Effect of selected agents for ochratoxin A biocontrol on the colour, texture and volatile profile of dry-cured fermented sausages.

BACKGROUND: Traditional dry-cured fermented sausages favour the growth of an autochthonous microbial population, which plays an important role in their sensory aspects. However, some moulds can produce mycotoxins such as ochratoxin A (OTA). The biocontrol agents (BCAs) Debaryomyces hansenii FHSCC 253H and Staphylococcus xylosus FHSCC Sx8 have been demonstrated to reduce OTA production in dry-cured meat products, but their influence in the sensory characteristics of sausages has to be tested. The aim of this study was to evaluate the effect of these BCAs on the colour, texture and volatile profile of dry-cured fermented sausages. RESULTS: D. hansenii caused few differences in the tested parameters with respect to the control batch. S. xylosus modified the texture and colour, although the values found were within the range expected for dry-cured fermented sausages 'salchichón'. Additionally, the volatile profile revealed the potential antioxidant effect of both BCAs and their ability to produce compounds associated with the ripened aroma that could increase product acceptability. CONCLUSION: The results indicate that there were no inconveniences in implementing both BCAs during the processing of dry-cured fermented sausages 'salchichón'. Moreover, D. hansenii FHSCC 253H could improve the volatile profile of this product. © 2023 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

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.

Deciphering Staphylococcus xylosus and Staphylococcus equorum mode of action against Penicillium nordicum in a dry-cured ham model system.

Penicillium nordicum is one of the major producers of ochratoxin A (OTA) in dry-cured ham. Staphylococcus xylosus Sx8 and Staphylococcus equorum Se31 have been previously proposed as biocontrol agents (BCAs) to prevent the OTA contamination, although their antifungal mode of action has not been established yet. Thus, the aim of this work was to elucidate their mode of action against P. nordicum in a dry-cured ham model system. For this, the effect of live cells, dead cells, and cell-free broth; the nutritional utilisation pattern, niche overlap index (NOI), interactions by dual-culture assays, antifungal effect of volatile compounds, OTA detoxification, and effect on fungal proteome were determined. No fungal growth was observed after 14 days of co-culture with live cells of each staphylococcus at 15 or 20 °C. However, such inhibition was not observed with either dead cells or extracellular extracts. The number of carbon sources utilised by P. nordicum was higher than those used by both cocci at 20 °C, whilst the opposite occurred at 15 °C. According to NOI, nutritional dominance depends on temperature, at 20 °C P. nordicum dominated the niche, but at 15 °C the mould is dominated by the BCAs. The volatile pattern generated by each coccus did not show antifungal effect, and both staphylococci failed to degrade or adsorb OTA. However, in the interaction assay, S. xylosus and S. equorum were able to decrease the fungal growth and its OTA production. In addition, proteomic analyses showed changes in the abundance of proteins related to the cell wall integrity (CWI), carbohydrate metabolism and the biosynthesis of secondary metabolites such as OTA. In conclusion, overall, the antagonistic effects of the two studied cocci against P. nordicum are greater at 15 °C than at 20 °C, being linked to competition for space and nutrients, triggering alterations in CWI pathway, OTA biosynthesis, and carbohydrate metabolism.

Biocontrol of Pathogen Microorganisms in Ripened Foods of Animal Origin.

Ripened foods of animal origin comprise meat products and dairy products, being transformed by the wild microbiota which populates the raw materials, generating highly appreciated products over the world. Together with this beneficial microbiota, both pathogenic and toxigenic microorganisms such as Listeria monocytogenes, Salmonella enterica, Staphylococcus aureus, Clostridium botulinum, Escherichia coli, Candida spp., Penicillium spp. and Aspergillus spp., can contaminate these products and pose a risk for the consumers. Thus, effective strategies to hamper these hazards are required. Additionally, consumer demand for clean label products is increasing. Therefore, the manufacturing sector is seeking new efficient, natural, low-environmental impact and easy to apply strategies to counteract these microorganisms. This review gathers different approaches to maximize food safety and discusses the possibility of their being applied or the necessity of new evidence, mainly for validation in the manufacturing product and its sensory impact, before being implemented as preventative measures in the Hazard Analysis and Critical Control Point programs.

Biocontrol of ochratoxigenic Penicillium nordicum in dry-cured fermented sausages by Debaryomyces hansenii and Staphylococcus xylosus.

Penicillium nordicum is the main ochratoxin A (OTA) producing mould in dry-cured meat products. The use of autochthonous microorganisms as protective cultures is a promising strategy to control this hazard. The aim of this work was to evaluate the effect of Debaryomyces hansenii and Staphylococcus xylosus isolated from dry-cured meat products as biocontrol agents (BCAs) against P. nordicum during the ripening of dry-cured fermented sausages. The BCAs were added to the dough, and P. nordicum were inoculated on the surface after stuffing. Then, the sausages were ripened following a traditional processing. The growth of the microorganisms was determined by plate count at the beginning and at the end of ripening. To assess the implantation of BCAs in the sausages, the yeasts and staphylococci isolated from the sausages at the end of processing were identified by sequencing the 16S and 18S rRNA respectively, and they were characterized by pulsed field gel electrophoresis (PFGE) of the chromosomal DNA. OTA was quantified by UHPLC-MS/MS. BCAs were able to colonise and develop throughout the processing. Although none of the BCAs reduced the growth of P. nordicum, a OTA decrease was observed in the sausages inoculated with D. hansenii individually or combined with S. xylosus. The drop of OTA amount was particularly marked in the portions where the casing was damaged allowing the mould to grow inside the sausages. In these areas, OTA was not detected in the inoculated batches. In conclusion, D. hansenii could be proposed as BCA individually or in combination with S. xylosus for the biocontrol of OTA hazard in dry-cured fermented sausages.

Development of a Methodology for Estimating the Ergosterol in Meat Product-Borne Toxigenic Moulds to Evaluate Antifungal Agents.

Antifungal agents are commonly used in the meat industry to prevent the growth of unwanted moulds, such as toxigenic ones, on dry-cured meat products. For enhancing the application of antifungals, their mode of action must be evaluated. Their effect on the mould ergosterol content is one of the most studied ones, since it is the target site of some commercialised antifungals or of those that are in development. The aim of this study was to develop a methodology for determining how the antifungal agents used in the meat industry work. A method for analysing ergosterol was firstly developed using high-performance liquid chromatography with fluorescence detection coupled to a diode array detector (HPLC-FLD/DAD). The chromatographically optimised conditions (gradient and mobile phases) allowed us to reduce the time per analysis with respect to previously published methods up to 22 min. Withing the six checked extraction methods, method 5, showing the best mean recovery values (99.51%), the shortest retention time (15.8 min), and the lowest standard deviation values (9.92) and working temperature (60 °C), was selected. The limit of detection and limit of quantification were 0.03 and 0.1 µg/mL, respectively. All the validation parameters corroborated the method's suitability. Finally, its feasibility for evaluating the effect of a commercial antifungal preparation (AP) and different herbs that are frequently added to meat products on the ergosterol content of several toxigenic moulds was studied. Differences at the strain level were obtained in the presence of AP. Moreover, the addition of herbs significantly reduced the ergosterol content in Penicillium nordicum up to 83.91%. The developed methodology is thus suitable for screening the antifungals' role in altering mould ergosterol biosynthesis before their application in real meat products.