The aim of this study was to examine the microbiological quality of raw donkey milk of an indigenous Serbian breed as well as the changes in the microbial populations during storage at 4 °C. In addition, antibacterial activity of donkey milk against E. coli, L. monocytogenes and S. aureus at 15 °C as well as the content of the two main antibacterial proteins lysozyme and lactoferrin were investigated. Microbiological examination of 137 individual milk samples collected over a period of 21 months showed good microbiological quality since foodborne pathogens such as Salmonella spp. and L. monocytogenes were not detected in any of the analyzed samples, while the number of E. coli, Enterobacteriaceae, total coliform bacteria, sulfite-reducing Clostridia and aerobic sporogenic bacteria was below the limit of quantification (<1 cfu mL⁻¹). During the six-days storage at 4 °C, total bacterial counts and the counts of lactic acid bacteria remained at the initial level while pathogenic bacteria were not detected. The strongest antibacterial activity of the tested milk was observed against E. coli, while S. aureus was the least sensitive to milk antibacterial compounds. Although further research is needed to fully elucidate the antibacterial mechanism and synergistic activity of different compounds in donkey milk, the high content lysozyme (2.63 ± 0.03 g L⁻¹) and lactoferrin (15.48 mg L⁻¹) observed in tested milk could contribute to its strong antibacterial activity and extension of the storage period during which it can be safely consumed.

The aim of this study is to compare the efficacy of selected food disinfectants on planktonic populations of Staphylococcus aureus and Escherichia coli and on the same microorganisms (MOs) incorporated in a biofilm. Two disinfectants were used for treatment: peracetic acid-based disinfectant (P) and benzalkonium chloride-based disinfectant (D). Testing of their efficacy on the selected MO populations was performed using a quantitative suspension test. The standard colony counting procedure was used to determine their efficacy on bacterial suspensions in tryptone soy agar (TSA). The germicidal effect (GE) of the disinfectants was determined based on the decimal reduction ratio. For both MOs, 100% GE was achieved at the lowest concentration (0.1%) and after the shortest exposure time (5 min). Biofilm production was confirmed with a crystal violet test on microtitre plates. Both E. coli and S. aureus showed strong biofilm production at 25 °C with E. coli showing significantly higher adherence capacity. Both disinfectants show a significantly weaker GE on 48 h biofilms compared to the GE observed after application of the same concentrations on planktonic cells of the same MOs. Complete destruction of the viable cells of the biofilms was observed after 5 min of exposure to the highest concentration tested (2%) for both disinfectants and MOs tested. The anti-quorum sensing activity (anti-QS) of disinfectants P and D was determined via a qualitative disc diffusion method applied to the biosensor bacterial strain Chromobacterium violaceum CV026. The results obtained indicate that the disinfectants studied have no anti-QS effect. The inhibition zones around the disc therefore only represent their antimicrobial effect.

Altogether, 39 cereal flour samples taken from a Serbian market were analyzed for mycobiota and mycotoxin content, among which were six Triticum aestivum specimens, five Triticum dicoccum specimens, four Hordeum vulgare specimens, five Fagopyrum esculentum specimens, three Secale cereale specimens, five Triticum spelta specimens, four Avena sativa specimens, two Oryza sativa specimens, two Zea mays specimens, and one specimen each of Panicum miliaceum, Triticum monococcum, and Triticum turgidum ssp. turanicum. To determine the mycobiota content using dilution techniques, the flour samples were transferred to a non-selective DG18 nutrient. The number of coloniforming units (CFU/g) varied from less than 100 (in the case of five samples, namely, two O. sativa, and one specimen each of S. cereale, H. vulgare, and T. aestivum) to as high as 5000 CFU/g (S. cereale), 6000 (A. sativa), 11,000 (T. aesticum), and 40,000 (Z. mays). The identification of fungal genera and species was performed on Czapex-Dox Agar and Potato dextrose Agar on the basis of the isolates’ colony characteristics and the morphology of the examined reproductive organs. The isolated fungi belonged to the following genera: Aspergillus, Penicillium, Alternaria, and Fusarium. Species from these genera are well-known mycotoxin-producing fungi. Among the identified species were A. candidus, A. flavus, A. carbonarius, A. ochraceus, A. oryzae, P. solitum, P. citrinum, P. griseofulvum, P. brevicompactum, A. alternata, F. avenaceum, and F. graminearum. The mycotoxin content was determined via the ELISA technique using Eurofins Technologies Hungary KFT kits for aflatoxin B1, deoxynivalenol, total aflatoxins, ochratoxin A, and zearalenone. In the case of eighteen samples, the total aflatoxin content was above the limit of detection, and seven of these samples were contaminated with aflatoxin B1, eight were contaminated with ochratoxin A, two were contaminated with dexynivalenol, and one was contaminated with zearalenon. Two samples of T. aestivum were contaminated with one or more toxins (33%), and the number of samples contaminated three for T. dicoccum (60%), one for H. vulgare (25%), four for F. esculentum (80%), one for S. cereale (33%), two for T. spelta (40%), three for A. sativa (75%), two for O. sativa (100%), two for Z. mays (100%), one for P. miliaceum (100%), one for T. monococcum (100%), and one for T. turgidum ssp. turanicum (100%).

Antimicrobial resistance (AMR) is a major global health challenge affecting human and animal health, food safety, and the effectiveness of antimicrobial therapy. Therefore, this study evaluated the antimicrobial activity of raw (RDM) and pasteurized donkey milk (PDM) against clinically relevant Gram-negative bacteria with different AMR profiles. Antimicrobial activity against ten clinical Enterobacterales isolates, including CTX-M-, OXA-48-, KPC-, NDM-, and VIM-producing strains, was assessed by monitoring bacterial growth kinetics in artificially contaminated milk samples during 8 h of incubation at 37 °C. Pasteurized cow milk (CM) and nutrient broth (NB) served as controls. The strongest antimicrobial effects were observed against OXA-48-producing Klebsiella pneumoniae A4 and CTX-M-producing Escherichia coli A1, whereas Citrobacter koseri F5 showed the lowest susceptibility. After 8 h, K. pneumoniae A4 counts remained below 2 log CFU/mL in RDM compared with >6 log CFU/mL in NB. Significant effects of milk type, incubation time, and their interaction on bacterial growth were confirmed (p < 0.001). Donkey milk significantly inhibited bacterial growth compared with NB and CM, while some differences between RDM and PDM were observed depending on the bacterial isolate. These findings support donkey milk as a bioactive matrix with potential antimicrobial applications.

Desugarized sugar beet molasses (DSBM), a nutrient-rich by-product of sugar production, is a promising and largely unexplored substrate for the production of microbiological media. This study aimed to develop and validate a ready-to-use (RTU) medium based on DSBM for the enumeration of yeasts and moulds in foods and feeds with water activity (a_w) less than or equal to 0.95. DSBM from three production seasons was characterized physicochemically and microbiologically and used to prepare three medium variants, evaluated for productivity and specificity, and evaluated in a single-laboratory method comparison study conducted in accordance with the method comparison protocol described in ISO 16140-2:2016. The three DSBM batches obtained from different production seasons covered dry matter contents of 49.33–52.00%, total sugar contents of 9.46–11.88%, and nitrogen contents of 1.68–1.95%. Potassium and sodium predominated among the minerals. Microbial counts were below the limit of quantification, with no detectable Salmonella spp. or Listeria monocytogenes. All medium variants met the productivity criterion specified in ISO 21527-2:2008 and completely inhibited the growth of 21 tested bacteria. They showed close agreement with the reference DG18 agar across six food and one feed category (0.60 < a_w ≤ 0.95), with mean biases within ±0.025 log₁₀ CFU/g and all results within the predefined acceptance limit of ±0.5 log₁₀ CFU/g. All 80% β-expectation tolerance intervals in the AP were within the ±0.5 log₁₀ CFU/g acceptance limit, and proficiency testing schemes confirmed acceptable performance. Therefore, the novel medium provides a new value-added application for DSBM, supporting the valorization of this underutilized resource from sugar beet processing.

Aflatoksin M1 (AFM1) je toksični metabolit aflatoksina B1 koji se u mleku i mlečnim proizvodima javlja kao posledica ishrane životinja kontaminiranom stočnom hranom. Kontaminacija AFM1 predstavlja kontinuiran i značajan izazov za bezbednost hrane, posebno u regionima u kojima klimatski uslovi pogoduju pojavi aflatoksina u sirovinama za proizvodnju stočne hrane. Cilj ovog istraživanja bio je da se ispita nivo kontaminacije AFM1 u mleku i mlečnim proizvodima dostupnim na tržištu Srbije tokom 2024. godine. Ukupno je analizirano 96 uzoraka mleka i mlečnih proizvoda, uključujući jogurt, kefir, kiselo mleko, kiselu pavlaku, sireve i mlečne namaze. Od ukupnog broja uzoraka, 19 proizvoda bilo je poreklom iz uvoza. Koncentracije AFM1 određene su primenom validovane ELISA metode. AFM1 je detektovan u svim analiziranim uzorcima mleka, jogurta i kefira, dok je prisustvo toksina utvrđeno u 86% uzoraka kiselog mleka, 78% uzoraka kisele pavlake i 50% uzoraka sireva i mlečnih namaza. Utvrđene koncentracije kretale su se od 0,012 do 0,573 µg/kg. Najviše vrednosti zabeležene su u grupi sireva i mlečnih namaza, pri čemu je maksimalna koncentracija AFM1 (0,573 µg/kg) utvrđena u uzorku mlečnog namaza. Iako su svi analizirani uzorci mleka bili u skladu sa važećim nacionalnim propisima (0,25 µg/kg), u 63% uzoraka utvrđene su koncentracije AFM1 iznad maksimalno dozvoljenog nivoa propisanog pravilnikom Evropske unije (0,05 µg/kg). Rezultati ove studije, zajedno sa nalazima prethodnih istraživanja iz Republike Srbije, potvrđuju da prisustvo AFM1 u mleku i mlečnim proizvodima predstavlja izazov koji traje duže od jedne decenije. Navedeni nalazi ukazuju na potrebu za kontinuiranim i pravovremenim monitoringom AFM1, unapređenjem kontrole stočne hrane kao primarnog izvora kontaminacije, kao i daljim usklađivanjem nacionalnog sistema bezbednosti hrane sa standardima Evropske unije.