Microbial Ecology of French Dry Fermented Sausages and Mycotoxin Risk Evaluation During Storage

Abstract

Dry fermented sausages are produced worldwide by well-controlled fermentation processes involving complex microbiota including many bacterial and fungal species with key technological roles. However, to date, fungal diversity on sausage casings during storage has not been fully described. In this context, we studied the microbial communities from dry fermented sausages naturally colonized or voluntarily surface inoculated with molds during storage using both culture-dependent and metabarcoding methods. Staphylococci and lactic acid bacteria largely dominated in samples, although some halotolerant genera (e.g., Halomonas, Tetragenococcus, and Celerinatantimonas spp.) were also frequently observed. Fungal populations varied from 7.2 to 9.8 log TFU/cm2 sausage casing during storage, suggesting relatively low count variability among products. Fungal diversity identified on voluntarily inoculated casings was lower (dominated by Penicillium nalgiovense and Debaryomyces hansenii) than naturally environment-inoculated fermented sausages (colonized by P. nalgiovense, Penicillium nordicum, and other Penicillium spp. and sporadically by Scopulariopsis sp., D. hansenii, and Candida zeylanoïdes). P. nalgiovense and D. hansenii were systematically identified, highlighting their key technological role. The mycotoxin risk was then evaluated, and in situ mycotoxin production of selected mold isolates was determined during pilot-scale sausage productions. Among the identified fungal species, P. nalgiovense was confirmed not to produce mycotoxins. However, some P. nordicum, Penicillium chrysogenum, Penicillium bialowienzense, Penicillium brevicompactum, and Penicillium citreonigrum isolates produced one or more mycotoxins in vitro. P. nordicum also produced ochratoxin A during pilot-scale sausage productions using “worst-case” conditions in the absence of biotic competition. These data provide new knowledge on fermented sausage microbiota and the potential mycotoxin risk during storage.

Domestication in dry-cured meatPenicilliumfungi: convergent specific phenotypes and horizontal gene transfers without strong genetic subdivision

“…Penicillium nalgiovense is the commonest species in the dry-cured meat food industry (Andersen & Frisvad, 1994; Larsen & Breinholt, 1999; Sunesen & Stahnke, 2003). Penicillium salamii was recently identified as an alternative ripening agent and has only been used or recovered from local dry-cured meat production sites, often producing naturally fermented meat products (M. Coton et al, 2021; Sinch & Dincho, 1994; Sunesen & Stahnke, 2003). This difference in usage was reflected in our samples from dry-cured meats, for which P. nalgiovense was the most frequently isolated species, followed by P. salamii.…”

“…None of the targeted extrolites, including mycotoxins and penicillin, were detected in P. nalgiovense or P. salamii dry-cured meat strains (M. Coton et al, 2021). However, the production of penicillin has been reported in some P. nalgiovense strains (Andersen & Frisvad, 1994) and the closely related species P. chrysogenum is known to produce mycotoxins (M. Coton et al, 2019; Frisvad et al, 2004).…”

“…Several traits have probably been subject to similar selection processes in both these fungi, either deliberately through human intervention or unwittingly, to enable these fungi to thrive in dry-cured meats and produce high quality products, with an appealing visual appearance and aroma, and a better shelf life and product safety. The fungi colonize the surface, thereby helping to preserve the dry-cured meat and to protect it against excessive desiccation, irradiation by light, oxygen and undesirable microorganisms, such as contaminating toxigenic molds 31,34,35 . These fungal species also help to improve the aroma of the dry-cured meat, through the metabolites they produce with their lipase, protease, transaminase and dehydrogenase activities 30,36 .…”

“…Like cheese fungi, Penicillium nalgiovense is an excellent model for studying domestication and adaptation. This species has been used to inoculate the surface of most dry-cured meat products for decades, to enhance the ripening process (M. Coton et al, 2021; Garcia et al, 2001; Ludemann et al, 2004; Sunesen & Stahnke, 2003). Another recently described fungus, Penicillium salamii , also occurs in dry-cured meats (Perrone et al, 2015) and may be included in starter cultures or form part of the natural mycobiota at local production sites (M. Coton et al, 2021; Magistà et al, 2016).…”

“…This species has been used to inoculate the surface of most dry-cured meat products for decades, to enhance the ripening process (M. Coton et al, 2021; Garcia et al, 2001; Ludemann et al, 2004; Sunesen & Stahnke, 2003). Another recently described fungus, Penicillium salamii , also occurs in dry-cured meats (Perrone et al, 2015) and may be included in starter cultures or form part of the natural mycobiota at local production sites (M. Coton et al, 2021; Magistà et al, 2016). Several traits have probably been subject to similar selection processes in both these fungi, either deliberately through human intervention or unwittingly, to enable these fungi to thrive in dry-cured meats and produce high quality products, with an appealing appearance and aroma, and a better shelf life and product safety.…”