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In addition to being a rich source of nutrients, autochthonous fermented foods are a reservoir of beneficial lactic acid bacteria (LAB) starter cultures and potential probiotics. The bacteriocin-based antibacterial capacity of the autochthonous LAB is investigated as a strategy for biopreservation. Therefore, the aim here was to screen the antibacterial activity in a selected group of Lactiplantibacillus plantarum strains belonging to different food microbiomes, autochthonous cheese or sauerkraut. Since Listeria monocytogenes and Staphylococcus aureus are frequently detected as foodborne pathogens, they have been applied as test microorganisms to demonstrate antibacterial activities. All tested L. plantarum strains exhibited antilisterial and antistaphylococcal activities when grown on a solid medium, and when the cell-free supernatant (CFS) was analysed, where D13 and SF15C showed the greatest inhibition. Antibacterial activity in broth cultures was re-established or enhanced by coculturing with sensitive bacterial strains S. aureus 3048 and L. monocytogenes ATCC®19111™ which can be associated with the induction of bacteriocin production typically present among LAB. Additionally, the proteinase nature of CFSs has been confirmed as another feature of plantaricins. PCR using total DNA from L. plantarum strains resulted in the amplification of plantaricin-related genes plnA, plnEF and plnJ of expected amplicon size, suggesting that the genome could harbour a pln gene clusters. Whole genome sequencing (WGS) revealed the existence of five plantaricin specific operons regulated by a three- component regulatory system, involved in a quorum sensing (QS) mechanism, of L. plantarum D13 and SF15C strain, with high similarity to L. plantarum WCFS1. The 3D structures of PlnEF and PlnJK, were predicted using AlphaFold Protein Structure Database, while physicochemical properties and amino acid sequence of α-helixes of Pln were determined by HeliQuest analysis server. Taken together, these findings contribute to the characterisation of plantaricins of L. plantarum D13 and SF15C, and their implementation as either a functional starter cultures or even probiotics. Their application could improve the quality of fermented foods, which could be achieved by controlling contamination by L. monocytogenes, which would extend the shelf-life of the final product.

Foods for special medical purposes (FSMPs) are unique therapeutic products formulated for the specific dietary management of disease-related conditions or specific medical disorders, for which distinctive nutritional requirements, based on recognized evidence-based scientific principles, are established. The human microbiome is composed of microbes that have a tremendous potential to impact our physiology, both in health and in disease. Since high-fiber diet impacts the entire gut microbe community and improves blood- glucose regulation, they can be used as FSMPs, in which they pose a beneficial effect in maintaining the gastrointestinal health.Therefore the aim of this research was to establish the lactogenic and bifidogenic effects of three different innovative fibers (K1, D1, D2), in order to develop innovative formulations of foods for diabetic medical purposes, using a diabetic mouse model. Standard diabetic fiber mixures and food without added fibers, were used as controls. All tested innovative dietary fibers showed in vivo lactogenic and in vitro bifidogenic and lactogenic effect, where the best effects were achieved with the addition of D2 innovative fiber mixture, respectively. Additionally, after feeding the mice, all innovative dietary fibers showed a positive impact on the growth of Lactococcus spp., with the best results upon the addition of D2 innovative fiber mixture. Innovative fiber mixture (D1) reduced the number of L. monocytogenes to undetectable cell count level, significantly decreased the number of S. aureus and Salmonella spp., while innovative fiber mixture (D2) significantly decreased the number of L. monocytogenes and S. aureus, in diabetic mice faeces samples in vivo. The reduction of L. monocytogenes was also achieved with innovative (K1) and standard (K0) fiber mixtures, to undetectable cell count level. Finally, innovative fiber mixtures (D1 and D2), along with the standard fiber mixtures (K0) showed the impact on the incresement of the total bacteria number in vivo. In conclusion, two innovative mixtures of dietary fibers, D1 and D2, can be used as nutritional supplementation for foods for diabetic medical purposes.

Uočeno je da neurohormoni i upale primarno posreduju u komunikaciji između crijeva i mozga, između središnjeg, autonomnog i crijevnog živčanog sustava. Crijevna mikrobiota utječe na funkciju mozga putem imunoloških, neuralnih, endokrinih i metaboličkih putova. Disregulacija ove osovine crijevne mikrobiote i mozga može uzrokovati različite kronične bolesti kao što su depresija, demencija i Parkinsonova bolest. Poremećaj sastava crijevne mikrobiote, najčešće uzrokovan antibiotičkom terapijom, izostankom dojenja, infekcijama i drugim bolestima te stresom, može utjecati na fiziologiju i ponašanje čovjeka. Određene bakterijske vrste bakterija kao lijekova, koje je američka FDA nazvala "živim bioterapijskim pripravcima" (LBPs), mogu imati pozitivan učinak na raspoloženje i depresiju, pri čemu se živac vagus identificira kao glavni komunikacijski put između crijevnih bakterija i mozga. Terapijski učinak bakterija kao lijekova zasniva se na imunomodulacijskom djelovanju i utjecaju njihovih bioaktivnih staničnih dijelova i metabolita kao što su kratkolančane masne kiseline, površinski S- proteini, egzopolisaharidi, bakteriocini i bioaktivni peptidi nastali djelovanjem proteolitičkih mikrobnih enzima, što intenzivno istražujemo na našim znanstvenim projektima, financiranim od strane HRZZ-a. Prvu terapiju bakterijama iz uzoraka stolice odobrila je US FDA krajem 2022. godine, za liječenje ponavljajućih Clostridioides difficile infekcija.

Mikrobna biomasa, nazvana humana mikrobiota, koja kolonizira površinu kože i sluznice ljudskoga tijela, ima temeljnu ulogu u ljudskom zdravlju i bolesti. Najviše je proučavana crijevna mikrobiota te je prepoznata kao jedan od glavnih ciljeva u terapiji različitih bolesti, uključujući metaboličke poremećaje, neurološke, autoimune i upalne bolesti crijeva. Analiza sastava crijevne mikrobiote temelji se na analizi mikrobioma – svih gena mikroorganizama prisutnih u crijevima. Već je prepoznata važnost filogenetičke raznolikosti crijevne mikrobiote na svim taksonomskim razinama, ali je važna i distribucija bakterijskih odjeljaka (phyla). Crijevna mikrobiota također se smatra epigenetičkim regulatorom fiziologije i imunosti domaćina kroz proizvodnju metabolita s pozitivnim utjecajem na zdravlje, kao što su kratkolančane masne kiseline (octena, propionska i maslačna kiselina). Ti se mikrobni metaboliti uglavnom proizvode od neprobavljivih polisaharida iz dijetalnih vlakana unesenih prehranom. Naime, prehrana ima najvažniji utjecaj na sastav, raznolikost i obilje crijevne mikrobiote, dok genetika i okolišni čimbenici imaju manji utjecaj. Terapija mikrobioma uključuje transplantaciju fekalne mikrobiote, kao i primjenu probiotika, prebiotika i njihove kombinacije – sinbiotika. Lijekovi za mikrobiotu vrlo će se vjerojatno razvijati kroz personalizirani terapijski pristup – kombinirajući lijekove i prehrambene smjernice, uz preporuku o zdravom načinu života.

Inovativni funkcionalni napitak proizveden je na bazi sirutke izdvojene nakon proizvodnje sušenog svježeg sira s probiotičkim bakterijama mliječne kiseline (BMK) specifičnih tehno-funkcionalnih svojstava značajnih za fermentaciju i funkcionalnost finalnog proizvoda. Fermentacija je provedena primjenom Levilactobacillus brevis ZG1 koji eksprimira S-protein, Lactiplantibacillus plantarum ZG1C koji sintetizira plantaricin, Limosilactobacillus fermentum D12 koji proizvodi egzopolisaharide (EPS), te Enterococcus faecium ZGZA7-10 s proteolitičkim djelovanjem. Nakon proizvodnje i tijekom skladištenja određen je značajno visok broj živih bakterijskih stanica dodanih BMK u uzorcima tekuće ili koncentrirane sirutke priređene liofilizacijom. Relativna zastupljenost dodatnih BMK u mikrobnoj populaciji sirutke ustanovljena je i primjenom tehnika nove generacije sekvencioniranja. Inovativni napitak na bazi sirutke je blago kiselih svojstava s niskim sadržajima masti i laktoze. Analize spregnutim tehnikama tekućinske kromatografije i spektrometrije masa potvrdile su prisutnost raznovrsnih peptida iz kazeina, pretpostavlja se uslijed proteazne aktivnosti primjenjene mješovite kulture BMK. Prema senzorskoj analizi, dodatkom arome vanilije i maline postignuta su poboljšana senzorska svojstva inovativnog napitka na bazi sirutke, ali i u formulaciji koncentrirane sirutke. Proizvedeni inovativni napitci u tekućoj i liofiliziranoj formi bogat su izvor nutrijenata, a dodana funkcionalna vrijednost postignuta je primjenom probiotičkih BMK s metaboličkim svojstvima sinteze bakteriocina, S-proteina i bioaktivnih peptida, odnosno EPS. Primjenom odabranih BMK moguće je utjecati i na održivost procesa, odnosno postići ekološki i ekonomski učinak uslijed iskorištavanja sirutke iz nusproizvoda u proizvod mliječne industrije.

Increased awareness about the importance of nutrition in health promotion and disease prevention has driven the development of nutraceuticals. In this research, probiotic strain Lacticaseibacillus casei 431® was incorporated into the food for special medical purposes to develop an innovative nutraceutical product. Therefore, the aim was to investigate the influence of the target food matrix on the specific probiotic properties of the strain in vitro. Both cultures, the pure lyophilized and the one isolated from food for special medical purposes, efficiently survived the adverse conditions in simulated gastrointestinal conditions, without significant effect of the food matrix. The observed effect of matrix on bile salts deconjugation activity was minimal, however, cholesterol assimilation was increased by 16.4%. Lb. casei 431® exhibited antibacterial activity against Gram-positive bacteria as revealed by the agar spot-test method, which was not impacted by the food matrix. Contrariwise, bacterial cells culture from food matrix showed a significantly higher inhibitory effect on test-microorganisms determined by the agar diffusion method. The result of the turbidimetric method indicated that the food for special medical purposes enhanced the antimicrobial effect of Lb. casei 431® probably due to the stimulation of metabolic activity and production of antimicrobial metabolites in the presence of matrix. The autoaggregation capacity of L. casei 431® cells was not affected by the food matrix. Furthermore, in adhesion 144 assays, the adherence of the bacterial cells of 431® to the extracellular matrix proteins, fibronectin, collagen and laminin found in intestinal tissues, was reduced after treatment with proteinase K where the highest adhesion to laminin was observed. Adhesion of Lb. casei 431® decreased the binding of E. coli 3014 by 1.81 log units and the binding of S. Typhimurium FP1 by 1.85 log units in model Caco-2 cell lines suggesting the potential of the pathogen competitive exclusion.

Food for Special Medical Purposes (FSMPs) is a new class of therapeutic products, specifically formulated for the dietary management of diseases, disorders or medical conditions of patients. Moreover, recent research pointed out the importance of the gut microbiota on host physiology, which composition can be highly influenced by dietary fiber intake. Therefore, the aim of this study was to examine the lactogenic and bifidogenic effects of different dietary fibers as nutritional supplementation of FSMPs in order to develop new innovative formulations by using mice faeces as a model system. All tested dietary fibers showed a lactogenic effect, which differed depending on dietary fiber applied. The largest increase in bacterial counts expressed as log CFU/mL was determined in the presence of soluble fibers, marked as SF 1, SF2 and SF3, as well as combination of soluble and insoluble fibers marked as CSIF 1 and insoluble fibers namely as IF 1. The highest bifidogenic effect was achieved by dietary fibers of soluble fibers SF 2 and combination of soluble and insoluble fibers CSIF 1. Tested mixtures of dietary fibers showed both lactogenic and bifidogenic effects, resulting out of the synergistic effect of component fibers, which differed from the lactogenic and bifidogenic effects achieved by individual dietary fibers. Further studies will be directed towards the determination of prebiotic effects on faecal microbiota of mice in vivo of dietary fiber mixtures created for different formulations of FSMPs.

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.

Otkriće da naše zdravlje ovisi o mikrobima prisutnim u probavnom traktu smatra se najvažnijim medicinskim otkrićem u zadnjih 100 godina, čak važnijim od probijanja šifre ljudskog genoma. Sprega mikrobiote, crijeva i mozga znači da crijevna mikrobiota i njeni metaboliti imaju ogroman utjecaj na rad mozga. Također je zanimljiva spoznaja da se raspoloženje može promijeniti iz depresije do dobrog raspoloženja, s promjenama u sastavu intestinalne mikrobiote. Terapijsko djelovanje probiotičkih bakterija i njihov utjecaj na crijevnu mikrobiotu, kao “živih bioterapijskih pripravaka” (eng. LBP prema US FDA) ili “farmabiotika” (prema industriji u EU) može biti: - zasnovano na djelovanju bioaktivnih mikrobnih metabolita na crijevnu mikrobiotu i zdravlje u cjelini, poput kratkolančanih masnih kiselina, površinskih S-proteina, egzopolisaharida, antimikrobnih peptida – bakteriocina i bioaktivnih peptida nastalih djelovanjem proteolitičkih mikrobnih enzima, što intenzivno istražujemo na našim znanstvenim projektima, financiranim od strane HRZZ-a. - mogu biti dizajnirani kao nosači za cjepiva, imunoglobuline i druge tzv. terapijske proteine. - ili su genetički modificirani tako da budu učinkoviti za terapiju različitih bolesti prema kojima dobivaju i specifične nazive: psihobiotici, onkobiotici, diabiotici, kolitobiotici i skinbiotici.

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.

Bakterije mliječne kiseline (BMK) su glavne zvijezde fermentiranih mliječnih proizvoda. Prepoznate su kao modulatori crijevne mikrobiote s pozitivnim utjecajem u prevenciji i liječenju mnogih metaboličkih poremećaja i kroničnih bolesti. Istraživanjima koja se provode na našim nacionalnim znanstvenim projektima, „Potencijalne terapijske biomolekule druge generacije probiotika“ i “Probiotici i starter kulture – površinski proteini i bakteriocini”, koje financira Hrvatska zaklada za znanost (HRZZ), ciljevi su odabir i funkcionalna karakterizacija probiotičkih BMK, iz fermentiranih proizvoda i majčinog mlijeka, sa specifičnim svojstvima proizvodnje potencijalnih terapijskih biomolekula kao što su egzopolisaharidi (EPS), površinski (S)- proteini, bakteriocini, bioaktivni peptidi u fermentiranim mliječnim proizvodima i funkcionalni metaboliti koji nastaju u gastrointestinalnom traktu (GIT-u) nakon njihove primjene. Funkcionalna i tehnološka svojstva površinskih S- proteina iz Levilactobacillus brevis sojeva i EPS- a iz Limosilactobacillus fermentum sojeva su prema rezultatima naših istraživanja: povećano preživljavanje tijekom gastrointestinalnog tranzita, kompetitivna ekskluzija patogena, dulje vrijeme zadržavanja u debelom crijevu domaćina i imunomodulacijsko djelovanje. Dodatno, EPS služe kao prebiotički supstrati za crijevnu mikrobiotu koja proizvodi kratkolančane masne kiseline i mliječnu kiselinu s antibakterijskim i imunomodulacijskim djelovanjem, te služe kao izvor energije intestinalnim epitelnim stanicama i pozitivno utječu na metabolizam kolesterola i lipida. Sojevi BMK s bakteriocinskom aktivnošću, potvrđenom nakon sekvencioniranja cijelih genoma i provedene anotacije njihovih gena, specifično inhibiraju bakterijske kontaminante mlijeka Staphylococcus aureus i Listeria monocytogenes. Združnom primjenom sojeva, producenta S-proteina Levilactobacillus brevis SF9B i producenta bakteriocina Lactiplantibacillus plantarum SF9C, postignuta je značajna promjena sastava crijevne mikrobiote štakora korištenih u eksperimentalnom modelu namijenjenom istraživanju Alzheimerove bolesti. Bioaktivni peptidi su detektirani nakon proteolitičke razgradnje mliječnih proteina s ispitivanim sojevima BMK, a kao bioaktivne molekule pokazuju antioksidacijsku, imunomodulacijsku, antitrombotičku, antimikrobnu i inhibicijsku aktivnost prema angiotenzin- konvertirajućem enzimu (ACE) što ima učinak snižavanja krvnog tlaka. Rezultati mnogih znanstvenih istraživanja upućuju na pozitivan učinak bioaktivnih peptida iz fermentiranih mliječnih proizvoda na probavni, endokrini, kardiovaskularni, imunosni i živčani sustav, što predstavlja veliki izazov za mljekarsku industriju. Naime, analitičari predviđaju vrijednost tržišta proizvoda s bioaktivnim peptidima i proteinima, bilo kao funkcionalne hrane, dodataka prehrani ili farmaceutskih lijekova, u ukupnom iznosu većem od 78, 6 milijardi EUR do 2027. godine.

Exopolysaccharides (EPS) are extracellular cell structures tightly bound to the cell envelope or loosely excreted in the microenviroment. They consist of linear or branched repeating units of sugars, but may also contain non-carbohydrate substituents. Depending on sugar composition, EPSs are classified as homo-EPS (HoEPS), made of one repeated monosaccharide unit, and hetero-EPS (HeEPS), made of more than one monosaccharide unit. Among microbial polysaccharides, EPS produced by generally regarded as safe (GRAS) lactic acid bacteria (LAB) have various applications. Most commonly, their role is recognized in improving the texture of the fermented food products. Also, their importance as functional molecules has been recently recognized due to the capacity of bifidogenic, and even antitumor, antiulcer, immunomodulating or cholesterol-lowering effects in vivo. The main challenge for large-scale industrial production is increasing the yield of synthesis. The total yield of EPS depends on several factors including producer strain, medium composition, growth conditions such as temperature, pH and incubation time. Therefore, the aim of this research is a structural and functional analysis of the EPS produced by MC1 strain isolated from mother´s milk. EPS-producer showing characteristic „ropy phenotype“ was identified as Limosilactobacillus fermentum MC1 by 16S RNA sequencing. Whole genome sequencing of the MC1 genome following functional annotation by RAST enabled the detection of the eps cluster for EPS biosynthesis. 1H NMR and 2D NMR analysis showed that EPS isolated from L. fermentum MC1 is composed of two structurally different polysaccharides – the main polysaccharide is composed of repeating units of 1, 6 linked galactofuranose with glucose branches on C2 of the galactose residue, while the determination of the structure of the second polysaccharide is still ongoing. L. fermentum MC1 has shown the ability to survive under simulated gastrointestinal tract conditions, as well as the potential to reduce adhesion of Escherichia coli 3014 via competitive exclusion mechanism. Fluorescence microscopy confirmed the adhesion of L. fermentum MC1 to the Caco-2 cell line. Moreover, addition of EPS further increased adhesion to Caco-2 cell line in vitro. Further experiments will be focused on the determination of the role of EPS related to effects of MC1 strain within the probiotic concept.

This study aimed to select the Lactobacillus strains, from human milk microbiota, with specific properties of synthesis of exopolysaccharides (EPS), S-proteins and bacteriocins with the potential to be characterised as therapeutic biomolecules. Detection of the „ropy“ phenotype was used for screening of EPS producers, while bacteriocinogenic activity was analysed by the determination of antagonistic activities in vitro. S-proteins of Lactobacillus brevis strains MB1, MB2, MB13 and MB20, were detected by SDS-PAGE analysis of GHCl extracted surfaceome and further analysed by LC-MS/MS using database search by Mascot. For the identification of the genes of interest, responsible for the expression of targeted biomolecules, PCR with specific primers was applied. 1H-NMR analysis confirmed that Lactobacillus fermentum MC1 strain produces EPS. PCR method with eps primers confirmed the genes of the eps cluster. Dendrograms of SDS-PAGE electrophoretic profiles, obtained using GelCompar II, showed characteristic bands of approximately 45 kDa in L. brevis strains identified by mass spectrometry. The spectrum of Lactobacillus strains showed bacteriocinogenic activity, however, the presence of plnJ, plnA and plnEF genes involved in the bacteriocin biosynthetic pathway was confirmed in the genome of six Lactobacillus plantarum strains. The comparative genomic analysis provided insights into the clusters related to the expression of potential biomolecules. The analysis of breast milk microbiota revealed the presence of Lactobacillus strains with the potential to express specific biomolecules. Further investigations will be directed towards deciphering mechanisms of probiotic actions triggered by these biomolecules in order to develop next-generation probiotics.

Brojni okolišni čimbenici utječu na sastav crijevne mikrobiote, a promjena u njenom sastavu, tzv. disbioza, povezana je s raznim poremećajima. Ta saznanja su dovela do povećane potražnje za probioticima, koji su definirani kao živi mikroorganizmi koji, primjenjeni u odgovarajućoj količini, imaju pozitivan učinak na zdravlje domaćina. Najnovija istraživanja usmjerena su na terapijsku primjenu probiotika kao “živih bioterapijskih pripravaka”, a odnose se na bakterije koje će liječiti ili spriječiti simptome bolesti. Pokazalo se da specifični ekstracelularni proizvodi metabolizma i određene komponente stanične ovojnice sudjeluju u interakcijama s mikrookolišem te da su uključene u probiotičko djelovanje bakterijskih sojeva producenata. Često se kao probiotici koriste bakerije mliječne kiseline (BMK) koje su u visokim koncentracijama zastupljene u fermentiranim prehrambenim proizvodima, ali je njihova prisutnost zabilježena i u majčinom mlijeku. Stoga je provedena funkcionalna karakterizacija probiotičkih sojeva BMK, izoliranih iz majčinog mlijeka, sa specifičnim svojstvima proizvodnje potencijalnih terapijskih biomolekula kao što su egzopolisaharidi (EPS) i S-proteini. Posjedovanje karakterističnog “ropy phenotype”-a pokazalo je da soj L. fermentum MC1 proizvodi EPS-e, te je, primjenom NMR spektroskopije, određena struktura glavnog polisaharida. Prisutnost S-proteina kod 4 L. brevis soja potvrđena je SDS-PAGE elektroforezom i LC-MS analizom.

Infant’s intestinal microbiome is believed to be highly influenced by their own mother’s breast milk microbiota. Mother’s milk is a rich source of nutrients, bioactive components, but also a diversity of microorganisms that are important for the health of a newborn. The focus of our research was screening of the Lactobacillus strains that rarely, but preferably, express S-proteins. Among over 100 strains, we have identified S-proteins solely in the 4 strains of Levilactobacillus spp. This is the first report that describes S-proteins of Levilactobacillus brevis strains isolated from mother’s milk. Four L. brevis strains, MB1, MB2, MB13 and MB20, were identified by whole genome sequencing. Gene annotation confirmed the presence of S-layer proteins, which were further identified by SDS-PAGE coupled to LC-MS/MS. The examined L. brevis strains possess S-proteins of 40-50 kDa. Further characterization revealed that S-proteins protect bacterial cells against adverse environmental stresses, such as those in gastrointestinal tract (GIT) or during freeze- drying, but also interact during cell adhesion to intestinal surfaces. This was observed through increased survival of the L. brevis strains in simulated GIT conditions, increased adhesion to Caco-2 cell line and stronger inhibition of adhesion of pathogenic bacterium Escherichia coli 3014 to Caco-2 cell line, as mentioned properties were negatively affected after the removal of S- layer proteins from the cell surface. Results imply that S-proteins mediate functional properties of the producer strains. Overall, S- layer proteins of investigated L. brevis strains have the potential to act as key mediators for their functionalities.

Ciljanom selekcijom bakterija mliječne kiseline odabrani su sojevi koji sintetiziraju bakteriocine i egzopolisaharide, te eksprimiraju proteaze za akumulaciju biopeptida. Istraživački cilj je strukturna i funkcionalna karakterizacija ovih biomolekula kao postbiotika. Producent plantaricina PlnJK i PlnEF, Lactiplantibacillus plantarum D13, iskazuje antilisterijsko i antistafilokokno djelovanje, a u genomu sadrži pln klaster. Limosilactobacillus fermentum D12, u genomu sadrži eps klaster i sintetizira glikogen i heteropolisaharid koji sadrži t-Glcp i 2, 6-vezanu Galf. EPS-i posreduju u probiotičkim svojstvima soja D12 te imaju zaštitne funcije. Enterococcus faecium ZGZA7-10 ima kazeinolitičko djelovanje. In vitro funkcionalnost konzorcija BMK dokazana je kroz kolonizacijski potencijal i učinak na modulaciju sastava intestinalnog mikrobioma in vivo. Uspostavljeni su i pristupi za dostavu probiotičkih bakterija in situ u obliku mikroformulacija te proizvodnja oplemenjenih fermentiranih proizvoda, optimiranjem sastava mješovite mikrobne starter kulture. Primjena konzorcija BMK doprinjela je proizvodnji funkcionalnog proizvoda i inovativnog napitka na bazi sirutke s povećanim sadržajem biopeptida. Zaključno, plantaricin D13, odnosno EPS D12, imaju potencijal probiotičkih molekula nove generacije te uz biokatalitički potencijal sustava proteaza obećavajući su postbiotici.

The awareness of the importance of the intestinal microbiota for host health has spurred efforts to develop strategies for its modulation during functional dysbiosis. Namely, the disruptions of the intestinal microbiome are linked not only with gastrointestinal but also extraintestinal disorders. Growing evidence suggests that intestinal microbiota biomodulators may again establish normal intestinal microbiome composition. Biomodulators, in addition to the almost conventional use of probiotics and prebiotics, also include new therapies proposed as the application of postbiotics, next-generation probiotics, and even faecal microbiota transplantation of a healthy donor. Though probiotic strains have been extensively investigated, the mechanistic basis of their health-promoting effects remains unclear. It is the surface-exposed or secreted cellular compounds that are the key molecules that interface bacteria to their environment while providing various functions. Therefore, the aim here is to present specific probiotic strains expressing trigger molecules that show the potential to act as postbiotics. In particular, case-studies related to the structural and/or functional characterization of S-layer proteins and exopolysaccharides released in the microenvironment will be presented. In addition, enzyme activities encompassed within the proteolytic system responsible for the accumulation of biopeptides in casein-rich sources will be discussed. Finally, the aim is to comment on the challenges in discovering the specific biomolecules, bacteriocins as another potential postbiotic. At first, the approaches in the screening and identification of novel lactic acid bacteria as probiotics and their target trigger biomolecules will be shown as well as evaluation of their importance in cell-to-cell or cell-to- host interactions. This implies their biological role in the functional properties of probiotic strains, specifically protective roles after exposure to stress conditions, immunomodulatory activity, antimicrobial activity, and adhesion. A metagenomics approach was applied to assess the capacity of probiotics to modulate the intestinal microbiome composition in vivo. Expanding knowledge on probiotics and trigger molecules thereof will induce further research related to both the improvement of bioprocess and new applications of probiotics as promising living drugs.

Additional file 6: Table S1. Genes of L. fermentum D12 involved in exopolysaccharides (EPSs) production and in the activation of the precursor molecules.

Majčino mlijeko sadrži mikrobiotu koja pozitivno utječe na uspostavljanje stabilne mikrobiote dojenčeta. Mikrobiota majčinog mlijeka sadrži bakterije iz obitelji Streptococcaceae, Pseudomonadaceae, Staphylococcaceae, Lactobacillaceae i Oxalobacteraceae. Posebno su značajni sojevi roda Lactobacillus koji posjeduju (gliko)proteine površinskog sloja (S-proteine) koji dodatno povećavaju probiotički kapacitet. Podjedinice S-proteina povezane su međusobno i s površinom stanice, nekovalentnim vezama. Veličina S-proteina je od 25 do 71 kDa. S-proteini sadrže dvije funkcionalne domene – vezujuću, odgovornu za vezanje proteina na staničnu stijenku, te kristalizacijsku, koja posreduje u združivanju S-proteina, odnosno u S-protein-S-protein interakcijama. Stoga je proveden odabir te genotipska i fenotipska identifikacija bakterija mliječne kiseline iz majčinog mlijeka koje proizvode S-proteine. Identificirana su 4 soja bakterije Levilactobacillus brevis koji na svojoj površini imaju S-proteine. Preliminarna istraživanja, unutar probiotičkog koncepta, funkcionalnih i tehnoloških svojstava izoliranih sojeva koje proizvode S-proteine su pokazala pozitivnu ulogu S-proteina u agregacijskim svojstvima bakterije i inhibiciji adhezije patogene bakterije Escherichia coli 3014 na Caco-2 staničnu liniju mehanizmom kompetitivne ekskluzije. Provedena istraživanja su pokazala bolje preživljavanje ovih sojeva u simuliranim uvjetima gastrointestinalnog trakta.

Mother's milk contains a diverse range of lactic acid bacteria (LAB) which have an emerging role in the establishment of the gut neonatal microbiota and have a huge potential as a next-generation of probiotics. Microbiomes analyses by QIIME2 bioinformatics platform, shown correlation between faeces microbiota composition of infant and their mother's milk. Unique autochthonous strains of LAB, isolated from the microbiota of mothers' milk, produce specific metabolites with potential therapeutic properties, such as surface S-layer proteins (Slps), bacteriocins and exopolysaccharides (EPS) which have an impact on their activity as probiotics in food supplements and as live biotherapeutics. According to our research results, Slps and EPS enhance survival of probiotics during biotechnological production by freeze-drying and gastrointestinal transit, prolong residence time in the colon of the host with potential immunomodulatory activity. Additionally, EPS are prebiotic supstrates for the gut microbiota - producers of short-chain fatty acids and lactate, metabolites with antibacterial activity, immune system modulation, enegry supply for the intestinal epithelial cells, and the modulation of cholesterol and lipid metabolism. Bacteriocins, as well as Spls and EPS, promote competitive exclusion od pathogens, whereas bacteriocins display antimicrobial activity. The huge potential of next-generation of probiotics as live biotherapeutics and food supplements is recognised for re-establishing of disturbed balance of intestinal microbiota, with potential therapeutic effect on obesity, mood, metabolic disorders, autoimmune diseases and female urogenital infections. Therefore, pharmaceutical and food companies founded large microbiome research centres for the development of next-generation of probiotics with predicted microbiota medicines market of over 500 million EUR by the year of 2030.

Breast milk not only provides a perfect balance of nutrients to meet all the needs of the infant in the first months of life, but also contains a variety of bacteria that play a key role in tailoring the neonatal gut microbiome. To investigate the impact of breast milk on the composition of the infant faecal microbiome, DNA was extracted from samples of breast milk (n=15) and infant faeces (n=15) collected from 5 mother-infant pairs at 3 different time points. The V1-V3 region of the 16S rRNA gene was amplified, and amplicon sequencing was performed using the Illumina MiSeq platform. Analysis of the raw sequencing data using the QIIME 2 platform revealed the unique composition of each breast milk and infant feaces microbiome and confirmed the correlation of their composition in each mother-infant pair. Firmicutes (64.28%) and Proteobacteria were the most predominant phyla in breast milk microbiome, whereas Firmicutes (32.92%) and Actinobacteria (31.86%) dominated the faecal microbiome. The analysis confirmed that the composition of the breast milk microbiome changes during lactation, as does the faecal microbiome during infant growth and development. α-diversity analysis revealed that the breast milk microbiota is more diverse, i.e. species-rich, than the infant faecal microbiota.

U svrhu odabira potencijalnih probiotika nove generacije, iz prikupljenih uzoraka majčinog mlijeka izolirano je 100 bakterijskih sojeva. Elektroforezom RAPD-PCR produkata, dobiveni su jedinstveni genetički profili svakog bakterijskog soja, koji su hijerarhijskom klaster analizom svrstani u 5 dendrograma. Analizom dendrograma, odabrano je 28 genetički različitih bakterijskih sojeva koji su fenotipski okarakterizirani nizom metoda i identificirani sekvenciranjem 16S rRNA gena. Usporedbom rezultata sekvenciranja s rezultatima hijerarhijske klaster analize, ustanovljeno je da 46 % sojeva pripada rodu Enterococcus, 27 % Staphylococcus, 22 % Lactobacillus i 5 % Streptococcus. SDS-PAGE analizom je utvrđeno da čak 4 Levilactobacillus brevis sojeva (MB1, MB2, MB13 i MB20) sadrže površinske (engl. Surface, S) proteine, po prvi put identificirane u sojevima izoliranim iz majčinog mlijeka. PCR analizom je ustanovljeno da 6 Lactiplantibacillus plantarum sojeva sadrže gene koji kodiraju za bakteriocine (plnJ, plnA i plnEF). Ispitivanjem antimikrobne aktivnosti, preživljavanja u nepovoljnim uvjetima te agregacije i adhezije na epitelne i subepitelne strukture intestinalnog trakta, ustanovljeno je da najveći probiotički potencijal među sojevima izoliranim iz majčinog mlijeka, imaju producenti potencijalnih terapijskih biomolekula S-proteina i bakteriocina.

Additional file 2: Table S1. Genes of Lb. plantarum SF9C involved in plantaricin production and their known or putative biochemical functions.

Biosinteza egzopolisaharida (EPOL) detektirana je pomoću specifičnog „ropy“ fenotipa bakterijskih kolonija Lactobacillus fermentum D12, autohtone bakterije mliječne kiseline izolirane iz svježeg dimljenog sira, poraslih na MRS agar hranjivoj podlozi obogaćenoj ugljikohidratima. Probiotička starter kultura Lb. fermentum D12 je identificirana pristupom potpunog sekvencioniranja genoma, te je genomska sekvenca porhranjena u NCBI računalnoj bazi podataka pod GenBank pristupnim brojem RHMA00000000. Kako se biosinteza EPOL može potaknuti dodatkom ugljikohidrata u suvišku, soj Lb. fermentum D12 inokuliran je u MRS hranjive podloge uz dodatak slijedećih izvora ugljika: glukoza, galaktoza, laktoza, fruktoza i saharoza. Najveći prinos EPOL-a od 200, 25 mg/mL, ostvaren je nakon uzgoja Lb. fermentum D12 u MRS hranjivoj podlozi uz dodatak glukoze kao indukcijskog faktora, što potvrđuje da soj D12 učinkovit mikroorganizam producent EPOL-a. 1H-NMR analiza je pokazala da odabir šećera dodanog u hranjivu podlogu ne utječe na konačnu kemijsku strukturu proizvedenog EPOL-a. Primjenom visokoučinkovite kromatografije HPSEC (eng. High Performance Size Exclusion Chromatography) ustanovljeno je da Lb. fermentum D12 proizvodi tri različita tipa EPOL-a koji se međusobno razlikuju u molekulskoj masi ; jedan je molekulske mase 400 kDa, i dva niskih molekulskih masa od 2 kDa. 1H-NMR, GC i GC-MS analiza pročišćenih EPOL-a pokazala je da soj Lb. ferementum D12 sintetizira jedan homopolisaharid, te dva različita heteropolisaharida. 2D-NMR analizom je ustanovljeno da je homopolisaharid molekulske mase od 400 kDa sastavljen od ponavljajućih jedinica α-1, 4 vezane glukoze, pri čemu je 20 % molekula šećera acetilirano na C-3 atomu. Budući eksperimenti će biti usmjereni s ciljem karakterizacije primarne strukture EPOL-a nižih molekulskih masa i potencijalnoj funkcionalnoj ulozi pojedinih EPOL-a u probiotičkoj aktivnosti soja producenta Lb. ferementum D12, posebice u adheziji na humane intestinalne epitelne Caco-2 stanice, kompetitivnoj ekskluziji patogena i potencijalnom imunomodulacijskom učinku.

Oksidativni stres (engl. xidative stress, OS) jest primarni uzrok patogeneze upale, djelomičnih ishemija, metaboličkih i neurodegenerativnih bolesti. Poremećaju homeostatskih procesa u mozgu doprinosi i crijevni mikrobiom. Os crijeva-mozak (engl. gut-brain axis, GBA) sastoji se od dvosmjernog komuniciranja između središnjeg i enteričnog živčanog sustava, povezujući emocionalne i kognitivne centre u mozgu s perifernom funkcijom crijeva. Mehanizmi komunikacije su brojni i uključuju neuralne, humoralne i upalne putove, ovisno o domaćinu i okolišnim čimbenicima. Na ovaj kompleksni simbiotski međuodnos između probavnog sustava, crijevnog mikrobioma i mozga domaćina snažno utječe prehrana ; optimalan odnos između prehrane i crijevnog mikrobioma može biti koristan za probavu hrane, unos hranjivih tvari, te zdravlje imunosnog i živčanog sustava. Temeljem navedenog cilj rada jest istražiti strukturne i funkcionalne promjene crijevnog mikrobioma u štakorskom modelu Alzheimerove bolesti (engl. Alzheimer disease, AD) ; točnije utvrditi da li je neravnoteža i promjena mikrobioma crijeva povezana s AD i da li primjenom potencijalno funkcionalne hrane možemo modulirati crijevnu mikrobiotu te smanjiti posljedice OS koje vode neurodegenerativnim bolestima. Istraživanje je provedeno na muškom spolu visokorodnih štakora Y59, starih 3 mjeseca, koji su podijeljeni u 6 skupina (zdrava kontrolna skupina HC - 0, 9% NaCl ; AD - AlCl3 10 mg/kg i D-gal 60 mg/kg ; AD + kvercetin 25 mg/kg (Qu25) ; AD + kvercetin 50 mg/kg (Qu50) ; Qu25 ; Qu50) svakodnevno obrađenih tijekom 28 dan intraperitonealnim (ip) injeciranjem navedenog. Sekvenciranjem V4 varijabline regije 16S rRNA dobivena je sveobuhvatna slika crijevnog mikrobioma. Statistički najznačajnije promjene (P≤0, 01) vidljive su u porodica čija je zastupljenost u rasponu 0, 1-1%, te u porodica čija je zastupljenost 1-10% (P≤0, 05). Dominantne porodice ne pokazuju statistički značajne (P≥0, 05) promjene. Daljnje analize su u tijeku. Istraživanje je financirano iz projekta: HRZZ- IP- 2014-09-9730 ; Analiza crijevnog mikrobioma u štakorskom modelu Alzheimerove bolesti (2017., br. projekta: 20282313) ; Molekularni mehanizmi kafeinske kiseline na angiogenezu i supresiju rasta Ehrlichovog ascitesnog tumora (2016., br. projekt: 20281220).

Zbirka autohtonih bakterija mliječne kiseline (BMK) Laboratorija za tehnologiju antibiotika, enzima, probiotika i starter kultura, Prehrambeno-biotehnološkog fakulteta Sveučilišta u Zagrebu, sadrži više od 200 sojeva BMK. Većina je bakterijskih sojeva izolirana iz tradicionalno proizvedenih fermentiranih mliječnih proizvoda, identificirana 16S rDNA sekvencioniranjem i selekcionirana na temelju strogih izbornih probiotičkih kriterija. Istraživanja koja se provode na nacionalnom znanstvenom projektu “Probiotici i starter kulture – površinski proteini i bakteriocini”, kojeg financira Hrvatska zaklada za znanost (HRZZ), imaju za cilj odabir i karakterizaciju „nove generacije probiotika“, bakterijskih sojeva sa specifičnim svojstvima, kao što su proizvodnja površinskih S-proteina, peptida s antibakterijskom aktivnošću - bakteriocina, egzopolisaharida i proteolitičkih enzima. Odabrani autohtoni sojevi BMK su okarakterizirani fiziološkim testovima, analizom sekvencioniranih genoma, te LC-MS/MS i NMR analizama, u svrhu definiranja njihovog specifičnog metabolizamskog potencijala. Prema dobivenim rezultatima, tradicionalno proizvedeni fermentirani mliječni proizvodi su bogat izvor korisnih bakterija mliječne kiseline koje mogu svojom specifičnom metabolizamskom aktivnošću pozitivno utjecati na raznolikost i sastav crijevne mikrobiote što je prepoznato kao preduvjet održavanja intestinalnog trakta zdravim. Velik potencijal probiotika kao “živih lijekova” prepoznat je u terapiji disbioze i ponovnom uspostavljanju narušene ravnoteže crijevne mikrobiote, koja je “zlatni cilj” terapije i “ključni senzor” okolišnih čimbenika koji uzrokuju metabolizamske poremećaje i kronične bolesti kod ljudi. Osim toga, promjene u sastavu intestinalne mikrobiote mogu utjecati na raspoloženje, a mogu uzrokovati i promjenu u fenotipu, od pretilosti do standardne tjelesne mase. Stoga prehrambene i farmaceutske tvrtke osnivaju istraživačke centre mikrobioma za ravoj probiotika kao lijekova koje US FDA naziva „živi bioterapijski pripravci“ (engl. „live biotherapeutic products (LBPs)“), a europska industrija „farmabiotici“ (engl. „pharmabiotics“). Analitičari predviđaju vrijednost tržišta „živih bioterapijskih pripravaka“ veću od 500 milijuna EUR do 2030. godine.

Tradicionalno proizvedeni sirevi su bogat izvor autohtonih sojeva bakterija mliječne kiseline (BMK) sa specifičnim metabolizamskim svojstvima, kao što su proizvodnja S-proteina, bakteriocina, egzopolisaharida i proteolitičkih enzima. Stoga su izolirani sojevi BMK odabrani prema strogo definiranom probiotičkim kriterijima, a njihova identifikacija je provedena 16S rDNA sekvencioniranjem i sekvencioniranjem cijelog genoma. S-proteini okarakterizirani su SDS-PAGE i LC-MS/MS analizama. Geni za proizvodnju bakteriocina su identificirani primjenom PCR-a (eng. Polymerase Chain Reaction) sa specifičnim početnicama, dok je bakteriocinska aktivnost ispitana metodom dvostrukog sloja i združenim uzgojem sa Staphylococcus aureus 3048 i Listeria monocytogenes ATCC19111. Soj Lactobacillus fermentum D12 proizvodi egzopolisaharide, a selekcioniran je na temelju specifičnog fenotipa „formiranja dugih rastezljivih niti“ (tzv. „ropy“ fenotip) poraslih kolonija na čvrstoj hranjivoj podlozi obogaćenoj ugljikohidratima. Sastav i struktura tri proizvedena egzopolisaharida određeni su GC, GC/MS, 1H-NMR i 2D-NMR analizama. Proteolitička aktivnost ispitana je Ansonovom, Tricine-SDS-PAGE i LC-MS/MS metodama, dok su geni za proteolitičke enzime detektirani primjenom PCR-a sa specifičnim početnicama. Na temelju provedenih istraživanja, odabrana su četiri soja BMK kao autohtone starter kulture za proizvodnju dimljenog sira ; Lactococcus lactis ZG7-10, Lactobacillus brevis D6, Lactobacillus plantarum D13 i Lactobacillus fermentum D12.

Aminoglikozidni antibiotici se upotrebljavaju u terapiji bolesti, kao aditivi stočnoj hrani, ili kao promotori rasta kako bi se poboljšalo iskorištavanje hrane za životinje te poticao rast životinja. Dio apliciranih antibiotika se akumulira unutar različitih tkiva, a dio se izlučuje kroz urin, izmet, mlijeko i jaja tretiranih životinja, bilo kao nemetabolizirani ishodišni spojevi, ili kao njihovi metaboliti. To može predstavljati stvarnu prijetnju potrošaču, kroz izloženost ostacima antibiotika, a moguć je razvoj i prijenos antibiotičke rezistencije ili pojava alergija i drugih zdravstvenih poremećaja. Negativni aspekti uporabe antibiotika kod životinja potvrđuju važnost kontinuiranog monitoringa ostataka antibiotika, kao i razvoja učinkovitih analitičkih tehnika za utvrđivanje njihove bioakumulacije u tkivima i precizne koncentracije ostataka antibiotika u proizvodima životinjskog podrijetla namijenjenih ljudskoj prehrani.

The aim of this study was to assess the probiotic potential of two Lactobacillus strains naturally occurring as a co-culture in the spontaneous fermented Croatian white cabbage cultivar Varaždinski. The automatic RAST annotation of their whole genome sequences obtained by Illumina MiSeq platform, and tblastn v.2.2.27 comparison of the assembled contigs with the sequences deposited in NCBI, revealed the presence of bacteriocin- encoding genes in the genome of L. plantarum SF9C and surface layer protein (Slp) genes in the genome of Lactobacillus brevis SF9B. Slp may act as an adhesin and contribute to the antimicrobial activity against the pathogens with whom the carrying strain competes for the same adhesion sites. The ability of SF9B cells to compete and exclude Gram-negative pathogens Escherichia coli 3014 and Salmonella enterica serovar Typhimurium FP1 using Caco-2 cell line was hereby confirmed. Moreover, the antimicrobial activity of L. plantarum SF9C against Gram- positive pathogens L. monocytogenes ATCC 19111 and S. aureus 3048 was verified using agar- spot, well diffusion and co- cultivation assay. Moreover, the combined probiotic effect of oral administration of the two Lactobacillus strains on fecal microbiota composition of rats was established.

Aminoglikozidni antibiotici se primjenjuju u kontroli bolesti, kao aditivi u hrani, ili sintetski promotori rasta u stočarstvu. Imaju širok antibakterijski spektar djelovanja, a najveću učinkovitost pokazuju u liječenju infekcija uzrokovanih aerobnim Gram-negativnim bakterijama. Ostaci aminoglikozidna mogu imati negativne posljedice na zdravlje potrošača kada njihove koncentracije prelaze definirane granice tolerancije. Uredbom Komisije (EU) 37/2010 definirane su najveće dopuštene količine (NDK) aminoglikozida u mlijeku. Svrha ovog rada je istraživanje koncentracija aminoglikozida u sirovom mlijeku primjenom tekućinske kromatografije ultravisoke djelotvornosti i višestruke spektrometrije masa (UHPLC-MS/MS). Metoda je validirana prema Odluci Komisije 2002/657/EC te su utvrđene granične koncentracije određivanja pojedinih aminoglikozida (CCα ; µg/kg): streptomicin 219, 5, dihidrostreptomicin 218, 4, spektinomicin 223, 2 ; kanamicin 158, 8 ; gentamicin 105, 1 ; neomicin 1591, 8. Utvrđena su iskorištenja u rasponu od 99, 0 do 102, 5 %. Granice kvantifikacije su se kretale od 2, 18 µg/kg za gentamicin do 351, 8 µg/kg za neomicin. Tijekom 2017. godine analizirano je ukupno 50 uzoraka sirovog kravljeg mlijeka na ostatke aminoglikozida. U jednom odnosno dva uzorka mlijeka utvrđene su koncentracije streptomicina odnosno dihidrostreptomicina ispod 100 µg/kg odnosno ispod NDK od 200 µg/kg. U jednom uzorku mlijeka određene su koncentracije dihidrostreptomicina iznad 300 µg/kg odnosno iznad NDK.

A rich collection of autochthonous lactic acid bacteria (LAB) was screened for the presence of S- layer proteins and production of bacteriocins. Analyses of bacterial surface proteins using SDS- PAGE, 2D-PAGE and PCR with specific primers for slp genes, discovered that only four Lactobacillus brevis strains express S-layer proteins, while using antibacterial activity assays and PCR with specific primers for genes encoding various bacteriocins, only three Lactobacillus plantarum strains were confirmed as bacteriocin producers. Biological functions of Lactobacillus S-layer proteins are still poorly understood, however, our investigations confirmed that they mediate bacterial adherence to intestinal epithelial cells and extracellular matrix proteins and also influence the immune response, which are important probiotic properties. When applied as mixed culture, S-layer protein carrying strain and bacteriocin producing strain made strong influence on microbiota composition of rats used as animal models in investigation of Alzheimer’s disease. Purified S-layer proteins of L. brevis SF9B strain, with MW of 50.9 kDa and pI of 9.54, were identified using LC/MS method, while the prediction of their secondary structure was generated using I-TASSER modelling. The fact that purified S-layers are stable toward non- physiological pH and that they protect the cell against various stress conditions, opens an interesting perspective in the development of vehicles for oral administration of drugs or vaccines, specially taking into account GRAS (Generally Regarded as Safe) status of LAB.

Cilj ovog rada je bio fenotipska i genotipska karakterizacija autohtone bakterije mliječne kiseline, Lactobacillus fermentum D12, koja proizvodi egzopolisaharide – funkcionalnu makromolekulu koja doprinosti probiotičkim svojstvima soja producenta. Proizvodnja egzopolisaharida soja L. fermentum D12 dodatno je potaknuta visokim koncentracijama glukoze, fruktoze, galaktoze, saharoze ili laktoze u hranjivoj podlozi i temperaturom uzgoja pri 30 °C, te je postignut prinos veći od 40 mg egzopolisaharida po litri hranjive podloge. Nakon izolacije i pročišćavanja, 1H-NMR analizom utvrđeno je da vrsta dodanog šećera u hranjivu podlogu ne utječe na kemijsku strukturu egzopolisaharida. Gel kromatografijom i plinskom kromatografijom je ustanovljeno da L. fermentum D12 proizvodi 3 tipa egzopolisaharida, a 1H-NMR analizom je dokazano da se radi o jednom homopolisaharidu i dva heterpolisaharida. sastava manoza, glukoza i galaktoza u omjeru 3, 3:2, 1:1. Dodatno je ispitana proteolitička aktivnost Ansonovom metodom i analizom razgradnje kazeina Tricine SDS-PAGE metodom, zbog potencijalne primjene ovog soja kao probiotičke starter kulture u proizvodnji sireva. S ciljem poboljšanja preživljavanja soja D12 tijekom proizvodnje suhog aktivnog probiotičkog pripravka, provedena je mikroinkapsulacija u alginatu uz dodatak različitih prebiotika. Sušenje mikroinkapsuliranih stanica soja D12 je provedeno liofilizacijom, uz dodatak obranog mlijeka kao najboljeg lioprotektivnog sredstva za preživljavanje stresnih uvjeta liofilizacije što je potvrđeno određivanjem broja živih bakterijskih stanica u mikroinkapsuliranim liofiliziranim pripravcima soja D12 neposredno nakon proizvodnje, te nakon 1, 2 i 3 mjeseca čuvanja. Kumulativni protektivni učinak obranog mlijeka i procesa mikroinkapsulacije je također dokazan tijekom izlaganja soja D12 simuliranim uvjetima gastrointestinalnog trakta.

Surface (S)-layer proteins (Slps) form crystalline arrays of protein subunits as the outermost component of the cell wall in only few strains of Lactobacillus genera. In the genome of Lactobacillus brevis SF9B strain, three regions showed high homology with following slp sequences of the reference strain L. brevis ATCC 14869: with SlpB and SlpC in contig 2 (percentage of identities 65% and 89%, respectively) and with SlpD in contig 32 (percentage of identities 99%). Using SDS-PAGE of GHCl-extracted surface proteins of SF9B, a 50 kDa protein band, indicating the expression of the presumed Slp, was detected. 2D- PAGE of the same protein sample revealed a spot with isoelectric point and molecular weight of approximately 10, and 50 kDa, respectively. Structure and function prediction of the isolated Slp was generated using I-TASSER server, based on the data available for L. brevis ATCC 14869. Since Slps are exposed at the cell surface of certain Lactobacillus strains, they represent an interface between the bacterium and its biotic and abiotic microenvironments. Considering the fact that these environments include stress conditions which can affect bacterial viability and activity in the intestinal tract, Slps must have great importance in probiotic properties of the bacterial cells which express them. Although Slps of Lactobacillus strains are of great research interest for number of years, their physiological function remained poorly understood. However, Slps of strain SF9B mediate bacterial adherence to mucin, Caco-2 cells and extracellular matrix proteins, increase survival of L. brevis SF9B cells in simulated gastrointestinal conditions and during freeze- drying, and enhance their aggregation and coaggregation ability, which are important probiotic properties. The above mentioned results indicate that Slps contribute to technological and probiotic properties of strain L. brevis SF9B and that they have great role in potential application of that strain as novel probiotic culture.