Three novel dehydrated wheat/rice cereal functional products with an addition of well documented probiotic Bifidobacterium animalis ssp. lactis BB-12® (BB-12®) were developed in Podravka factory for the infants older than 4 months: instant rice cereal, instant rice cereal with fruits and instant wheat cereal with vanilla. Notably, the number of viable BB-12® cells in each of the novel products was higher than the required minimal number of probiotic cells per gram of product (106 CFU/g) during the storage period of 106 weeks. Therefore, BB-12® strain recovery and genome stability were evaluated by strain-specific polimerase chain reaction and amplified fragment length polymorphism fingerprinting analysis. Further aim was to evaluate the influence of these three different cereal food matrices on specific probiotic properties of BB-12® strain in vitro. Applied food matrices positively influenced the survival in the simulated conditions of the gastrointestinal tract and antagonistic activity against undesirable microorganisms, while no influence on auto- and coaggregation ability of B. animalis ssp. lactis BB-12® was observed. Adhesion to extracellular matrix proteins and intestinal epithelial Caco-2 cells together with antibacterial activity emphasized competitive pathogen exclusion from Caco-2 cells by probiotic strain BB-12®.

Lactiplantibacillus plantarum D13 shows antistaphylococcal and antilisterial activity, probably due to the synthesis of a presumptive bacteriocin with antibiofilm capacity released in the cell-free supernatant (CFS), whose inhibitory effect is enhanced by cocultivation with susceptible strains. An in silico analysis of the genome of strain D13 confirmed the pln gene cluster. Genes associated with plantaricin biosynthesis, structure, transport, antimicrobial activity, and immunity of strain D13 were identified. Furthermore, the predicted homology-based 3D structures of the cyclic conformation of PlnE, PlnF, PlnJ, and PlnK revealed that PlnE and PlnK contain two helices, while PlnF and PlnJ contain one and two helices, respectively. The potential of the strain to modulate the intestinal microbiota in healthy or dextran sulphate sodium (DSS)-induced colitis mouse models was also investigated. Strain D13 decreased the disease activity index (DAI) and altered the gut microbiota of mice with DSS-induced colitis by increasing the ratio of beneficial microbial species (Allobaculum, Barnesiella) and decreasing those associated with inflammatory bowel disease (Candidatus Saccharimonas). This suggests that strain D13 helps to restore the gut microbiota after DSS-induced colitis, indicating its potential for further investigation as a probiotic strain for the prevention and treatment of colitis.

Bioactive peptides (BPs) are molecules of paramount importance with great potential for the development of functional foods, nutraceuticals or therapeutics for the prevention or treatment of various diseases. A functional BP-rich dairy product was produced by lyophilisation of bovine milk fermented by the autochthonous strains Lactococcus lactis subsp. lactis ZGBP5-51, Enterococcus faecium ZGBP5-52 and Enterococcus faecalis ZGBP5-53 isolated from the same artisanal fresh cheese. The efficiency of the proteolytic system of the implemented strains in the production of BPs was confirmed by a combined high-throughput mass spectrometry (MS)-based peptidome profiling and an in silico approach. First, peptides released by microbial fermentation were identified via a non-targeted peptide analysis (NTA) comprising reversed-phase nano-liquid chromatography (RP nano-LC) coupled with matrix-assisted laser desorption/ionisation-time-of-flight/time-of-flight (MALDI-TOF/TOF) MS, and then quantified by targeted peptide analysis (TA) involving RP ultrahigh-performance LC (RP-UHPLC) coupled with triple-quadrupole MS (QQQ-MS). A combined database and literature search revealed that 10 of the 25 peptides identified in this work have bioactive properties described in the literature. Finally, by combining the output of MS-based peptidome profiling with in silico bioactivity prediction tools, three peptides (⁷⁵QFLPYPYYAKPA⁸⁶, ⁴⁰VAPFPEVFGK⁴⁹, ¹¹⁷ARHPHPHLSF¹²⁶), whose bioactive properties have not been previously reported in the literature, were identified as potential BP candidates.

Research background. Increasing awareness of the importance of nutrition in health promotion and disease prevention has driven to the development of foods for special medical purposes (FSMPs). In this study, the probiotic strain Lacticaseibacillus paracasei ssp. paracasei (Lacticaseibacillus casei 431®) was incorporated into FSMPs to develop an innovative product. The aim was to investigate the influence of the FSMP matrix on the specific probiotic properties of L. casei 431® in vitro. Experimental approach. A series of in vitro experiments were performed as part of the probiotic approach. After evaluation of antibiotic susceptibility profiles, functional properties such as survival under simulated gastrointestinal tract (GIT) conditions, bile salt deconjugation activities, cholesterol assimilation, antagonistic activity against spoilage bacteria and adhesion to Caco-2 cell line monolayers and extracellular matrix proteins were investigated. Results andconclusions. The L. casei 431® strain, both the lyophilised strain and the strain isolated from the FSMP matrix, effectively survived the simulated adverse gastrointestinal conditions without significant effects of the food matrix. The effect of the FSMP matrix on the deconjugation activity of the bile salts of L. casei 431® was minimal; however, cholesterol assimilation was increased by 16.4 %. L. casei 431® had antibacterial activity against related lactic acid bacteria regardless of whether it was used in FSMPs or not. Conversely, the probiotic strain isolated from FSMP matrix had significantly higher inhibitory activity against six potential pathogens than the lyophilised culture. The autoaggregation ability of the L. casei 431® cells was not affected by the FSMP matrix. The adhesion of L. casei 431® bacterial cells to the extracellular matrix proteins was reduced after treatment with proteinase K, with the highest adhesion observed to laminin. The adhesion of L. casei 431® reduced the binding of E. coli 3014 by 1.81 log units and the binding of S. Typhimurium FP1 to Caco-2 cell lines by 1.85 log units, suggesting the potential for competitive exclusion of these pathogens. Novelty and scientific contribution. The results support the positive effect of the FSMP matrix on the specific probiotic properties of L. casei 431®, such as antibacterial activity, bile salt deconjugation and cholesterol assimilation, while the incorporation of this probiotic strain adds functional value to the FSMPs. The synergistic effect achieved by the joint application of L. casei 431® and innovative FSMP matrix contributed to the development of the novel formulation of an improved functional food product with added value.

Although rare, the ability to produce surface S-layer proteins is beneficially associated with particular Lactobacillus strains being investigated as probiotics. Therefore, this work aimed to study specific probiotic functionalities of selected Levilactobacillus brevis strains MB1, MB2, MB13 and MB20, isolated from human milk microbiota, and to assess the contribution of S-proteins. Firstly, Rapid Annotation using Subsystem Technology revealed that cell wall-related genes were abundant in analysed L. brevis genomes. Furthermore, the results demonstrated that S-proteins mediate aggregation capacity and competitive exclusion of selected pathogens by L. brevis strains. The improvement of Caco-2 epithelial monolayer barrier function was demonstrated by the increase in JAM-A and occludin expressions when L. brevis strains or S-proteins were added, with the effect being most pronounced after treatment with MB2 and S-proteins of MB1. L. brevis strains, especially MB20, exerted the potential to adhere to recombinant human ZG16. Strain MB2 and MB20-S-proteins improved the barrier function of HT29 epithelial monolayer, as evidenced by increased ZG16 expression. Analysed L. brevis strains and S-proteins differentially affected the protein expression of IL-1β, IL-6 and IL-8, and IL-10 cytokines. The most prominent effect was observed by S-proteins of MB20, since IL-1β production was decreased while IL-10 production was significantly increased.

The presence of surface (S)-layer proteins as a rare property of lactic acid bacteria plays an important role in the expression of the probiotic properties of the producer strain and protects the cells from unfavourable environmental conditions. Therefore, the aim of this work was to evaluate the role of S-layer proteins of four Levilactobacillus brevis MB1, MB2, MB13, and MB20 strains, previously isolated from human milk. The results showed better survival under simulated gastrointestinal tract (GIT) conditions and better adhesion to Caco-2 cells, achieved when S-proteins were present on the surface of the examined probiotic Levilactobacillus brevis strains. Free cells and cells microencapsulated in alginate, with or without galactooligosaccharides (GOS) and fructooligosaccharides (FOS), survived in high numbers during freeze-drying and one-month storage at 4 °C, as well as during exposure to simulated GIT conditions (>108 CFU/g), thus fulfilling the primary technological criterion for the selection of probiotic strains. Higher survival of freeze-drying of encapsulated cells than free cells, can be attributed to the protective effect of alginate, GOS and FOS as prebiotic agents. However, GOS was more protective than FOS in maintaining the viability of microencapsulated Levilactobacillus brevis MB1, MB2, MB13 and MB20 cells in alginate during storage and under the harsh conditions of GIT and freeze-drying.