Control of toxigenic Aspergillus spp. in dried figs by volatile organic compounds (VOCs) from antagonistic yeasts.

Aspergillus flavus and Aspergillus niger are fungi which can contaminate dried figs before and after harvest and consequently produce aflatoxins (AFs) and ochratoxin A (OTA). Many approaches have been applied to minimise the growth of these filamentous fungi, mainly involving the use of synthetic fungicides which are limited due to their negative impact on human health and the environment. In this context, biocontrol is a recent approach that needs to be explored. This study evaluated the potential of three volatile organic compounds (VOCs), octanoic acid (OA), 2-phenylethyl acetate (2PEA) and furfuryl acetate (FA), produced by Hanseniaspora uvarum and Hanseniaspora opuntiae yeasts on the growth, germination, gene expression and production of AFs and OTA by A. flavus M144 and A. niger M185 on dried fig-based agar and the incidence rates in dried figs. Two of the three VOCs evaluated (2PEA and FA) effectively controlled A. flavus M144 and A. niger M185 by using at least amounts of 50 µL (715 µL/L in the headspace) for FA and 100 µL (1430 µL/L in the headspace) for 2PEA in dried figs. One of the mode of actions of both compounds consists in early repressing the expression of genes involved in the biosynthesis of AFs (aflR) and OTA (pks) of A. flavus and A. niger, respectively. The results of this study support the application of 2PEA and FA at the early post-harvest stages of dried figs to control mycotoxin accumulation.

In Vitro Biological Control of Aspergillus flavus by Hanseniaspora opuntiae L479 and Hanseniaspora uvarum L793, Producers of Antifungal Volatile Organic Compounds.

Aspergillus flavus is a toxigenic fungal colonizer of fruits and cereals and may produce one of the most important mycotoxins from a food safety perspective, aflatoxins. Therefore, its growth and mycotoxin production should be effectively avoided to protect consumers' health. Among the safe and green antifungal strategies that can be applied in the field, biocontrol is a recent and emerging strategy that needs to be explored. Yeasts are normally good biocontrol candidates to minimize mold-related hazards and their modes of action are numerous, one of them being the production of volatile organic compounds (VOCs). To this end, the influence of VOCs produced by Hanseniaspora opuntiae L479 and Hanseniaspora uvarum L793 on growth, expression of the regulatory gene of the aflatoxin pathway (aflR) and mycotoxin production by A.flavus for 21 days was assessed. The results showed that both yeasts, despite producing different kinds of VOCs, had a similar effect on inhibiting growth, mycotoxin biosynthetic gene expression and phenotypic toxin production overall at the mid-incubation period when their synthesis was the greatest. Based on the results, both yeast strains, H. opuntiae L479 and H. uvarum L793, are potentially suitable as a biopreservative agents for inhibiting the growth of A. flavus and reducing aflatoxin accumulation.

Effect of Debaryomyces hansenii and the antifungal PgAFP protein on Alternaria spp. growth, toxin production, and RHO1 gene expression in a tomato-based medium.

Tomato fruit is susceptible to Alternaria spp. spoilage, which poses a health risk due to their mycotoxin production. Biopreservation relies on the use of whole microorganisms or their metabolites to manage spoilage microorganisms including filamentous fungi. However, the use of treatments at fungistatic level might activate intracellular pathways, which can cause an increment in mycotoxin accumulation. The objective of this work was to evaluate the effect of two strains of Debaryomyces hansenii and the antifungal protein PgAFP at 10 and 40 µg/mL. Both growth and production of two of the most common mycotoxins (tenuazonic acid and alternariol monomethyl ether) by Alternaria tenuissima sp.-grp. and Alternaria arborescens sp.-grp. on a tomato-based matrix, were analysed at 12 °C. Additionally, the impact of these biocontrol agents on the stress-related RHO1 gene expression was assessed. All treatments reduced mycotoxin accumulation (from 27 to 92% of inhibition). Their mode of action against Alternaria spp. in tomato seems unrelated to damages to fungal cell wall integrity at the genomic level. Therefore, the two D. hansenii strains (CECT 10352 and CECT 10353) and the antifungal protein PgAFP at 10 µg/mL are suggested as biocontrol strategies in tomato fruit at postharvest stage.

Competitiveness of three biocontrol candidates against ochratoxigenic Penicillium nordicum under dry-cured meat environmental and nutritional conditions.

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.

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.

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 ß-oxidation, and aflP, which is required for aflatoxin biosynthesis, were evaluated. The aflatoxin overproduction induced by PgAFP seems not to be linked to peroxisomal ß-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.

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.

Inhibition of ochratoxigenic moulds by Debaryomyces hansenii strains for biopreservation of dry-cured meat products.

The ability of the osmotolerant yeast Debaryomyces hansenii to inhibit Penicillium nordicum, the most common ochratoxigenic mould encountered in dry-cured meat products, was evaluated. The antagonistic effect of ten D. hansenii strains isolated from dry-cured ham was screened in vitro using malt extract media and meat extract peptone media with the water activity (a(w)) adjusted to 0.97 and 0.90. A significant inhibition of the two tested P. nordicum strains by D. hansenii cells and cell-free supernatants was observed. At 0.97 a(w), increasing D. hansenii inoculum concentrations significantly improved the inhibition of mould growth on solid medium, whereas at 0.90 a(w) this was not always the case. As observed by bright field microscopy, most D. hansenii strains were able to delay P. nordicum spore germination when co-cultured in malt extract broth. D. hansenii FHSCC 253H showed the highest overall in vitro inhibition of ochratoxigenic mould growth, and was therefore chosen for co-cultivation assays in dry-cured ham slices incubated at 0.94 and 0.84 a(w) simulating ham ripening. Regardless of the experimental conditions tested, lower levels of the inoculated P. nordicum strain were detected in co-cultivation batches compared with batches without D. hansenii. The highest level of mould growth inhibition was observed in batches at 0.94 a(w). Ochratoxin A (OTA) production in ham samples was detected by HPLC-MS. Co-culturing of P. nordicum with D. hansenii FHSCC 253H resulted in lower OTA levels compared with control samples without D. hansenii. The decrease of the mycotoxin presence due to D. hansenii FHSCC 253H was more efficient at 0.94 a(w) (OTA was below the detection limit). In conclusion, D. hansenii is potentially suitable as a biopreservative agent for preventing ochratoxigenic mould growth and OTA accumulation in dry-cured meat products. The inoculation of D. hansenii should be made at the beginning of processing (at the end of post salting) when the a(w) of the product is still high (near 0.94). This action in addition to application of appropriate hygienic actions and control of temperature and relative humidity throughout ripening is required to reduce health risks due to OTA exposure.