Bojana Bogovič Matijašić

Background/Objectives: Diaper dermatitis (DD) is a common dermatological condition that is often exacerbated by antibiotics. Probiotics may restore microbiota balance and influence the incidence of DD. This study aimed to evaluate whether 6 weeks of Limosilactobacillus (L.) reuteri DSM 17938 supplementation during and after antibiotic treatment affects the incidence of DD. Methods: A secondary analysis of a randomised, double-blind, placebo-controlled trial that included 89 term neonates treated with antibiotics during the first 3 weeks of life was conducted. Neonates were assigned to receive either a probiotic supplement or a placebo for a total of 6 weeks. DD incidence, onset of DD, and skin care data were collected for 87 infants. Faecal samples were collected 6 weeks after study enrolment to analyse the composition of gut microbiota. Results: Twenty-four (28%) infants developed DD, of whom six (25%) had fungal DD. There was no significant difference in the incidence of DD or fungal DD between the probiotic and placebo groups (30% vs. 25% and 7% in both groups, respectively). Probiotic supplementation had no significant effect on the gut microbiota composition or diversity. In an exploratory binary logistic regression analysis based on six fungal DD events, probiotic supplementation was not associated with fungal DD, whereas breastfeeding was associated with lower odds of fungal DD; however, the overall model was not statistically significant, and this finding should be interpreted with caution because of the small number of events. Conclusions: In this exploratory secondary analysis, supplementation with L. reuteri DSM 17938 was not associated with a reduced incidence of DD or alterations in gut microbiota composition in antibiotic-treated neonates. Breastfeeding was associated with lower odds of fungal DD; however, this finding should be confirmed in larger, adequately powered studies.

Lactoferrin (Lf) and Lf-derived peptides are multifunctional milk components with potential applications in food preservation due to their antibacterial and antioxidant properties. In this study, the antibacterial and antioxidant activities of bovine lactoferrin and Lf-derived peptides obtained by enzymatic hydrolysis with pepsin, trypsin, and chymotrypsin were evaluated. Antibacterial activity was assessed against four foodborne pathogens and spoilage microorganisms (Escherichia coli, Listeria monocytogenes, Staphylococcus epidermidis, and Latilactobacillus sakei), while antioxidant activity was determined using four complementary assays. Lf showed stronger antibacterial activity than the corresponding hydrolysates against all tested strains, while the hydrolysates notably inhibited Listeria monocytogenes and Latilactobacillus sakei. Both Lf and its peptides showed lower antioxidant capacity than Trolox, although native Lf and its peptides markedly inhibited lipid peroxidation. Lf peptides demonstrated greater antioxidant activity in the superoxide scavenging and FRAP assays. Low-molecular-weight peptides (<10 kDa) contributed most to antioxidant activity, while mass spectrometry analysis revealed peptide sequences rich in hydrophobic and electron-donating amino acid residues, providing mechanistic insight into the observed activities. Overall, these findings highlight the potential of lactoferrin and its enzymatic hydrolysates as natural antimicrobial and antioxidant agents for food preservation.

Gassericins K7 are heterodimeric bacteriocins produced by Lactobacillus paragasseri K7. Their biological characterisation has been limited by challenges in obtaining these compounds in sufficient purity and quantity. In this study, we used a preparative isolation and purification workflow for gassericins K7, combining ammonium sulphate precipitation, Amberlite XAD-16 extraction, and reversed-phase chromatography. Mass spectrometry confirmed the presence of all four peptide components GasK7A α, GasK7A β, GasK7B α, and GasK7B β in the pooled preparation, including GasK7A β, identified here for the first time in natural isolates of this strain. The purified bacteriocin preparation exhibited strong antibacterial activity against indicator strain Latilactobacillus sakei NCDO 2714. Cytotoxicity testing in RAW 264.7 macrophages revealed minimal effects at concentrations up to 50 µg/ml, with only a modest reduction in cell viability observed at 100 µg/ml. Subsequent evaluation of immunomodulatory properties demonstrated a clear dose-dependent anti-inflammatory effect in LPS-stimulated macrophages. Treatment with gassericins K7 reduced nitric oxide (NO) production, downregulated inducible nitric oxide synthase (iNOS) expression, and suppressed secretion of the pro-inflammatory cytokines TNF-α and IL-6. Importantly, exposure of macrophages to gassericins alone did not elicit any inflammatory response. These findings provide the first evidence of anti-inflammatory activity of purified gassericins K7 in eukaryotic immune cells and highlight their potential as postbiotic components or biotherapeutic candidates for inflammatory disorders. The preparative method used in this study enables the production of gassericins K7 in quantities sufficient for advanced in vitro and in vivo investigations, facilitating future development of bacteriocin-based therapeutics.

The growing demand for sustainable bioprocessing has positioned whey, a side stream of the dairy industry, as a valuable substrate for producing high-value compounds. This study investigated the production and purification of nisin, a potent lantibiotic, using permeate (10 kDa cut-off) of ultrafiltered acid whey (PUFAW) devoid of lactoferrin as growth medium. Three strains of Lactococcus lactis subsp. lactis , including two artisanal isolates (IM 143 and IM 145) and a reference strain (IM 407), were evaluated for nisin production in PUFAW supplemented with yeast extract (YE) and mineral salts. Strain IM 145 showed superior growth and antimicrobial activity in PUFAW with YE (30 g/L) and produced up to 51,200 BA/mL of nisin. The bacteriocin was purified by ammonium sulphate precipitation followed by preparative RP-HPLC, yielding a product with 79.2% chromatographic purity. Tricine-SDS-PAGE and mass spectrometry confirmed the identity of the purified compound as nisin Z. This study presents a cost-effective, circular economy approach for the production of high-purity nisin from dairy side streams, with potential applications in food preservation and biomedicine.

The adequate survival of probiotics in the harsh environment of the gastrointestinal (GI) tract plays a crucial role in the expression of their functional properties. The aim of the present study was to evaluate the survival of commercial probiotics during digestion using a standardised INFOGEST 2.0 model extended with three food matrices simulating three scenarios for the consumption of probiotics: on an empty stomach, with juice, or with food (porridge). All eight products matched the bacterial content stated on the label. After simulated digestion, we observed an average decrease in viability of 1.6 log10 colony forming units (CFU) when the product was co-digested with water, a 2.5 log10 CFU decrease in the presence of juice, and a 1.2 log10 CFU decrease in the presence of porridge. The survival rate of the probiotics was statistically higher in the test samples with porridge (91.8%) than in those with juice (79.0%). For two products, the number of lactobacilli and bifidobacteria strains after digestion was less than 5 CFU, which can be considered insufficient. The present study has shown that the survival of probiotic strains during GI passage depends not only on their ability to withstand these harsh conditions but may also be influenced by the manufacturing process and by the foods consumed together with the probiotics.

Probiotics are live microorganisms that can have beneficial effects on humans. Encapsulation offers them a better chance of survival. Therefore, nozzle-free electrospinning was introduced for their embedding in nanofibrous material. Probiotic Lactobacillus paragasseri K7 in lyophilized and fresh form, with and without inulin as prebiotic, was added to a polymer solution of sodium alginate (NaAlg) and polyethylene oxide (PEO). Conductivity, viscosity, pH, and surface tension were determined to define the optimal concentration and volume ratio for smooth electrospinning. The success of the formed nanoscale materials was examined by scanning electron microscope (SEM), while the entrapment of probiotics in the nanofibrous mats was detected by attenuated total reflection—Fourier transform infrared spectroscopy (ATR-FTIR) and X-ray photoelectron spectroscopy (XPS). Spontaneous diffusion of bacteria from electrospun samples in PBS buffer pH 7.4 was studied by plate counting on MRS agar. By exposing polymer solutions containing L. paragasseri K7 and inulin to a high electric field, the nanofilm was formed on a polypropylene substrate, used as collecting material. When polymer solutions without inulin were used, the bead-like nanofibers may have become visible. The SEM results suggest that inulin, in addition to K7 strain, additionally lowers the conductivity of spinning macromolecular solution and hinders the nanofiber formation. The results of ATR-FTIR confirmed the presence of L. paragasseri K7 embedded in nanocomposites by the appearance of characteristic peaks. The samples containing the probiotic regardless of its form with inulin had similar surface composition, except that the sodium content was higher in the samples with fresh probiotic, probably due to greater and thus less easy embedding of the bacteria in NaAlg. Within 2 h, the largest amount of probiotic strain K7 was spontaneously released from the electrospun sample containing the inulin and probiotic in freeze-dried form (44%), while the amount released from the nanofibrous sample, which also contained the inulin and probiotic in fresh form, was significantly lower (21%). These preliminary results demonstrate the potential of nozzle-free electrospinning technology for the development of probiotic delivery systems for short-term use, such as feminine hygiene materials (tampons, pads, napkins).