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Synthesis of zinc oxide (ZnO) nanoparticles (NPs) was mediated by plant extracts to assist in the reduction of zinc atoms during the synthesis and act as a capping agent during annealing.The preparation used ethanolic extracts from the roots of Japanese knotweed (Fallopia japonica). Two major outcomes could be made. (i)A synergistic effect of multiple polyphenolic components in the extract is needed to achieve the capping effect of crystallite growth during thermal annealing at 450 °C characterized by an exponential growth factor (n) of 4.4 compared to n = 3 for bare ZnO. (ii) Synergism between the ZnO NPs and plant extracts resulted in superior antimicrobial activity against both Gram-positive bacteria, e.g., Staphylococcus aureus, and Gram-negative bacteria, e.g., Escherichia coli and Campylobacter jejuni. The materials were also tested for their antimicrobial activity against S. aureus under ultraviolet (UV) illumination. Also here, the photocatalyst prepared with plant extracts was found to be superior. The residues of the plant extract molecules on the surface of the catalyst were identified as the main cause of the observed differences, as proved by thermal gravimetry. Such a preparation using ethanolic extract of Fallopia japonica could serve as a more controlled synthesis of ZnO and potentially other metal oxides, with low environmental impact and high abundance in nature. Soavtorji: Ajda Kunčič, Lev Matoh, Sonja Smole Možina, Martin Šala, Andraž Šuligoj. Nasl. z nasl. zaslona. Opis vira z dne 10. 11. 2022. Objavljeno na spletu: 1. nov. 2022. Bibliografija: str. 31244-31245. Abstract.

Campylobacter jejuni is the leading cause of bacterial gastroenteritis, or campylobacteriosis, in humans worldwide, and poultry serves as a major source of infection. To reduce the risk associated with C. jejuni transmission via poultry meat, effective interventions during poultry production are needed, and the use of probiotics is a promising approach. In this study, 15 Bacillus subtilis strains were initially screened for their anti-Campylobacter activities. B. subtilis PS-216 strain demonstrated the best anti-Campylobacter activity against 15 C. jejuni isolates when examined using in vitro co-cultures. To evaluate the suitability of B. subtilis PS-216 for probiotic use, its susceptibility to eight clinically important antimicrobials and simulated gastric conditions was investigated. B. subtilis PS-216 was sensitive to all of the tested antibiotics. Although vegetative cells were sensitive to gastric conditions, B. subtilis PS-216 spores were highly resistant. We further evaluated the use of a B. subtilis PS-216 spore preparation (2.5 × 106 CFU/mL water) to prevent and/or reduce C. jejuni colonization in broiler chickens in vivo. Compared to the untreated group, significantly lower Campylobacter counts were detected in caeca of broilers continuously treated with B. subtilis PS-216 spores in their drinking water. Furthermore, broilers continuously treated with B. subtilis PS-216 spores showed improved weight gain, compared to the control group. Together, these results demonstrate the potential of B. subtilis PS-216 for use in poultry to reduce C. jejuni colonization and improve weight gain.

Tropaeolum majus L. is a plant belonging to the Tropaeolaceae family, and is rich in benzyl glucosinolate [1]. The degradation product of benzyl glucosinolate, benzyl isothiocyanate (BITC), exhibits various biological activities, such as antiproliferative and antimicrobial activities [1]. Two modern extraction techniques (microwave-assisted distillation (MAD), and microwave hydrodiffusion and gravity (MHG)) were used to prepare essential oil (EO) and extract from T. majus seeds, afterwards their chemical composition of volatile compounds was investigated with GC-MS/MS present in. The biological activity of samples (EO, extract and pure compounds) was focused on the antiproliferative effect against different cancer cell lines: cervical cancer cell line (HeLa), human colon cancer cell line (HCT116), and human osteosarcoma cell line (U2OS), while antibacterial activity was evaluated by determination of minimal inhibitory concentration (MIC) and anti-adhesion effect on polystyrene surface of T. majus samples against Staphylococcus aureus and Escherichia coli. EO of T. majus enriched with BITC showed higher antiproliferative activity (IC50 < 5 µg/mL) than T. majus extract (IC50 < 27 µg/mL) against three cancer cell lines: HeLa, HCT116, and U2OS. BITC showed significantly higher inhibitory effect on the proliferation rate of all tested cancer cells compared to BCN. T. majus EO and extract showed strong antibacterial activity against S. aureus and E. coli, with MIC values ranging from 0.06 mg/mL to 1 mg/mL, respectively. Among tested formulations, BITC had the best anti-adhesion activity, even in subinhibitory concentration against both bacteria. Studies on the antiproliferative and antimicrobial activities of EOs and extracts rich in isothiocyanates, are of great importance as they have the potential to target different types of carcinomas or to extend the shelf life of food as natural preservatives in the food industry. References: [1] I. Vrca, F. Burčul, I. Blažević, A. Bratanić, T. Bilušić. Croat. J. Food Sci. Technol. 13 (2021) 160-166.

Kontaminacija s plesnimi je ena od pogostih težav v živilski verigi od polja do mize. Plesni povzročajo bolezni kmetijskih pridelkov in kvarjenje hrane, njihovi mikotoksini so škodljivi za zdravje ljudi in živali. Spore plesni so prisotne povsod v naravi, omogočajo preživetje v različnih stresnih razmerah, hitro širjenje in rast plesni na različnih substratih (1).

Moulds that cause food spoilage are a huge problem in the food industry, especially mycotoxin- producing moulds, which pose a major health risk to food consumers. Penicillium verrucosum is one of the major producers of ochratoxin A (OTA) in food. International Agency for Research on Cancer (IARC) classifies OTA as a nephrotoxic, neurotoxic, hepatoxic, teratogenic, genotoxic, immunotoxic, embryotoxic, and carcinogenic toxin. Traditionally, mould growth and OTA production in foods have been controlled by the use of various chemical additives, but nowadays there is an increasing demand from consumers to avoid chemical food additives, which has prompted researchers to search for new safe and biodegradable preservatives. Many studies indicate that essential oils (EOs) and volatile isolates (VIs) rich in low molecular weight phenolic and terpenoid compounds are promising alternative antifungal agents, but there haven’t been many studies showing the antifungal activity of EOs and VIs rich in glucosinolate breakdown products, such as isothiocyanates, nitriles, epithionitriles, and thiocyanates. For that purpose, the antifungal activity of nasturtium (Tropaeolum majus L.) VI was tested. The nasturtium seeds powder was subjected to microwave hydrodiffusion and gravity (MHG) for 15 min at a power of 500 W using an ETHOS X device, and further hydrodistillated with Clevenger system for 2.5 hours to obtain a VI. The chemical composition of nasturtium VI was determined by GC-MS/MS, while the minimal inhibitory concentrations (MIC) of nasturtium VI and its pure compounds (benzyl isothiocyanate, 2- phenylacetonitrile) against P. verrucosum were determined by microdilution method. GC-MS/MS analysis revealed that nasturtium VI was mainly composed of benzyl isothiocyanate (54.35%), and 2- phenylacetonitrile (37%). Among these compounds, nasturtium VI was the most effective with MIC value of 0.05 mg/mL. Excellent antifungal activity was also found for pure benzyl isothiocyanate with MIC value of 0.10 mg/mL, while pure 2- phenylacetonitrile was 8 fold lower with MIC value of 0.8 mg/mL. For comparison, Jeršek et al. (2014) found that oregano EO, mainly composed of phenolic compounds thymol (4.5%) and carvacrol (53.4%), had a MIC of 1.09 mg/mL. The most potent of these compounds was thymol with a MIC of 0.12 mg/mL, while carvacrol had a MIC of 0.38 mg/mL. Thus, it can be concluded that nasturtium VI, which is rich in glucosinolate breakdown products (benzyl isothiocyanate, 2-phenylacetonitrile), has ca. 20 times better antifungal activity against P. verrucosum than oregano EO. It can be assumed that isothiocyanates are the main compounds that confer excellent antifungal activity to nasturtium VI. This result indicates that VIs rich in glucosinolate breakdown products, which are not typically used in mould control studies, are a promising alternative to phenol-rich EOs. Isothiocyanates are lipophilic and therefore can react with membrane-bound enzymes, inhibit fungal growth and cause cell death. In addition, benzyl isothiocyanate is known for its inhibitory effect on mycotoxin production, such as AME, AOH, ALT and TEN (Wang et al., 2020). Therefore, it is necessary to determine the effect of nasturtium VI, benzyl isothiocyanate and 2-phenylacetonitrile on mycotoxin production of P. verrucosum. It is also important to test synergism between the glucosinolate breakdown products in the further studies.

The biomass per plant and total phenolics (TPH) of ethanol extracts of 3 species of thyme, Thymus vulgaris, T. serpyllum,and T. citriodorus, and 3 varieties of basil (Ocimumbasilicum), Genovese, Thai, and Cinnamon, were investigated. All were grown organically. The 2 herbs that showed the greatest biomass were T. citriodorus (26.5 g/plant) and O.basilicum var. Cinnamon (105 g/plant). For these, total flavonoids (TFL), total flavones and flavonols (TFF), and total flavanones and dihydroflavonols (TFDH) were also determined for their ethanol extracts. Furthermore, the antioxidant potential (AOP) and antibacterial activity were determined against foodborne bacteria: Staphylococcus aureus, Listeria monocytogenes, Campylobacter jejuni, and verotoxin-producing Escherichia coli(VTEC). The highest TPH (36 mg chlorogenic acid equivalents per gram of fresh weight) was seen for T. citriodorus collected in 2010. The thyme ethanol extracts contained more TPH and TFL, and their subgroups of TFF and TFDH, than the ethanol extracts from basil, and had AOP comparable to the ethanol extracts from basil, although all of them were lower than the synthetic antioxidant, t-butylated hydroxytoluene. Drying of these herbs decreased TFF and TFDH, while TPH and TFL remained unchanged (for T. citriodorus) or even increased (for Cinnamon basil), and AOP was higher than that of the ethanol extracts from the frozen herbs. The antimicrobial activities of these ethanol extracts depended mainly on the bacterial target. They were weak against gram-negative VTEC, while their effects against C. jejuni, S. aureus, and L. monocytogenes correlated with their TPH and chemical compositions.

The prevalence of antimicrobial resistance of foodborne pathogen Campylobacter jejuni increases, so new natural antimicrobials are becoming attractive alternatives. One of such compounds is pinocembrin, present in honey and therefore a part of normal healthy diet in low concentrations. First, anti-Campylobacter activity of pinocembrin was studied in vitro. Beside minimal inhibitory concentrations (MICs), the whole-genome transcriptomic response od C. jejuni to pinocembrin with the microarray approach was determined, as well as the effects on bacterial virulence potential in systemic infection in BALB/c mice model. MICs of pinocembrin were found at the median concentration of 39 mg/L on 31 Campylobacter isolates. The culture mimicking the traits of multidrug-resistant phenotype cmeR was used in further experiments. The 2-h pinocembrin pre-tretment of C. jejuni with sub-inhibitory concentration resulted in bacterial decrease in mice liver 8 days post infection as well as in decreased expression of ribosomal proteins and up-regulation of several NADH dehydrogenase sub-units and iron uptake proteins, suggesting disrupted the protein production, redox cycle and iron metabolism, which may cause disadvantage in host environment during the infection.

Namen: Da bi preverili inhibicijo adhezije C. jejuni, smo v poskusih in vitro testirali različne rastlinske ekstrakte na celičnem monosloju celic PSI cl1, in sicer ekstrakt tropin sorte Modri pinot (GSS), ekstrat oljčnih listov (OE) in timijana (TE) ter njegov preostanek po hidrodestilaciji eteričnega olja (TE-R) ter ekstrakt iz semen rastline Alpinia katsumadai (SEE) in njegov preostanek po hidrodestilaciji eteričnega olja (hdSEE-R). Želeli smo preveriti uporabnost odpadnega materiala in stranskih proizvodov agro-živilstva pri preprečevanju adhezije C. jejuni na celično linijo PSI cl1. Metode: Pred poskusom protiadhezijske učinkovitosti ekstraktov smo preverili njihovo protimikrobno delovanje ter citotoksičnost na celični liniji PSI cl1 in s tem določili koncentracijsko območje za testiranje protiadhezijske aktivnosti uporabljenih ekstraktov. Rezultati: Najučinkovitejša ekstrakta pri preprečevanju adhezije C. jejuni na celice PSI sta bila SEE in hdSEE- R, sledila sta TE in TE-R, ki sta do 30 % zmanjšala adhezijo bakterije na celično linijo. Najslabšo protiadhezijsko učinkovitost je imel ekstrakt GSS. Zaključek: Odpadni materiali (TE%R in hdSEE%R) ter OE, ekstrakt iz stranskega proizvoda agro%živilske industrije, so se pokazali kot zelo uspešni pri preprečevanju adhezije C. jejuni tudi pri zelo nizkih koncentracijah. Obetavni rezultati študije kažejo možnosti uporabe ekstraktov iz odpadnih rastlinskih materialov na različnih področjih, vključno z industrijo in skrbjo za zdravje ljudi in živali.

Recent advances in nutrition science encourage fast development of new attractive, safe, health protective and stable food products. This is a technological challenge also for introducing biologically active, natural additives of plant origin with delaying microbiological and oxidative spoilage and food quality and safety losses. Research and development is strongly oriented in production and characterization of plant materials with good antioxidant and antimicrobial activity. The aim of the bilateral project called "Natural, biologically active additives for healthy and stable food" between Biotechnical Faculty, UL and the Department of Food Technology and Biotechnology, Faculty of Chemistry and Technology, University of Split was the investigation of extracts from different Mediterranean herbs and vinification by-products ; determination of their phenolic profile, antioxidative and antimicrobial properties in "in vitro" and "in vivo" systems. Selected plant extracts with good biological properties and high share of phenolics could be considered as a source of powerful antioxidants and antimicrobials which can be used as substitute for synthetic additives in order to prevent/delay degradation of food products.

Bakterija Campylobacter jejuni je najpogostejši povzročitelj okužb s hrano v razvitem svetu. Porast tako števila okužb kot tudi odpornosti proti antibiotikom narekujeta razvoj novih metod za zmanjševanje kontaminacije s to bakterijo in ena izmed njih so bakteriofagi. Ugotovili so, da lahko z uporabo bakteriofagov zmanjšamo število bakterij vrste C. jejuni v piščančjem črevesju za faktor med 1 in 4 log10 (Connerton in sod., 2011). V prispevku bomo predstavili sodobne pristope in poglede na bakteriofage kot protimikrobna sredstva ter ugotovitve, ki opisujejo odnos med bakterijo in bakteriofagom ter omogočajo njihovo uporabo. Specifične bakteriofage za C. jejuni smo izolirali iz piščančjega in prašičjega slepega črevesa ter mesa. Pripadajo družini Myoviridae, imajo dvojnovijačno DNA dolžine 130-135kb in litično razkrajajo predvsem seve človeškega izvora, medtem ko so perutninski nanje manj občutljivi. Genetske lastnosti pridobljene s sekvenciranjem izbranih bakteriofagov nakazujejo značilno organizacijo genomov, lahko smo identificirali strukturne in nestrukturne gene, zapisov za toksine ipd. pa nismo našli. Z mutagenezo gostiteljev smo ugotovili, da se bakteriofagi specifično vežejo na komponente kapsule, medtem ko so flagele drugotnega pomena. Specifičnost te interakcije se odraža delno na genetskem nivoju in sposobnosti inaktivacije bakteriofagov s protitelesi. Za potrebe obsežnejših študij učinkovitosti in nadaljnje karakterizacije bakteriofagov smo optimizirali proces priprave bakteriofagov v bioreaktorju ter zaključne procese. Kot prvi smo ugotovili, da proces namnoževanja bakteriofagov bakterijske vrste Campylobacter lahko spremljamo v realnem času z merjenjem deleža koncentracije kisika, ki sovpada s potekom infekcije gostitelja. Te bakteriofage smo primerno koncentrirali in očistili na CIM Multus koloni (Bia Separations, Slovenija) ter jih uporabili v študijah stabilnosti in učinkovitosti v mišjem in celičnem modelu. Stabilnost bakteriofagov in vitro je v večji meri odvisna od pH in temperature, in vivo pa se na bakteriofage odzovejo tudi z nevtralizacijskimi protitelesi, vendar fagi ostanejo infektivni vsaj 12 ur.

Responsibility for food safety must be taken through the entire food28 production chain, to avoid consumer cross-contamination. The antimicrobial activities of an 29 Alpinia katsumadai seed extract and epigallocatehin gallate (EGCG), and their combination, 30 were evaluated against individual food-borne pathogenic strains of Listeria monocytogenes, 31 Escherichia coli and Campylobacter jejuni, individually and as a cocktail, in chicken-meat 32 juice and sterile minced meat as food models, and in minced meat with the naturally present 33 microflora, as an actual food sample. 34 35 The antimicrobial combination of the A. katsumadai extract and EGCG was the 36 most efficient for C. jejuni growth inhibition, followed by inhibition of L. monocytogenes, 37 which was reduced more efficiently in the bacterial cocktail than as an individual strain. The 38 antimicrobial combination added to minced meat at refrigeration temperatures used in the 39 food chain (8 °C) revealed inhibition of these pathogens and inhibition of the naturally 40 present bacteria after 5 days. 41 42 The antibacterial efficiencies of the tested combinations are influenced by 43 storage temperature. Food safety can be improved by using the appropriate combination of 44 natural antimicrobials to reduce the microbiological risk of minced meat.

Thermotolerant campylobacters frequently cause bacterial gastroenteritis in humans who are usually infected through consumption of undercooked poultry meat.We examined Campylobacter jejuni heat-stress responses in vitro, after exposure to 48°C and 55°C. Also in vivo modulation of its pathogenicity was investigated, using BALB/c mice intravenously infected with stressed C. jejuni. Regardless of the bacterial growth phase, their culturability and viability in vitro was reduced after exposure to 55°C. This correlated with the altered protein profile and decreased virulence properties seen in vivo. Heat stress at 48°C elicited transition to more resistant bacterial forms, independent of morphological changes or appearance of shorter spiral and coccoid cells. This treatment did not cause marked changes in bacterial virulence properties in vivo. The results indicate that C. jejuni characteristics and pathogenicity in response to heat stress differ depending on temperature. Findings relating C. jejuni response to stresses used during food processing, and modulation of virulence are important to provide a better understanding its contamination and infective cycle and thus contribute to improved safety in food production chain.

Introduction: Campylobacters have developed a number of mechanisms for responding to environmental conditions. However, the different virulence properties of these cells following exposure to stress are still poorly understood. Methods: We analysed in vitro response to environmental stress (starvation, oxidative stress, heat shock) and the consequent modulation of Campylobacter jejuni pathogenicity in eukaryotic cell (Caco-2, J774 murine macrophages) and in vivo in a murine model. Results: In vitro, the influence of starvation and oxidative stress has milder effect than that of heat shock, although all of the stress conditions influenced the survival of C. jejuni. As published, we proved that environmental stresses have influenced the adhesion, invasion and intraepithelial survival of C. jejuni in eukaryotic cell models as well as the course of the infection of BALB/c mice. The systemic infection of mice occurred no matter what stress was investigated, but with different bacterial load in their livers and spleens as well as different production dynamics of the cytokines investigated (interleukins 6 and 10, tumor necrosis factor- α, interferon-γ) in plasma and liver homogenates. The most pronounced differences were in interferon-γ and interleukin 10 productions, indicating their roles in the immune response to C. jejuni infection. Impact of research: The study of environmental impact on bacterial virulence reveals that microbial adaptation during stress challenge is crucial not just for pathogen survival out of the host, but also during host-pathogen interactions, and thus for the bacterial pathogenicity.

Introduction: Campylobacter jejuni has become the leading cause of human inflammatory enteritis and of the most important zoonosis worldwide. despite of high prevalence of human campylobacteriosis, mainly transmitted by food, extraintestinal survival and virulence properties of thermotolerant campylobacters are still poorly understood, also due to the lack of useful models for such studies. Methods and materials: We confirmed previously bacterial environmental stresses to be implicated in the adhesion, invasion and intraepithelial survival of C. jejuni in Caco-2 cells, J774 murine macrophages and PSI cell lines in in vitro models. In this work BALB/c mice were experimentally infected with stressed C. jejuni cells to mimic the infection in vivo. After bacterial spreading and tissue invasion we followed the survival of stressed cammpylobacters (exposed to starvation or short term oxidative stress) at 1, 3 and 8 days post infection in the organs (liver, spleen) of infected animals. Results: Colonisation of untreated as well as stressed campylobacters occured in the livers and spleens of BALB/c mice. Starvation impaired the ability of Campylobacter to persist in organs, however, short term exposure to oxygen enhanced its survival and therefore also the duration of infection with Campylobacter pre-exposed to short term oxidative stress. Conclusions: C. jejuni can adapt to environmental oxidative stress and modulate its intestinal epithelial cell interactions, such as adherence, invasion, and intraepithelial survival. Adaptive stress response in food or in food processing environment is thus crucial not only for extra-intestinal survival of Campylobacter cells, but also during host pathogen interaction and thus for its pathogenicity. In vivo studies have confirmed our current knowledge about virulence properties of C. jejuni 49/4 isolate from in vitro tests in conventional and also new functional model of the intestinal epithelium, e.g. noncarcinogenic small intestinal epithelial cells (PSI).

Campylobacter jejuni causes food-borne illnesses worldwide due to contaminated food, mainly poultry. This is partly the consequence of microbial adaptation to environmental stresses, which has resulted in increased survival and modulation of virulence properties. In addition, stress has an impact on increased/ reduced sensitivity of the host cells. C. jejuni has evolved several survival strategies which also contribute to its persistence/ spreading in the environment. In the present study we analysed in vivo modulation of C. jejuni virulence in response to environmental stress (starvation, oxygen, temperature) on survival and infectivity in BALB/c mice. The majority of the stress conditions more or less significantly influenced the survival of C. jejuni. We investigated the bacterial numbers at different times in the liver and spleen of the infected mice and the levels of several cytokines in plasma and liver homogenates from these mice. Colonization of untreated and stressed campylobacters can occur in the liver and spleen of BALB/c mice. There were noticed different patterns of production dynamics of the cytokines investigated, although not all were significant. As we show here, in vivo studies of environmental regulation of bacterial virulence are needed to provide important observations regarding the strategies used by pathogens to respond and survive under changing environmental conditions. Adaptive stress responses during stress challenge are crucial for survival of microbial cells during host-pathogen interactions and thus for their pathogenicity.

Campylobacter as a foodborne pathogen developed survival mechanisms to overcome extra-intestinal environmental stresses including food processing. Despite the prevalence of human campylobacteriosis, the pathogenesis of infection is still poorly understood. We studied stress-mediated resistance to evaluate the involvement of stress response mechanisms in survival and virulence modulation of campylobacters. We determined the presence of virulence gene (cdtB, cdtC, virB11, ceuEJ, ceuEC, cadF) and assessed the physiological and morphological changes upon stresses (culturability, viability, morphology). Beside physico – chemical stress conditions, also the activity of alternative antimicrobial agent was tested according to bacterial survival and morphology changes. In our previous work we confirmed environmental stress factors may have implications in the pathogenesis of campylobacteriosis. Using in vitro cell culture models for studying adhesion, invasion and intracellular survival by Caco-2 cells or J774 murine macrophages we indicated oxygen exposure may be involved. In this study we focus in animal model on the kinetics of bacterial clearance from different organs (liver, spleen), using campylobacters exposed to selected stresses and rosemary plant extract. BALB/c mice were intravenously injected with stressed campylobacters and bacterial invasion into their livers and spleens was followed. At different time points after infection, organs were aseptically removed, dissected and the number of CFU was determined. Bacteriological and histopathological analyses of mice organs were performed. Plasma from the infected animals was tested for systemic cytokine production (IL-6, IL-10, IL-12, TNF-alpha, IFN-gamma). The results will be discussed to point on the fact that understanding of involvement and interactions of different defense mechanism (stress response, resistance) on bacterial survival and infectivity is vital for quantitative microbial risk assessment purposes and improved food safety. Non-lethal stresses induce higher resistance to subsequent stresses and can eventually change also bacterial virulence properties.

Campylobacters are the most common bacteria causing human gastroenteritis and are mainly transmitted through foods. As foodborne pathogens they have developed survival mechanisms to overcome the stresses they face in the environment and during food processing. However, the link between environmental stress exposure and its virulence properties of Campylobacter is not often investigated. The aim of our study was to evaluate the effect of selected environmental stress factors: temperature shift, starvation and atmospheric oxygen exposure on the ability of Campylobacter to cause disseminated infection in mice. BALB/c mice were intravenously injected with stressed campylobacters and bacterial invasion into their livers and spleens was followed. At different time points after infection, organs were asseptically removed, dissected and the number of CFU was determined. Simultaneously, paraffin sections were prepaired for histopathological evaluation. According to our results, nutrient insufficiency was the most powerful stress factor which significantly decreased infectious potential of C. jejuni, according to the lowest bacterial load in examined organs. Heat shock was also capable to modulate the in vivo virulence of C. jejuni, but in a lesser extent then starvation. To the opposite, oxidative stress increased survival of C. jejuni inside the livers and spleens of infected mice.

Campylobacters, gram negative spiral shaped, microaerophylic and termophilic bacteria, counter several survival strategies which contribute to their persistence and spreading in the environment. These bacteria cause food- borne illnesses worldwide due to contaminated food and cross-contamination. This is at least partly the consequence of bacterial adaptation to environmental stresses they face in the environment and during food processing. The results are increased survival and virulence properties of these bacteria. Stress has also an impact on increased/reduced sensitivity of the host (intestinal) cells to infection. The aim of our study was to evaluate the effect of environmental stress factors: temperature, starvation and atmospheric oxygen exposure on the ability of C. jejuni to cause systemic infection in experimental animals. BALB/c mice were intravenously injected with stressed campylobacters and at different time points after infection, the number of bacterial CFU in their livers and spleens were determined. At the same time paraffin sections were prepaired for histopathological evaluation. The experiments showed that nutrient insufficiency was the most powerful stress factor. Starvation significantly decreased infectious potential of C. jejuni, seen as the lowest bacterial load in examined organs. Heat shock exposure of C. jejuni also caused modulation of its virulence in vivo, but in a lesser extent than starvation. To the opposite, oxidative stress enhanced C. jejuni virulence according to the highest bacterial load in the livers and spleens of infected mice

Consumer demands for safe, stable and naturally preserved food products have intensified research of different phenolic compounds and plant phenolic extracts which are known to possess antioxidant and antimicrobial activity and offering a potential alternative to synthetic preservatives. By using broth microdilution method combined with TTC, INT or ATP signal measurement we determined minimal inhibitory concentrations (MICs) of several hydroxybenzoic and hydroxycinnamic acids, carnosic and rosmarinic acid and laboratory extracted or commercially produced extracts from different Lamiaceae species. They are among most popular medicinal plants used in phytotherapy as well as widely used flavouring agents in foods. Their extracts are also used as anti-oxidant preservatives. Since they are known to be a rich source of phenolic acids, we tested their antimicrobial activity against gram-positive (Bacillus and Staphylococcus) and gram-negative (Campylobacter, Salmonella and Escherichia) bacterial reference strains and against antibiotic susceptible and resistant food isolates. The antimicrobial activity depended on the concentration and chemical structure of the phenolic compounds and their composition in the extracts. When the activity of the extracts is expressed in MICs of total phenols, e.g. gallic acid (GAE) equivalents per mL of medium and compared to pure phenolic acids, much higher efficiency of the extracts was confirmed against all tested bacteria, indicating synergistic antibacterial activity of phenolic compounds in the mixtures. Carnosic acid and the extracts with higher content of carnosic acid were very efficient against gram-positive strains, but not against Enterobacteriaceae (Salmonella and E. coli). For pure phenolic acids, the structure-activity relationship was expected, but no significant differences were confirmed among hydroxybenzoic and hydroxycinnamic acids against most tested bacteria. However, the latter group (e.g. p-coumaric, ferulic, sinapic and chlorogenic acid) were most efficient against all Campylobacter strains.

Bacteria are exposed to different stress conditions in the environment, or during food production as well as in the host. Temperature changes, availability of nutrients or the presence of toxic substances and antimicrobials are some of them. Despite the lack of many stress response mechanisms that other food-borne bacteria possess, campylobacters display a distinct survival potential in the extraintestinal environment and consequently also high incidence as a cause of human infections. This is at least partially the consequence of microbial adaptation to environmental stresses (1, 2). Consequently, discovering the link between survival and infectivity is vital for risk assessment purposes and for establishing the dynamics of potential food-borne infection. Since there are not many publications in this field, we investigated C. jejuni following heat shock, starvation and oxidative stress. The environmental impact on physiology, survival and virulence properties were studied by culturability, viability, as well as the ability of adhesion, invasion and intracellular survival within in vitro cell culure model using Caco-2 intestinal cells and murine macrophages J774 (3). With additional in vivo experiments in the murine model we investigated the influence of Campylobacter stress response on its virulence potential. At different time points after infection bacterial spreading and tissue invasion of infected BALB/c mice was determined (2, 4). (1) Klancnik, A. et al.(2006):Int.J.Food Microbiol., 112:200-207. (2) Klancnik, A. et al.(2009):Res.Microbiol. (Article in press) (3) Sikic Pogacar, M. et al.(2009):Int.J.Food Microbiol., 129:68-73. (4) Vuckovic, D. et al.(1998):Microb.Pathog., 24:263-268.

C. jejuni infects humans by colonizing the mucus layer of the intestine followed by adherence and invasion of epithelial cells. In order to survive and adapt to unfavorable conditions, C. jejuni has evolved systems to sense and respond environmental stimuli. Since there are not many publications documenting the links between environmental stress responses and virulence in gram-negative bacteria we investigated food and clinical isolates of C. jejuni after heat shock, starvation and oxidative stress exposure. Survival and virulence properties were studied by culturability, viability as well as the ability of adhesion, invasion and intracellular survival within ‘ in vitro’ cell culture model using Caco-2 intestinal cells and murine macrophages J774. Starvation severely impaired C. jejuni culturability but not viability. In starved C. jejuni, adhesion and invasion ability were mostly reduced but intracellular survival varied. While starved cells survived within Caco-2 cells up to 4 days, they were already killed by macrophages 10 h post infection. Heat shock reduced culturability, viability and virulence properties of C. jejuni. Physiological stress response after short oxidative stress increased internalization and intracellular survival of C. jejuni, although the bacteria could not remain viable for extended period within murine macrophages. With additional ‘ in vivo’ experiments in the murine model we investigated the influence of stress response of campylobacters to their virulence potential. We infected BALB/c mice intravenously with C. jejuni following above mentioned stresses and focused on bacterial spreading and tissue invasion. At different time points after infection the number of C. jejuni CFU in liver and spleen of infected mice was determined. Our findings about ‘ in vitro’ and ‘ in vivo’ modulation of C. jejuni virulence in response to environmental stress factors during food processing may be important for better understanding its contamination and infective cycle and thus contribute to improved safety in food supply.

Campylobacter is currently recognized as a leading cause of bacterial gastroenteritis worldwide. The species of Campylobacter which are responsible for food poisoning are classified as thermophilic as they grow at 42o but not at 25oC and microaerophilic, requiring a low oxygen content for growth. However, during transmission through the contamination cycle, Campylobacter is constantly exposed to stressful conditions such as changes in temperature, nutrient availability and oxygen saturation. In an in vitro model using Caco-2 cells, the cell adherence and invasion abilities of two Campylobacter jejuni isolates (poultry and clinical origin) were studied. Additionaly, the influence of environmental stress on adhesion and invasion properties was investigated. The present study showed that both C. jejuni strains adhered to and invaded Caco-2 cells. The human clinical isolate appeared to be more invasive than the animal isolate, but the difference did not reach statistical significance. The binding and invasion of C. jejuni in Caco-2 cells seemed to be enhanced by 5 hours exposure to atmospheric oxygen, especially when the human isolate was used. The growth pattern of oxygen stressed C. jejuni was characterized by a rapid intracellular replication in Caco-2 cells, reaching maximal values at 72h post infection. At the completition of the assay (5 days) a significant population of cells still remained viable, as confirmed by cultivation on blood agar plates. The consequence of C. jejuni exposure to environmental stresses and subsequent changes in physiological state and virulence properties of the bacteria will be discussed.

There is an increasing interest in development of rapid methods for microbial viability determination, which is crucial for detection and enumeration of spoilage and pathogenic microorganisms, evaluation of antimicrobial efficiency of food preservation treatments etc. Traditional plate counting underestimates the real viable portion of microbial population, especially when cells are exposed to environmental stresses resulting in sublethal injuries of the cells - these could remain still virulent but undetected by routine plating methods. Campylobacter jejuni is currently recognized as a leading foodborne pathogen in many countries. Its ability of transforming into a viable but nonculturable (VBNC) state, undergoing alteration in cell morphology, is well documented. The aim of this study was to follow up survival of C. jejuni cells from exponential or stationary growth phase after exposure to different environmental stresses. Starvation of cells in combination with heat or oxidative shock in laboratory model system was tested. Cell morphology and membrane integrity changes were evaluated using the LIVE/DEAD Baclight bacterial viability kit (BacLightTM) and subsequent fluorescent microscopy. In addition flow cytometric measurement after SYTO 9 and propidium iodide staining was folowed. The results of fluorescent measurements were comparativelly evaluated to CFU counts detected on selective and non-selective Campylobacter agar plates. It was confirmed that Campylobacter jejuni cells shift from actively growing spiral cells to noncultivable coccoid forms and remain viable during prolonged exposure to stress conditions. Comparatively, microbial survival was the best in 5h-starved cells from exponential growth phase. Important differences in viability during applied stresses were detected and will be presented. Flow cytometry analysis showed that new population of cells appeared in starved cells exposed to oxidative stress. The consequences of the exposure of Campylobacter cells to environmental stresses and subsequent changes in physiological state of the cells will be discussed. The impact of these changes on Campylobacter cells infectivity and virulence properties are further aims of our study.

Namen magistrskega dela je bil preizkusiti delovanje protimikrobno učinkovitih fenolnih izvlečkov v kombinaciji s biopolimerom hitozanom za funkcionalizacijo aktivne embalaže živil. Delce izvlečkov smo kapsulirali v delce hitozana in jih naknadno dispergirali v makromolekularni raztopini hitozana ter formulacijo kot zunanjo plast dvoslojnega hitozanskega premaza nanesli na polimlečno-kislinske (PLA) folije z metodo valjčkanja. Fizikalni in kemijski nanos formulacij na površino folije smo spremljali z analizama ATR-FTIR in XPS, ki sta jasno pokazali prisotne funkcionalne skupine in kemijsko sestavo izvlečkov ter hitozana na površini. Omočljivost smo spremljali z analizo kontaktnega kota, ki se je pri vseh vzorcih zmanjšal in s tem pripomogel k zmanjšanju potencialne kondenzacije, kar je izjemnega pomena za praktično uporabo. Pri poskusu desorpcije z analizama ATR-FTIR in XPS smo zaznali migracijo elementov s površine folij, kar nakazuje na nestabilnost premazov. Rezultati analize SEM potrjujejo pokritost folij z različnimi nanosi, hkrati pa razkrivajo njihovo nehomogenost, kar bi lahko izboljšali s spremembo tehnologije nanosa. Novo razvite funkcionalizirane folije izkazujejo dobre antioksidativne lastnosti in več kot 95,0 % protimikrobno aktivnost proti patogeni bakteriji Staphylococcus aureus. Funkcionalizacija folij PLA s hitozanskimi premazi, obogatenimi z izbranimi izvlečki je bila uspešna ob sočasnem zagotavljanju hidrofilnih, antioksidativnih in protimikrobnih lastnosti. The aim of this work was to test the performance of antimicrobial-effective phenolic extracts in combination with biopolymer chitosan for the functionalization of active food packaging material. Particles of the extracts were encapsulated in chitosan biopolymer particles and dispersed in a macromolecular chitosan solution. Formulations were then applied as two-layer chitosan coatings of polylactic acid (PLA) films with the rolling method. The physical and chemical deposition of the formulations to the foil surface was monitored by ATR-FTIR and XPS analyses, which clearly showed the presence of functional groups and the chemical composition of the extracts and chitosan on the surface. The wettability was monitored with contact angle measurements, which decreased in all cases and thus contributed to the anti-fog foil properties, which are extremely important for practical use. In the desorption experiment with ATR-FTIR and XPS analysis, we detected the migration of elements from the surface of the foils, which indicates the instability of the coatings. The results of the SEM analysis confirm the coverage of the foils with different coatings, but simultaneously reveal the inhomogeneity of the coating, which could be improved by changing the coating technology. The newly developed functionalized films show good antioxidant properties and more than 95,0 % antimicrobial activity against the pathogenic bacteria Staphylococcus aureus. Functionalization of PLA films with biopolymer coatings with chitosan enriched with selected phenolic extracts was successful while providing hydrophilic, antioxidant, and antimicrobial properties.

Novi pristopi obvladovanja bakterij Campylobacter jejuni v živilski industriji so predmet številnih raziskav. Rastlinski izvlečki predstavljajo obetavne alternativne protimikrobne učinkovine za nadzor biofilma. V tej doktorski nalogi smo pripravili in testirali različne pripravke sivke vrst L. angustifolia in L. x intermedia proti filmotvornosti bakterij C. jejuni na abiotskih površinah. S filmotvornostjo smo se spoprijeli preko tarč, kot so gibljivost, pritrjevanje in medcelična komunikacija bakterij C. jejuni. Optimizirali smo metodo gojenja in preučevanja biofilma bakterij C. jejuni na abiotski površini in sicer na interfazi trdna površina/tekočina s konfokalno vrstično lasersko mikroskopijo. Poleg vpeljave visoko tlačne tekočinske kromatografije v kombinaciji s fluorescenčnim detektorjem (HPLC-FLD) smo razvili, optimizirali in nadgradili metodo preučevanja medcelične komunikacije bakterij C. jejuni z biosenzorjem V. harveyi MM30. Nova metoda je v primerjavi s HPLC-FLD natančnejša in občutljivejša. Vsi pripravki sivke so uspešno odstranili zreli biofilm bakterij C. jejuni z abiotske površine. Prav tako so vsi pripravki sivke uspešno zavrli gibljivost, pritrjevanje bakterij C. jejuni na abiotske površine in filmotvornost. Pripravki sivke niso značilno znižali koncentracije signalnih molekul avtoinduktor-2 (AI-2) v izrabljenem gojišču bakterij C. jejuni. Rezultati metode rt-PCR niso pokazali vpliva eteričnega olja sivke na izražanje gena luxS pri bakterijah C. jejuni. Transkriptomska analiza planktonske kulture bakterij C. jejuni po izpostavljenosti eteričnemu olju sivke je pokazala spremembo izražanja številnih genov, pomembnih za gibljivost, filmotvornost, metabolizem železa in stresni odziv bakterij C. jejuni. Te lastnosti predstavljajo nove, obetavne tarče za obvladovanje biofilma bakterij C. jejuni v živilski industriji. New approaches are being studied intensively to control Campylobacter jejuni in the food industry. Plant extracts represent promising alternative antimicrobial agents for biofilm control. In this doctoral thesis, we prepared and tested different preparations of Lavandula angustifolia and L. x intermedia against biofilm formation of C. jejuni on abiotic surfaces. Biofilm formation was challenged through targets such as motility, attachment and intercellular communication of C. jejuni. We optimized the method of culturing and studying the biofilm of C. jejuni on an abiotic surface, specifically at the solid/liquid interface, using confocal laser scanning microscopy. We developed, optimized and upgraded a method for studying the intercellular communication of C. jejuni with the V. harveyi MM30 biosensor strain, which proved to be accurate and more sensitive compared to the high pressure liquid chromatography with fluorescence detector (HPLC-FLD) method. All lavender preparations successfully removed the mature biofilm of C. jejuni from the abiotic surface. Likewise, all lavender preparations successfully inhibited the motility, attachment of C. jejuni to abiotic surfaces and biofilm formation of C. jejuni. Lavander preparations did not lower significantly the concentration of autoinducer-2 (AI-2) signalling molecules in the spent medium of C. jejuni. The rt-PCR did not show the significant effect of lavender essential oil on the expression of the luxS gene in C. jejuni. Transcriptomic analysis of the planktonic culture of C. jejuni after exposure to lavender essential oil showed that the expression of many genes important for motility, biofilm formation, iron metabolism and stress response of C. jejuni has been changed. These traits represent new, promising targets for C. jejuni biofilm control.

Cilj dela je bila priprava gotovega živila iz hitro pokvarljivih mediteranskih surovin in testiranje protimikrobnega delovanja bioaktivnih izvlečkov in vitro in v modelih živila - homogenatu in namazu iz jajčevca. Testirali smo protimikrobno aktivnost fenolnega izvlečka lista robide (Rubus fruticosus), izvlečka odpadnega destilata navadnega brina (Juniperus communis), njune kombinacije, izrabljenega gojišča (CFSM) in zaščitne kulture Lb. paraplantarum ter embalažnih folij PLA s premazom hitozana in izvlečki ter njihovih kombinacij proti patogenim mikroorganizmom, povzročiteljem kvara in naravno prisotni mikrobioti namaza. S procesi priprave namaza in njegovim skladiščenjem pri realnih pogojih hranjenja v hladilniku 4 - 5 °C smo zavrli rast aerobnih sporogenih bakterij. Dodatki v namazu niso zmanjšali skupnega števila aerobnih mezofilnih bakterij. Kombinacija izvlečka robide in brina ter CFSM Lb. paraplantarum je bila učinkovita v obremenitvenem testu s P. fragi in L. innocua, tako ima največji potencial za uporabo v živilih. Uporaba zaščitne kulture je smiselna pri živilih, ki so namenjena daljšemu shranjevanju, njihova protimikrobna aktivnost se razvije kasneje in bolj zakisa izdelek. Funcionalizirana folija PLA s premazom hitozana in/ali dodanimi rastlinskimi izvlečki protimikrobne aktivnosti kombinacije dodatkov v živilo ni dodatno izboljšala, kljub izkazani protimikrobni učinkovitosti folij v in vitro testih. Aim of this work was preparation of food from quickly perishable Mediterranean ingredients and to test antimicrobial activity of bioactive extracts in vitro and in food models - eggplant homogenate and spread. We tested antimicrobial activity of the phenolic leaf extract from blackberry (Rubus fruticosus), extract of the waste distillate of common juniper (Juniperus communis), their combination, cell free spent medium (CFSM) of Lb. paraplantarum, protective culture Lb. paraplantarum and packaging PLA foil with chitosan coating and extract combinations against pathogenic and food spoilage microorganisms and natural microbiome of spread. With preparation processes of spread and its storage under realistic storage conditions in the refrigerator 4 - 5 °C, we inhibited the growth of aerobic sporogenic bacteria. Additives in the spread did not reduce the total number of aerobic mesophilic bacteria. The combination of blackberry and juniper extracts and Lb. paraplantarum CFSM was effective in challenge test with P. fragi and L. innocua, thus it has the greatest potential for use in foods. The use of a protective culture makes sense for foods that are intended for long-term storage, as their antimicrobial activity develops over time and makes the product more acidic. Functionalized PLA film with chitosan coating and/or added plant extracts did not further improve the antimicrobial activity of the combination of food additives, despite the demonstrated antimicrobial efficacy of the films in in vitro tests.

Razvoj aktivnega pakiranja je posledica potreb sodobnega časa. Koncept aktivne embalaže vključuje interakcijo le-te z živilom preko aktivne spojine v ali na površini embalaže za izboljšanje obstojnosti in kakovosti živila. Cilj dela je bil izboljšati funkcionalnost inertne polipropilenske folije z dvoslojnimi premazi, s prvim slojem na osnovi primarnega ali kvartarnega hitozana in drugim slojem na osnovi naravnih izvlečkov, bodisi rjave alge (Padina pavonica), iglic brina ali listov robide. Pripravili smo folije z različnimi kombinacijami premazov za pridobitev podatkov o vplivu posameznih nanosov ter njihovem učinku pri dvoslojni aplikaciji. Vezavo premazov na folije smo potrdili s tehtanjem folij in z XPS analizo. Določili smo fizikalno-kemijske lastnosti folij, ki so pomembne z vidika pakiranja živil (omočljivost, prepustnost kisika), ter testirali antioksidativni potencial in protimikrobno aktivnost proti S. aureus in E. coli. Prisotnost hitozana ter izvlečkov listov robide in iglic brina je izboljšala antioksidativni potencial folij, medtem ko so veliko slabši antioksidativni potencial izkazale folije s slojem izvlečka alge. Kvartarni hitozan in izvlečka listov robide in iglic brina so izkazali odlično protimikrobno delovanje proti bakteriji S. aureus, proti bakteriji E. coli pa le folija s samostojnim slojem kvartarnega hitozana. Prisotnost tako hitozana kot izvlečkov je pomembna, saj v dvosloju vpelje dobre bioaktivne lastnosti, ključne za koncept aktivne embalaže, prav tako izboljša omočljivost folije oz. zmanjša stični, kot tudi prepustnost kisika, kar je pomembno z vidika funkcionalnosti živilske embalaže. The development of active packaging is a result of the needs of modern times. The concept of active packaging involves its interaction with food through an active compound in or on the surface of the packaging material to improve the shelf-life and quality of food. The goal was to improve the functionality of an inert polypropylene (PP) film with two-layer coatings, with the first layer based on primary or quaternary chitosan and the second layer based on natural extracts, either brown algae (Padina pavonica), juniper needles or blackberry leaves. We prepared films with different combinations of coatings to obtain data on the impact of individual coatings and their effect in a two-layer application. We confirmed the presence of coatings on the films by weighing and by an XPS analysis. We determined the physico-chemical properties of foils, important for food packaging (wettability, oxygen permeability), and tested the antioxidant potential and antimicrobial activity against S. aureus and E. coli. Chitosan and the blackberry leaves and juniper needles extracts improved the antioxidant potential of films, while foils with a layer of algae extract showed a much lower antioxidant potential. Quaternary chitosan and extracts proved excellent antimicrobial activity against S. aureus. Only films with an individual layer of quaternary chitosan proved good antimicrobial activity against E. coli. The presence of both chitosan and extracts is important, as it not only introduces good bioactive properties into the bilayer and is key for the concept of active packaging, but it also improves wettability, i.e. reduces the contact angle, as also oxygen permeability, which are important for the functionality of food packaging.

Slovenija ima edinstveno in raznoliko floro, posledično pa proizvodnjo številnih vrst medu z različnimi fizikalno-kemijskimi in bioaktivnimi lastnostmi. V študiji smo z določanjem minimalne inhibitorne koncentracije (MIK) proti izbranim mikroorganizmom raziskali 8 vrst medu, zbranih med leti 2020-2022 iz različnih regij Slovenije. Razdelili smo jih na temne medove (ajdov, kostanjev, hojev, gozdni med) in svetle medove (akacijev, cvetlični, lipov in med oljne ogrščice). Testirali smo njihovo protimokrobno aktivnost z mikrodilucijsko metodo proti petim bakterijskim vrstam: Staphylococcus aureus, Listeria monocytogenes, Bacillus cereus, Escherichia coli in Pseudomonas aeruginosa. Rezultate skupine temnih medov s skupno 60 vzorci in skupine svetlih vrst medov s skupno 58 vzorci smo analizirali z Mann-Whitney U testom, s katerim smo potrdili, da imajo temne vrste medu boljše protibakterijsko delovanje. Najboljšo protibakterijsko aktivnost smo določili hojevemu medu, sledila sta mu gozdni in kostanjev med. Pokazali smo torej, da je protibakterijsko delovanje slovenskih vrst medu pogojeno z izvorom medu, saj so imeli manini medovi manjše vrednosti MIK in zato boljše protibakterijsko delovanje. Izmerjene vrednosti MIK pa so bile odvisne tudi od vrste testne bakterije. Največjo občutljivost smo potrdili pri bakteriji Staphylococcus aureus, z najpogosteje izmerjeno vrednostjo MIK 31,3 mg/ml, največjo odpornost pa pri bakteriji Bacillus cereus. Slovenia has a unique and diverse flora and consequently production of several types of honey, with different physico-chemical and bioactive properties. In this study, we investigated 8 types of honey, divided into dark honeys (buckwheat, chestnut, fir, forest) and light honeys (acacia, flower, linden, and rapeseed), obtained from different regions of Slovenia between 2020-2022, by determining the minimum inhibitory concentration (MIC). The selected honeys were tested against five bacterial species: Staphylococcus aureus, Listeria monocytogenes, Bacillus cereus, Escherichia coli, Pseudomonas aeruginosa. The results of a group of dark honeys with a total of 60 samples and a group of light honeys with a total of 58 samples were analysed using the Mann-Whitney U test to confirm that dark honeys have better antibacterial activity. The best best antibacterial activity was found for hoja honey, followed by forest and chestnut honeys. We have therefore shown that the antibacterial activity of Slovenian honeys is conditioned by the origin of the honey, as manna honeys had lower MIC values and therefore better antibacterial activity. However, the measured MIK values also depended on the type of test bacterium. The highest sensitivity was confirmed for Staphylococcus aureus, with the most frequently measured MIK value of 31,3 mg/ml, while the highest resistance was found for Bacillus cereus.

Namen magistrske naloge je bil iz narave izolirati kvasovke rodu Saccharomyces, ki so sorodne vrstam S. uvarum in S. eubayanus, in rodu Hanseniaspora. Vzorčili smo drevesno lubje različnih vrst drevesnih sestojev predalpskega sveta in plodove sadnih dreves na odročnih kmetijah Koroške in Gorenjske. Vzorčili smo tudi lubje oljčnih dreves Slovenske Istre in grozdje iz vinorodnih okolišev po celi Sloveniji. V prvem delu naloge smo z uporabo selektivnih gojišč izolirali seve kvasovk, katerim smo izolirali DNA in na podlagi nukleotidnega zaporedja notranjih prepisujočih distančnikov (regije ITS) in regije D1/D2 ribosomskega gena le-te identificirali. Skupno smo izolirali 213 sevov kvasovk, ki so pripadali 26 različnim vrstam. Izmed teh sevov smo jih 19 uvrstili v rod Saccharomyces in 58 v rod Hanseniaspora. Vrste rodu Saccharomyces, S. cerevisiae, S. jurei in S. paradoxus, smo najpogosteje izolirali iz skorje dreves in s plodov gozdnih sestojev. Vrsti rodu Hanseniaspora, H. uvarum in H. vineae, pa smo najpogosteje izolirali iz plodov drevesnih in grmovnih vrst, ter iz grozdja v vinogradih. Kvasovke obeh rodov smo najpogosteje izolirali z uporabo selektivnega gojišča, ki vsebuje kvasno dušikovo osnovo (YNB) z dodanim etanolom in glukozo. Drugi del naloge je obsegal kemijsko-fizikalno analizo fermentirajočega grozdnega soka iz treh slovenskih vinorodnih dežel, od koder smo pretežno izolirali kvasovke rodu Hanseniaspora. Ugotovili smo, da te vrste počasi fermentirajo grozdni sok, ter da ne dokončajo fermentacije, saj so v njem ostanki sladkorjev. Prav tako te vrste tvorijo velike količine hlapnih kislin. The aim of the master's thesis was to isolate wild yeasts of the genus Saccharomyces, related to the species S. uvarum and S. eubayanus, and Hanseniaspora from the natural environment. We sampled tree bark and fruits from fruit trees in remote parts of the Koroška and Gorenjska regions, as well as from various tree species of the pre-alpine world. We sampled bark from olive trees in Slovenian Istria and grapes from vine-growing areas throughout Slovenia. In the first part of the thesis, yeasts were isolated on selective media. DNA was isolated from the pure cultures and the yeasts were identified using sequences of the internal transcribed region (ITS) or the D1/D2 regions of the ribosomal gene. In total, we isolated 196 yeast strains, which were accomodated to 31 different species: 18 isolates to the genus Saccharomyces and 58 to the genus Hanseniaspora. The species of the Saccharomyces genus, S. cerevisiae, S. jurei, and S. paradoxus, were most frequently isolated from tree bark and from fruits of forest stands. Isolates of the genus Hanseniaspora (H. uvarum and H. vineae) were most frequently isolated from fruit of tree and shrub species and from grapes in vineyards. Yeasts of both genera were most frequently isolated using a yeast nitrogen base (YNB) selective medium with the addition of ethanol and glucose. The second part of the thesis included the chemical-physical analysis of fermenting grape juice from three Slovenian wine-growing regions, from which mainly yeasts of the genus Hanseniaspora were isolated. We found out that these species ferment grape juice slowly and do not complete fermentation, as the fermented juice contains large amounts of residual sugar. These species also produce large amounts of volatile acids.

Globalizacija in ozaveščenost potrošnikov pospešeno spodbujata industrijo konzerviranja in pakiranja živil k uporabi okolju in zdravju prijaznih naravnih virov protimikrobnih materialov in dodatkov. Eden izmed uspešnih biorazgradljivih materialov so folije na osnovi polimlečne kisline. Ker folije same nimajo protimikrobnega učinka, jim za izboljšanje te funkcije dodajajo različne protimikrobne in antioksidativne polimere. Hitozan je eden izmed najpogosteje uporabljenih polimernih dodatkov zaradi dobrih mehanskih ter fizikalno-kemijskih lastnosti. Za izboljšanje protimikrobne aktivnosti mu še dodajajo bioaktivne snovi, kot so eterična olja ali izvlečki alg in rastlin. Z diplomsko nalogo smo preverili protiglivno aktivnost različnih oblik hitozanskega premaza (primarni in kvarterni) z vgrajenim dodatkom izvlečka rjave alge Padina pavonica proti glivi Aspergillus flavus. Pri testiranju smo ugotavljali protimikrobno in antioksidativno aktivnost štirih različnih formulacij folij z metodo ISO 22196. Z eksperimentalnim delom smo potrdili boljši protimikrobni učinek, če je bil izvleček alge kapsuliran s kvarterno obliko hitozana (69,91 in 69,12 %). Vzorci, kapsulirani s primarno obliko hitozana, so dosegli 45,25 in 40,34 % aktivnost. Pri antioksidativnem učinku izstopa le formulacija, kjer je kvarterni hitozan uporabljen tako pri osnovnem premazu, kot pri kapsulaciji izvlečka (25,22 % SA). Ostale formulacije so imele statistično neznačilne antioksidativne lastnosti (16,48 %, 14,05 % in 13,38 % SA). Globalization and consumer awareness are rapidly pushing the food preservation and packaging industry towards the environment and health less harmful natural sources of antimicrobial materials and additives. One of the most successful biodegradable material is based on polylactic acid foil. The foil itself does not have any antimicrobial effect, therefore various antimicrobial and antioxidant polymers are added for functionality improvement. Chitosan is one of the extensively used polymer additives due to its effective mechanical and physicochemical properties. To improve its antimicrobial activity, bioactive substances such as essential oils or extracts of algae and plants are added. Within our work we tested antifungal activity against the Aspergillus flavus with various forms of chitosan coatings (with primary and quaternary chitosan formulations and encapsulated extract of brown algae Padina pavonica. Throughout the experiment, we determined antimicrobial and antioxidant activity of four different foil coating formulations by ISO 22196 method. Experimental results revealed better antimicrobial and antioxidant effect in samples with quaternary chitosan used for algae extract encapsulation (69,91 % and 69,12 % activity). Formulations with primary chitosan for encapsulation expressed 45,25 and 40,34 % activity, recpectively. Antioxidant test showed significant difference only in formulation where quaternary chitosan was used in base coating as well as for extract encapsulation (25,22 % SA). Other formulations showed slight antioxidant activity, which was not statisticaly significant (16,48 %, 14,05 % and 13,38 % SA, respectively).

Phenolic profiles, antioxidant, and antimicrobial activities of hydroethanolic olive leaf extracts from six Mediterranean olive cultivars (Croatian: Lastovka, Levantinka, Oblica; Italian: Moraiolo, Frantoio, Nostrana di Brisighella) were investigated. As expected, various distributions of phenolic levels were observed for each cultivar and the total phenolic content showed high variability (ranging from 4 to 22 mg GAE/g of dry extract), with the highest amount of phenolics found in the Oblica sample, which also provided the highest antiradical (ORAC) and reducing activity (FRAP). The screening of individual compounds was performed by HPLC-PDA-ESI-QTOF-MS and the main detected compounds were oleuropein, hydroxytyrosol, oleoside/secologanoside, verbascoside, rutin, luteolin glucoside, hydroxyoleuropein, and ligstroside. While the antioxidant activity of the samples was relatively high, they showed no bactericidal and bacteriostatic activity against E. coli and S. Typhimurium; weak activity against Staphylococcus aureus, Bacillus cereus, and Listeria innocua; and inhibitory effects against Campylobacter jejuni at 0.5 mg dry extract/mL. The obtained results support the fact that olive leaf extracts, and especially those from the Oblica cultivar, could potentially be applied in various industries as natural preservatives and effective and inexpensive sources of valuable antioxidants., PRIMA program under project BioProMedFood 1467, European Commission, MCIN/AEI RTI2018-099835-A-I00

The consumers’ demand for safe foods without chemical additives increased the research for green solutions, based on natural antimicrobials. Plants can be an important source of bioactive compounds able to prevent the development of foodborne pathogens and spoilage microflora. This paper aimed to characterize phenolic extracts (PEs) and essential oils (EOs) obtained from Mediterranean Rubus fruticosus leaves and Juniperus oxycedrus needles and to evaluate their antimicrobial effects against Listeria monocytogenes Scott A. The growth dynamics with sub-lethal concentrations of plant derivatives were modeled and flow cytometry was used to better evidence the effect on cell viability and culturability. The results showed that these plant derivatives affected the growth of L. monocytogenes, increasing lag phase (about 40 h in the presence of PEs vs. 8 h in the control) and decreasing the final cell load of at least 1 log cycle with respect to the control. R. fruticosus EO was the most effective, determining an initial decrease of cell counts of about 6 log cycles, followed by a restart of growth after 10 h, with rate similar to the control (0.08 with R. fruticosus EO vs. 0.09 ((log CFU/ml)/h in the control) but significantly lower final cell load (7.33 vs. 8.92 log CFU/ml). According to flow cytometry, only R. fruticosus EO induced a relevant increase of dead cells, while the other plant derivatives determined different extent of sub-lethal cell injury. The discrepancy observed in some cases between viability and culturability could indicate the presence of cells not able to grow in culture media, whose fate needs to be further investigated to assess their potential recovery, thus bringing to an overestimation of the antimicrobial effect of these substances. This research contributed to increase the knowledge of these underused raw materials such as blackberry leaves and juniper needles that can be exploited in food and other industries.

Spomladanska resa (Erica carnea) je zimzelena rastlina, ki ji pripisujejo številne biološke aktivnosti, med njimi tudi protibakterijsko delovanje. V diplomski nalogi smo preverili protibakterijsko in protiadhezijsko delovanje etanolnih izvlečkov spomladanske rese in kemijsko čistih učinkovin na izbrane testne mikroorganizme. Uporabili smo grampozitivni bakteriji Staphylococcus aureus in Listeria innocua ter gramnegativni bakteriji Escherichia coli in Pseudomonas fragi. Določili smo minimalno inhibitorno koncentracijo (MIK) in minimalno baktericidno koncentracijo (MBK) etanolnih izvlečkov in nekaterih kemijsko čistih učinkovin, ki jih vsebujejo etanolni izvlečki spomladanske rese. Rezultati so pokazali najboljši protibakterijski učinek etanolnih izvlečkov proti bakteriji S. aureus, MIK je znašala 1 mg/ml. Najboljši protibakterijski učinek kemijsko čiste učinkovine je imel kvercetin, z najnižjimi vrednostmi proti grampozitivnim bakterijam (MIK 0,25 mg/ml MBK 1 mg/ml). Ugotovili smo tudi relativno nizke vrednosti MIK etanolnih izvlečkov proti bakteriji P. fragi (1-2 mg/ml). Te vrednosti so primerljive, ali v primeru kemijsko čistih učinkovin celo nižje od tistih za grampozitivne bakterije, na kar predvidoma vpliva specifična encimska aktivnost bakterij P. fragi. Določili smo tudi dobro protiadhezijsko aktivnost etanolnega izvlečka spomladanske rese, ki je adhezijo bakterije P. fragi na polistiren statistično značilno zmanjšal, povprečno za 52,5 %. Na podlagi rezultatov smo potrdili protibakterijsko delovanje etanolnih izvlečkov spomladanske rese proti izbranim testnim bakterijam, pri čemer so dobljene vrednosti MIK in protiadhezijske aktivnosti na bakterijo P. fragi zanimiva tema nadaljnjih študij. Spring heath (Erica carnea) is an evergreen plant, with many biological activities, including antibacterial activity. In this thesis we checked the antibacterial and antiadhesion effect of ethanolic extracts of spring heath and some chemically pure active ingredients of these ethanolic extracts on selected target microorganisms. We used two gram positive target bacteria, Staphylococcus aureus and Listeria innocua, and two gram negative bacteria, Escherichia coli and Pseudomonas fragi. We determined the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of ethanolic extracts and some of their chemically pure active ingredients. The results showed the best antibacterial effect of ethanolic extracts against S. aureus (MIC 1 mg/ml) and the best antibacterial effect of quercetin, the chemically pure active substance, with the lowest values for gram positive bacteria (MIC 0,25 mg/ml MBC 1 mg/ml). The results for gram negative P. fragi are interesting, due to relatively low MIC values (1-2 mg/ml), which are comparable or, in case of chemically pure active compounds, even lower than the values obtained for gram positive bacteria. Presumably, this is influenced by specific enzymatic activity of P. fragi. We also determined a statistically significant reduction (52,5 %) in the adhesion of P. fragi to polystyrene, after the addition of ethanol extract. We have confirmed the antibacterial effect of spring heath extract on selected target microorganisms and found that the obtained MIC values and antiadhesion activity for P. fragi are an interesting topic for additional studies.

The aim of this study was to determine the technological and sensory properties of m. longissimus dorsiin lambs. The Lička pramenka lambs were reared traditionally and fed with voluminous and concentrate feed with the addition of the dry or chopped fresh Agraricus bisporus as a supplement in different percentages. A feeding experiment was conducted on 30 lambs divided into three groups. The carcasses were sampled 0 and 24h after slaughter. The av erage slaughter weight was 10.40 kg - in the control group, 12.29 kg in group 2 and 11.99 kg in group 3. The pH ranged from an average of 6.95 in control and group 3 to 6.72 in group 2. After 24 hours, a decrease in pH was recorded (control group 5.38, gro up 2 5.52 and group 3 5.74). The values of the colour parameters (CIE L * a * b *) were - L* control group 61.20 ; group 2. 55.92 and group 3. 59.53, a* parameter was 5.22 in the control, 3.95 in group 2 and 4.68 in group 3, while b* was 3.46 in the control , 1.24 in group 2 and 1.39 in group 3. The sensory evaluation confirmed that the meat showed the characteristics of colour, smell and taste of young lamb meat. The meat was light pink in colour, sparsely covered with adipose tissue. The smell and taste in the cooking and roasting samples were specific to the lamb meat. For determination of oxidative stability with thiobarbiturate test (TBARS) and fat content, samples were collected on days 0, 3, and 6. As anticipated TBARS values were increased from day 0 to day 6, and there were no significant differences between groups 2 and 3. Cholesterol in the meat of the control group was high (31.95 mg/100g), which is significantly higher than the values found in the meat of group 2 (9.63 mg/100g) and group 3 (95.2 mg /100 g). In conclusion, by adding this specific supplement to the lambs' diet, the meat retained all the positive sensory and technological properties with reduced cholesterol levels

In recent years, the demand for novel antioxidant agents derived from natural products is rising in the food industry. Medicinal plants such as bay laurel (Laurus nobilis L), sage (Salvia Officinalis L.) and thyme (Thymus vulgaris L.) contain a variety of bioactive molecules that contribute to the plants' antioxidant potential. While the antioxidant potential of plant extracts and essential oils has been well researched, the potential of hydrosols secondary products of hydrodistillation process, has recently raised research interest. Hydrosols consist of distillation water with small amounts of dispersed essential oils which provide them antioxidant and antimicrobial properties potentially applicable in the food industry. The aim of this study was to evaluate the antioxidant potential of hydrosols of bay laurel, sage and thyme, and their mixture (1:1:1) obtained during hydrodistillation on Clevenger apparatus, and to monitor their antioxidant stability through 28 days of storage at room temperature by DPPH Radical Scavenging and Ferric Reducing Antioxidant Power (FRAP) assays. Bay laurel hydrosol showed the highest antioxidant activity on the first day with a value of 2000 µM of Trolox equivalents (TE) as determined by both DPPH and FRAP followed by the plant mixture (DPPH 1000 µM TE, FRAP 621 UM TE), thyme (DPPH and FRAP 370 µM TE) and sage whose antioxidant activity was at the bottom of detection level. Both FRAP and DPPH showed that the antioxidant activity of all hydrosols has gradually decreased down to approximately 50% after 28 days. Nevertheless, bay laurel hydrosol has shown the highest potential for application in the food industry, for example in minimal processing of fruits and vegetables with the goal of prolonged shelf life, since the DPPH and FRAP values were still relatively high (1000 µM TE) after 28 days.

Confectionery cakes are products obtained by mixing, shaping, baking, or other suitable processing procedures of two or more ingredients, giving the characteristic sensory properties of the product. The chemical composition and high content of water make the confectionery cakes suitable for growth and multiplication of various microorganisms. Contamination can occur at all stages of the production process thus conducting good hygiene practices is necessary to obtain a product safe for consumption. The aim of this two years’ study was to provide insight into the contamination of confectionery cakes in the Republic of Croatia by microorganisms, either potentially pathogenic or indicators of hygienic production. The results on the number and type of Enterobacteriaceae in samples in 2018. indicated the presence of certain types of bacteria from the family of Enterobacteriaceae, not listed in Regulation 2073/2005. The number of Enterobacteriaceae above 102 CFU/g was detected in 22.5% of creamy, 22.6% of chocolate, and in 34.4% of fruit cakes. Further bacteria determination was done by the MALDI-TOF method. Exposure assessment was performed by a qualitative approach. Medium to high risk of exposure was found for bacteria Enterobacter (E.). asburiae and Klebsiella (K.). oxytoca in chocolate cakes, for E. cloacae, Serratia (S.) liquefaciens and Buttiauxella (B.) gaviniae in fruit cakes and for E. kobei, E. cloacae, Pantoea (P.) agglomerans, S. liquefaciens, E. asburiae, K. oxytoca, B. warmboldiae and Ratinella in creamy cakes. Following the obtained results, it is recommended that in case of increased number of Enterobacteriaceae, food should be further analyzed for the presence of pathogens and then its safety should be assessed.

The aim of this paper was to replace traditionally used pork and lard with meat from specially reared lambs and olive oil, in order to produce a dry sausage with a preferable fatty acid composition while retaining positive sensory characteristics. Lička pramenka lambs were fed with a specific feed supplement containing various percentages of Agraricus bisporus . Three recipes of dry sausages were produced – 1 Beef/Lamb/Lard ; 2 Lamb/Beef/Lard/Olive oil/Bread ; 3 Po rk/Beef/Lamb/Pork backfat. All three sausages underwent the same technological production process. The pH decreased with ripening time, ranging from 5.54 to 5.92 depending on the sausage, while water activity decreased to 0.756 and 0.824 respectively. Sens ory analysis was performed by professional evaluators. The parameters that were evaluated were: appearance (0 - 15), cross - section (0 - 20) and smell and taste (0 - 25) with a possible maximum number of 60 points. The results of the sensory analysis were as foll ows: sausage 1 - 52.36 ; sausage 2 - 56.06 ; and sausage 3 - 56.32 points. Volatile aroma compounds were identified by the solid‐phase microextraction (SPME) combined with gas chromatography - mass spectrometry (GC‐MS). A total of approximately 60 volatile com pounds were detected by GC‐MS, of which nearly 50 per cent were major volatile compounds mutual for all sausage samples. The most of identified compounds are complex terpenoids like aroma compounds such as α - Phellandrene, Linalool, γ - Terpinene, p - Cymene, D - Limonene, 3 - Carene and Humulene. These compounds are characteristic volatile aroma compounds of numerous herbs and spices and contribute to the overall aroma profile of prepared lamb sausages. The cholesterol level was affected by the raw material composi tion of sausages. The lowest value was 64 mg/100 g in the sausage sample 2. To conclude, the substitution of meat and pork/lard with lamb and olive oil enhanced the sensory of the dry sausage, and at the same time, it successfully improved the fatty acid composition of the product.

Innovative technologies are being widely studied for their application in the production of virgin olive oil. Flash thermal treatment is one of these techniques, since the regulation of temperature allows control over complex enzyme systems that affect the nutritional value, oxidative stability and sensory profile of virgin olive oil [1-3]. The aim of this study was to determine the effects of flash thermal treatment (both cooling and heating) on the antioxidant activity of virgin olive oil from four different varieties (Oblica, Rosulja, Istarska bijelica and Levantinka). Flash thermal treatment was applied at 6 different temperatures in the range of 15-40 °C. It was used as a malaxation pretreatment for the production of virgin olive oil on a laboratory scale using the Abencor system. The antioxidant activity of the produced oils was determined using the DPPH method previously described by Kraljić et al [4]. The results show a significant influence of the variety and pretreatment on the antioxidant activity of virgin olive oil, as well as their interaction. Rosulja had the highest antioxidant activity (90.8% on average) compared to the other three varieties (84.3, 75.6 and 74.8% for Oblica, Istarska bijelica and Levantinka, respectively). In relation to the applied flash thermal treatment, Oblica, Rosulja and Levantinka showed significantly higher antioxidant activity at the highest temperature (40 °C), while Istarska bijelica responded better to mild temperatures (20-30 °C).

Rice (Oryza sativa L.) is one of the most important and widely grown staple crop. It represents a major part of the diet for more than a half of the world’s population. Due to its high carbohydrate content (mainly starch) it represents important source of energy, but it is also a valuable source of micronutrients, including minerals. It`s usually recommended as wheat alternative for persons who suffer from various allergies, intolerances and diseases such as celiac disease. Despite its high nutritional value, rice may contain undesirable levels of toxic elements, mainly As and Cd. In this study, aim was to determine the elemental composition of 58 rice samples (20 brown, 31 white and 7 parboiled) purchased from the Croatian market using ICP-MS method, and to investigate effects of processing type, cultivation method and the origin on element levels. Additionally, the consumers benefit and health risks from element exposure were quantified based on mean consumption of one serving of cooked rice [1]. Elements in rice were estimated in the order: P > K > Mg > Ca > Fe > Mn > Zn > Cu > Mo > As > Se > Cd > Co. Brown rice contained 2 to 4-fold higher levels of essential elements Mg, P, K, Fe, Mn, Co, Zn and Se compared to white rice. Mean toxic element level in all samples were 0.142 ± 0.057 mg/kg for As and 0.038 ± 0.035 mg/kg for Cd, with highest values observed in conventionally produced integral rice sample (As: 0.259 mg/kg ; Cd: 0.207 mg/kg). Only one sample exceeded the European Commission limit for Cd of 0.20 mg/kg. Rice originated from the European Union had higher As but lower Se than those from Asia. Among integral rice, non-organically grown rice contained more P, K, Mg, Fe and Zn compared to organic ones. Moreover, negative relationship (r=-0, 47, p=0.0002) between As and Se in rice was found. According to data obtained in this study, rice, especially integral one, may be considered as an important source of P, Mg and Cu. Observed low toxic element levels in all rice samples along with the latest recorded consumption rate of 2.7 kg rice per capita among Croatian population [2] indicate that rice is, with regard to toxic element, safe for consumption. References: [1] M. Rogić, R. Kovačević, B. Horvat, in Dietary reccomendations for people with diabetes, 11th ed., Zagreb, Tiskara Varteks, 2003. [2] Statistic reports for rice consumption per capita in Croatia, https://www.helgilibrary.com/indicators/rice-consumption-per-capita/croatia/ (accessed 21 April 2022).

This study aimed to investigate the effect of treatment with laurel (Laurus nobilis) essential oil (LEO) on the shelf life of fresh-cut potatoes (FCP). Peeled and sliced potatoes were dipped in the aqueous solution of different LEO concentrations (25, 125 and 250 mg/L) and water (control) for 15 minutes, then drained, vacuum packed and stored at 7 ᵒC for 14 days. The CIELAB color parameters and aerobic mesophilic bacteria count (AMBC) of the raw FCP and the sensory characteristics of the raw, cooked and fried samples were determined. With the increase of LEO concentration, AMBC decreased and, in general, this trend persisted during storage. Slight negative changes in color were observed with storage time. The characteristics of laurel flavor and odor in raw and cooked potatoes increased with the increase of LEO, while LEO aroma was not pronounced after frying. Despite the antibacterial activity of LEO, spoilage of FCP was not prevented during storage, as AMBC increased in all samples after 6 days of storage. However, in the samples treated with LEO, this AMBC increase was slightly pronounced in a dose-dependent manner. FCP treated with 125 and 250 mg/L LEO, after 6 days of storage, showed the best results in terms of absence of browning and off-odour of raw and off- taste and sour taste of cooked and fried FCP.

The effects of PEF technology on the content of anthocyanins (ANT), hydroxycinnamic acids (HCA) and flavonols (FL) in strawberry juices from the strawberry (Fragaria x ananassa Duch.) cultivar 'Albion' harvested at 75 % maturity (J1) and 100 % maturity (J2) and stored at +4 °C for 7 days were evaluated. The parameters of the PEF treatment were: (i) electric field strength (40 and 50 kV/cm), (ii) frequency (100 and 200 Hz), and (iii) treatment duration (3 and 6 min). All results were analyzed by multifactorial analysis of variance (p≤0.05). The untreated juice J2 had higher ANT, HCA and FL values than the untreated juice J1. After 7 days of storage at 4 °C, the control samples showed decreasing values of FL and increasing values of ANT and HCA. The treated juice samples of both maturity levels stored at 4 °C for seven days had significantly lower FL, while the values of ANT and HCA were significantly higher on the first day of storage. The electrical strength of 50 kV showed a statistically significant effect on the increase of ANT, HCA and FL content compared to the 40 kV strength. In contrast to the electrical strength, increasing the frequency from 100 Hz to 200 Hz resulted in a statistically significant decrease in ANT, HCA, and FL. Treatment duration did not show a statistically significant effect on ANT and HCA, with the exception of FL, where an increase in treatment duration resulted in a decrease in the levels of FL. PEF-treated J2 samples had significantly higher ANT, HCA and FL than J1. Regardless of maturity, storing treated samples at a temperature of 4 °C for seven days resulted in a statistically significant increase in ANT, HCA, and a decrease in FL. In general, it can be concluded that PEF technology positively affects the stability of ANT and HCA in strawberry juices during seven days of storage at 4 °C. Among the PEF process parameters, electric field strength and frequency had a statistically significant effect on the content of ANT, HCA and FL in the juices.

Lactic acid bacteria (LAB) produce a wide variety of exopolysaccharides (EPSs), high-molecular-mass carbohydrate polymers that are classified as homopolysaccharides or heteropolysaccharides, depending on the sugar composition. LAB-derived EPSs are used as texturizing agents in fermented dairy products. However, novel perspectives are emerging because of their potential functional properties such as protective effects on producer cells, immunomodulatory, anticancer or cholesterol-lowering effects in the host or prebiotic potential among the intestinal microbiota. Therefore, among the 100 bacterial strains isolated from human milk microbiota, we searched for EPS-producing LAB to further characterise their probiotic properties. Only MC1 strain, identified by 16S RNA sequencing as Limosilactobacillus fermentum, showed a ropy phenotype typical for EPS synthesis. Whole-genome analysis of the MC1 genome confirmed the identification, while functional annotation by RAST revealed the genes responsible for EPS production. EPSs were isolated from L. fermentum MC1 strain grown on MRS agar plates for 4 days at 30°C under anaerobic conditions, purified using affinity chromatography and subjected to 1D and 2D NMR spectroscopy for structural analysis. It was shown that MC1 strain produces EPSs that consist of two polysaccharides. The main polysaccharide is composed of -1, 6-linked galactofuranoses with α- glucopyranosyl branched on the second carbon of the main chain, while the determination of second polymer structure is still in progress. L. fermentum MC1 has shown ability to survive under simulated gastrointestinal tract (GIT) conditions. Adhesion of L. fermentum MC1 to the Caco-2 cell monolayer was confirmed by fluorescence microscopy, as well as the potential to reduce adhesion of Escherichia coli 3014 via competitive exclusion mechanism. Further studies will be directed towards the determination of EPS functions related to probiotic effects of MC1 strain.

The strawberry tree (Arbutus unedo L.) is one of the most common species with fleshy fruits in the Mediterranean region [1]. In local agricultural communities, the mealy and sweetish but not very aromatic fruits are mainly used for the production of jams, marmalades and alcoholic beverages. Also, the chemical composition indicates that the fruits are a good source of minerals and have high content of phenols and antioxidant capacity [2]. The aim of our work was to investigate the antioxidant and cytotoxic potential of A. unedo fruits on human squamous cell carcinoma cell line (CAL 27), human gastric adenocarcinoma cell line (AGS), colorectal adenocarcinoma cell line (Caco-2) and hepatocellular carcinoma cell line (HepG2). Furthermore, the DNA-protective effect and antimicrobial activity against Escherichia coli were investigated. A concentration range of 0.01-100 mg/mL was used in the experiments, with 1 mg/mL and 10 mg/mL being the recommended daily dose of polyphenols based on human blood volume and average human weight, respectively. Strawberry tree fruits at the recommended daily doses had a proliferative effect on AGS and CAL 27 cells without recovery and with 24-hour cell recovery, respectively. In CAL 27 cells, antioxidant activity was shown during 24-hour cell recovery. In Caco-2 and HepG2 cells, A. unedo fruit was shown to induce reactive oxygen species (ROS). The concentrations tested showed no antimicrobial activity against E. coli, but a dose-dependent protective effect on DNA was demonstrated. Additionally, further studies on individual bioactive compounds from strawberry tree fruit are needed to provide more detailed information on their potential use.

Grape brandies are one of the most popular alcoholic beverages in the world, and the most popular are Cognac and Armagnac. Besides these two brandies, which are subject to stricter regulations, grape brandy is produced all over the world, Spanish brandy from the Jeres region, South American brandy Pisco from Peru and Chile, German brandy called Weinbrand and so on.. According to the European Union legislation, brandy is a spirit drink (alcoholic beverage) produced from wine spirit and aged for at least six months in oak casks smaller than 1000 litres in capacity or twelve months with oak receptacles. Esters represents one of the most important group of volatile compounds influencing brandy aroma. During ageing, the content of esters increases as a consequence of the slow esterification of different organic acids with ethanol. The predominant esters in Cognac and Armagnac are isoamyl acetate, hexyl acetate, ethyl hexanoate, ethyl octanoate, ethyl decanoate, and ethyl laurate. Accordingly, the aim of this study was to investigate the effect of 3 L barrique oak barrel on the predominant esters during six months of ageing of Cabernet Sauvignon grape brandy. The esters were analysed by gas chromatography with a mass spectrometer (GC/MS). The highest concentration of investigated predominant esters during ageing is determined for ethyl octanoate (95.41 - 135.67 mg/L), followed by ethyl decanoate (51.61 - 106.86 mg/L) and ethyl hexanoate (38.99 - 104.39 mg/L). Together these three esters accounted for 78.36 - 82.55 % of investigated esters.

Research of the human gut microbiome has confirmed the presence of phylogenetically diverse bacterial species with health-promoting effects and potential of faecal microbiota transplantation as long-term engraftment of the patients gut microbiota. Microbiota present on mucosal and skin surfaces also impacting the interactions along the gut-liver-brain axis. According to the results of our research, selected and functionally characterized autochthonous strains of lactic acid bacteria (LAB), isolated either from the breast milk microbiota or artisanal fermented food, have a huge potential as a next-generation of probiotics [1]. They produce specific macromolecules such as surface S-layer proteins (Slps), bacteriocins, exopolysaccharides (EPS) and proteolytic enzymes, which are associated with their probiotic activities. Slps and EPS contribute to the stress-resistant properties of the producer cells during biotechnological production by freeze-drying as well as during gastrointestinal transit, and adherence in the colon, with potential immunomodulatory activity. Additionally, EPS are also prebiotic substrates for the gut microbiota – producers of short-chain fatty acids and lactate, metabolites with antibacterial and immunomodulatory activity, energy supply for the enterocytes, and the modulation of cholesterol and lipid metabolism. Bacteriocins, Slps and EPS promote competitive exclusion of pathogens, whereas bacteriocins and biopeptides-released upon the proteolytic activity, display antimicrobial activities. Biopeptides have also shown anti-inflammatory, ACE-inhibitory and antioxidant activities. Selected LAB strains have promising applications, ranging from the design of future functional food, food supplements or live biotherapeutic products. The impact of next-generation probiotics and prebiotics on human microbiota balance will be further defined through an advanced meta-omics approach and data analysis by new bioinformatics algorithms and analysis tools.