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Lanac hrane predstavlja jedan od mogućih puteva transmisije bakterija rezistentnih na antibiotike između populacija životinja i ljudi. Komensalne bakterije, poreklom od životinja, predstavljaju deo mikrobiote hrane životinjskog porekla, i često se prepoznaju kao „rezervoari“ gena za rezistenciju na antibiotike. Rezistencija na tetraciklin kod komensalnih bakterija je dobro dokumentovana i s obzirom da je prenosive prirode, koristi se kao model za praćenje ekologije rezistencije na antibiotike. Homoljski, zlatarski i sjenički sir su na nacionalnom nivou zaštićeni oznakom geografskog porekla. Proizvode se na tradicionalan način, od sirovog mleka, te bakterije mlečne kiseline, poreklom iz sirovine i/ili procesnog okruženja čine značajan deo mikrobiote tradicionalnih sireva Srbije. U naučnoj literaturi postoje brojni podaci o tome da bakterije mlečne kiseline mogu biti nosioci gena za rezistenciju na antibiotike. Ciljevi ove doktorske disertacije bili su: karakterizacija biodiverziteta tradicionalnih sireva Srbije, utvrđivanje fenotipa i prevalencije rezistencije na antibiotike i genetske baze rezistencije na tetraciklin kod izolata bakterija mlečne kiseline poreklom iz tradicionalnih sireva, kao i mogućnosti prenosa tet gena u uslovima in vitro. Ukupno 48 uzoraka tradicionalnih sireva (13 uzoraka homoljskog sira, 20 zlatarskog i 15 uzoraka sjeničkog sira), zasejano je na MRS-S agar radi utvrđivanja broja BMK i na MRS-S agar sa dodatkom tetraciklina u dvostrukorastućim koncentracijama (1-256 μg/ml), kako bi se utvrdila zastupljenost populacije BMK rezistentne na tetraciklin. Prosečan broj BMK u uzorcima homoljskog sira iznosio je 7,65±1,09 log CFU/g, u uzorcima zlatarskog sira 7,74±0,6 log CFU/g i u uzorcima sjeničkog sira 7,47±0,71 log CFU/g. Pri koncentraciji tetraciklina od 64 μg/ml primećeno je veće smanjenje broja BMK... Food chain may represent a possible route for the transmission of antibiotic resistant bacteria between human and animal populations. Commensal bacteria originating from food-producing animals can contaminate food and by that means become a part of the complex food microbiota. Commensal bacteria can also serve as reservoirs for the antibiotic resistance genes. Tetracycline resistance in commensal bacteria is well documented and is coded with transmissible genes. Therefore, it is recognized as a model for the monitoring of antibiotic resistance ecology. Homolj, Zlatar and Sjenica cheese have protected geographical indication on the national level. As Homolj, Zlatar and Sjenica cheese are raw milk cheeses produced in the traditional manner, LAB originating from the raw milk and the environment constitute an important part of Serbian traditional cheeses’ microbiota. There are many data in the scientific literature that LAB can carry transmissible antibiotic resistance genes. The aims of this doctoral dissertation were to: characterize biodiversity of traditional Serbian cheeses; determine phenotype and prevalence of antibiotic resistance; define genetic basis of tetracycline resistance in the LAB isolates and to investigate possibility for in vitro transmission of the tet genes. Total of 48 samples traditional Serbian cheeses (13 samples of Homolj cheese, 20 samples of Zlatar cheese and 15 samples of Sjenica cheese) were analyzed. All samples were plated on MRS-S agar plates in order to determine LAB count and on MRS-S agar plates with added tetracycline in doubling concentrations (1-256 μg/ml), in order to determine tetracycline resistant LAB subpopulation count. Average count of LAB in Homolj cheese was 7,65±1,09 log CFU/g, in Zlatar cheese 7,74±0,6 log CFU/g and in Sjenica cheese 7,47±0,71 log CFU/g. Addition of tetracycline in concentration of 64 μg/ml has considerably reduced LAB count, which was in Homolj cheese 3,98±2,28 log CFU/g, in Zlatar cheese 3,35±2,02 log CFU/g and in Sjenica cheese 3,93±2,24 log CFU/g...

Food chain may represent a possible route for the transmission of antibiotic resistant bacteria between human and animal populations. Commensal bacteria originating from food-producing animals can contaminate food and by that means become a part of the complex food microbiota. Commensal bacteria can also serve as reservoirs for the antibiotic resistance genes. Tetracycline resistance in commensal bacteria is well documented and is coded with transmissible genes. Therefore, it is recognized as a model for the monitoring of antibiotic resistance ecology. Homolj, Zlatar and Sjenica cheese have protected geographical indication on the national level. As Homolj, Zlatar and Sjenica cheese are raw milk cheeses produced in the traditional manner, LAB originating from the raw milk and the environment constitute an important part of Serbian traditional cheeses’ microbiota. There are many data in the scientific literature that LAB can carry transmissible antibiotic resistance genes. The aims of this doctoral dissertation were to: characterize biodiversity of traditional Serbian cheeses; determine phenotype and prevalence of antibiotic resistance; define genetic basis of tetracycline resistance in the LAB isolates and to investigate possibility for in vitro transmission of the tet genes. Total of 48 samples traditional Serbian cheeses (13 samples of Homolj cheese, 20 samples of Zlatar cheese and 15 samples of Sjenica cheese) were analyzed. All samples were plated on MRS-S agar plates in order to determine LAB count and on MRS-S agar plates with added tetracycline in doubling concentrations (1-256 μg/ml), in order to determine tetracycline resistant LAB subpopulation count. Average count of LAB in Homolj cheese was 7,65±1,09 log CFU/g, in Zlatar cheese 7,74±0,6 log CFU/g and in Sjenica cheese 7,47±0,71 log CFU/g. Addition of tetracycline in concentration of 64 μg/ml has considerably reduced LAB count, which was in Homolj cheese 3,98±2,28 log CFU/g, in Zlatar cheese 3,35±2,02 log CFU/g and in Sjenica cheese 3,93±2,24 log CFU/g... Lanac hrane predstavlja jedan od mogućih puteva transmisije bakterija rezistentnih na antibiotike između populacija životinja i ljudi. Komensalne bakterije, poreklom od životinja, predstavljaju deo mikrobiote hrane životinjskog porekla, i često se prepoznaju kao „rezervoari“ gena za rezistenciju na antibiotike. Rezistencija na tetraciklin kod komensalnih bakterija je dobro dokumentovana i s obzirom da je prenosive prirode, koristi se kao model za praćenje ekologije rezistencije na antibiotike. Homoljski, zlatarski i sjenički sir su na nacionalnom nivou zaštićeni oznakom geografskog porekla. Proizvode se na tradicionalan način, od sirovog mleka, te bakterije mlečne kiseline, poreklom iz sirovine i/ili procesnog okruženja čine značajan deo mikrobiote tradicionalnih sireva Srbije. U naučnoj literaturi postoje brojni podaci o tome da bakterije mlečne kiseline mogu biti nosioci gena za rezistenciju na antibiotike. Ciljevi ove doktorske disertacije bili su: karakterizacija biodiverziteta tradicionalnih sireva Srbije, utvrđivanje fenotipa i prevalencije rezistencije na antibiotike i genetske baze rezistencije na tetraciklin kod izolata bakterija mlečne kiseline poreklom iz tradicionalnih sireva, kao i mogućnosti prenosa tet gena u uslovima in vitro. Ukupno 48 uzoraka tradicionalnih sireva (13 uzoraka homoljskog sira, 20 zlatarskog i 15 uzoraka sjeničkog sira), zasejano je na MRS-S agar radi utvrđivanja broja BMK i na MRS-S agar sa dodatkom tetraciklina u dvostrukorastućim koncentracijama (1-256 μg/ml), kako bi se utvrdila zastupljenost populacije BMK rezistentne na tetraciklin. Prosečan broj BMK u uzorcima homoljskog sira iznosio je 7,65±1,09 log CFU/g, u uzorcima zlatarskog sira 7,74±0,6 log CFU/g i u uzorcima sjeničkog sira 7,47±0,71 log CFU/g. Pri koncentraciji tetraciklina od 64 μg/ml primećeno je veće smanjenje broja BMK...

Lactic acid bacteria (LAB) are widely used in the food industry, especially in the pro­duction of fermented dairy products and meat. The most studied species among LAB is Lactococcus lactis. L. lactis strains are of great importance in the production of fermented dairy products such as yogurt, butter, fresh cheese and some kind of semi-hard cheese. Although L. lactis acquired the 'Generally Regarded As Safe' (GRAS) status, many investigations indicated that lactococci may act as reservoirs of antibiotic resistance genes, which could be transferred to other bacterial species in human gastrointestinal tract includ­ing pathogens. The genome analysis of L. lactis indicated the presence of at least 40 putative drug transporter genes, and only four multidrug resistance (MDR) transporters are functional­ly characterized: LmrA, LmrP, LmrCD i CmbT. LmrA is the first described MDR transport­er in prokaryotes. LmrCD is responsible for resistance to cholate, which is an integral part of human bile and LmrCD is important for intestinal survival of lactococci that are used as probiotics. Secondary multidrug transporter LmrP confers resistance to lincosamides, mac­rolides, streptogramins and tetracyclines. CmbT protein has an effect on the host cell re­sistance to lincomycin, sulfadiazine, streptomycin, rifampicin, puromycin and sulfametox­azole. Since the food chain is an important way of transmitting resistance genes in human and animal population, it is of great importance to study the mechanisms of resistance in lactococci and other LAB, intended for the food industry. Bakterije mlečne kiseline (BMK) imaju veliku primenu u prehrambenoj industriji, posebno u proizvodnji fermentisanih mlečnih proizvoda i mesa. Najviše izučavana vrsta u okviru BMK je Lactococcus lactis. Sojevi koji pripadaju vrsti L. lactis su od izuzetnog značaja u proizvodnji fermentisanih mlečnih proizvoda poput kiselog mleka, maslaca, svežih sireva i nekih vrsta polutvrdih sireva. Iako se generalno laktokoke smatraju bezbednim mikroorganizmima, istraživanja ukazuju da mogu biti rezervoari gena za rezistenciju na antibiotike, koji se horizontalnim transferom mogu preneti na druge bakterijske vrste, uključujući i patogene. Pokazano je da se u membrani bakterija vrste L. lactis nalaze efluksne pumpe koje obezbeđuju rezistenciju na strukturno različite supstrate (eng. multidrug resistance transporters-MDR). Analizom genoma L. lactis detektovano je prisustvo 40 potencijalnih gena koji kodiraju MDR transportere, od čega su do danas svega četiri funkcionalno okarakterisana: LmrA, LmrP, LmrCD i CmbT. LmrA je prvi uopšte opisan primarni transporter kod prokariota. LmrCD obezbeđuje rezistenciju na holat koji ulazi u sastav žuči, što je značajno za preživljavanje laktokoka koje se primenjuju kao probiotici u digestivnom traktu ljudi i životinja. LmrP je sekundarni transporter i obezbeđuje rezistenciju na neke linkozamide, makrolide, streptogramine I tetracikline. CmbT je poslednji okarakterisan MDR transporter kod L. lactis i doprinosi rezistenciji na linkomicin, sulfonamidne antibiotike, sulfametoksazol i sulfadiazin, aminoglikozidni antibiotik streptomicin, aminonukleozidni antibiotik puromicin i semisintetski antibiotik rifampicin. Kako lanac hrane predstavlja važan put prenosa gena za rezistenciju u okviru ljudske i životinjske populacije, neophodno je intenzivno izučavanje mehanizama rezistencije kod laktokoka i drugih BMK namenjenih za upotrebu u prehrambenoj industriji.

Knowledge on the antibiotic resistance of lactic acid bacteria is still limited, possibly because of the large numbers of genera and species encountered in this group, as well as variances in their resistance spectra. The EFSA considers antibiotic resistances, especially transferable resistances, an important decision criterion for determining a strain's QPS status. There are no approved standards for the phenotypic or genotypic evaluation of antibiotic resistances in food isolates. Also, the choice of media is problematic, as well as the specification of MIC breakpoint values as a result of the large species variation and the possible resulting variation in MIC values between species and genera. The current investigations in this field showed that we might end up with a range of different species- or genus-specific breakpoint values that may further increase the current complexity. Another problem associated with safety determinations of starter strains is that once a resistance phenotype and an associated resistance determinant have been identified, it becomes difficult to show that this determinant is not transferable, especially if the resistance gene is not located on a plasmid and no standard protocols for showing genetic transfer are available. Encountering those problems, the QPS system should allow leeway for the interpretations of results, especially when these relate to the methodology for resistance phenotype determinations, determinations of MIC breakpoints for certain genera, species, or strains, the nondeterminability of a genetic basis of a resistance phenotype and the transferability of resistance genes. Saznanja o rezistenciji na antimikrobne lekove bakterija mlečne kiseline su još uvek ograničena, verovatno zbog velikog broja rodova i vrsta koje postoje kod ove grupe, kao i zbog razlika u njihovom spektru rezistencije. Evropska agencija za bezbednost hrane EFSA smatra rezistentnost na antimikrobne lekove, posebno prenosive rezistencije, važnim kriterijumom kod donošenja odluke o kvalifikovanoj proceni bezbednosti -QPS statusu određenog soja. Ne postoje prihvaćeni standardi za fenotipsku ili genotipsku evaluaciju rezistencije na antimikrobne lekove kod izolata hrane. Takođe, izbor medija je problematičan, kao i specifikacija minimalne inhibitorne koncentracije MIC vrednosti kao posledica velikih varijacija u vrsti i moguće rezultirajuće varijacije u MIC vrednostima između vrsta i roda. Tekuća istraživanja u ovoj oblasti pokazala su da bi se na kraju mogao dobiti niz različitih MIC vrednosti specifičnih za vrstu ili rod koje bi mogle još više da prodube sadašnju kompleksnost. Još jedan problem koji postoji u vezi sa određivanjima bezbednosti starter sojeva jeste da, kada se jednom identifikuju fenotip rezistencije i asocirana determinanta rezistencije, postaje teško da se pokaže da ta determinanta nije prenosiva, posebno ukoliko se gen rezistencije ne nalazi na plazmidu i ukoliko nema standardnih protokola za prikazivanje genetskog transfera. Usled postojanja takvih problema, sistem o kvalifikovanoj proceni bezbednosti QPS treba da ostavi prostora za interpretaciju rezultata, posebno kada se oni odnose na metodologiju određivanja fenotipa rezistencije, određivanje MIC vrednosti za određeni rod, vrstu, ili soj, na nemogućnost ustanovljavanja genetske baze fenotipa rezistencije, kao i prenosivost gena rezistencije.

Količina proizvoda od surutke na tržištu je zanemarljivo mala u odnosu na količinu fermentisanih mlečnih proizvoda. To navodi na zaključak da bi se unapređenjem procesa fermentacije surutke mogli dobiti novi nekonvencionalni proizvodi koji bi zauzeli značajnije mesto u paleti mlečnih proizvoda namenjenih širokoj potrošnji. U tom smislu, cilj ove doktorske disertacije je bio proizvodnja i karakterisanje funkcionalnih fermentisanih napitaka na bazi surutke. U cilju proizvodnje funkcionalnih fermentisanih napitaka na bazi surutke izvršena je selekcija odgovarajuće kulture mikroorganizama, optimizacija procesa fermentacije i karakterizacija proizvedenog fermentisanog napitka. Tokom selekcije, optimizacije i karakterizacije praćeno je: vreme trajanja fermentacije, sadržaj mlečne kiseline, promena pH, preživljavanje mikroorganizama, sadržaj aminokiselina, sadržaj antioksidanasa, viskozitet, sinerezis kao i nutritivni sastav proizvedenog napitka. Takođe je izvršeno in vitro karakterisanje probiotske aktivnosti odabranih mikroorganizama praćenjem sposobnosti preživljavanja u uslovima simuliranog gastričnog soka, antimikrobne aktivnosti i rezistencije na antibiotike primenom klasičnih mikrobioloških tehnika. U sklopu nutritivne karakterizacije praćena je razgradnje proteina surutke nakon fermentacije. Proizvedeni napitak je nutritivno okarakterisan preračunavanjem nutritivne vrednosti proizvoda, što je predstavljalo polaznu tačku njegovog deklarisanja u skladu sa Pravilnikom o deklarisanju RS, označavanju i reklamiranju hrane 85/2013, ali i EU Regulative 1169/2011. Pored toga, u skladu sa nutritivnim sastavom, napitku su dodeljene nutritivne i/ili zdravstvene izjave odobrene od strane EFSA (European Food Safety Authority), prema EU Regulativi 11924/2011 i Direktivi 432/2012. Proizvod je upakovan u specijalno kreiranu ambalažu, koja je u skladu sa propisima za pakovanje mlečnih proizvoda, čime je dobijeni napitak u potpunosti pripremljen za plasman na tržište. Ispitivanje mogućnosti proizvodnje funkcionalnih fermentisanih napitaka na bazi surutke, primenom različitih sojeva bakterija mlečne kiseline pokazalo je da svi sojevi bakterija mlečne kiseline (ukupno 15) testirani u ovom istraživanju mogu biti primenjeni u procesu... Number of whey based products on the market is negligible compared to the number of fermented dairy products, which suggests that the improvement of the whey fermentation process could result in new unconventional products that take up a significant place in the wide range of dairy products intended for direct consumption. In accordance with that, the aim of this PhD thesis was the production and characterisation of functional fermented whey based beverages. In order to produce the functional fermented whey based beverages, selection of microorganisms, optimisation of the fermentation process and the characterisation of the produced beverages were carried out. During the selection, optimisation and characterisation, fermentation time, titratable acidity, pH value, amino acid content, antioxidative activity, viscosity, syneresis, as well as nutritional composition of produced beverages were followed. In vitro characterisation of probiotic character of the selected microorganisms, by tracking ability to survive under simulated gastric juice, antimicrobial activity and resistance to antibiotics using conventional microbiological techniques were also performed. Within the nutritional characterisation, percentage of whey protein degradation obtained after the fermentation, was examined. Manufactured beverages were nutritionally characterised by recalculating the nutritional value, which was the starting point of its declaration in accordance with the Serbian Regulations on declaration, labelling and advertising of food 85/2013. Also, in accordance with the nutritional composition of the beverages, nutritional and/or health claims approved by EFSA (European Food Safety Authority) and according to EU Regulation 1169/2011 and Regulation 432/2012 were given. The product was packaged in a specially created packaging that complies with the regulations for dairy products, and thus obtained beverage was fully prepared for the market. When examining the possibilities of whey based beverages production using different strains of lactic acid bacteria, it has been shown that all strains of lactic acid bacteria (total of 15) tested in this study can be applied in the production of functional fermented whey based...

”Pirot`s kachkaval” is a traditional cheese produced only on the territory of Pirot municipality. It belongs to the pasta filata cheeses and can be made of cow`s, ewe`s or the mixture of these milks. In order to determine the changes in the lactic acid bacteria microbiota during 60 days of the ripening of the ”Pirot`s kachkaval” 5 samples of cow`s milk kachkaval and 5 samples of ewe`s milk kachkaval were analyzed. A total number of 315 LAB strains were isolated and subjected to physiology tests and FTIR analysis for preliminary characterization and grouping. Representatives of each group were identified by the use of molecular methods. Same strains were subjected to technological characterization. Additionally, samples of the kachkaval up to 30 days of ripening were investigated for the concentration of organic acids and volatile compounds. The lactic acid bacteria strains isolated from ewe`s milk kachkaval belong to the species Streptococcus macedonicus, Enterococcus faecium, Pediococcus acidilactici, Pediococcus pentosaceus, Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus casei/ rhamnosus, Lactobacillus casei and Lactobacillus paracasei, while strains isolated from cow`s milk kachkaval belong to the species Enterococcus faecium, Pediococcus acidilactici, Pediococcus pentosaceus, Lactobacillus plantarum, Lactobacillus rhamnosus and Lactobacillus casei. The presence of lactic (in the highest concentration), malic, citric and acetic acid was confirmed. Also, pyruvic acid was detected in traces. Volatile compounds detected in the samples of kachkaval belonged to monocarboxylic acids, alcohols, ketones, aldehydes, terpenes and hydrocarbons. Acids and ketons were the most abundant, while esters were the most numerous group. Technological characterization of the isolates indicated that 40% of the isolates can use citrates and 32% can produce diacetyl. Also, 12% of the isolates produce exopolysacharides and 7% produced bacteriocines which were active against used test microorganisms Lаctococcus lactis subsp. lactis BGMNI-596, Lc. lactis subsp. lactis NP-45 and pathogenic Listeria sp. and Listeria monocytogenes.

Bakteriocini bakterija mlečne kiseline predstavljaju antimikrobne komponente proteinske prirode koje se primenjuju u prehrambenoj industriji (kao konzervansi), a takođe imaju potencijalnu primenu u veterini i medicini (kao alternativa ili dodatak antibiotskoj terapiji). Takođe, ove antimikrobne komponente danas se izučavaju i sa fundamentalnog aspekta, pre svega se izučava njihova struktura i mehanizam delovanja na senzitivne ćelije. Za bakteriocine klase I (nizin), pokazano je da koristi lipid II (prekursor sinteze ćelijskog zida) kao ciljni vezujući molekul za svoje delovanje. Do nedavno je definisano samo nekoliko membranskih proteina kao bakteriocinski receptori za bakteriocine klase II. Okarakterisane membranske komponente koje imaju ulogu receptora uključene su u transport šećera (komponente man-PTS sistema i maltoza-ABC transporter), takođe je nedavno okarakterisan UppP protein, receptor za dvokomponentni bakteriocin, laktokokcin G. LsbB bakteriocin je jedan od bakteriocina koje produkuje prirodni izolat Lactococcus lactis subsp. lactis BGMN1-5. U cilju identifikacije receptora za LsbB bakteriocin, urađena je nasumična mutageneza Lc. lactis BGMN1-596, soja senzitivnog na LsbB, nakon čega su selekcijom na šoljama koje su sadržale LsbB bakteriocin dobijene dve kategorije mutanata (BGMN1-596R2 i BGMN1-596R3), dok su analizom stepena tolerancije polaznog soja BGMN1-596 na hemijski sintetisanom LsbB bakteriocinu dobijeni spontano rezistentni mutanti kategorije BGMN1-596SR. U cilju komplementiranja senzitivnosti mutanata na LsbB konstruisana je kozmidna biblioteka soja BGMN1-596, gde je dobijeno 148 kozmidnih klonova, od kojih je svaki nosio fragmente EcoRI parcijalo isečene hromozomalne DNK veličine oko 40 kb. Kozmidna DNK je izolovana iz svakog klona i njom su transformisani mutanti. Kozmid pAZILcos/MN2 je komplementirao LsbB rezistentne mutante sve tri kategorije, odnosno vratio senzitivnost mutanata na LsbB bakteriocin... Bacteriocins of lactic acid bacteria are antimicrobial peptides used in food industry as preservatives, but also they could be used in veterinary and medicine (as alternatives to antibiotics). These antimicrobial components are also studied from a fundamental aspect, with a primarily focus on structure and mechanisms of action. The class I bacteriocin, nisin and some closely related lantibiotics have all been shown to employ lipid II, a cell wall precursor molecule, as a docking site. Until recently, only a few membrane proteins were defined as receptors for class II bacteriocins. Membrane receptors for these bacteriocins are involved in sugar transport (man-PTS system and maltose-ABC transporter). Another recently characterized protein, UppP protein was identified as receptor for two component bacteriocin, lactococcin G. Lactococcus lactis subsp. lactis BGMN1-5 among others produces a leaderless class II bacteriocin called LsbB. In order to identify the receptor for LsbB, MNNG was used for random mutagenesis of the LsbB sensitive strain Lc. lactis BGMN1-596. Two categories of resistant mutants were obtained (BGMN1-596R2 and BGMN1-596R3) by selection on LsbB bacteriocin contining plates, whereas spontaneously resistant mutants (BGMN1-596SR) were obtained by selection on plates containing synthesized LsbB bacteriocin. In order to complement sensitivity in mutants cosmid library of the LsbB-sensitive strain BGMN1-596 was constructed in E. coli, and 148 cosmid clones (EcoRI partially digested chromosomal DNA, each clone contain about 40 kb of genomic DNA) were obtained. Cosmid DNA was individually isolated and transferred to LsbB-resistant mutants of BGMN1-596. Cosmid pAZILcos/MN2, carrying a 40-kb insert, was found to be able to restore LsbB sensitivity in LsbB-resistant mutants. In order to identify the minimal genetic unit on the cosmid pAZILcos/MN2, which could transfer sensitivity to LsbB, pAZILcos/MN2 was digested with different restriction enzymes and subcloned into pAZIL vector...

Number of whey based products on the market is negligible compared to the number of fermented dairy products, which suggests that the improvement of the whey fermentation process could result in new unconventional products that take up a significant place in the wide range of dairy products intended for direct consumption. In accordance with that, the aim of this PhD thesis was the production and characterisation of functional fermented whey based beverages. In order to produce the functional fermented whey based beverages, selection of microorganisms, optimisation of the fermentation process and the characterisation of the produced beverages were carried out. During the selection, optimisation and characterisation, fermentation time, titratable acidity, pH value, amino acid content, antioxidative activity, viscosity, syneresis, as well as nutritional composition of produced beverages were followed. In vitro characterisation of probiotic character of the selected microorganisms, by tracking ability to survive under simulated gastric juice, antimicrobial activity and resistance to antibiotics using conventional microbiological techniques were also performed. Within the nutritional characterisation, percentage of whey protein degradation obtained after the fermentation, was examined. Manufactured beverages were nutritionally characterised by recalculating the nutritional value, which was the starting point of its declaration in accordance with the Serbian Regulations on declaration, labelling and advertising of food 85/2013. Also, in accordance with the nutritional composition of the beverages, nutritional and/or health claims approved by EFSA (European Food Safety Authority) and according to EU Regulation 1169/2011 and Regulation 432/2012 were given. The product was packaged in a specially created packaging that complies with the regulations for dairy products, and thus obtained beverage was fully prepared for the market. When examining the possibilities of whey based beverages production using different strains of lactic acid bacteria, it has been shown that all strains of lactic acid bacteria (total of 15) tested in this study can be applied in the production of functional fermented whey based... Količina proizvoda od surutke na tržištu je zanemarljivo mala u odnosu na količinu fermentisanih mlečnih proizvoda. To navodi na zaključak da bi se unapređenjem procesa fermentacije surutke mogli dobiti novi nekonvencionalni proizvodi koji bi zauzeli značajnije mesto u paleti mlečnih proizvoda namenjenih širokoj potrošnji. U tom smislu, cilj ove doktorske disertacije je bio proizvodnja i karakterisanje funkcionalnih fermentisanih napitaka na bazi surutke. U cilju proizvodnje funkcionalnih fermentisanih napitaka na bazi surutke izvršena je selekcija odgovarajuće kulture mikroorganizama, optimizacija procesa fermentacije i karakterizacija proizvedenog fermentisanog napitka. Tokom selekcije, optimizacije i karakterizacije praćeno je: vreme trajanja fermentacije, sadržaj mlečne kiseline, promena pH, preživljavanje mikroorganizama, sadržaj aminokiselina, sadržaj antioksidanasa, viskozitet, sinerezis kao i nutritivni sastav proizvedenog napitka. Takođe je izvršeno in vitro karakterisanje probiotske aktivnosti odabranih mikroorganizama praćenjem sposobnosti preživljavanja u uslovima simuliranog gastričnog soka, antimikrobne aktivnosti i rezistencije na antibiotike primenom klasičnih mikrobioloških tehnika. U sklopu nutritivne karakterizacije praćena je razgradnje proteina surutke nakon fermentacije. Proizvedeni napitak je nutritivno okarakterisan preračunavanjem nutritivne vrednosti proizvoda, što je predstavljalo polaznu tačku njegovog deklarisanja u skladu sa Pravilnikom o deklarisanju RS, označavanju i reklamiranju hrane 85/2013, ali i EU Regulative 1169/2011. Pored toga, u skladu sa nutritivnim sastavom, napitku su dodeljene nutritivne i/ili zdravstvene izjave odobrene od strane EFSA (European Food Safety Authority), prema EU Regulativi 11924/2011 i Direktivi 432/2012. Proizvod je upakovan u specijalno kreiranu ambalažu, koja je u skladu sa propisima za pakovanje mlečnih proizvoda, čime je dobijeni napitak u potpunosti pripremljen za plasman na tržište. Ispitivanje mogućnosti proizvodnje funkcionalnih fermentisanih napitaka na bazi surutke, primenom različitih sojeva bakterija mlečne kiseline pokazalo je da svi sojevi bakterija mlečne kiseline (ukupno 15) testirani u ovom istraživanju mogu biti primenjeni u procesu...

The aim of our study was the effect of salting and marinating on microbiological status and chemical composition of mackerel packed in the modified atmosphere. For the purposes of the testing, mackerel were divided into two groups where the first group consisted of salted mackerel packed in MAP and the other group was marinated mackerel packed in the same way. Marinades and salt primarily have impact on microbiological status of meat and the aim of this study was to identify certain groups of bacteria (total bacteria, lactic acid bacteria and the number of Enterobacteriaceae) during the fifty days of storage. Salting and especially marinating, significantly affect the change in the chemical composition of fish meat, since it leads to a significant reduction in water content, an increase in fat content and increased ash content. These processes affect the reduction of aw value of fish meat. In the samples of marinated fish, especially fish samples packaged in the modified atmosphere, significantly lower bacteria count was determined in the tested group relative to the samples which were treated with brine, and were packed under modified atmosphere. Cilj našeg rada je bio uticaj soljenja i mariniranja na mikrobiološki status i hemijski sastav skuše upakovane u modifikovanu atmosferu. Za potrebe ispitivanja skuša je podeljena u dve grupe, gde je prva grupa bila soljena skuša pakovana u MAP, a druga grupa je bila marinirana skuša pakovana u MAP. Marinade i so imaju pre svega uticaj na mikrobiološki status mesa pa je i cilj ovog istraživanja bio utvrđivanje pojedinih grupa bakterija (ukupan broj bakterija, bakterije mlečne kiseline i broj enterobakterija) tokom pedeset dana skladištenja. Soljenje, a posebno mariniranje, značajno utiče na promenu hemijskog sastava mesa ribe, budući da dolazi do značajnog smanjenja sadržaja vode, povećanja sadržaja masti i povećanja sadržaja pepela. Ovi postupci utiču i na smanjenja aw vrednosti mesa ribe. U uzorcima marinirane ribe, utvrđen je značajno manji broj svih ispitivanih grupa bakterija, u odnosu na uzorke koji su bili tretirani slanim rastvorom.

Lactic acid is a significant chemical for food, cosmetic, pharmaceutical and chemical industry. Currently, Serbia imports lactic acid and utilization of distillery stillage, a waste water from bioethanol production, as a cheap substrate for lactic acid fermentation could be an efficient and environmentally friendly approach. The main goal of this work was to investigate the possibilities of integrated production of lactic acid and animal feed on an industrial distillery stillage from bioethanol production on starch feedstock. Chemical analysis of the stillage has shown high content of proteins and valuable composition of minerals for the growth of LAB. In the selection process, Lactobacillus rhamnosus ATCC 7469 was chosen as the most promising strain for integrated production of lactic acid and biomass suitable for animal feed. The kinetics of lactic acid and biomass production in the batch fermentation and the impact of temperature, inoculum concentration, shaking and pH control were evaluated. The temperature of 41°C, shaking (90 rpm) and inoculum concentration of 5% (v/v) were selected for the fermentation. Among two different neutralizing agents (powdered CaCO3 and 30% solution of NaOH) a solution of 30% NaOH was selected for pH adjustment in four hour intervals during the fermentation. This system enabled the most optimal pH control and resulted in high lactic acid productivity and extensive growth of Lb. rhamnosus ATCC 7469. Significant improvements of the process were achieved in fed-batch fermentation where the final concentration of lactic acid was increased for 47.6 % and volumetric productivity for 21 % over the batch process. Maximal lactic acid productivity in fed-batch fermentation was 1.80 g L-1 h-1, with lactic acid yield of 0.87 g g-1 and lactic acid concentration of 97.1 g L-1. The number of viable cells at the end of fermentation was 109 CFU ml-1. The fermentation with a recirculation of immobilized Lb. rhamnosus ATCC 7469 onto zeolite was studied as a fermentation strategy. The strong adsorption of Lb. rhamnosus ATCC 7469 cells onto the zeolite surface allowed easy cell separation from the fermentation media, dense growth and high stability of immobilized cells for reuse in repeated batch cycles. A number of viable cells of over 1010 CFU per g of zeolite was achieved at the end of fourth fermentation cycle. A maximal process productivity of 1.69 g L-1, maximal lactic acid concentration of 42.19 g L-1 and average yield coefficient of 0.96 g g-1 were reached in the fermentation of stillage without mineral or nitrogen supplementation. However, in accordance to mineral requirements of Lb. rhamnosus ATCC 7469 and chemical content of minerals in stillage, exchange of Na+ ions in zeolite structure with selected Mg was performed. Exchange of Na+ with Mg2+ ions had improved immobilization of bacterial cells and increased lactic acid production for approximately 10%... Mlečna kiselina je važna supstanca za prehrambenu, farmaceutsku i hemijsku industriju. Srbija trenutno uvozi mlečnu kiselinu pa bi korišćenje destilerijske džibre, otpadne vode iz procesa proizvodnje bioetanola, kao jeftinog supstrata u proizvodnji mlečne kiseline moglo biti efikasna i ekološki povoljna strategija. Osnovni cilj ove disertacije je bio da se ispita mogućnost integrisane proizvodnje mlečne kiseline i stočne hrane na industrijskoj destilerijskoj džibri iz proizvodnje bioetanola na skrobnim sirovinama. Hemijskom karakterizacijom džibre je pokazano da je džibra bogata proteinima i da sadrži odgovarajući sastav jona metala za rast bakterija mlečne kiseline. U toku selekcije mikroorganizama odabran je soj Lactobacillus rhamnosus ATCC 7469, kao odgovarajući soj za paralelnu proizvodnju mlečne kiseline i biomase pogodne za stočnu ishranu. U šaržnom sistemu je ispitan uticaj temperature, koncentracije inokuluma, mešanja i kontrole pH. Nakon optimizacije, odabrana je temperature od 41 °C, mešanje od 90 obrt/min, koncentracija inokuluma 5 % (v/v). Ispitana je mogućnost korišćenja CaCO3 i 30% NaOH kao sredstava za neutralizaciju i kontrolu pH vrednosti i odabran je dodatak 30% NaOH u četvorosatnim intervalima kao najpovoljniji za integrisani postupak proizvodnje mlečne kiseline i stočne hrane. Primenom NaOH je ostvarena visoka produktivnost i intenzivan rast Lb. rhamnosus ATCC 7469. Značajno unapređenje procesa je postignuto primenom dolivnog postupka gde je konačna koncentracija mlečne kiseline u medijumu povećana za 47,6% a zapreminska produktivnost za 21% u odnosu na šaržni postupak. Maksimalna postignuta produktivnost mlečne kiseline je iznosila 1,80 g L-1 h-1, sa prinosom mlečne kiseline od 0,87 g g-1 i koncentracijom mlečne kiseline od 97,1 g L-1. Broj vijabilnih ćelija po završetku fermentacije je bio 109 CFU ml-1. Recirkulacioni šaržni postupak sa imobilisanim Lb. rhamnosus ATCC 7469 na zeolitu je takođe isptivan. Snažna adsorpcija ćelija na površinu zeolita je omogućila lako odvajanje imobilizata od fermentisanog medijuma, gust rast ćelija u biofilmu i visoku stabilnost imobilisanih ćelija za recirkulaciju. Na kraju četvrtog ciklusa broj vijabilnih imobilisanih ćelija iznosio je više od 1010 CFU g-1 zeolitnog nosača. U šaržnom procesu sa recirkulacijom na tečnoj džibri bez dodatka izvora azota i minerla najviša postignuta produktivnost je iznosila 1,69 g L-1 h-1, uz maksimalnu koncentraciju mlečne kiseline 42,19 g L-1 i prosečni koeficijent prinosa od 0,96 g g-1. Ipak, u skladu sa potrebama Lb. rhamnosus ATCC 7469 i mineralnim sastavom džibre izvršena je i modifikacija zeolita, izmenom jona Na+ jonima Mg2+, što je rezultovalo povećanom adsorpcijom bakterija na površinu modifikovanog zeolita, kao i povećanjem proizvodnje mlečne kiseline za oko 10%...

Aerobic stability is a term that indicates the length of the time that silage remains cool and does not spoil after it is exposed to air. Therefore, rapid removal of air from forage mass and prevention of air infiltration into the silage during storage and feed out are the important factors that determine the duration of the silage aerobic stability. The measurement of CO2 production, separately or in combination with other measurements such as temperature, pH, lactic acid and volatile fatty acids and microbial examination, can serve as a reliable laboratory method to determine the duration of silages aerobic stability in silages. This paper work has aim to point on the importance of determination the duration of aerobic stability of silages, because quality changes and losses in the case of aerobic degradation. Aerobna stabilnost je termin kojim se definiše dužina vremena za koje silaža ostaje hladna i ne kvari se po izlaganju vazduhu. Zbog toga, brzo otklanjanje vazduha iz silaže tokom siliranja i smanjeno izlaganje vazduhu po otvaranju silosa su dva važna faktora koji određuju dužinu aerobne stabilnosti silirane stočne hrane. U proceni aerobnog kvarenja,merenje proizvodnje CO2, odvojeno ili u kombinaciji sa drugim parametrima kao što je temperatura silaže, pH vrednost, sadržaj mlečne i sirćetne kiseline i mikrobiološka analiza, mogu se koristiti kao pouzdani laboratorijski metod. Ovaj rad ima za cilj da ukaže na značaj procene vremena trajanja aerobne stabilnosti stočne hrane,zbog promena i gubitaka kvaliteta pri aerobnoj degradaciji.

Cilj rada je bio ispitivanje funkcionalnih karakteristika kombuha napitkaod melise (Melissa officinalis L.). Antibakterijska aktivnost kombuhanapitaka optimalne konzumne i većih kiselosti ispitana je premabakterijama izolovanim iz hrane i vode za piće. Nosilac antimikrobneaktivnosti je sirćetna kiselina, a na ostale nosioce ukazuje delovanjeneutralisane kombuhe i čajnog napitka prema nekim test bakterijama.Spektrofotometrijskom metodom određen je sadržaj ukupnih fenolnihjedinjenja, a HPLC analizom određen je kvalitativni i kvantitativni sastavfenolnih jedinjenja u fermentacionim tečnostima, čajnim i kombuhanapicima od melise i crnog čaja. Antioksidativna aktivnost istih uzorakaispitana je na DPPH i OH radikale ESR spektralnom metodom. Uzorcifermentacione tečnosti i kombuha napitka od melise imali su većuantioksidativnu aktivnost prema oba radikala u odnosu na uzorke sacrnim čajem. Konzumna kombuha od melise imala je većuantioksidativnu aktivnost od čajnog napitka. Aktivne komponentekombuha napitka od melise su verovatno ruzmarinska kiselina ikvercetin. U ispitivanju antiproliferativne aktivnosti konzumnogkombuha napitka i čajnog napitka od melise na tri ćelijske linije humanihkarcinoma: HeLa (epitelni karcinom grlića materice), MCF-7(adenokarcinom dojke) i HT-29 (adenokarcinom debelog creva) utvrđenoje da nije došlo do stimulacije proliferacije ispitanih ćelijskih linija prikoncentracijama većim od 100 μg/ml. Pored istraživanja biološkeaktivnosti ispitana je mogućnost simultane mlečno-kiselinske i kombuhafermentacije. Dodatkom starter kultura i Lactobacillus spp. izolata ufermentacionu tečnost dolazi do povećanja sadržaja L- i D- mlečnekiseline, iako su ćelije bakterija mlečne kiseline, osim izolata iz kiselogtesta (L. hilgardii), pokazale malu otpornost na uslove tokomfermentacije i čuvanja pripremljenih napitaka. Izvršena je i identifikacijabakterija sirćetnog vrenja izolovanih iz lokalnih čajnih gljiva PCRmetodom. Dva izolata verovatno pripadaju vrsti Gluconobacter oxydans,a treći vrsti Gluconacetobacter hansenii., The aim of this study was to investigate functional characteristics of akombucha beverage from lemon balm (Melissa officinalis L.) tea. Antibacterial activity of kombucha beverages with optimum and higheracidities was tested against bacteria isolated from food and drinking water. The main active component of antibacterial activity was acetic acid, while slight activity of neutralized kombucha and unfermented tea against some test bacteria indicated presence of other antibacterial components. Total phenol concentration in unfermented tea samples, fermentation broths and kombucha beverages from lemon balm and black tea was determined spectrophotometrically whereas qualitative and quantitative concentration of polyphenolic compounds was determined by HPLC method. Antioxidant activity on DPPH and hydroxyl radical in the same samples was determined on an ESR spectrometer. Fermentation broth and kombucha beverage from lemon balm had higher antioxidant activity against both radicals than the samples from black tea. Kombucha beverage from lemon balm with optimum acidity had higher antioxidant activity than unfermented lemon balm tea. The main active components of antioxidant activity were probably rosmarinic acid and quercetin. Antiproliferative activity of lemon balm tea and kombucha was measured by sulforhodamine B colorimetric assay on HeLa (cervix epitheloid carcinoma), HT-29 (colon adenocarcinoma), and MCF-7 (breast adenocarcinoma) cell lines. By applying concentrations higher than 100 μg/ml, tested samples did not stimulate proliferation of cell lines. The possibility of simultaneous lactic acid and kombucha fermentation was tested as well. When starter cultures and Lactobacillus spp. isolates were applied, the content of Land D- lactic acid increased during fermentation, although lactic acid bacteria (except L. hilgardii isolated from sour dough) showed low resistance to the conditions during fermentation and beverages storage. Acetic acid bacteria isolated from local tea fungus were identified by PCR method. Two isolates might be Gluconobacter oxydans and one - Gluconacetobacter hansenii.

In this article the prevalence of lactic acid bacteria through the production and ripening period of autochthonous Sjenica cheese was reviewed. The activity of main LAB group was evaluated by pH changes and evolution of ripening coefficient. U radu je prikazana prevalenca bakterija mlečne kiseline tokom procesa proizvodnje i zrenja autohtonog sjeničkog sira. Aktivnost bakterija mlečne kiseline je procenjena na osnovu promene pH vrednosti i koeficijenta zrelosti tokom perioda zrenja od 90 dana.

Autochthonous cheese manufacturing within the area of Serbia has very long tradition. These cheeses are produced without using any known starter culture. Therefore, studying of all aspects of such cheese manufacturing is very important. This approach will assure continuation of traditional way of manufacturing cheeses at the places of their origin. Moreover, research of this kind will open possibility for introduction of autochthonous cheese production at either semi-industrial or industrial level, or their presentation on different markets as well. Golija cheese belongs to the group of white soft cheeses. It is produced from nonpasteurized raw cow milk immediately after milking. Normally it is kept on the mountain for one to two months for ripening. Cheese is traditionally cut into slices and packed in distribution packaging. This production is mostly carried out by small manufacturers, what results in uneven quality and ungraded value of cheese. However, due to production in reserved region of origin, demands for Protected Denomination of Origin (PDO) were satisfied. Aim of this work was to investigate autochthonous cheese manufacturing, chemical composition and microbiological quality of cheese produced on mountain Golija. Investigation of the fluctuation of lactic acid bacteria (LAB) population in Golija cheese was made by analysis of the presence of LAB in the milk of which the cheese was made, as well as in the cheese during ripening. The composition of cheese microflora was recorded in cheeses after 1, 10, 20, 30, 45 and 60 days of ripening. Autohtona proizvodnja sireva na području Srbije ima veoma dugu tradiciju. Ovi sirevi se proizvode bez korišćenja starter kulture. Iz tog razloga, proučavanje svih aspekata proizvodnje takvih sireva je veoma važno. Ovakav pristup će obezbediti nastavak tradicionalnog načina proizvodnje sireva na područjima odakle i potiču. Osim toga, ovakva istraživanja stvoriće mogućnost predstavljanja proizvodnje autohtonih sireva, bilo na poluindustrijskom ili industrijskom nivou, kao i njihovo prezentovanje na različitim tržištima. Golijski sir pripada grupi mekih sireva. Proizvodi se od nepasterizovanog (sirovog) kravljeg mleka, koje se u tu svrhu koristi odmah posle muže. Uobičajeno je da sir ostaje u planinskom domaćinstvu jedan ili dva meseca dok traje period zrenja. Sir se tradicionalno seče u kriške i pakuje u odgovarajuću ambalažu. Ovu proizvodnju uglavnom vrše mali proizvođači, što ima za posledicu neujednačen kvalitet i nestandardizovanu vrednost sira. Međutim, proizvodnjom u zaštićenim regionima, zadovoljeni su zahtevi za oznaku geografskog porekla. Cilj ovog rada bio je istraživanje autohtone proizvodnje, hemijski sastav i mikrobiološki kvalitet sira proizvedenog na planini Golija. Ispitivanje promena u populaciji bakterija mlečne kiseline (BMK) u siru sa Golije izvršeno je analizom na prisustvo BMK u mleku za proizvodnju sira, kao i u siru za vreme zrenja. Sastav mikroflore sira praćen je posle 1, 10, 20, 30, 45 i 60 dana zrenja sira.

The antibiotic resistance is an ecological problem generated by the world-wide use of antibacterial agents. At present, there is great concern that commensal bacterial populations from food, such as lactic acid bacteria (LAB) and bifidobacteria could act as a reservoir for antibiotic resistance genes. Resistance could ultimately be transferred to human pathogenic and opportunistic bacteria either during food manufacture or during passage through the GIT. LAB species have traditionally been used as starter cultures in the production of fermented food. The genes conferring resistance to several antimicrobials (chloramphenicol, erythromycin, streptomycin, tetracycline and vancomycin) located on transferable genetic element have already been described in lactic acid bacteria. This work reports the antibiotic resistance profiles of several LAB species including lactobacilli, lactococci, enterococci, Bifidobacterium spp., Leuconostoc spp. and Str. thermophilus. Rezistencija na antibiotike predstavlja ekološki problem uslovljen, najvećim delom, primenom enormnih količina antibiotika u humanoj i veterinarskoj medicini. U sadašnje vreme, interesovanje naučne javnosti pobuđuje činjenica da populacija komenzalnih bakterija, kao što su bakterije mlečne kiseline i bifidobakterije mogu predstavljati rezervoar gena rezistencije na antibiotike. Svojstvo rezistencije se može preneti putem mehanizama horizontalnog transfera gena na patogene i oportunističke mikroorganizme bilo tokom procesa proizvodnje ili tokom pasaže kroz gastrointestinalni trakt. Determinante rezistencije odgovorne za rezistenciju na nekoliko antibiotika (hloramfenikol, eritromicin, streptomicin, tetraciklin i vankomicin) lokalizovane na prenosivim genetskim elementima utvrđene su kod bakterija mlečne kiseline. Rad izveštava o profilima rezistencije na antibiotike kod nekoliko vrsta bakterija mlečne kiseline, uključujući laktobacile, laktokoke, enterokoke, bifidobakterije, leukonostok vrste i Str. Thermophilus.

Sombor cheese is a soft cheese traditionally produced from raw sheep milk in the southern part of Serbia. The knowledge of the microbial diversity of this traditional product is in turn fundamental for the attainment of its unique sensory properties. A study of the resident microbial population of Somborcheese has been undertaken. The main groups of native lactic acid bacteria (lactococci, lactobacilli, enterococci) were determined following conventional microbiological procedures. The Lactoccoccus spp. reached level of 106- 108 cfu/g, and the microbial counts for presumptive lactobacilli and enterococci attained 104-107cfu/g and 103-107cfu/g, respectively. Isolated strains of lactococci, lactobacilli and enterococci were further characterized regarding their technological properties such as acidification proteolytic ability and production of diacetyl in order to evaluate their possible application as a co-culture in cheese-making practice. Somborski sir je meki sir koji se tradicionalno proizvodi iz ovčijeg mleka u severnom delu Srbije. Poznavanje diverziteta mikrobne populacije ovog tradicionalnog proizvoda jeste od posebnog značaja za održavanje jedinstvenih senzornih karakteristika sira. Ispitivana je autohtona mikroflora bakterija mlečne kiseline somborskog sira. Glavne grupe autohtone mikroflore bakterija mlečne kiseline (laktokoke, laktobacili, enterokoke) određene su primenom uobičajenih metoda mikrobiološke analize. Broj Lactococcus spp.u somborskom siru je 106- 108 cfu/g, Lactobacillus spp. 104-107cfu/g sira a Enterococcus spp. 103-107cfu/g. Izolovani sojevi laktokoka, laktobacila i enterokoka su dalje okarakterisani u odnosu na tehnološke karakteristike kao što su acidifikacija, proteolitička aktivnost i sposobnost stvaranja diacetila a u cilju njihove moguće primene kao pomoćnih kultura u procesu proizvodnje sira.

Autochthonous isolates of lactic acid bacteria and enterococci, from Sjenica cheese had been isolated and identified. From 10 samples of Sjenica cheese that were traditionally produced without starter cultures, 53 thermophile mesophile and citrate (+) isolates were isolated using selective media (MRS agar, M17 agar, LDCagar i LDV agar). According to Gram staining, cell morphology and catalase test, it was determined that all isolates were pure culture of G (+) and catalase (-) bacteria, 21 bacilli and 35 cocci. For the purpose of diferentiation enterococci and lactococci, growth of all cocci bacteria was examined on 6,5% NaCI, pH 9,6 and heat treatment 60°C 20 min. Out of 35 cocci isolates, 7 isolates were able to grow at these conditions. Identification by API CHL 50 test for lactobacilli and RAPID ID 32 Strep for cocci, showed that isolates were Lactococcus lactis ssp. lactis Lactobacillus paracasei ssp. paracasei, Lactobacillus plantarum, Lactococcus lactis ssp. lactis boiv. dicetylactis, Lactobacillus curvatus, Lactobacillus brevis, Leuconostoc sp. i Enterococcus sp. Dominant lactococci strain was Lactococcus lactis ssp. lactis (35,85%) and for lactobacilli Lactobacillus paracasei ssp. paracasei (18,86%). Strains from genus Enterococcus sp. had the rate of 13,20%. U radu su izdvojeni i identifikovani izolati bakterija mlečne kiseline i enterokoka iz sjeničkog sira, proizvedenog na tradicionalan način, bez dodavanja starter kulture. Iz 10 uzoraka sjeničkog sira izdvojeno je 53 termofilna, mezofilna i citrat+ izolata na odgovarajućim selektivnim podlogama (MRS agar, M17 agar, LDCagar i LDV agar). Postupkom bojenja po Gramu, mikroskopskim pregledom i katalaza testom je utvrđeno da svi izolati predstavljaju čiste kulture Gram-pozitivnih i katalaza-negativnih bakterija pri čemu je utvrđeno da je 21 izolat štapićastog, a 35 okruglastog oblika. Radi razdvajanja enterokoka od laktokoka, za sve okruglaste forme je ispita-na sposobnost rasta pri 6,5% NaCI, pH 9,6 i nakon termičkog tretmana na 60°C 20 minuta je utvrđeno da od 35 izolata okruglastog oblika, 7 izolata podnosi navedene uslove. Determinacijom dobijenih izolata primenom API sistema i to za štapičaste oblike API CHL50testa, a za okruglaste oblike RAPID ID 32 Strep sistema, utvrđeno je da izolati pripadaju vrstama Lac-tococcus lactis ssp. lactis, Lactobacillus paracasei ssp. paracasei Lactobacillus plantarum, Lactococcus lactis ssp. lactis boiv. dicetylactis Lactobacillus curvatus, Lactobacillus brevis, Leuconostoc sp. i Enterococcus sp. Ubedljivo najveću zastupljenost je imala okruglasta vrsta Lactococcus lactis ssp. lactis (35,85%), a od štapićastih, vrsta Lactobacillus paracasei ssp. Paracasei (18,86%). Vrste roda Enterococcus sp. bile su zastupljene sa 13,20 %.

Autochthonous isolates of lactic acid bacteria and enterococci from Kashkaval cheese had been isolated and identified. From 6 samples of Kashkaval cheese that were traditionally produced without starter cultures, 27 isolates were isolated using selective media (MRS agar, M17 agar) and two incubation temperatures (37ºC and 43ºC). According to Gram staining, cell morophology and catalase test, it was determined that all isolates were pure culture of G (+) and catalase (-) bacteria, 12 bacilli and 15 cocci. For the purpose of diferentiation between enterococci and lactococci, growth of all cocci bacteria was examined on 6,5% NaCl, pH 9,6 and heat treatment 60ºC 20 min. Out of 15 cocci isolates, 6 isolates were able to grow at these conditions. Acidogenic activity was determinated by measuring pH values of 1% inoculated reconstituted skim milk during 24 hours. Researched isolates showed significant difference in lactic acid production. Ten isolates had good acidogenic activity (pH 4,12-4,90) and seventeen isolates were weak producers (pH 5,10 - 6,48). Identification by API CHL 50 test for lactobacilli and RAPID ID 32 Strep for cocci, showed that isolates were Lactobacillus delbrueckii ssp. delbrueckii, Lactobacillus casei, Lactobacillus plantarum Lactobacillus fermentum, Lactobacillus curvatus, Streptococcus salivarius ssp. thermophilus, Lactococcus lactis ssp. lactis, Lactococcus lactis ssp. cremoris, Enterococcus faecium i Enterococcus durans. U radu su izdvojeni i okarakterisani izolati bakterija mlečne kiseline i enterokoka iz kačkavalja, proizvedenog bez dodavanja starter kulture. Iz 6 uzoraka kačkavalja različite starosti izdvojeno je 27 izolata na odgovarajućim selektivnim podlogama MRS i M17 agaru i temperaturama rasta od 37ºC i 43ºC. Postupkom bojenja po Gramu, mikroskopskim pregledom i katalaza testom je utvrđeno da svi izolati predstavljaju čiste kulture Gram-pozitivnih i katalaza-negativnih bakterija, pri čemu je utvrđeno da je 12 izolata štapićastog, a 15 okruglastog oblika.. Radi razdvajanja enterokoka od bakterija mlečne kiseline, za sve okruglaste forme je ispitana sposobnost rasta pri 6,5% NaCl, pH 9,6 i nakon termičkog tretmana na 60ºC 20 minuta utvrđeno je da od 15 izolata okruglastog oblika, 6 izolata podnosi navedene uslove. Acidogena aktivnost je određena merenjem pH vrednosti rekonstituisanog obranog mlaka sa 1 % inokuluma u toku 24 sata, pri čemu je utvrđeno da se izolati značajno razlikuju u sposobnosti stvaranja kiseline. Deset izolata je pokazalo dobru acidogenu sposonost (pH 4,12-4,90), dok je 17 izolata slabo produkovalo kiselinu (pH 5,10 - 6,48). Determinacijom dobijenih izolata primenom API sistema i to za štapićaste oblike API CHL 50 testa, a za okruglaste oblike RAPID ID 32 Strep sistema, utvrđeno je da izolati pripadaju vrstama: Lactobacillus delbrueckii ssp. delbrueckii, Lactobacillus casei Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus curvatus Streptococcus salivarius ssp. thermophilus, Lactococcus lactis ssp. lactis Lactococcus lactis ssp. cremoris, Enterococcus faecium i Enterococcus durans.

Kajmak was produced according to the production procedure proposed by Puđa Starčević and Radovanović, which enables standard composition and characteristics, as well as high microbiological quality of the product. Directly after collection, kajmak was inoculated with the mixture of lactic acid bacteria which are used as starter cultures: Lactococcus lactis ssp. lactis, Lc. lactis ssp. cremoris, Lc. lactis ssp. lactis bv. diacetylactis and Leuconostoc mesenteroides ssp. cremoris. As protective cultures, the following species were used: Lactobacillus rhamnosus and Propionbacterium freudenreichii ssp. shermanii. Maturation of kajmak took place on 12°C and 18°C. Kajmak surface was protected by coating with the wax layer. Kajmak composition and dynamics of starter growth were analyzed during 30 days of ripening. pH value of kajmak was decreasing in the first days of maturation and draped to 4.7 for 7 and 15 days when maturation took place at 18°C and 12"C, respectively. Growth of aroma producing bacteria was increasing from 106 cfu/g after inoculation to max. value of 108 cfu/g of kajmak. The kajmak samples which were ripened at the 12°C showed max. number of aroma producing bacteria at the 15th day of ripening, while at 18°C max. number was achieved after two days of maturation. Proizvodnja kajmaka obavljena je po tehnološkom postupku Puđa, Starčević Radovanović (12) koji obezbeđuje standardni sastav i karakteristike, kao i visoku mikrobiološku ispravnost proizvoda. Kajmak je neposredno po izuzimanju inokulisan smešom starter kultura bakterija mlečne kiseline Lactococcus lactis ssp. lactis, Lc. lactis ssp. cremoris, Lc. lactis ssp. lactis bv. diacetylactis i Leuconostoc mesenteroides ssp. Cremoris, a kao protektivne kulture korišćene su vrste Lactobacillus rhamnosus i Propionbacterium freudenreichii ssp. shermanii. Zrenje kajmaka je obavljeno na temperaturama 12°C i 18°C u ambalaži pri čemu je po površini kajmaka nanesen sloj zaštitnog premaza u cilju zaštite od atmosferskih uticaja. Kajmak je ispitivan u toku jednomesečnog zrenja u pogledu hemijskog sastava i dinamike razvoja startera. pH vrednost kajmaka je opadala intenzivno u početku zrenja i ustalila se na vrednosti od oko 4,7 posle 7 dana zrenja na 18°C, odnosno 15 dana zrenja na 12°C. Razvoj aromogenih bakterija mlečne kiseline imao je trend porasta i povećavao se od 106 cfu/g nakon inokulacije do maksimalne vrednosti od oko 108 cfu/g kajmaka. U kajmaku koji je imao zrenje na 12°C maksimalni broj aromogenih bakterija postignut je 15. dana zrenja, dok je na 18°C maksimalan broj postignut već posle dva dana zrenja kajmaka.

Lactic acid bacteria strains isolated from autochthonous cheeses were researched. In products such as these, where starters were not added fermentation occurs as a result of natural flora present in the surrounding environment. Thirty thermophilic, mesophilic and citrate+ LAB strains were isolated by selective medium (LM17 agar and LDC agar) from 5 samples of autochthonous cheeses. They were exposed to further analysis and identification. After examination by Gram test and catalase test, 12 strains of LAB, were selected for further analysis. On the basis of lactose fermentation and acid production in 1% reconstituted skim milk, 6 homofermentative strains were selected, that also had good acidogenic activity. Their proteolytic ability to hydrolyze B-casein was examined by SDS polyacrilamid electrophoresis, where these strains showed different proteolytic activity. Determination of these strains by API Rapid ID 32 Strep tests showed that 4 strains belong to the Lactococcus lactis ssp. tech's and 2 strains to the Lactococcus lactis ssp. lactis biovar. diacetylactis. Rad je obuhvatio karakterizaciju bakterija mlečne kiseline izolovanih iz autohtonih sireva proizvedenih na tradicionalan način, bez dodavanja starter kultura. Iz 5 uzoraka sira izvršena je izolacija 30 termofilnih mezofilnih i cit-rat+ sojeva BMK na odgovarajućim selektivnim podlogama (LM17 agar i LDC agar). Od izdvojenih čistih kultura, nakon određivanja Gram reakcije i katalaza testa, izabrano je 12 sojeva za dalja ispitivanja. Na osnovu načina fermentacije laktoze (homo-heterofermentacija)i acidogene sposobnosti praćene u 1% rekonstituisanom obranom mleku u toku 24 sata, selekcionisano je 6 sojeva koji su pokazali homofermentativnu aktivnost i dobru acidogenu sposobnost. Za ove sojeve je ispitana proteolitička aktivnost u odnosu na β-kazein, pomoću SDS poliakrilamidne elektroforeze, koja je pokazala međusobne razlike u sposobnosti razgradnje β-kazeina. Na kraju je izvršena njihova identifikacija pomoću API Rapid ID 32 Strep testa za laktokoke, i utvrđeno je da 4 soja pripadaju vrsti Lactococcus lactis ssp. lactis, a dva soja vrsti Lactococcus lactis ssp. lactis biovar. diacetylactis.

Lactic acid bacteria strains isolated from autochthonous cheeses of Sjenica were investigated. In those products, where starters were not added fermentation occurred as a result of natural flora present in the surrounding environment. Forty thermophilic, mesophilic and citrat+ LAB strains were isolated by selective mediums (MRS agar, LM17 agar and LDC agar) from 5 samples of Sjenica-cheeses. They were exposed to further analysis and identification. After examination by Gram test and catalase test, 13 strains of LAB, were selected for further analysis. On the basis of lactose fermentation way and acid production in 1% reconstituted skimed milk, 6 strains were selected. Determination of these strains by API 50 CHL and Rapid ID 32 Strep tests showed that 2 strains belonged to the Lactococcus lactis ssp. lactis, 2 strains to the Lactococcus lactis ssp. cremoris and 2 strains to the Lactobacillus para.paracasei. U radu su izolovani i karakterisani sojevi bakterija mlečne kiseline izdvojeni iz sjeničkog sira, proizvedenog autohtonom tehnologijom, bez dodavanja starter kultura. Iz 5 uzoraka sjeničkog sira izvršena je izolacija 40 termofilnih, mezofilnih i citrat+ sojeva BMK na odgovarajućim selektivnim podlogama (MRS agar, LM17 agar i LDC agar). Ovi sojevi su dalje podvrgnuti ispitivanju na Gram reakciju i katalaza test, posle čega je izabrano 13 sojeva za dalja ispitivanja. Na osnovu načina fermentacije laktoze i acidogene sposonosti praćene u 1% rekonstituisanom obranom mleku selekcionisano je samo 6 sojeva. Determinacijom ovih sojeva pomoću API CHL 50 i Rapid ID 32 Strep testa, pokazalo se da 2 soja pripadaju vrsti Lactococcus lactis ssp. lactis, 2 soja Lactococcus lactis ssp. cremoris i 2 soja vrsti Lactobacillus para.paracasei.

Analysis of the presence of lactic acid bacteria in home-made white-soft cheese manufactured at mountain Zlatar was performed. Samples of cheese are collected from fifteen different manufacturers. In addition, one of the cheese's sample that showed the best sensory characteristics were used to test the fluctuation of microflora during cheese ripening. The fluctuation of microflora was followed after one, five, ten, twenty and thirty days of ripening. Results showed that the number of Lactoccocus cells decreased during ripening period. It is not possible to detect Lactoccocus cells after twenty days of cheese ripening. After thirty days of cheese ripening mesophilic lactobacilli, especially Lactobacillus paracasei and Lactobacillus plantarum, were dominant microflora Interestingly Enterococcus feacalis took a considerably numerous population of microflora during the whole period of cheese ripening. Da bi se dobila što bolja slika o bakterijama mlečne kiseline zastupljenim u zlatarskom siru analizirano je 15 uzoraka sira proizvedenih u različitim domaćinstvima na području Zlatara. Sasatav mikroflore jednog od uzoraka je praćen nakon jednog, pet, deset, dvadeset i trideset dana zrenja sira. Analizom mikroflore je zapaženo da broj vrsta roda Lactoccocus opada i da skoro potpuno nestaje posle 20 dana zrenja. Nakon 30 dana zrenja u uzorcima dominiraju mezofilne vrste roda laktobacila i to Lactobacillus paracasei i Lactobacillus plantarum. Tokom posmatranog perioda zrenja znatnu populaciju u uzorcima sira čini i vrsta Enterococcus feacalis.

Lactic acid bacteria play a central role in manufacturing fermented milk products. The most common cause of failure of this process is bacteriophage infection, which cause lysis of the starter bacteria. They inhibit or completely prevent acidification by the starter culture. A number of measures to prevent phage infection, including implementation of hygienic procedures, rotation of starter cultures, isolation of resistant mutants and using of media to suppress phage multiplication have been instituted The early detection of phage contamination in dairy fermentation could be important strategy to prevent and control phage development. Bakterije mlečne kiseline imaju osnovnu ulogu u proizvodnji fermentisanih proizvoda od mleka. Upotrebom starter kultura usmerava se tok fermentacije Međutim, često dolazi do neželjenih promena, usporavanje acidifikacije smanjenja viskoziteta proizvoda, nedostatka arome, preterane ili nedovoljne proizvodnje gasa, potpunog prestanka fermentacije i gubitka u prinosu Smatra se da je za 70-80% ovih problema odgovorna infekcija bakteriofagima koji izazivaju lizis bakterija. Identifikovana su dva osnovna izvora kontaminacije fagima u mlekarama: prisustvo bakterija mlečne kiseline u sirovom mleku i lizogene starter kulture. U nastojanjima da se uspešno resi problem bakteriofagnih infekcija razrađeno je više mera zaštite: fizičke (bolja sanitacija, čista okolina, zaštićene sirarske kade), fiziološke (upotreba podloga koje sprečavaju rast bakteriofaga starter kulture bez prisutnih bakteriofaga, rotacija starter kultura) i genetički program kojim se obezbeđuju starter kulture povećane otpornosti na infekciju bakteriofagima. Zbog stalne pretnje od ozbiljnih fagnih infekcija u mlekarama je potrebno neprekidno praćenje prisustva faga, što istovremeno predstavlja i osnovnu zaštitnu meru.

Criteria for selection of probiotic lactic acid bacteria are presented in this review paper. General characteristics, strain stability as well as functional and physiological aspects are pointed out in details. . U ovom preglednom radu prikazani su kriterijumi za selekciju bakterija mlečne kiseline sa probiotičkim delovanjem. Među brojnim kriterijumima detaljno su tretirani sledeći: opšte karakteristike, stabilnost sojeva i funkcionalni i fiziološki aspekti.

The aim of this work was to obtain the stable - powdered polycomponent preparation based on fermented soybean extract that is supplemented with beetroot juice and yeast autolysate with preserved biological activity. Such a preparation is more convenient for broad application. The fermentation was carried out with intestinal isolate Bifidobacterium sp. A71. The drying was performed in spout-fluid bed drier at two different tempera­tures (working temperature of 75ºC and 85ºC). Viable cell number and inhibitory activity were determined as parameter of the biological activity of powdered preparation. The results of this investigation showed that the greater viable cell number was in powdered preparation obtained at lower drying temperature. Beside the fact that the temperature influenced the viability of starter culture, the utilization of spout-fluid bed drier can ensure the preservation of biological activity of poly-component preparation at satisfactory level. . Cilj rada je da se višekomponentni proizvod na bazi fermentisanog ekstrakta soje, soka cvekle i autolizata pivskog kvasca prevede u stabilniju formu - prah, koji je pogodniji za široku upotrebu, i da se sušenjem očuvaju njegove biološke aktivnosti, Fermentacija je vršena prirodnim intestinalnim izolatom Bifidobacterium sp. A71. Sušenje je vršeno u fontansko-fluidizovanom sloju uz primenu dva temperaturna režima (radna temperatura 75°C i 85°C). Očuvanje biološke aktivnosti praškastog proizvoda je praćeno brojem preživelih ćelija korišćenog startera i njegovom inhibitornom aktivnošću. Rezultati ovih ispitivanja su pokazali da je broj ćelije soja Bifidobacterium sp. A71 veći u praškastom proizvodu dobijenom pod blažim temperaturnim režimom. Pored činjenice da primenjeni temperaturni režimi imaju uticaja na vijabilnost korišćenih startera u praškastom proizvodu, optimizacijom parametara sušenja u fontansko-fluidizovanom sloju, može se dobiti praškasti multikomponentni proizvod sa, u velikoj meri, očuvanom biološkom aktivnošću.

The identification and classification of microorganisams are of crucial importance in environmental. Industrial, medical and agricultural microbiology and microbial ecology. For a long time, microbial taxonomy was considered as one of the most boring fields, in microbiology. Basic research on lactic acid bacterial and their application in industrial food fermentations required reliable and simple methods for identification, because many lactic acid bacteria have similar nutritional and growth requirements and it is very difficult to identify them by classical methods. The conventional methods for identification of lactic acid bacteria, such as morphology, biochemical tests are time-consuming and sometimes lead to ambiguous results. Spectacular developments in the last 10 years in the field of sequencing of rRNA and genes coding for rRNA allow reliable identification and determination microbes by using different milecular biology methods. Each of these methods permits certain level of phylogenetic classification, from the genus, species, subspecies, biovar to the strain specific level. . Identifikacija i klasifikacija mikroorganizama zauzima veoma važno mesto u industrijskoj, medicinskoj, poljoprivrednoj i ekološkoj mikrobiolgoiji. Dugo vremena je taksonomija mikroorganizama smatrana dosadnom oblašću mikrobiologije. Mnoge bakterije mlečne kiseline imaju slične zahteve u pogledu ishrane i uslova uzgajanja, što dodatno otežava njihovu identifikaciju do sada poznatim klasičnim metodama. Klasični biohemijski testovi vremenski dugo traju, a tumačenje tako dobijenih rezultata ponekad dovodi do pogrešne determinacije. U poslednjih 10 godina, sekvenciranje rRNK i gena koji kodiraju rRNK, omogućena je identifikacija i klasifikacija mikroorganizama korišćenjem molekularnih metoda. Svaka od molekularnih metoda omogućava određen nivo filogenetske klasifikacije do vrste, podvrste, biovariteta i soja. .