Perspectives on the Probiotic Potential of Indigenous Moulds and Yeasts in Dry-Fermented Sausages.

The role of indigenous fungi in the appropriate development of sensory properties and the safety of dry-fermented sausages has been widely established. Nonetheless, their applications as probiotic agents have not been elucidated in such products yet, despite their promising functional features. Thus, it should be interesting to evaluate the probiotic potential of native Debaryomyces hansenii isolates from dry-fermented sausages and their application in the meat industry, because it is the most frequently isolated yeast species from these foodstuffs and its probiotic effects for animals as well as its possible probiotic activity for human beings have been demonstrated. Within the functional ability of foodborne yeasts, anti-inflammatory, antioxidant, antimicrobial, antigenotoxic, and immunomodulatory properties have been reported. Similarly, the use of dry-fermented sausages as vehicles for probiotic moulds remains a challenge because the survival and development of moulds in the gastrointestinal tract are still unknown. Nevertheless, some moulds have been isolated from faeces possibly from their spores as a form of resistance. Additionally, their beneficial effects on animals and humans, such as the decrease in lipid content and the anti-inflammatory activity, have been reported, although they seem to be more related to their postbiotic capacity due to the generated bioactive compounds with profunctional attributes than to their role as probiotics. Therefore, further studies providing knowledge useful for generating dry-fermented sausages with improved functionality are fully necessary.

Proteomic evaluation of the effect of antifungal agents on Aspergillus westerdijkiae ochratoxin A production in a dry-cured fermented sausage-based medium.

Aspergillus westerdijkiae may produce large amounts of ochratoxin A (OTA) in dry-cured meat products. Natural strategies to control ochratoxigenic moulds using biocontrol agents (BCAs) are currently in the spotlight. The aim of this study was to test the effects of Debaryomyces hansenii and its combination with rosemary derivatives and with a commercial antifungal preparation composed by natamycin and potassium sorbate (AP) against A. westerdijkiae in a dry-cured fermented sausage based-medium. The yeast and rosemary leaves were added to the medium, and rosemary essential oil and AP were added on the casings put on the medium surface to simulate the real product. The growth rate, OTA production and comparative proteomics were analysed. The mould growth in the presence of each treatment was not indicative of their efficiency on OTA repression. The treatment with AP did not affect to the OTA concentration, maybe as consequence of the stressful stimulation of the subinhibitory doses used. D. hansenii added alone or with rosemary showed the best results, decreasing the OTA production >80 %, suggesting that it can be useful as preservative agent during industrial processing. Attending to the proteomic results, its antifungal activity seems to be based on the reduction in abundance of proteins involved in OTA biosynthesis and in the cell wall integrity pathway.

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.

Effects of Preservative Agents on Quality Attributes of Dry-Cured Fermented Sausages.

Enterococcus faecium SE920, Debaryomyces hansenii FHSCC 253H, Penicillium chrysogenum CECT 20922, producer of the antifungal protein PgAFP, and this protein itself have previously been proposed to control toxigenic molds in dry-cured meat products. However, their effects on the usual microbial population, and the sensory characteristics of these foods, have not yet been evaluated. The aim of this study was to assess the viability of the inoculation of these protective cultures, and their impact on the quality of dry-cured fermented sausages. These microorganisms were co-inoculated with a native desirable population (Penicillium nalgiovense, P. chrysogenum, D. hansenii, and Staphylococcus vitulinus) in a dry-cured fermented sausage (salchichón)-based medium in the presence and absence of PgAFP. Macroscopically, the biocontrol candidates did not produce relevant changes in the growth of the native population, enabling their coexistence. However, PgAFP causes the alteration of the hyphae structure in desirable molds. Thus, PgAFP was discarded for use on the surface of raw dry-cured fermented sausages (salchichón) in the pilot plant. The used biocontrol agents did not negatively affect the physico-chemical parameters of the dry-cured fermented sausages (salchichón) after ripening, which showed the typical volatile profile and odor. Thus, the application of E. faecium SE920, D. hansenii FHSCC 253H, and P. chrysogenum CECT 20922 as protective cultures against toxigenic molds during the ripening of dry-cured fermented sausages does not modify their typical sensorial quality.

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.

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.

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.

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.

Efficiency of mitochondrial DNA restriction analysis and RAPD-PCR to characterize yeasts growing on dry-cured Iberian ham at the different geographic areas of ripening.

The efficiency of mitochondrial DNA (mtDNA) restriction analysis and random amplification of polymorphic DNA (RAPD)-PCR to characterize yeasts growing on dry-cured Iberian ham was evaluated. Besides, the distribution of the main species and biotypes of yeasts in the different ripening areas of this product was investigated. MtDNA restriction analysis allowed yeast characterization at species and strain level. RAPD-PCR with the primers (GACA)(4) and (GAC)(5) was inappropriate for characterization at species level. Most of the mtDNA restriction patterns detected in dry-cured Iberian ham were consistent with Debaryomyces hansenii. Several yeasts biotypes were associated to specific geographic areas of dry-cured Iberian ham ripening.