A volume of 5 mL of the overnight cultures of the LAB strains was centrifuged using an Eppendorf 5804 R centrifuge (Eppendorf, Hamburg, Germany) at 8000×g for 10 min at 4 °C. The cells were washed twice with sterile phosphate-buffered saline (PBS, pH=7.4). A volume of 200 µL of each cell suspension was inoculated into 10 % (m/V) skimmed milk (Sigma-Aldrich, Merck) and incubated at 37 °C for 16 h.

The ability of the LAB to coagulate milk was determined according to Hebert et al. (18). Depending on the rate of milk coagulation, the results were interpreted as follows: no coagulation, or as low, moderate, good or excellent coagulation.

Acidification capacity was measured in the supernatants by monitoring the pH change with a pH meter (Metrohm, Herisau, Switzerland) and by the titration method with 0.1 M NaOH (Carlo Erba, Milan, Italy) with the addition of phenolphthalein indicator (Kemika, Zagreb, Croatia) until a pink colour appeared. The titratable acidity expressed as °SH (1 °SH=0.0225 % lactic acid) was determined as follows:

where V is the volume (in mL) of 0.1 M NaOH and f_NaOH is the correction factor of NaOH (1).

Mass per volume ratio of 10 % skimmed milk (Sigma-Aldrich, Merck) was used to observe the proteinase phenotype of the strains according to Raveschot et al. (19) with slight modifications. Solid skimmed milk agar plates were routinely prepared by adding 1 % (m/V) agar (Biolife) to the medium. After solidification, sterile wells with a diameter of 7 mm were drilled and 50 µL of cell suspension or the supernatant of the overnight grown bacterial culture was added. Transparent halos were an indicator of proteolytic activity.

The proteolytic activity of selected LAB strains was determined using the Anson method according to Beganović et al. (16). A volume of 1 mL of the supernatant filtrate of the overnight culture of each strain was suspended with 5 mL of a 0.65 % (m/V) casein solution in PBS (pH=7.2). After a 10-minute incubation at 37 °C, the reaction was halted by adding 5 mL of trichloroacetic acid (Thermo Fischer Scientific, Waltham, MA, USA), which led to precipitation of the non-hydrolysed proteins. After another incubation (30 min, 37 °C) and filtration, 5 mL of 0.4 M Na₂CO₃ solution (Kemika, Zagreb, Croatia) and 1 mL of Folin-Ciocalteu phenol reagent (Merck, Darmstadt, Germany) were added to 2 mL of filtrate. The sample was incubated again for 30 min at 37 °C and filtered. The absorbance was measured at 670 nm on an LKB 5060-006 microplate reader (LKB Vertriebs GmbH, Vienna, Austria). The blank contained a pre-incubated casein solution to which trichloroacetic acid was added at the beginning of the experiment, followed by the addition of the supernatant and all the procedures described above.

Based on the linear equation, the amount (nmol) of l-tyrosine (Merck) released was calculated by measuring the absorbance (A_{670 nm}), resulting from the hydrolysis of casein by proteases in the samples (Fig. 1).

The caseionolytic activity of selected bacterial strains was tested according to El-Ghaish et al. (20) with modifications. Overnight cultures were centrifuged using an Eppendorf 5804 R centrifuge at 3600×g for 10 min and then washed with phosphate buffer (pH=7.4). The resulting biomass was suspended in a 2 % (m/V) skimmed milk solution (Sigma-Aldrich, Merck) and incubated at 37 °C for 48 h. Casein degradation was then monitored using the Tris-tricine sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) method. A non-inoculated skimmed milk solution was used as a control. The gel for Tris-tricine SDS-PAGE was prepared according to Haider et al. (21).

Isolation of LAB with desired functional properties from human sources is a tantalising approach to select potent probiotics for health-promoting applications (24). Selected LAB strains isolated from the human milk of Croatian women were previously identified by 16S RNA and whole genome sequencing, deposited in the NCBI database and characterised as producers of potential biotherapeutic molecules such as S-layer proteins (L. brevis MB1, MB2, MB13 and MB20), exopolysaccharides (L. fermentum MC1) and plantaricins (L. plantarum KR19, MC19 and MB18) (3). These strains showed good survival under simulated gastrointestinal tract conditions, aggregation and adhesion to various epithelial and subepithelial structures of the intestinal tract (11). The aim of this study was therefore to assess the antimicrobial activity of selected LAB strains using the turbidimetric method in an appropriate liquid growth medium. The results were expressed as growth inhibition (%) of the tested bacteria compared to the control growth. The turbidimetric method was chosen due to the higher sensitivity than agar methods and due to the potential application of selected LAB strains isolated from human milk as functional starter cultures or even probiotics in fermented functional products. According to the results, after 24 h of incubation, the strains L. plantarum MC19 and L. fermentum MC1 showed the strongest (p<0.05) antimicrobial activity against the test microorganisms, while the antagonistic activity against the related LAB strains was depleted. L. plantarum MC19 strongly inhibited S. aureus ATCC^®25925^™, S. Typhimurium ATCC^®14028^™, L. monocytogenes ATCC^®19111^™ and E. coli ATCC^®25922^™ with inhibition rates of more than 80 %. A similar antimicrobial activity was observed for L. fermentum MC1, with a significantly higher inhibition against L. monocytogenes ATCC^®19111^™ (p=0.004) than strain MC19 (Fig. 2a). A slightly lower, but still very high antimicrobial activity was observed for the strains L. plantarum MB18 and KR19 against both the test microorganisms and related LAB strains (Fig. 2b). In contrast, L. brevis MB13 showed no activity, while other S-layer expressing strains (L. brevis MB1, MB2 and MB20) showed lower activity than other strains tested. The antimicrobial effect of LAB against common foodborne pathogens such as E. coli, L. monocytogenes and Salmonella spp. results from the lowering of the pH of the medium due to the organic acids formed and the activity of synthesised bacteriocins, vitamins, EPSs and other metabolites with proven antimicrobial activity (25, 26). While bacteriocins directly kill competing, closely related bacteria or pathogens or inhibit their growth by various mechanisms such as forming pores in the target cell membrane, disrupting ion gradients or inhibiting cell wall synthesis, S-layer proteins form a crystalline layer that covers the surface of the producing bacteria and contributes to structural integrity, mediates interactions with host tissue and modulates host immune responses (11). As a result, S-layers significantly enhance the adhesion of the producer to host cells and prevent pathogen adhesion without having direct bactericidal or bacteriostatic properties. This is probably the reason why S-layer producers have a less potent antimicrobial effect than bacteriocin producers. The pln loci of three investigated L. plantarum strains were disclosed by the detection of plnEF, plnA and plnJ genes responsible for the production of the bacteriocins PlnEF and PlnA, and the peptide PlnJ of the plantaricin PlnJK, using gene-specific primers (3). On the other hand, EPSs have health-promoting and rheological properties in the food, pharmaceutical and nutraceutical industries by exerting antimicrobial, antioxidant, immunomodulatory and many other biological functions. Strain L. fermentum MC1 biosynthesises a mixture of three different polymers and harbours the genes involved in EPS production and transport, as well as a gene cluster related to bacteriocin production (12), which is a functional property contributing to antimicrobial activity. Strain MC1 also showed excellent adhesion properties, which are important for its probiotic activity. It can therefore be surmised that the strong antimicrobial activity of the plantaricin-producing L. plantarum strains and the EPS-producing L. fermentum MC1 is due to the biomolecules they produce.

The main component of human and bovine milk is β-casein, a protein that is a rich source of bioactive and antimicrobial peptides contributing to the endogenous peptidome of milk (6, 27). Therefore, bovine skimmed milk was used as a milk model system for the evaluation of proteolytic capacity. Preliminary analyses of the selected autochthonous strains from human milk have shown that their ability to efficiently coagulate milk and exhibit a fast milk coagulation (Fmc) phenotype is a strain-dependent trait (Fig. 3a). L. plantarum MB15 showed a low coagulation efficiency, L. brevis MB20 a moderate one, the strains L. plantarum KR19, L. plantarum MB18, L. brevis MB13, L. brevis MB1 and L. brevis MB2 a good one, while the strain L. fermentum MC1 showed an excellent coagulation efficiency. Determination of the fast milk acidification rate showed that the strains decreased the pH of the cultivation medium after overnight growth to values between (4.12±0.01) for L. fermentum MC1 and (3.81±0.08) for L. plantarum MB18, which is consistent with the degree of acidity (Fig. 3b). Cervantes-Elizarrarás et al. (28) have reported that organic acids generated by LAB can suppress the growth of Gram-negative bacteria by penetrating their cell membranes and thus impairing their function, leading to acidification of the cytoplasm and inhibition of acid-sensitive enzymes. Although L. fermentum MC1 decreased the pH of the medium the least, it exhibited the strongest Fmc+ phenotype as determined by the fastest coagulation rate of the milk. This discrepancy can be explained by the fact that rapid milk coagulation does not necessarily require high acid production, as LAB can possess various traits and mechanisms, such as high enzymatic activity, which allow them to effectively coagulate milk proteins independently of acid production. Presumably, strain MC1 produces proteolytic enzymes that directly cleave milk proteins and cause effective coagulation without significantly lowering the pH of the medium.

Cell-envelope proteinases (CEPs) play a crucial role in the proteolytic system of LAB, as they are required to degrade proteins into peptides and/or amino acids, serving as a nitrogen source for LAB. Therefore, the presence of potential proteinase activity was tested in concentrated bacterial cell suspensions and in cell-free culture supernatants. The result showed that both the strains and their cell-free supernatants were able to hydrolyse the skimmed milk proteins as evident from the appearance of the transparent halos around the wells in the agar, indicating casein hydrolysis (Fig. 3c). The mean diameter of the transparent halos was 10.8±1.2 for the LAB cell suspensions and 10.2±0.7 for the cell-free supernatants, indicating a slightly stronger caseinolytic activity of the LAB cells, albeit without significant meaning (p>0.05). However, the cell suspension of strain L. brevis MB20 showed significantly stronger (p=0.036) caseinolytic activity than its cell-free supernatant, suggesting that its proteolytic activity may be due to CEPs. These results are consistent with the research of Novak et al. (6), in which caseinolytic activity was detected in concentrated cell biomass of lactobacilli and lactococci strains isolated from various autochthonous fermented products.

Representative exopolysaccharide (L. fermentum MC1), bacteriocin (L. plantarum MB18) and S-protein (L. brevis MB1 and L. brevis MB2) producers, whose cell-free culture supernatants and concentrated bacterial cell suspensions showed the highest proteinase activity, were further selected for evaluation of casein degradation potential. Human milk contains two classes of proteins, casein and whey proteins (29). In this study, the hydrolysis of casein and whey proteins into protein fragments and peptides was studied using Tris-tricine SDS-PAGE (Fig. 4a). This resulted in the appearance of lower-intensity bands, implying that casein and whey proteins were partially degraded, i.e. a smaller amount of intact proteins remained. The strain L. plantarum MB18 showed the highest proteinase activity, which is consistent with the quantitative analysis of proteinase activities, where the amount of l-tyrosine released was (46.0±8.8) nmoL (Fig. 4b). According to the literature, many LAB belonging to the L. plantarum strains produce peptides with numerous bioactive effects such as anti-inflammatory, antihaemolytic, antioxidant, antimutagenic or antimicrobial effects through the fermentation of milk (30). Since infant formulas are highly rich in casein, which makes them difficult to digest compared to human milk, supplementation with strains expressing active proteinase could eventually contribute to improved casein digestibility (31). This feature is attractive from various aspects of the application of Lactobacillus strains, whether as a probiotic supplement in cow's milk-based infant formula or as a starter culture in fermented food products as this can lead to the accumulation of health-promoting bioactive peptides.

An imbalance in the body can be caused by oxidative stress, which leads to damage to cells and tissues, triggered by the excessive production of reactive oxygen species (ROS) and reactive nitrogen species (RNS). As a result, various diseases can develop, such as diabetes, cancer, cardiovascular problems and inflammatory and neurological diseases. For this reason, it is necessary to develop supplements with antioxidant effect in order to reduce oxidative stress, and here the potential of probiotics has also gained tremendous scientific importance (13, 32). During food fermentation, the antioxidant activity of LAB can be attributed to bioactive peptides, EPSs, organic acids and a change in the pH of the environment, which can lead to an increase in their bioavailability (5, 33).

Therefore, the antioxidant activity of the selected strains was evaluated using DPPH radical scavenging activity. Strain L. brevis MB2 showed the highest and strain L. plantarum KR19 the lowest radical scavenging activity, while strains MB1, MB13, MB20, MB18, MC1 and MC19 showed similar DPPH radical scavenging activity of about 50 % (Fig. 5). Using the same method, Vougiouklaki et al. (34) reported that Lactobacillus gasseri ATCC 33323 removed 78 % of DPPH radicals, while the values for Lacticaseibacillus rhamnosus GG ATCC 53103, Levilactobacillus brevis ATCC 8287 and Lactiplantibacillus plantarum ATCC 14917 were between 33 and 39 %.

Among all the strains tested, the EPS-producing L. fermentum MC1 and the plantaricin-producing L. plantarum MB18 strains can find potential application due to their desirable properties. Proteolytic activity is a prerequisite for starter culture application, which can result in production of bioactive peptides with numerous functional properties (5). Due to their ability to inhibit the growth of a wide spectrum of bacteria, they can serve as biopreservatives that can extend the shelf life of the consumed product. In addition to antimicrobial properties, antioxidant activity may also be important in extending the shelf life of functional products as well as for protection from oxidative damage in the human body after consumption, along with their functional role, while EPS production can have a positive impact on rheological properties. Overall, all these effects can additionally improve the nutritional relevance of functional products with functional roles for host health, such as gut microbiota balance.