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Page 2 of 15 Kamilari et al. Microbiome Res Rep 2025;4:3 https://dx.doi.org/10.20517/mrr.2024.47

region of Cyprus were differentiated by the presence of the species Lactobacillus helveticus, whereas industrially
produced sausages were differentiated by the species Leuconostoc mesenteroides. Focusing mainly on Lactobacillus
and Leuconostoc, negative associations with pathogenic bacteria, such as Salmonella, and spoilage fungi, such as
Fusarium poae, were revealed.

Conclusion: The present metataxonomic analysis provided insights into the microbial communities that
characterize Cypriot and Mytilini sausages. The findings provide an indication that the microbial diversity might be
applied as an additional marker of Cypriot sausages’ authenticity.

Keywords: Sausages, fermented, Mediterranean, 16S rRNA gene, ITS loci, high-throughput sequencing, interaction
networks

INTRODUCTION
Fermented meat products, such as sausages, have been an important part of the gastronomical culture of
East Mediterranean countries. Traditional Cypriot sausages are highly favored locally, with the regional
Pitsilia sausages recently registered as protected geographical indications (PGI) by the European
Commission (EU No: PGI-CY-02369 - 15.9.2017). Their distinctive sensory characteristics arise from their
specific processing conditions. Specifically, ground pork is mixed with salt, spices, and herbs such as
[1]
coriander, and then stuffed into casings made from pork intestines . The fermentation process involves
soaking the sausages in local, red dry wine for eight days, followed by smoking over wood from indigenous
bushes or trees for two to five days. Pitsilia is a mountainous region with a cold climate, which enhances the
preservation of meat products. Traditional Mytilini sausages are also made from pork meat, salt, herbs, and
spices, all encased in a casing derived from pork intestine. Mytilini sausages are also smoked in beech or
other type of wood for seven hours. Their main difference from traditional Cypriot sausages is the absence
of red wine. Industrialized sausages can also be found in the Cypriot and Mytilini markets. Following wine’s
addition, industrialized sausages are treated with cold smoking, and preservatives such as phosphates and
nitrate are added.

The microbial diversity of sausages is influenced by a combination of contributors, including their origin,
the type of meat, processing and fermentation conditions, temperature, ripening stage, the addition of
[2-6]
additives, salt, herbs, spices, and preservatives, the storage conditions, the use of starter cultures, etc. . The
combinatory effect of these factors selects for the development of a unique microbial pattern that affects the
sensorial and qualitative characteristics of the final product. In spontaneously fermented sausages, the
fermentation initiates with a pool of microbes shaped by microorganisms existing in the meat, in the other
added ingredients, and other microbes from the environment . Previous studies have shown that pre-
[7]
fermentation sausages’ microbiota is composed of several unwanted microbes, such as representatives of the
families Lactobacillaceae, Bacillaceae, Actinomycetaceae, Enterobacteriaceae, and Pseudomonadaceae, yeasts,
[8,9]
and molds . Post-fermentation, the microbiota is dominated by coagulase-negative staphylococci and
lactic acid bacteria (LAB), including representatives of Lactobacillaceae, Leuconostocaceae, and
Streptococcaceae. Additionally, the spore-forming Bacillaceae, and spoilage microorganisms, such as
Enterobacteriaceae, Pseudomonadaceae, and Actinomycetaceae, can also be found [10-12] .

To prevent the growth of pathogenic and spoilage microorganisms, a common practice involves the
addition of preservatives, previously synthetic and nowadays natural [13,14] . More recently, an alternative
practice to control the quality of fermented sausage, recognized by the US Food and Drug Administration
(FDA), is called biopreservation . Biopreservation involves the administration of Generally Recognized as
[15]
Safe (G.R.A.S) organisms, such as LAB and/or their metabolites, including bacteriocins, bacteriophages, and