0.94 and 0.87) and temperature (20-25 °C), to simulate conditions usually found during ripening of dry-cured meat products. Mould growth was evaluated by measuring the diameter of the colony every 24 h, and the production of mycotoxins by UHPLC-MS/MS every 2 days, during 10 days of incubation. Addition of paprika favours growth of the two mould species tested. However, the synthesis of mycotoxins was reduced at 0.94 and 0.98 aw when at least a 2% of paprika was added. Therefore, the addition of Spanish smoked paprika at 2-3% in the formulations may help to minimize AFs and OTA production in dry-cured meat products such as loins or "chorizo" sausages.

Effects of environmental conditions and substrate on growth and ochratoxin A production by Penicillium verrucosum and Penicillium nordicum: Relative risk assessment of OTA in dry-cured meat products.

Ecological conditions during ripening of dry-cured meat products favour the growth of an uncontrolled mould population that could suppose a risk of ochratoxin A (OTA) production. In this work the influence of water activity (aw), temperature and substrate composition on fungal growth and OTA production by Penicillium nordicum and Penicillium verrucosum isolated from dry-cured meat products have been studied. In addition, the relative risk of OTA presence on dry-cured meat products has been evaluated using the Risk Ranger software. Fungal growth was observed in the range of 0.99-0.90 aw and 15-25 °C being mainly temperature-dependent. P. nordicum and P. verrucosum were able to produce OTA in every substrate in these ranges of aw and temperature. The production of OTA by P. verrucosum was mainly influenced by temperature and media composition. However, P. nordicum it is affected mainly by substrate or temperature depending on the strain studied. Both species produce a large amount of OTA on dry-cured ham and on dry-cured fermented sausage "salchichón" in environmental conditions usually found throughout the ripening of these products. The Risk Ranger software reveals that the relative risk of OTA on dry-cured meat products is 75%. Thus, control measures during dry-cured meat products processing to prevent OTA risk should be established.

Relationship between cyclopiazonic acid production and gene expression in Penicillium griseofulvum under dry-cured ham processing environmental conditions.

Cyclopiazonic acid (CPA)-producing Penicillium griseofulvum is usually found on the dry-cured ham surface during its ripening. The objective of this work was to evaluate the effect of temperature and water activity (aw) of dry-cured ham processing on growth, CPA production, and temporal relative expression of genes involved in CPA biosynthesis on dry-cured meat-based media. P. griseofulvum CECT 2919 grew faster than P. griseofulvum IBT 14319 in all conditions tested, although no growth occurred at 0.85 aw. Besides, the dry-cured ham-based medium favoured CPA synthesis for both strains compared to the meat-based medium. For the strain CECT 2919, the expression of the mfs-1 and pks-nrps genes were stimulated at 0.90 and 0.95 aw, respectively, while the dmaT gene expression was inhibited during the incubation time. By contrast, the strain IBT 14319 showed that the dmaT gene expression was stimulated at 0.90 aw, while the pks-nrps and mfs-1 genes were repressed throughout incubation time. In conclusion, it is necessary to reduce aw on the surface of the hams below 0.85 during ripening before to increase temperature to reduce growth of P. griseofulvum and CPA production. This information may be useful to design preventive and corrective actions to minimise risks associated with the presence of CPA in dry-cured ham.

Biocontrol of aflatoxigenic Aspergillus parasiticus by native Debaryomyces hansenii in dry-cured meat products.

Dry-cured meat products, such as dry-cured ham or dry-fermented sausages, are characterized by their particular ripening process, where a mould population grows on their surface. Some of these moulds are hazardous to the consumers because of their ability to produce mycotoxins including aflatoxins (AFs). The use of native yeasts could be considered a potential strategy for controlling the presence of AFs in dry-cured meat products. The aim of this work was to evaluate the antagonistic activity of two native Debaryomyces hansenii strains on the relative growth rate and the AFs production in Aspergillus parasiticus. Both D. hansenii strains significantly reduced the growth rates of A. parasiticus when grown in a meat-model system at different water activity (aw) conditions. The presence of D. hansenii strains caused a stimulation of AFs production by A. parasiticus at 0.99 aw. However, at 0.92 aw the yeasts significantly reduced the AFs concentration in the meat-model system. The relative expression levels of the aflR and aflS genes involved in the AFs biosynthetic pathway were also repressed at 0.92 aw in the presence of both D. hansenii strains. These satisfactory results were confirmed in dry-cured ham and dry-fermented sausage slices inoculated with A. parasiticus, since both D. hansenii strains significantly reduced AFs amounts in these matrices. Therefore, both tested D. hansenii strains could be proposed as biocontrol agents within a HACCP framework to minimize the hazard associated with the presence of AFs in dry-cured meat products.

Biocontrol of Penicillium griseofulvum to reduce cyclopiazonic acid contamination in dry-fermented sausages.

Dry-fermented sausages are very appreciated by consumers. The environmental conditions during its ripening favor colonization of their surface by toxigenic molds. These molds contribute to the development of sensory characteristics; however, some of them could produce mycotoxins such as cyclopiazonic acid (CPA). CPA is mainly produced by Penicillium commune and Penicillium griseofulvum which have been found in dry-cured meat products. Thus, strategies to prevent the CPA contamination in dry-fermented sausages are needed. The objective of this work was to evaluate the ability of P. griseofulvum to produce CPA in dry-fermented sausage during its ripening as well as to test different strategies to prevent CPA production. The ability of PgAFP antifungal protein-producing Penicillium chrysogenum, Debaryomyces hansenii and Pediococcus acidilactici for inhibiting CPA production by P. griseofulvum was tested on dry-fermented sausage-based medium. Only P. chrysogenum inhibited the CPA production, so this mold was co-inoculated with P. griseofulvum on sausages whose ripening was performed at low temperature. CPA reached around 800 ng/g in the control batch, being reduced to 20 ng/g by the presence of P. chrysogenum. This work demonstrates the risk posed by CPA on dry-fermented sausages, and provides a successful strategy to prevent this hazard.

Sensitive determination of cyclopiazonic acid in dry-cured ham using a QuEChERS method and UHPLC-MS/MS.

An extraction method and an UHPLC-MS/MS method for the quantification of CPA in dry-cured ham were developed and validated. To optimise detection and quantification of CPA, the composition of mobile phase, flow rates, gradient-related factors and solvents used for resuspension of dry extracts were evaluated. Besides, four extraction methods were tested. The best peak shape and resolution were obtained by eluting the mobile phase consisting in acetic acid-ammonium acetate buffer pH 5.75/methanol in gradient mode at a flow rate of 0.2 mL/min. The method 4 relied on the QuEChERS methodology was the most effective one. Almost half of the 61 dry-cured ham samples examined were contaminated with CPA, with values ranging from 36.1 to 540.1 ng/g. The combination of a QuEChERS-based extraction method and analysis by UHPLC-MS/MS allows highly sensitive, fast, reliable and cheap detection and quantification of CPA for routine analysis in ham.

Potential of yeasts isolated from dry-cured ham to control ochratoxin A production in meat models.

The environmental conditions reached during the ripening of dry-cured meat products favour the proliferation of moulds on their surface. Some of these moulds are hazardous to consumers because of their ability to produce ochratoxin A (OTA). Biocontrol using Debaryomyces hansenii could be a suitable strategy to prevent the growth of ochratoxigenic moulds and OTA accumulation in dry-cured meat products. The aim of this work was to evaluate the ability of two strains of D. hansenii to control the growth and OTA production of Penicillium verrucosum in a meat model under water activities (aw) values commonly reached during the dry-cured meat product ripening. The presence of D. hansenii strains triggered a lengthening of the lag phase and a decrease of the growth rate of P. verrucosum in meat-based media at 0.97 and 0.92 aw. Both D. hansenii strains significantly reduced OTA production (between 85.16 and 92.63%) by P. verrucosum in the meat-based medium at 0.92 aw. Neither absorption nor detoxification of OTA by D. hansenii strains seems to be involved. However, a repression of the expression of the non-ribosomal peptide synthetase (otanpsPN) gene linked to the OTA biosynthetic pathway was observed in the presence of D. hansenii. To confirm the protective role of D. hansenii strains, they were inoculated together with P. verrucosum Pv45 in dry-fermented sausage and dry-cured ham slices. Although P. verrucosum Pv45 counts were not affected by the presence of D. hansenii in both meat matrices, a reduction of OTA amount was observed. Therefore, the effect of D. hansenii strains on OTA accumulation should be attributed to a reduction at transcriptional level. Consequently, native D. hansenii can be useful as biocontrol agent in dry-cured meat products for preventing the hazard associated with the presence of OTA.

Gene expression as a good indicator of aflatoxin contamination in dry-cured ham.

Aspergillus flavus and Aspergillus parasiticus are mould species producers of aflatoxins (AFs) and may grow on dry-cured ham during the ripening process. In this study, the influence of different water activity (aw) and temperatures on the temporal relative expression of three genes involved in AFs biosynthesis and their relationship with AFs production on dry-cured ham-based medium were evaluated. In general, the regulatory aflR and aflS genes showed similar expression patterns, and the expression of the structural aflP gene was much higher than that obtained for aflR and aflS genes. Regarding A. flavus, a decrease of aw regardless of temperature caused an increase of the expression of the regulatory aflR and aflS genes. Concerning A. parasiticus, the highest and lowest expression values of the regulatory aflR and aflS genes were found at 0.95 aw and 0.85 aw, respectively. The expression of the structural aflP gene of both species was stimulated at low temperature and aw. The PCA analysis indicated that both toxigenic species showed a strong correlation between the relative expression of the aflR and aflS genes and the concentration of AFs under conditions which simulate dry-cured ham ripening. This suggests that an early detection of the expression of regulatory genes can be a good indicator of possible AFs contamination of dry-cured ham through ripening.

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.

Selection and evaluation of Debaryomyces hansenii isolates as potential bioprotective agents against toxigenic penicillia in dry-fermented sausages.

Biocontrol using autochthonous Debaryomyces hansenii isolates is a potentially suitable strategy for inhibiting toxigenic moulds in dry-cured meat products. The antifungal activity of 280 D. hansenii isolated from dry-cured meat products as well as the mode of action of the most active isolates against toxigenic penicillia were evaluated in this work. A 13.9% of the D. hansenii isolates showed inhibitory activity in a radial inhibition assay. The effects on penicillia growth of both the cell-free culture filtrate and volatile compounds from active yeast isolates were analysed. Penicillia growth inhibition by D. hansenii was probably based on additive or synergistic effects of several inhibiting factors such as competition for nutrient and space, and production of soluble or volatile compounds. When four D. hansenii isolates were tested on dry-fermented sausage, two of them produced a significantly growth reduction of the ochratoxigenic Penicillium verrucosum, keeping its counts under the level considered as hazardous for the mycotoxin presence. Therefore, the use of these two D. hansenii isolates during the processing of dry-fermented meat product could be a promising tool to control toxigenic moulds in the meat industry.