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Probiotic fermented milks are a group of functional food that, in addition to their nutritional value, contain certain components considered beneficial to human health. For their production, besides probiotic bacterial strains, yeasts - most commonly Saccharomyces boulardii (S. boulardii), are used too in the form of an secondary culture. The biotherapeutic effect of this yeast on the human body has been most studied. S. boulardii is also considered a potential natural preservative due to its antagonistic effect against other microorganisms. Its survivability and its effects on organoleptic properties have been studied in the production of yogurt from cow’s and goat’s milk, kefir, and acidophilic fermented milk. Based on all these factors, these products can be considered as new functional dairy products.

Introduction: Spontaneously fermented game meat sausages produced by traditional techniques are complex microbial ecosystems that can be the sources of beneficial or spoilage and pathogenic microorganisms. Aim: This study aimed to investigate the fate of indigenous microbiota of spontaneously fermented game meat sausages in order to estimate microbiological hazards as well to identify and preserve the microbial pool that could serve as a source of strains relevant for standardization of sausages production. Material and Methods: Three wild boar and three dear meat sausages, produced by five different manufacturers following traditional techniques, were physicochemically and microbiologically investigated at different time points of their production. Results: Total of 917 isolates were collected from different media used for cultivation of lactic acid bacteria (LAB) and identified to species and strain level by PCR tools. Although commonly used for the isolation of specific groups of LAB, most of the applied media exhibited poor selectivity and supported the growth of diverse LAB or even non-LAB species. In total, Leuconostoc mesenteroides was identified as the most frequently isolated species (28.24%), followed by Lactobacillus sakei (20.72%) and Enterococcus casseliflavus (11.55%), however, strong differences were observed in distribution profiles of LAB among investigated sausages. Cluster analysis revealed strong sausage-specific clustering of strains and remarkable diversity with 280 genotypes detected. High count of spoilage and/or pathogenic bacteria was noted for two sausages, making them potentially hazardous for human health. Conclusions: The vast differences between sausages in prevailing microbiota indicated the need for exploring the possibilities of product standardization, likely to select starter/bioprotective cultures from indigenous microbiota.

Sausages from red deer (Cervus elaphus) meat are manufactured at small scale in Croatia following traditional techniques. As such, these artisanal products have increased value due to their specific sensory properties and authenticity. However, factors such as anatomical shooting location, rifle i.e. munitions type, shooting – evisceration time and geographical location may influence the hygienic conditions of game meat, and consequently the hygienic conditions of game meat sausages. In order to evaluate their microbiological status, samples were collected from 3 different sausage producers (J1, J2 and J3) after 0, 4, 7, 10, 20 days and at the end of the ripening. Samples were screened for the presence of L. monocytogenes according to ISO standard. Pathogenic and spoilage bacteria, such as Staphylococcus aureus, Enterobacteriaceae, Bacillus cereus and E. coli were isolated and enumerated using BP, VRBG, MYP and CCA media, respectively. Yeasts and moulds were isolated and their number was determined using DRBC media while KAA media was used for enterococci. For further analysis and possible application as starter or bioprotective cultures, lactic acid bacteria (LAB) strains were isolated and collected using MRS media supplemented with cycloheximide. Collected LAB isolates were grouped using the rep – PCR method, after which representative strains were selected and identified using 16s rRNA gene sequencing. Our results showed that the number of LAB was low in the initial period (2, 39 log CFU / g), after that they proliferated and reached the maximum value after 7 days (8.3 log CFU / g) and their growth stabilized through the end of ripening (7.82 log CFU / g). Among 139 MRS isolates collected, Lactobacillus sakei was the most dominant species (47.48 %) followed by Lactobacillus curvatus.(14.38 %) and Leuconostoc mesenteroides (7.91 %). Enterococci were detected only in J1. Moulds were not detected while the growth trends of yeasts differed between J1, J2 and J3. The number of L. monocytogenes, E. coli, Staphylococcus aureus and Bacillus cereus was below the detection limit in ready-to-eat sausages. Enterobacteriaceae were not detected at the end stages of ripening in J1 and J3 while their number was high in J2 (4.52 log CFU/g). The difference in results obtained between the three deer meat sausages indicate the need for further molecular and microbiological characterization and moreover to explore the possibilities of product standardization.

Natural fermentation of game meat sausages has a long tradition in various countries for thousands of years, and nowadays still represents most commonly used practice for game meat preparation and preservation. Artisanal sausages prepared from wild boar meat are highly appreciated due to their authenticity and specific sensory qualities. However, there are certain concerns regarding the safety and hygienic conditions of ready-to-eat products. The current study aimed to explore the microbial diversity of traditionally produced wild boar sausages in order to estimate microbiological hazards as well as to obtain the collection of indigenous lactic acid bacteria (LAB) strains that could potentially be used as a starter and/or bioprotective cultures. Samples were collected from 3 different sausage producers (F1, F2, F3) after 0, 4, 7, 10, 20 days, and at the end of ripening. Total of 531 LAB isolates were collected using KAA, MRS, and MSE selective media and grouped using the rep – PCR method. After the cluster analysis, the representative members of each cluster group were identified based on 16S rRNA gene sequencing and based on PCR tools for the identification at the genus and species level. The prevalent isolates were identified as Leuconostoc mesenteroides (23.35%), followed by Lactobacillus sakei (18.08%) and Enterococcus casseliflavus (13.75%). Relevant strains were selected for further technological characterization. One out of three ready-to-eat sausages (F3) indicated safety risks to the consumers due to the high incidence of E.coli, with cell counts of 3.04 log CFU/g. This finding was supported by detected high numbers of B. cereus (3.86 log/CFU/g), Enterobacteriaceae (3.86 log CFU/g) and Staphylococcus aureus (3.15 log CFU/g). Spontaneously fermented wild boar sausages not only gave rise to safety concerns but also represent a valuable source of new presumptive starter strains.

Fermented foods produced by traditional techniques represent an important vehicle for live bacteria to human gastrointestinal tract (GIT). These bacteria may carry genes coding for antibiotic resistance which may be transferred to the other commensal or pathogen bacteria in GIT. It has been reported that indigenous bacteria found in artisan products may possess intrinsic multi-resistance to antibiotics, a trait that has evolved in bacteria before antibiotics were even applied in medicine or husbandry. Furthermore, bacteria possess a remarkable ability to develop new resistances (acquired resistance) through many different mechanisms. As the data for antibiotic resistances of bacteria and interspecies diversity of enterococci from traditional fermented sausages is scarce, one of the main aims of our study was to identify enterococci of traditional sausages produced from deer (Cervus elaphus) and wild boar (Sus scrofa) meat and to analyse their susceptibility to different clinically important antibiotics. Many studies have based the identification of enterococci with 16S rRNA gen-specific primers. However, due to the fact that non-human sources may be atypical and usually do not meet the criteria used for standard characterization we have used the superoxide dismutase gene (sodA) for interspecies differentiation of enterococci. To test their antimicrobial susceptibility, agar disc diffusion method against 6 antibiotics was applied. By sodA PCR approach, we were able to confirm and to determine Enterococcus faecalis, E. faecium, E. durans, E. casseliflavus and E. gallinarum isolated on kanamycine esculine azid agar (KAA) from game meat sausages during the fermentation and ripening (0, 4, 7, 10, 20, 40 days). From total of 280 isolates, the resistance towards tetracycline 30 µg with 4 %, erythromycin 15 µg with 2.5 % and vancomycin 5 with 3 % was observed, as well as intermediate resistance for erythromycin 15 µg (57 %), chloramphenicol 30 µg (5 %) and tetracycline 30µg (2 %). In order to detect genes and where they are located (plasmid or chromosome), further study is necessary. Identification of enterococci on species level by multiplex PCR method with primers for unique sodA gene was a valuable tool for reliable identification of indigenous enterococci from game meat sausages.

Tradicionalne kobasice od divljači proizvode se u Hrvatskoj u domaćoj radinosti i prema tradicionalnoj recepturi, bez dodatka starter kultura. Takvi visoko vrijedni proizvodi odlikuju se svojom autentičnošću i senzornim svojstvima. Poznato je da fermentirana hrana predstavlja način prijenosa živih bakterija u ljudskom gastrointestinalnom (GI) traktu. Bakterije unesene hranom mogu imati gene koji kodiraju za antibiotičku rezistenciju, koje potom mogu, najčešće horizontalnim transferom gena, prenijeti na ostale bakterije, uključujući patogene vrste. Zbog toga je od iznimne važnosti detaljno karakterizirati autohtonu mikrobiotu tradicionalnih kobasica proizvedenih od mesa jelena (Cervus elaphus) i divlje svinje (Sus scofa) kao i utvrditi obrazac njihove antibiotičke rezistencije. Osim nasljedne rezistencije, gram-pozitivne i gram- negativne bakterije posjeduju izuzetnu sposobnost stjecanja rezistencije na antibiotike različitim mehanizmima. Budući da podatci o antibiotičkoj rezistenciji bakterija izoliranih iz tradicionalnih fermentiranih kobasica nedostaju, cilj ovog istraživanja bio je istražiti osjetljivost gram-pozitivnih i gram-negativnih bakterija, izoliranih iz tradicionalnih kobasica od divljači, na različite klinički važne antibiotike. Antibiotička rezistencija gram-negativnih bakterija (E. coli i Enterobacteriaceae) istražena je prema 9 antibiotika, a gram- pozitivnih bakterija (Enterococcus spp.) prema 6, metodom difuzije diska u agaru. Svi izolati bakterije E. coli (51 izolat) bili su osjetljivi na sve testirane antibiotike. Bakterije roda Enterobacteriaceae (186 izolata), pokazale su rezistentnost na ampicilin (20 %), cefoksitin (7 %) i ampicilin/sulbaktam (1 %). Kod bakterija roda Enterococcus (280 izolata), utvrđena je rezistentnost na tetraciklin (4 %), eritromicin (2, 5 %) i vankomicin (3 %) te srednja osjetljivost na eritromicin (57 %), kloramfenikol (5 %) i tetraciklin (2 %).

Several studies have indicated that intensive tillage has notable effect on properties of the soil microbiota that may influence numerous important soils functions, e.g. mobilization of nutrients or change of the overall emission rates of greenhouse gases. Therefore, the aim of our study was to investigate dynamic of microbial communities in soil planted with soybean under different tillage systems. Moreover, abundance of populations harboring the nitrous- oxide reductase gene (nosZ) as an indicator for potential shift s in N2O emission rates was studied. The study was established at chernozem soil of Northern Baranja region in Republic of Croatia as completely randomized block design of four replicate plots for each tillage system in three years experiment. The soil was managed as followed: CT - conventional tillage (moldboard ploughing at 25-30 cm depth), DH - multiple discs harrowing (10-15 cm depth), and NT – no-tillage system. Soil samples were collected in summer and autumn in year 2003. Our results suggested that the reduction of tillage had no effects on the bacterial community structure. This might be a result of the very dry climatic conditions at the investigated site and /or a result of plant species effect (soybean). Slight effects of the tillage management became visible at least when samples were taken in autumn for microbes harboring the N2O reductase gene, indicating that there might be shift s in denitrification pattern in response to changes in tillage practice.

Spontano fermentirane trajne kobasice, koje se proizvode u domaćoj radinosti, prepoznati su i cijenjeni tradicionalni proizvodi. Odlikuju se specifičnim senzornim karakteristikama i izvor su vrijednih hranjivih sastojaka. Međutim, različit izbor sirovine i dodataka te varijabilni uvjeti proizvodnje dovode do neujednačene mikrobiološke i senzorne kvalitete tradicionalnih kobasica. Ključnu ulogu u osiguravanju zdravstvene ispravnosti takvih proizvoda imaju prirodno prisutne, autohtone populacije bakterija mliječne kiseline (BMK). Selektirane i aplicirane kao starter kulture autohtone populacije BMK mogle bi smanjiti rizike uzorkovane mikrobiološkom kontaminacijom za vrijeme klanja, odnosno lova i tijekom prerade mesa te povoljno utjecati na organoleptičke karakteristike krajnjeg proizvoda. Stoga je cilj ovog rada pokazati specifičnosti bakterija mliječne kiseline i mogućnosti njihove aplikacije u fermentaciji tradicionalnih suhih kobasica proizvedenih od mesa domaćih ili divljih životinja.

Stalan rast populacije uzrokuje zabrinutost vezanu uz energetsku krizu, proizvodnju hrane, bolesti, globalno zagrijavanje i ekološka pitanja koja zahtijevaju održiva rješenja iz prirode. Cijanobakterije se nalaze u gotovo svim staništima na Zemlji i mogu se naći u okruženjima podložnim stresnim uvjetima, kao što su pustinjska tla, ledenjaci i topli izvori. Zbog svoje velike prilagodljivosti, svestranosti, karakterističnoj strukturi stanica i proizvodnji brojnih metabolita, danas su glavni predmet istraživanja u svrhu njihove primjene u biotehnologiji. S druge strane, cijanobakterije mogu ispuštati niz cijanotoksina različitih struktura i učinaka na zdravlje ljudi i životinja. Stoga ovaj rad predstavlja pregled literatura koje opisuju upotrebu i njihov potencijal u poljoprivrednoj proizvodnji, zaštiti okoliša, prehrambenoj, farmaceutskoj industriji te u biorafineriji. U ovom radu su opisani i najvažniji rodovi za svaku pojedinu granu biotehnologije. The constant growth of the population causes concerns related to the energy crisis, food production, diseases, global warming and environmental issues that require sustainable solutions from nature. Cyanobacteria are located in almost all Earth’s habitats and can be found in stress-prone environments such as desert soils, glaciers and hot springs. Due to their high adaptability, ubiquity, the characteristic structure of cells and the production of numerous metabolites, they are now the main subject of research for their application to biotechnologies. On the other hand, cyanobacteria may release a range of cyanotoxins of different structures and effects on human and animal health. Therefore, this thesis presents an overview of literature that describes the use and their potential in agricultural production, environmental protection, food production, pharmaceutical industry and biorefinery. This thesis also describes the most important genera for each branch of biotechnology.

Curcuma longa L. (porodica Zingiberaceae) i njen polifenolski spoj kurkumin ima raznovrsne antimikrobne utjecaje, a upotreba mu je u svakodnevnom životu sve opširnija i češća. Razultati antibakterijskog djelovanja brojnih provedenih istraživanja pokazuju kako kurkuma ima obećavajuće rezultate pri djelovanju na različite patogene bakterije (MRSA, L. monocytogenes, Salmonella), viruse (HIV, Influenza) i gljive (C. neoformans, C. albicans, F. solani). Kurkuma ima poprilično širok raspon farmakoloških svojstava u relativno netoksičnim dozama. Kao što je dokazano brojnim istraživanjima, kurkumin djeluje jače kada je u kombinaciji sa nekim drugim već postojećim ljekovima u slučaju bolesti uzrokovanih bakterijama, virusima ili gljivama te u kombinaciji sa fungicidima u slučaju fitopatogenih bolesti biljaka. Kurkumin je prirodni proizvod sa velikim ljekovitim značajkama koji će zbog snažnog sinergistički učinka upotpuniti djelovanje antibiotika. Curcuma longa L. (family Zingiberaceae) and its polyphenolic compound curcumin have various antimicrobial effects, and its use in everyday life is becoming more extensive and more frequent. The results of the antibacterial activity of numerous studies show that turmeric has promising results in its action on various pathogenic bacteria (MRSA, L. monocytogenes, Salmonella), viruses (HIV, Influenza) and fungi (C. neoformans, C. albicans, F. solani). Turmeric has a fairly wide range of pharmacological properties in relatively non-toxic doses. As proven in many scientific researches, curcumin works better combined with some other already existing medicines in case of disease caused by bacteria, viruses or fungi, and in combination with fungicides in case of phytopathogenic disease of plants. Curcumin is a natural product with great medicinal properties that will complement the action of antibiotics due to its strong synergistic effect.

U ovom radu sakupljena su i interpretirana istraživanja vezana uz primjenu ne-Saccharomyces kvasaca u modernom vinarstvu i vinogradarstvu. Tijekom 20. stoljeća smatrano je da negativne karakteristike ovih kvasaca prevladavaju nad njihovom korisnošću, ali su se iscrpnim istraživanjima utvrdila i njihova izrazito pozitivna i komplementarna svojstva sa inače korištenim Saccharomyces kvascima u proizvodnji vina. Tržište i konzumacije vina usmjeravaju ka novim trendovima u proizvodnji vina sa kompleksnijim aromatskim profilima te smanjenim količinama alkohola. Upotrebom ne-Saccharomyces kvasaca postiže se i povratak izvornom mikrobiomu vinograda i podruma kao i izbjegavanje primjene različitih sintetičkih pripravaka za poboljšanje tijeka fermentacije, a upravo njihov iznimni potencijal je nešto što je jedna od središnjih tema istraživanja enologa današnjeg vremena. In this paper, gathered and interpreted is the research related to the application of non-Saccharomyces yeasts in modern winemaking and viticulture. During the 20th century, the negative characteristics of these yeasts were considered to outweigh their usefulness, but with the development of technology, the perspective also changed. In order to satisfy the increasingly demanding desires of consumers, the production of wines that have a more complex aromatic profile and lower amounts of alcohol has proven to be one of the ways to go. With the use of non-Saccharomyces yeasts the return to the original microbiome of vineyards and cellars is being accomplished and at the same time the use of various synthetic preparations to improve the course of fermentation is being avoided. It is their exceptional potential that is one of the central topics of research by oenologists today.

Zelena biotehnologija bavi se uporabom ekološki prihvatljivih rješenja kao alternativa tradicionalnoj poljoprivredi te na taj način nastoji smanjiti ovisnost poljoprivrede o mehaničkim i kemijskim inovacijama. Primjer zelene biotehnologije je proizvodnja transgenih biljaka uz pomoć mikroorganizama kao što su to agrobakterije. Rod Agrobacterium obuhvaća Gram-negativne mikroorganizme koji su poznati po svojoj patogenosti. Naime, vrste ovoga roda posjeduju Ti-plazmid uz pomoć kojeg stvaraju tumore vrata korijena ili Ri plazmid koji uzrokuje pojačani rast korijenovih dlačica. Infekcija nastaje preko ozljede biljke, nakon čega dolazi do transformacije biljnih stanica u tumorne stanice koje se dalje samostalno razmnožavaju. Svrha infekcije je da agrobakterija kontrolira mehanizme domaćina za sintezu nekih esencijalnih metabolita za vlastiti rast. Jedinstvenost roda Agrobacterium leži u mogućnosti genetske transformacije domaćina prenošenjem dobro definiranog segmenta DNA iz Ti-plazmida u genom stanice domaćina. Regija T-DNA je mobilni element koji je odgovoran za stvaranje tumora i biosintezu opina u biljci. Kako bi postali pogodni za uporabu u biotehnologiji, nekada korišteni sojevi Agrobacterium trebali su biti konstruirani od odabranih sojeva divljeg tipa. Neke prirodne značajke Ti-plazmida morale su se u potpunosti ukloniti (npr. geni odgovorni za stvaranje tumora), dok su karakteristike nekih komponenata mehanizama za transformaciju morale biti poboljšane. Uz poboljšane karakteristike Ti-plazmida vrste roda Agrobacterium postale su pogodne za izradu vektora, čime se mogu dobiti transgene biljke koje su rezistentne na štetočine, tolerantne na herbicide, te imaju poboljšani nutritivni sastav. Green biotechnology deals with the use of environmentally-friendly solutions as an alternative to traditional agriculture and in that way seeks to reduce the dependence of agriculture on mechanical and chemical innovations. An example of green biotechnology is the production of transgenic plants with the help of microorganisms such as agrobacteria. The genus Agrobacterium includes Gram-negative microorganisms that are known for their pathogenicity. Moreover, species of this genus possess Ti-plasmid which causes crown gall or Ri-plasmid which causes increased growth of root hairs. Infection occurs through injury to the plant, followed by the transformation of plant cells into tumor cells that further proliferate independently. The purpose of infection is for the agrobacterium to control host mechanisms for the synthesis of some essential metabolites for its own growth. Uniqueness of the genus Agrobacterium lies in the possibility of genetic transformation of the host by transferring a well-defined segment of DNA from the Ti-plasmid into the genome of the host cell. The T-DNA region is the mobile element responsible for tumor formation and opin biosynthesis in the plant. To become suitable for use in biotechnology, once-used Agrobacterium strains had to be constructed from selected wild-type strains. Some natural features of Ti-plasmids had to be completely removed (e.g. genes responsible for tumor formation), while the characteristics of some components of the transformation mechanisms had to be improved. With the improved characteristics of Ti-plasmids species of the genus Agrobacterium have become suitable for vector construction, which can provide transgenic plants that are resistant to pests, tolerant to herbicides, and have improved nutritional composition.

Bakterija Deinococcus radiodurans spada u skupinu organizama koji su se prilagodili životu u ekstremnim uvjetima. Izuzetno je otporna na razne faktore koji uzrokuju oštećenja molekule DNA poput UV zračenja, ionizacijskog zračenja, isušivanja te izlaganja kemijskim mutagenim spojevima. Ono što najviše plijeni pažnju kod bakterije D. radiodurans je njena otpornost na visoke doze ionizacijskog zračenja, točnije x-zraka i gama zraka; može preživjeti dozu zračenja od 20000 Gy, što je 3000 puta veća doza od one koja se smatra letalnom za čovjeka. Najteži tip oštećenja genoma nakon zračenja predstavljaju dvolančani lomovi DNA. Oni se u bakteriji D. radiodurans popravljaju posebnim mehanizmom koji se bazira na homolognoj rekombinaciji te uključuje opsežnu sintezu DNA. Ključnu ulogu u ovom popravku ima protein RecA. U bakterijama mutiranima u genu recA odvija se RecA-neovisni popravak koji je, međutim, slabo učinkovit, neprecizan i dovodi do velikih genomskih rearanžmana. Cilj ovog rada bio je istražiti učinak mutacije recG na popravak DNA i genomsku nestabilnost recA mutanata bakterije D. radiodurans. Ispitali smo preživljenje mutanata recA i recA recG nakon UV i gama zračenja, a metodom elektroforeze u promjenjivom električnom polju analizirali smo DNA stanica koje su preživjele 2 kGy gama zračenja. Dobiveni rezultati pokazuju da su mutanti recA recG podjednako osjetljivi na zračenje kao i mutanti recA, no za razliku od njih, ne stvaraju velike genomske rearanžmane. Bacterium Deinococcus radiodurans belongs to the group of organisms adapted to the life in extreme conditions. It is extremely resistant to various agents that cause damage to DNA molecules such as UV radiation, ionizing radiation, desiccation and exposure to various mutagenic compounds. The most interesting feature of D. radiodurans is its resistance to high doses of ionizing radiation, namely x-rays and gamma rays; it can survive the dose of 20,000 Gy, which is 3,000 fold higher than the dose that is lethal for a human. The most severe form of genomic damage after radiation is double-strand DNA breaks. In D. radiodurans double-strand DNA breaks are repaired by a special mechanism that is based on homologous recombination, and which involves extensive DNA synthesis. The key role in this mechanism is played by the RecA protein. In D. radiodurans cells mutated in the recA gene, a RecA-independent repair takes place, which is, however, less efficient, inaccurate and leads to gross genome rearrangements. The aim of this work was to investigate the effect of a recG mutation on DNA repair and genomic instability of D. radiodurans recA mutants. We examined survival after UV and gamma radiation in recA and recA recG mutants and analyzed their DNA by pulsed-field gel electrophoresis. Our results showed that the recA recG mutants are as sensitive to radiation as the recA mutants, but do not produce gross genome rearrangements.