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The beneficial effects of probiotics on host health have been widely documented. Although the market is flooded with probiotic products, studies often show that the available probiotic strains do not meet the requirements for probiotics. One of the main criteria is a high survival rate during passage through the host gastrointestinal tract in order to exert beneficial effects at the target site. Because probiotics must survive these stressful conditions, techniques that protect the cells with non-toxic biopolymers are often used. An example of the use of such a matrix is microencapsulation in an alginate carrier. In addition, it is important to extend the shelf life of probiotic products. Therefore, freeze-drying techniques are used to allow longer storage. In addition to basic probiotic properties, certain functional properties are often desired. For example, recent studies recommend the consumption of products with high antioxidant potential, as they have many beneficial effects on health. In this work, probiotic bacteria (Lactobacillus plantarum M2 and Lactobacillus plantarum KO9) were encapsulated and freeze-dried. Freeze-dried microcapsules of these strains showed a high survival rate under simulated gastrointestinal tract conditions and were a suitable carrier for L-ascorbic acid, which increased the antioxidant capacity of the final product. The basic antioxidant activity of formed capsules, expressed as the ability to remove DPPH radicals, was determined. Since L-ascorbic acid has potential antimicrobial activity, its effect on the growth of selected pathogenic bacteria, such as Salmonella typhimurium, Escherichia coli, Staphylococcus aureus, and Listeria monocytogenes was investigated. This study shows how microencapsulation of probiotics with the addition of desired biological molecules can enhance the beneficial functional properties of probiotics.

The human exposome represents an intriguing gene-environment interactions. Since the food is one of the most easily manageable human exposure, the serach for food items that might have beneficial properties to human health is of high importance. This study aimed to assess the probiotic potential and the antioxidant properties of a new indigenous probiotic bacteria isolated from equid (ili donkey) milk, known for its beneficial properties for ages. DPPH scavenging ability of live cells was determined and compared to antioxidant standard (L-ascorbic acid). Furthermore, the antioxidant activity of extracellular bacterial metabolites, with molecular mass smaller than 2000 daltons (Da) that can cross the epithelial barrier was assessed accompanied by the investigation of cytotoxic and genotoxic effect of metabolites on peripheral blood lymphocytes. The battery of tests showed excellent probiotic potential. The DPPH free radical scavenging activity of L. plantarum M2 cells was 7.25%. The tested bacterial media fraction removed 4.84% for L. plantarum M2 and L-ascorbic acid (20 mg/L) as a positive standard removed 56.41% of free radicals. Examined metabolites exhibited no cytotoxic effects on peripheral blood mononuclear cells which was detected by acridine orange/ethidium bromide vital staining compared to controls. The results of comet assay also did not show inducton of primary DNA damage of peripheral blood mononuclear cells exposed to bacterial extracellular metabolites. Taken together, we managed to present a new bacteria from donkey milk with excellent probiotic potential. Furthermore it does not posses any toxic properties so we confirmed Qualified Presumption Safety Status. The next steps in research will try to elucidate its possibility of minimising effects of mutagens found in food.

Beneficial health effects of lactic acid bacteria (LAB) used in functional foods and pharmaceutical products are based on the ability and capacity of these microorganisms in stimulation of host immune system. Health improving immune modulation and production of bioactive substances are restricted only to some LAB strains. In this study, peripheral blood mononuclear cells (PBMC) were stimulated with bacterial lipopolysaccharide (LPS) to produce pro-inflammatory cytokine tumor necrosis factor alpha (TNF-α). The aim of this study was to determine in vitro if extracellular bacterial metabolites, with molecular mass smaller than 2000 Da, from four selected LAB strains, suppress TNF-α production in LPS stimulated PBMC and therefore exhibit anti-inflammatory effect. Furthermore, cytotoxicity and genotoxicity of used bacterial culture media fractions containing these metabolites on PBMC was determined. Bacterial fractions showed no cytotoxicity or genotoxicity on PBMC, and all selected strains showed significant suppression of TNF-α production in LPS stimulated cells.

The influence of the oral treatment with probiotic strain Lactobacillus plantarum 1K on an oral infection of mice by Salmonella enterica serovar Typhimurium was investigated. Mice were fed with milk-based diets supplemented with L. plantarum 1K for three days prior and following an oral challenge with S. Typhimurium. Survival, competition, adhesion and colonization of L. plantarum 1K and influence on infection with S. Typhimurium were monitored in the gastrointestinal tract. After the oral treatment of mice with L. plantarum 1K in combination with S. Typhimurium, the total number of lactic acid bacteria in faeces and in intestinal homogenates was increased, contrary, the total number of enterobacteria and Salmonella sp. was reduced. These results demonstrate that L. plantarum 1K can reduce the severity of infection due to the pathogenic S. Typhimurium, and suggest that this reduction is associated with competitive exclusion in the intestinal tract conferred by the L. plantarum 1K

The influence of the oral treatment with probiotic strain Lactobacillus plantarum 1K isolated from „Slavonski kulen“ on an oral infection of mice by Salmonella sp. was investigated. Mice were fed with milk-based diets supplemented with L. plantarum 1K for three days prior and following an oral challenge with Salmonella sp. Survival, competition, adhesion and colonization of L. plantarum 1K and influence on infection with Salmonella sp. were monitored in the gastrointestinal tract of mice. After the oral treatment of mice with L. plantarum 1K in combination with Salmonella sp., the total number of lactic acid bacteria in faeces and in intestinal homogenates was increased, contrary, the total number of enterobacteria and Salmonella sp. was reduced. These results demonstrate that L. plantarum 1K can reduce the severity of infection due to the pathogenic Salmonella sp., and suggest that this reduction is associated with competitive exclusion in the intestinal tract. Considering that L. plantarum 1K has demonstrated the basic functional criteria for the selection of probiotic strains, as such, it can be used as functional autochthonous starter culture for fermented meat products.

Lactobacillus helveticus M92 (designated initially as Lactobacillus acidophilus M92) was previously selected as probiotic starter culture based on in vitro and in vivo selection criteria. Besides its health promoting effects as probiotic, there is also a potential of this strain as a carrier for oral and mucosal vaccine due to the presence of S-layer surface proteins. These proteins are responsible for the survival of this strain in the gastrointestinal tract of mice and for adhesion to intestinal epithelial cells. The objective of this work was to screen proteins exposed on cell envelope that mediate these processes. Hence, the cell envelope associated proteome profile of L. helveticus M92 was studied by 1D SDS-PAGE LC/MS-MS. As the genome of L. helveticus M92 was not sequenced, we used that of the reference strain L. acidophilus NCFM to generate a protein database used for interrogation in mass spectrometry experiment. Protein identification was performed with Bioworks 3.2 software. 396 different proteins were identified and among them, 121 were cell envelope associated. Prominent proteins were different sugar and amino acid transporters. In addition, a protein band, containing SlpA protein, was analysed with PEAKS software and searched against a database built from all available Lactobacillacea genome sequence. This resulted in higher S-layer protein coverage, and high homology with L. helveticus S-layer protein.

The protective effects of the oral treatment with Lactobacillus helveticus M92 in an intragastric challenge by Salmonella enterica serovar Typhimurium were investigated. Mice were fed with milk-based diets supplemented with L. helveticus M92, for seven days prior to and following oral challenge with S. Typhimurium. Survival, competition, adhesion and colonization of L. helveticus M92 in combination with S. Typhimurium were monitored in the gastrointestinal tract of mice, as well as their immunomodulating capability. After feeding of mice with L. helveticus M92 the total number of lactic acid bacteria in faeces was increased, contrary the reduction of total number of enterobacteria was observed, compared to non-probiotic-fed mice (mice fed with S. Typhimurium). These results were in accordance with results obtained in homogenates of small and large mice intestine 7th day after feeding with L. helveticus M92. Following the oral immunization of mice, with a daily dose of 1 x 1011 viable L. helveticus M92 cells, the concentration of serum anti-S. Typhimurium IgA antibodies from all immunized mice was significantly higher compared to non-probiotic-fed mice i.e. mice fed with S. Typhimurium and to the control group of mice. Interestingly, results showed that, after the challenge, L. helveticus M92 – fed mice exhibited lower cumulative morbidity and bacterial translocation rates, compared to non-probiotic-fed mice. These results demonstrate that L. helveticus M92 can reduce the severity of infection due to the pathogen S. Typhimurium, and suggest that the reduction may be associated with enhanced immune protection conferred by the probiotic.

The ability of probiotic strain L. acidophilus M92 to assimilate the various kinds of prebiotic substrates (inulin, lactulose and raffinose) in vitro and in vivo, on Swiss albino mice as animal model, was investigated. Survival, competition, adhesion and colonization of L. acidophilus M92 in combination with prebiotics (synbiotic effect) were monitored in the gastrointestinal tract of mice, as well as their immunomodulating capability. After the feeding of mice with synbiotics (2 x 1010 probiotic cells in combination with 10% of prebiotic substrates) the number of lactic acid bacteria in faeces was increased and reduction of enterobacteria and sulphite-reducing clostridia was observed. The similar results were obtained in homogenates of small and large intestine of mice in the 1st and 14th day after feeding with synbiotics. After oral immunization of mice with viable cells of L. acidophilus M92, with a daily dose of 2 x 1010 cells, alone and in combination with prebiotic substrates, the concentration of faecal SIgA and total serum IgA antibodies from all immunized mice were significantly higher in comparison to the control. They didn’ t evoke the specific humoral immune response after oral application and they are as such suitable for synbiotic application. L. acidophilus M92 in combination with inulin has shown the best synbiotic effect on intestinal and faecal microflora of Swiss albino mice, and their immune system.

Sirutka je važan prehrambeni proizvod s pozitivnim učincima na zdravlje. Brojna istraživanja ukazuju na njena protuupalna, antikancerogena i antioksidativna svojstva te da je spontano fermentirana sirutka potencijalan izvor probiotičkih bakterija. Cilj ovog rada bio je istražiti antioksidativni potencijal probiotičke bakterije Lactobacillus plantarum S1 izolirane iz sirutke. Određena je sposobnost uklanjanja 2,2-difenil-1-pikrilhidrazil (DPPH) slobodnih radikala kao ukupna antioksidativna sposobnost te je određena unutarstanična koncentracija glutationa. Istražena je i korelacija broja stanica i sposobnosti uklanjanja DPPH slobodnih radikala kako bi preporučili optimalnu koncentraciju za ispoljavanje zdravstveno korisnih učinaka. Rezultati su ukazali na visoku sposobnost uklanjanja DPPH slobodnih radikala i umjerenu prisutnost glutationa u neinduciranim uvjetima. Također, ukupna antioksidativna aktivnost istraživanog probiotika ovisi o koncentraciji bakterijskih stanica. Whey is a food product that has a positive effect on health. Various studies indicate its anti-inflammatory, anticancer and antioxidant properties and that spontaneously fermented whey is a potential source of probiotic bacteria. The goal of this thesis was to investigate the antioxidative potential of probiotic bacteria Lactobacillus plantarum S1 isolated from whey. Ability to remove 2,2-diphenyl-1-picrylhydrazyl free radicals (DPPH) was determined as the total antioxidative capability and the intracellular glutathione concentration was also determined. The correlation between the number of cells and the ability to remove DPPH free radicals was researched to recommend optimal concentration for health effects. The results point to a high ability of removing DPPH free radicals and a moderate presence of glutathione under controlled conditions. Also, the total antioxidative activity of the researched probiotic depends on the concentration of bacterial cells.