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Probiotic fermented milks are a group of functional food that, in addition to their nutritional value, contain certain components considered beneficial to human health. For their production, besides probiotic bacterial strains, yeasts - most commonly Saccharomyces boulardii (S. boulardii), are used too in the form of an secondary culture. The biotherapeutic effect of this yeast on the human body has been most studied. S. boulardii is also considered a potential natural preservative due to its antagonistic effect against other microorganisms. Its survivability and its effects on organoleptic properties have been studied in the production of yogurt from cow’s and goat’s milk, kefir, and acidophilic fermented milk. Based on all these factors, these products can be considered as new functional dairy products.

Introduction: Spontaneously fermented game meat sausages produced by traditional techniques are complex microbial ecosystems that can be the sources of beneficial or spoilage and pathogenic microorganisms. Aim: This study aimed to investigate the fate of indigenous microbiota of spontaneously fermented game meat sausages in order to estimate microbiological hazards as well to identify and preserve the microbial pool that could serve as a source of strains relevant for standardization of sausages production. Material and Methods: Three wild boar and three dear meat sausages, produced by five different manufacturers following traditional techniques, were physicochemically and microbiologically investigated at different time points of their production. Results: Total of 917 isolates were collected from different media used for cultivation of lactic acid bacteria (LAB) and identified to species and strain level by PCR tools. Although commonly used for the isolation of specific groups of LAB, most of the applied media exhibited poor selectivity and supported the growth of diverse LAB or even non-LAB species. In total, Leuconostoc mesenteroides was identified as the most frequently isolated species (28.24%), followed by Lactobacillus sakei (20.72%) and Enterococcus casseliflavus (11.55%), however, strong differences were observed in distribution profiles of LAB among investigated sausages. Cluster analysis revealed strong sausage-specific clustering of strains and remarkable diversity with 280 genotypes detected. High count of spoilage and/or pathogenic bacteria was noted for two sausages, making them potentially hazardous for human health. Conclusions: The vast differences between sausages in prevailing microbiota indicated the need for exploring the possibilities of product standardization, likely to select starter/bioprotective cultures from indigenous microbiota.

Sausages from red deer (Cervus elaphus) meat are manufactured at small scale in Croatia following traditional techniques. As such, these artisanal products have increased value due to their specific sensory properties and authenticity. However, factors such as anatomical shooting location, rifle i.e. munitions type, shooting – evisceration time and geographical location may influence the hygienic conditions of game meat, and consequently the hygienic conditions of game meat sausages. In order to evaluate their microbiological status, samples were collected from 3 different sausage producers (J1, J2 and J3) after 0, 4, 7, 10, 20 days and at the end of the ripening. Samples were screened for the presence of L. monocytogenes according to ISO standard. Pathogenic and spoilage bacteria, such as Staphylococcus aureus, Enterobacteriaceae, Bacillus cereus and E. coli were isolated and enumerated using BP, VRBG, MYP and CCA media, respectively. Yeasts and moulds were isolated and their number was determined using DRBC media while KAA media was used for enterococci. For further analysis and possible application as starter or bioprotective cultures, lactic acid bacteria (LAB) strains were isolated and collected using MRS media supplemented with cycloheximide. Collected LAB isolates were grouped using the rep – PCR method, after which representative strains were selected and identified using 16s rRNA gene sequencing. Our results showed that the number of LAB was low in the initial period (2, 39 log CFU / g), after that they proliferated and reached the maximum value after 7 days (8.3 log CFU / g) and their growth stabilized through the end of ripening (7.82 log CFU / g). Among 139 MRS isolates collected, Lactobacillus sakei was the most dominant species (47.48 %) followed by Lactobacillus curvatus.(14.38 %) and Leuconostoc mesenteroides (7.91 %). Enterococci were detected only in J1. Moulds were not detected while the growth trends of yeasts differed between J1, J2 and J3. The number of L. monocytogenes, E. coli, Staphylococcus aureus and Bacillus cereus was below the detection limit in ready-to-eat sausages. Enterobacteriaceae were not detected at the end stages of ripening in J1 and J3 while their number was high in J2 (4.52 log CFU/g). The difference in results obtained between the three deer meat sausages indicate the need for further molecular and microbiological characterization and moreover to explore the possibilities of product standardization.

Natural fermentation of game meat sausages has a long tradition in various countries for thousands of years, and nowadays still represents most commonly used practice for game meat preparation and preservation. Artisanal sausages prepared from wild boar meat are highly appreciated due to their authenticity and specific sensory qualities. However, there are certain concerns regarding the safety and hygienic conditions of ready-to-eat products. The current study aimed to explore the microbial diversity of traditionally produced wild boar sausages in order to estimate microbiological hazards as well as to obtain the collection of indigenous lactic acid bacteria (LAB) strains that could potentially be used as a starter and/or bioprotective cultures. Samples were collected from 3 different sausage producers (F1, F2, F3) after 0, 4, 7, 10, 20 days, and at the end of ripening. Total of 531 LAB isolates were collected using KAA, MRS, and MSE selective media and grouped using the rep – PCR method. After the cluster analysis, the representative members of each cluster group were identified based on 16S rRNA gene sequencing and based on PCR tools for the identification at the genus and species level. The prevalent isolates were identified as Leuconostoc mesenteroides (23.35%), followed by Lactobacillus sakei (18.08%) and Enterococcus casseliflavus (13.75%). Relevant strains were selected for further technological characterization. One out of three ready-to-eat sausages (F3) indicated safety risks to the consumers due to the high incidence of E.coli, with cell counts of 3.04 log CFU/g. This finding was supported by detected high numbers of B. cereus (3.86 log/CFU/g), Enterobacteriaceae (3.86 log CFU/g) and Staphylococcus aureus (3.15 log CFU/g). Spontaneously fermented wild boar sausages not only gave rise to safety concerns but also represent a valuable source of new presumptive starter strains.

Fermented foods produced by traditional techniques represent an important vehicle for live bacteria to human gastrointestinal tract (GIT). These bacteria may carry genes coding for antibiotic resistance which may be transferred to the other commensal or pathogen bacteria in GIT. It has been reported that indigenous bacteria found in artisan products may possess intrinsic multi-resistance to antibiotics, a trait that has evolved in bacteria before antibiotics were even applied in medicine or husbandry. Furthermore, bacteria possess a remarkable ability to develop new resistances (acquired resistance) through many different mechanisms. As the data for antibiotic resistances of bacteria and interspecies diversity of enterococci from traditional fermented sausages is scarce, one of the main aims of our study was to identify enterococci of traditional sausages produced from deer (Cervus elaphus) and wild boar (Sus scrofa) meat and to analyse their susceptibility to different clinically important antibiotics. Many studies have based the identification of enterococci with 16S rRNA gen-specific primers. However, due to the fact that non-human sources may be atypical and usually do not meet the criteria used for standard characterization we have used the superoxide dismutase gene (sodA) for interspecies differentiation of enterococci. To test their antimicrobial susceptibility, agar disc diffusion method against 6 antibiotics was applied. By sodA PCR approach, we were able to confirm and to determine Enterococcus faecalis, E. faecium, E. durans, E. casseliflavus and E. gallinarum isolated on kanamycine esculine azid agar (KAA) from game meat sausages during the fermentation and ripening (0, 4, 7, 10, 20, 40 days). From total of 280 isolates, the resistance towards tetracycline 30 µg with 4 %, erythromycin 15 µg with 2.5 % and vancomycin 5 with 3 % was observed, as well as intermediate resistance for erythromycin 15 µg (57 %), chloramphenicol 30 µg (5 %) and tetracycline 30µg (2 %). In order to detect genes and where they are located (plasmid or chromosome), further study is necessary. Identification of enterococci on species level by multiplex PCR method with primers for unique sodA gene was a valuable tool for reliable identification of indigenous enterococci from game meat sausages.