Microbiota of autochthonous fermented product Sjenica sheepʼs ham

Sjenička ovčija stelja je autohtoni fermentisani proizvod od ovčijeg mesa koji se tradicionalno proizvodi u seoskim domaćinstvima na teritorijama opština Sjenica i Tutin. Proizvod se priprema prema jedinstvenoj recepturi i tehnologiji, od iskošćenih i oblikovanih ovčijih trupova autohtone rase ovaca sjenička pramenka koje su gajene na sjeničko-pešterskoj visoravni, bez dodataka starter kultura i aditiva. Za potrebe eksperimenta je korišćena sjenička ovčija stelja proizvedena u izabranim domaćinstvima sela Blato (A), Krajinoviće (B) i Rasno (V) sa područja Peštera u tri proizvodne sezone (2016/17 - 1; 2017/18 - 2; 2018/19 - 3), krajem jesenjeg i početkom prolećnog perioda, u toku 120 dana zrenja. U doktorskoj disertaciji definisani su parametri kvaliteta sjeničke očije stelje kroz ispitivanje hemijskog sastava i senzornih karakteristika proizvoda kao i kroz ispitivanje autohtone mikrobiote proizvoda i poređenje svih parametara kroz različite sezone, periode zrenja i domaćinstva. Ispitivanje sastava mikrobiote autohtonog proizvoda sjenička ovčija stelja je podrazumevalo ispitivanje prisustva i brojnosti aerobnih mezofilnih bakterija, bakterija mlečne kiseline (BMK), koagulaza-negativnih stafilokoka (KNS), bakterija iz familija Enterobacteriaceae i Pseudomonadaceae, vrsta iz roda Salmonella i Listeria monocytogenes kao i plesni. Identifikacija BMK i KNS je izvršena korišćenjem biohemijskih testova i MALDI-TOF masene spektrofotometrije. Izolacija plesni je izršena na Dihloran 18% glicerol agaru (DG18) sa površine uzoraka ovčije stelje, dok je identifikacija sprovedena prema ključevima za determinaciju. Ispitane su tehnološke osobine izolata BMK, KNS i plesni. Antimikrobni potencijal izolata BMK i KNS u odnosu na rast Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, Listeria monocytogenes ATCC 19115, Pseudomonas aeruginosa ATCC 27853 i Bacillus cereus ATCC 14579 je ispitan metodom difuzije iz bunarića. Izolati sa najboljim antimikrobnim potencijalom su odabrani za analizu uticaja enzima, pH, temperature i hemijskih jedinjenja na antimikrobnu aktivnost poluprečišćenih bakteriocina i kinetike rasta i biosinteze antimikrobnih jedinjenja. Za procenu probiotskog potencijala, odabrano je 42 BMK izolata. Izolati su ispitani na sposobnost tolerancije na uslove GIT-a (tolerancija na nisku pH, prisustvo pepsina, pankreatina i žučnih soli, prisustvo fenola), hidrofobnost, sposobnost autoagregacije i koagregacije i urađena je bezbednosna procena izolata kroz ispitivanje sposobnosti hemolize na krvnom agaru i rezistencije na antibiotike. Hemijskom analizom sjeničke ovčije stelje utrđeno je da uzorci ispunjavaju zahteve koji definišu propisi o kvalitetu proizvoda od mesa, kao i elaborata o zaštiti geografskog porekla sjenička očija stelja u pogledu sadržaja vode, masti, proteina mesa, soli i pepela. Promene pH vrednosti tokom tehnološkog procesa su odgovarale vrsti proizvoda. Na kraju zrenja pH vrednosti su se kretale u intervalima od 5,15 do 5,6, što ukazuje na odgovarajuću zrelost proizvoda. Tokom zrenja sjeničke ovčije stelje došlo je do promene aw vrednosti u proizvodu koje su ukazivale na mikrobiološku stabilnost tradicionalnog proivoda. Senzornom analizom nisu zabeležena oštećenja, mrlje i diskoloracije proizvoda, pri čemu je istaknuta specifičnost proizvoda po svim istraživanim senzornim parametima Rezultati ispitivanja mikrobiote istakli su dominaciju BMK, koje je pratilo prisustvo KNS i plesni. Prisusto bakterija iz familije Enterobacteriaceae i Pseudomonadaceae je potrđeno u toku procesa proizvodnje, dok na kraju procesa zrenja nisu detektovane. Prisustvo vrsta iz roda Salmonella i Listeria monocytogenes, nije detektovano ni u jednoj fazi proizvodnog procesa ovčije stelje u svim ispitivanim uzorcima iz svih sezona. Od ukupno 432 BMK izolata, identifikovano je ukupno 6 vrsta, od kojih je 3,83% pripadalo rodu Leuconostoc, 8,35% rodu Enterococcus, 2,03% rodu Lactiplantibacillus, a najveći procenat (87,58%) je pripadao rodu Lactobacillus. Od ukupno 376 izolata KNS identifikovano je 152 izolata (40,42%) kao Staphylococcus equorum, 103 izolata (27,39%), kao Staphylococcus xylosus, 64 izolata (17,2%) kao Staphylococcus carnosus i 46 izolata (12,23%) je identifikovano kao Staphylococcus saprophyticus. Značajan deo mikrobiote sjeničke očije stelje su činile plesni, ukupno 221 izolata, pri čemu je karakterizacijom i identifikacijom determinisano ukupno 4 roda: Aspergillus (2,71%), Eurotium (10,86%), Penicillium (83,71%) i Mucor (2,71%). Rast plesni iz ovčije stelje je bio pod direktnim uticajem aktivnosti vode u supstratu. Tehnološkom karakterizacijom izolata je utvrđeno da izolati iz roda Lactobacillus pokazuju sposobnost rasta na temperaturi do 45ºC kao i na svim testiranim koncentracijama soli i različitim pH vrednostima, bez proteolitičke i lipolitičke aktivnosti. Izolati L. plantarum i L. mesenteroides su uspešno rasli na temperaturi do 15ºC, koncentracijama soli do 6,5% i pri pH vrednostima iznad 5. Izolati L. mesenteroides su pokazali proteolitičku aktivnost. Izolati koji su pripadali vrstama E. faecium i E. faecalis su rasli na temperaturama do 15ºC i na svim koncentracijama soli, i pokazali su proteolitičku i lipolitičku aktivnost. E. faecium izolati su rasli na svim testiranim pH vrednostima, dok su izolati E. faecalis rasli na pH vrednostima većim od 6. Antimikrobnu aktivnost prema testiranim indikatorskim vrstama pokazalo je 47,61% testiranih BMK izolata, sa najvećom zonom inhibicije prema E. coli ATCC 25922. Najveću antilisterijsku aktivnost je pokazao E. faecium Ios4 sa zonom inhibicije od 25 mm. KNS izolati nisu pokazali antimikrobnu sposobnost prema odabranim indikatorskim vrstama. Antimikrobna aktivnost poluprečišćenih bakteriocina izolata L. curvatus IIos4, L. curvatus IIos6, L. sakei IIb11, L. sakei IIa13, L. mesenteroides IIos4i i E. faecium Ios4 se potpuno izgubila pod delovanjem proteolitičkih enzima, dok se dobra antimikrobna aktivnost polipeptida zadržala na pH 7. Prisustvo hemijskih jedinjenja nije uticalo na antimikrobnu aktivnost poluprečišćeih bakteriocina. Proizvodnja antimikrobnih jedinjenja je zapažena u eksponencijalnoj fazi rasta. Tokom produžene inkubacije u stacionarnoj fazi (nakon 30 h) aktivnost supernatanta je značajno opala, da bi nakon inkubacije od 48 h antimikrobna aktivnost prečišćenih bakteriocina nestala. Testirani izolati BMK su pokazali dobar probiotski potencijal. Izolati Lactobacillus spp. su pokazali visok nivo tolerancije na nisku pH (2 i 3), kao i sposobnost rasta u prisustvu žučnih soli. Izolati L. plantarum i E. faecalis su pokazali slabu toleranciju na nisku pH (2 i 3) i prisustvo žučnih soli. Odabrani izolati su pokazali dobru sposobnost preživljavanja u simuliranim uslovima želudca i tankog creva i visok nivo sposobnost rasta u prisustvu fenola. Hidrofobnost prema hloroformu, n-heksadekanu i ksilolu se kretala u rasponu od 5,2% do 80,9%. Sposobnost autoagregacije i koagregacije sa E. coli ATCC 25922, testiranih izolata BMK izolata je bila izolat specifična. Testirani izolati nisu pokazali sposobnost sinteze biogenih amina kao ni hemolizu na krvnom agaru. Apsolutnu rezistenciju na antibiotike nije pokazao ni jedan testirani izolat. Osetljivost na istraživane antibiotike zabeležena je kod 69,04% testiranih izolata. Rezultati dobijeni ovom disertacijom ukazuju na činjenicu da se autohtoni fermentisani proizvod sjenička očija stelja odlikuje svim specifičnostima kvaliteta suvomesnatog proizvoda, koji u mnogome zavisi od mikrobiote koja se u toku fermentacije razvija u proizvodu. Rezultati probiotskih i bezbedonosnih svojstava BMK izolata iz sjenčike ovčije stelje otvaraju mogućnost daljih istraživanja potencijalne primene određenih sojeva u mesnoj industriji kao startera za proizvodnju suvomesnatih proizvoda.Sjenica’s sheep ham is an autochthonous fermented sheep meat product that is traditionally produced in rural households in the municipalities of Sjenica and Tutin. The product is prepared according to a unique recipe and technology, from boneless and shaped sheep carcasses of the indigenous breed of sheep called Sjenička pramenka which were raised on the Sjenica-cave plateau, without the addition of starter cultures and additives. For the purposes of the experiment, Sjenica’s sheep ham produced in selected households in the villages of Blato (A), Krajinoviće (B), and Rasno (V) from the Pešter area in three production seasons was used (2016/17 - 1; 2017/18 - 2; 2018/19 - 3), at the end of autumn and the beginning of spring, during 120 days of ripening. This dissertation defines the quality parameters of Sjenica’s sheep ham by examining the chemical composition and sensory characteristics of the product as well as by examining the indigenous microbiota of the product and comparing all parameters through different seasons, maturation periods, and households. Examination of the microbiota composition of the autochthonous product Sjenica’s sheep ham included examination of the presence and abundance of aerobic mesophilic bacteria, lactic acid bacteria (LAB), coagulase-negative staphylococci (CNS), bacteria from the families Enterobacteriaceae and Pseudomonadaceae, species of the genera Salmonella and Listeria monocytogenes as well as molds. Identification of LAB and CNS was performed using biochemical tests and MALDI-TOF mass spectrophotometry. Mold isolation was performed on Dichloran 18% glycerol agar (DG18) from the surface of sheep litter samples, while identification was performed using identification keys. The technological properties of LAB and CNS isolates and molds were examined. The antimicrobial potential of LAB and CNS isolates against the growth of Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, Listeria monocytogenes ATCC 19115, Pseudomonas aeruginosa ATCC 27853 and Bacillus cereus ATCC 14579 was examined by well diffusion method. Isolates with the best antimicrobial potential were selected to analyze the influence of enzymes, pH, temperature, and chemical compounds on the antimicrobial activity of semi-purified bacteriocins and the kinetics of growth and biosynthesis of antimicrobial compounds. To assess the probiotic potential, 42 LAB isolates were selected. Isolates were tested for their ability to tolerate GIT conditions (tolerance to low pH, presence of pepsin, pancreatin, and bile salts, presence of phenols), hydrophobicity, ability to autoaggregation and co-aggregation, and safety assessment of isolates was performed by testing blood agar hemolysis and resistance on antibiotics. Chemical analysis of Sjenica’s sheep ham determined that the samples meet the requirements defined by the regulations on the quality of meat products, as well as the study on the protection of geographical origin of Sjenica’s sheep ham in terms of water, fat, meat protein, salt, and ash. Changes in pH values during the technological process correspond to the type of product. At the end of ripening, the pH values ranged from 5.15 to 5.6, which indicates the appropriate maturity of the product. During the ripening of the Sjenica’s sheep ham, there was a change in aw value of the product, which indicated the microbiological stability of the traditional product. The sensory analysis did not show damage, stains, and discoloration of the product, emphasizing the specificity of the product for all examined sensory parameters. The results of microbiota testing highlighted the dominance of LAB, which was accompanied by the presence of CNS and mold. The presence of bacteria from the families Enterobacteriaceae and Pseudomonadaceae was confirmed during the production process, while at the end of the maturation process, they were not detected. The presence of species from the genera Salmonella and Listeria monocytogenes was not detected at any stage of the production process of sheep ham in all tested samples from all seasons. Out of a total of 432 LAB isolates, a total of 6 species were identified, of which 3.83% belonged to the genus Leuconostoc, 8.35% to the genus Enterococcus, 2.03% to the genus Lactiplantibacillus and the largest percentage (87.58%) belonged to the genus Lactobacillus. Out of a total of 376 CNS isolates, 152 isolates (40.42%) were identified as Staphylococcus equorum, 103 isolates (27.39%) as Staphylococcus xylosus, 64 isolates (17.2%) as Staphylococcus carnosus and 46 isolates (12.23 %) was identified as Staphylococcus saprophyticus. A significant part of the microbiota of the Sjenica’s sheep ham consisted of molds, a total of 221 mold isolates, with the characterization and identification of a total of 4 genera: Aspergillus (2.71%), Eurotium (10.86%), Penicillium (83.71%) and Mucor (2.71%). The growth of mold from sheep ham was directly affected by water activity in the substrate. Technological characterization of isolates has determined that isolates of the genus Lactobacillus show the ability to grow at temperatures up to 45ºC as well as at all tested salt concentrations and different pH values, without proteolytic and lipolytic activity. Isolates of L. plantarum and L. mesenteroides successfully grew at temperatures up to 15ºC, salt concentrations up to 6.5%, and pH values above 5. Isolates of L. mesenteroides showed proteolytic activity. Isolates belonging to E. faecium and E. faecalis species grew at temperatures up to 15ºC and at all salt concentrations and showed proteolytic and lipolytic activity. E. faecium isolates grew at all tested pH values, while E. faecalis isolates grew at pH values greater than 6. Antimicrobial activity according to the tested indicator species was shown by 47.61% of tested LAB isolates, with the highest zone of inhibition according to E. coli ATCC 25922. The highest antilisterial activity was shown by E. faecium Ios4 with a zone of inhibition of 25 mm. CNS isolates did not show antimicrobial ability according to the selected indicator species. The antimicrobial activity of semi-purified bacteriocins of L. curvatus IIos4, L. curvatus IIos6, L. sakei IIb11, L. sakei IIa13, L. mesenteroides IIos4i, and E. faecium Ios4 isolates was completely lost by proteolytic enzymes, while the good antimicrobial activity of the polypeptide was maintained at pH 7. The presence of chemical compounds did not affect the antimicrobial activity of semi-purifying bacteriocins. The production of antimicrobial compounds was observed in the exponential growth phase. During the prolonged incubation in the stationary phase (after 30 h) the activity of the supernatant decreased significantly, and after the incubation of 48 h, the antimicrobial activity of the purified bacteriocins disappeared. LAB isolates testing showed good probiotic potential. Isolates of Lactobacillus spp. showed a high level of tolerance to low pH (2 and 3), as well as the ability to grow in the presence of bile salts. Isolates of L. plantarum and E. faecalis showed poor tolerance to low pH (2 and 3) and the presence of bile salts. Selected isolates showed good survival ability in simulated gastric and small intestine conditions and a high level of growth ability in the presence of phenol. Hydrophobicity towards chloroform, n-hexadecane, and xylene ranged from 5.2% to 80.9%. The ability of auto-aggregation and coaggregation with E. coli ATCC 25922, tested isolates of LAB isolates was isolate specific. The tested isolates did not show the ability to synthesize biogenic amines or hemolysis on blood agar. None of the tested isolates showed absolute antibiotic resistance. Susceptibility to the studied antibiotics was observed in 69.04% of the tested isolates. The results obtained by this dissertation indicate the fact that the autochthonous fermented product Sjenica’s sheep ham is characterized by all the specifics of the quality of cured meat product, which largely depends on the microbiota that develops in the product during fermentation. The results of probiotics and safety properties of LAB isolates from the shade of sheep litter open the possibility of further research into the potential application of certain strains in the meat industry as a starter to produce meat products

In vitro procjena antimikrobnog potencijala bakterije Streptococcus uberis izolirane iz autohtonog sira jugoistočne Srbije

Streptococcus uberis is an environmental bacterium responsible for bovine mastitis. It is occasionally described as a human pathogen. In our study, the isolation was undertaken of lactic acid bacteria from a local cheese from Southeastern Serbia, produced in a traditional way. S. uberis (7 isolates) and S. agalactiae (1 isolate) were isolated from the cheese samples taken in the summer. The biochemical and physiological characteristics of the isolates were examined. Using tetracycline, chloramphenicol, novobiocin and rifampicin, the antibiotic susceptibility of the isolates was evaluated. The results demonstrated that all the isolates were susceptible to all the tested antibiotics, with a growth inhibition zone from 36-48 mm. Also, the antagonism was examined of S. uberis KGPMF1-7 and S. agalactiae KFPMF8 isolates on the growth of Escherichia coli ATCC 25922, Proteus mirabilis ATCC 12453, Klebsiella oxytoca KGPMF1, Klebsiella ornithinolytica KGPMF8 and Aeromonas hydrophila, as indicator stains. The results were compared with the activities of chloramphenicol, streptomycin and tetracycline on the tested indicator stains. The strongest antagonism was demonstrated by all Streptococcus isolates on the growth of K. oxytoca KGPMF1 (growth inhibition zone from 12-20 mm) and the A. hydrophila (growth inhibition zone from 13-20 mm). When these results were compared with the results of the sensitivity of tested indicator stains to antibiotics, S. uberis KGPMF1-7 and S. agalactiae KGPMF8 isolates showed a moderate antagonistic effect. Due to the specific way cheese is made in from Sokobanja, these isolates probably originate from cows’ udders.Streptococcus uberis jest okolišna bakterija odgovorna za mastitis kod goveda, a povremeno se opisuje kao patogen u ljudi. U ovom su istraživanju izolirane bakterije mliječne kiseline iz autohtonog sira jugoistočne Srbije, proizvedenog na tradicionalni način. S. uberis (7 izolata) i S. agalactiae (1 izolat) izolirani su iz uzoraka sira prikupljenih tijekom ljeta. Analizirane su biokemijske i fiziološke značajke izolata. Koristeći se tetraciklinom, kloramfenikolom, novobiocinom i rifampicinom, procijenjena je osjetljivost izolata na antibiotike. Rezultati su pokazali da su svi izolati osjetljivi na sve istražene antibiotike, sa zonom inhibicije rasta od 36 do 48 mm. Također, istražen je antagonizam S. uberis KGPMF1-7 i S. agalactiae KFPMF8 izolata na rast Escherichia coli ATCC 25922, Proteus mirabilis ATCC 12453, Klebsiella oxytoca KGPMF1, Klebsiella ornithinolytica KGPMF8 i Aeromonas hydrophila kao indikatorskih sojeva. Rezultati su uspoređeni s aktivnostima kloramfenikola, streptomicina i tetraciklina na testiranim indikatorskim sojevima. Najjači antagonizam pokazali su svi Streptococcus izolati na rast K. oxytoca KGPMF1 (zona inhibicije rasta od 12 do 20 mm) i A. hydrophila (zona inhibicije rasta od 13 do 20 mm). Usporedbom tih rezultata s rezultatima osjetljivosti istraženih indikatorskih sojeva na antibiotike, za S. uberis KGPMF1-7 i S. agalactiae KGPMF8 izolate utvrđen je umjeren antagonistički učinak. Ti izolati vjerojatno potječu iz vimena krava i u siru iz Sokobanje prisutni su zbog specifičnosti njegove pripreme

Antagonistički potencijal i sposobnost stvaranja biofilma bakterija Enterococcus spp. izoliranih iz sira u Srbiji

In this paper, the presence, biochemical and physiological characteristics of the members of genus Enterococcus, isolated from traditionally made Serbian cheese, were investigated. The members were identified as: Enterococcus faecium (8 isolates), E. faecalis (14 isolates), E. hirae (4 isolates) and E. durans (4 isolates), using the biochemical tests and MALDI-TOF mass spectrometry. All the tested isolates showed good acidification ability in pure and enriched milk. The antagonism of enterococci on the growth of Escherichia coli ATCC 25922, Proteus mirabilis ATCC 12453, Klebsiella oxytoca KGPMF1, Klebsiella ornithinolytica KGPMF8 and Aeromonas hydrophila, as well as their ability to form biofilms, were examined. The tested isolates showed moderate inhibitory activity (10-22 mm) on the growth of the indicator strains. Among all the isolates, only E. hirae KGPMF9 and E. faecium KGPMF14 showed the ability of biofilm formation. The results provide a basis for further research into the possible practical application of the isolated enterococci.U ovom je radu istražena prisutnost te biokemijske i fiziološke značajke bakterija iz roda Enterococcus, izoliranih iz domaćeg sira u Srbiji, proizvedenoga na tradicionalan način. Biokemijskim testom i masenom spektrometrijom MALDI-TOF dokazani su Enterococcus faecium (8 izolata), E. faecalis (14 izolata), E. hirae (4 izolata) i E. durans (4 izolata). Svi su analizirani izolati pokazali dobru sposobnost acidifikacije u sirovu i obogaćenom mlijeku. Istraženi su antagonizam enterokoka na rast bakterija Escherichia coli ATCC 25922, Proteus mirabilis ATCC 12453, Klebsiella oxytoca KGPMF1, Klebsiella ornithinolytica KGPMF8 i Aeromonas hydrophila te njihova sposobnost stvaranja biofilma. Analizirani izolati pokazali su umjerenu inhibicijsku aktivnost (10 - 22 mm) u rastu navedenih sojeva. Među izolatima samo su E. hirae KGPMF9 i E. faecium KGPMF14 pokazali sposobnost stvaranja biofilma. Rezultati ove studije daju temelj za daljnja istraživanja mogućnosti praktične primjene izoliranih enterokoka

Quality and autochthonous microbiota of dry-cured sheep ham from western Balkans

Dry-cured sheep ham is a traditional product of Western Balkans. It is prepared by dry curing specially treated whole sheep carcasses which are smoked for a short time and spontaneously fermented in air under uncontrolled conditions. Lactic acid bacteria (LAB) play a key role in defining the quality and organoleptic characteristics of dry-cured sheep ham. The aim of this research was to investigate the chemical parameters of drycured sheep ham quality as well as the isolation and preliminary categorization of LAB. To this end, samples of dry-cured sheep ham were obtained from nine sheep of average age of about five years, from three households from the geographical area Sjenica (Western Serbia). Physicochemical analysis has determined the content of water, protein, fat, mineral matter, water activity and pH values in the product. Phenotypic characterization of LAB isolated from dry-cured sheep ham was based on the general morphology of the cell, physiological tests and sugar fermentation patterns of LAB isolates. 124 isolates of LAB were preliminary identified as Lactobacillus curvatus , Lactobacillus sakei and Enterococcus faecium . Chemical analysis confirmed a harmonious relationship between the quality parameters of dry-cured sheep ham.Author's versio

Organic sunflower honey from the area of Banat (northeastern Serbia) - physicochemical and microbiological characterizations

Today, special attention is paid to organic honey due to increasing pollution and numerous toxins, and certified organic honey is described as having no chemical pollution, including that related to bee migration in search of good flowers that beekeepers do not directly control. This study aimed to characterize through analysis 5 samples of organic sunflower honey from Banat (Serbia): physicochemical quality parameters (moisture, HMF, diastase activity, free acidity, glucose and fructose content, sucrose and ash) and microbiological safety (presence of aerobic mesophilic bacteria, coliforms, lactic acid bacteria, molds and yeasts). Mean values obtained for physicochemical parameters are: 16.52% humidity, 8.17 mg/kg HMF, 10.8 diastasis activity, 24.07 meq/kg of free acidity, 60.8% of glucose and fructose 0.5% sucrose and 0.01% ash. The microbiota that was isolated from all the samples tested consisted of Bacillus spp. ranging from 0.50-0.55 x10-2 cfu/g and Saccharomyces spp., ranging from 0.31-0.44 x10-2 cfu/g. MALDI TOF confirmed preliminary identification to be Bacillus pumilus, while yeasts were identified as Saccharomyces cerevisiae. This research could contribute to the valorization of sunflower honey, which would lead to standardization and increase the production of this product

The influence of environmental factors on the planktonic growth and biofilm formation of Escherichia coli

In this study, the effects of environmental factors (different media, temperature, pH, salt and sugar concentrations) on the planktonic growth, biofilm formation and formed biofilm of Escherichia coli KGPMF 16 and Escherichia coli KGPMF 17 were investigated. Tested bacteria were isolated from traditionally made cheese produced in Southeastern Serbia (Sokobanja region). The influence on planktonic growth, biofilm formation and formed biofilm was determined using spectrophotometric method. The limiting factors for the planktonic growth and biofilm formation were temperature of 4 °C and all tested concentrations of salt. The growth of tested bacteria was higher in media enriched with lactose than in media containing glucose. TSB was more congenial media to the planktonic growth of bacteria than MHB broth. None of the tested bacteria demonstrated the ability to form biofilm at 4 °C and 44 °C. Only E. coli KGPMF 17 showed ability to form biofilm in TSB at 37 °C. Different concentrations of salt, glucose and lactose exhibited inhibitory effect on biofilm formation, but all tested concentrations of lactose showed stimulating effect on formed biofilm of E. coli KGPMF 17. These results contribute to better understanding of the effects of environmental factors on the development of E. coli in cheese

Dry-fermented sausage as probiotic carrier food

The aim of this research is to explore the potential use of Lactobacillus curvatus and Enterococcus faecium isolated from traditional fermented sausage from Southeast Serbia as probiotics. Analytic criteria for determining the probiotic potential included haemolysis, autoagreggation and coaggregation tests as well as the test for hydrophobicity of natural isolates. The best autoaggregation ability was observed in Lb. curvatus, sk217 and sk65 (73%) while the lowest level of grouping was shown by the isolate sk110 (68%), as the coaggregation ability ranged from 34% to 58%. The value of autoaggregation of examined strains of enterococci ranged from 47% to 55.2%, while coaggregation ranged from 31.9% to 45.2%. Haemolytic reaction was not observed in researched isolates. Hydrophobicity was determined by bacterial adherence to hydrocarbons, nhexadecane, xylene and chloroform. All isolates possessed a high level of hydrophobicity. The results of this study of probiotic characteristics of Lactobacillus curvatus and Enterococcus faecium indicate that the tested strains can be recommended as good candidates for use in the food industry.Publishe

Lactobacillus curvatus from fermented sausages as new probiotic functional foods

This research examined in vitro probiotic potential of Lactobacillus curvatus isolates from fermented sausage originating in Southeast Serbia which was prepared using traditional production techniques. Strains of L. curvatus were isolated and identified at the level of species using API 50CHL sets and 16s rDNA sequencing, after which their probiotic potential was measured. The research consisted of the examination of isolate survival rates in the gastrointestinal tract, synthesis of biogenic amines, the growth on the media with different quantities of phenol, antibiotic resistance test, and antimicrobial activity against clinical strains of Pseudomonas spp., Proteus spp., Enterobacter spp., Escherichia coli and Listeria monocytogenes. The examined isolates have shown a good survival rate at low pH in the simulated stomach and small intestine conditions. Synthesis of biogenic amines was exhibited by the isolate sk1-10, which had a positive reaction to histidine. Analyzed isolates grew well on media with 0.1% and 0.2% of phenol, L.curvatus sk1-8 and sk5-2 have grown in media with 0.3% phenol. All tested isolates have exhibited high sensitivity to most analyzed antibiotics (tetracycline, amoxicillin, ceftriaxone, erythromycin, penicillin), resistance to ofloxacin, and cephalexin isolates sk4-3a and sk4-15. The examined isolates have shown a broad inhibitory spectrum against pathogens.Publishe

Microbiota of autochthonous fermented product Sjenica sheepʼs ham

Sjenička ovčija stelja je autohtoni fermentisani proizvod od ovčijeg mesa koji se tradicionalno proizvodi u seoskim domaćinstvima na teritorijama opština Sjenica i Tutin. Proizvod se priprema prema jedinstvenoj recepturi i tehnologiji, od iskošćenih i oblikovanih ovčijih trupova autohtone rase ovaca sjenička pramenka koje su gajene na sjeničko-pešterskoj visoravni, bez dodataka starter kultura i aditiva. Za potrebe eksperimenta je korišćena sjenička ovčija stelja proizvedena u izabranim domaćinstvima sela Blato (A), Krajinoviće (B) i Rasno (V) sa područja Peštera u tri proizvodne sezone (2016/17 - 1; 2017/18 - 2; 2018/19 - 3), krajem jesenjeg i početkom prolećnog perioda, u toku 120 dana zrenja. U doktorskoj disertaciji definisani su parametri kvaliteta sjeničke očije stelje kroz ispitivanje hemijskog sastava i senzornih karakteristika proizvoda kao i kroz ispitivanje autohtone mikrobiote proizvoda i poređenje svih parametara kroz različite sezone, periode zrenja i domaćinstva. Ispitivanje sastava mikrobiote autohtonog proizvoda sjenička ovčija stelja je podrazumevalo ispitivanje prisustva i brojnosti aerobnih mezofilnih bakterija, bakterija mlečne kiseline (BMK), koagulaza-negativnih stafilokoka (KNS), bakterija iz familija Enterobacteriaceae i Pseudomonadaceae, vrsta iz roda Salmonella i Listeria monocytogenes kao i plesni. Identifikacija BMK i KNS je izvršena korišćenjem biohemijskih testova i MALDI-TOF masene spektrofotometrije. Izolacija plesni je izršena na Dihloran 18% glicerol agaru (DG18) sa površine uzoraka ovčije stelje, dok je identifikacija sprovedena prema ključevima za determinaciju. Ispitane su tehnološke osobine izolata BMK, KNS i plesni. Antimikrobni potencijal izolata BMK i KNS u odnosu na rast Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, Listeria monocytogenes ATCC 19115, Pseudomonas aeruginosa ATCC 27853 i Bacillus cereus ATCC 14579 je ispitan metodom difuzije iz bunarića. Izolati sa najboljim antimikrobnim potencijalom su odabrani za analizu uticaja enzima, pH, temperature i hemijskih jedinjenja na antimikrobnu aktivnost poluprečišćenih bakteriocina i kinetike rasta i biosinteze antimikrobnih jedinjenja. Za procenu probiotskog potencijala, odabrano je 42 BMK izolata. Izolati su ispitani na sposobnost tolerancije na uslove GIT-a (tolerancija na nisku pH, prisustvo pepsina, pankreatina i žučnih soli, prisustvo fenola), hidrofobnost, sposobnost autoagregacije i koagregacije i urađena je bezbednosna procena izolata kroz ispitivanje sposobnosti hemolize na krvnom agaru i rezistencije na antibiotike. Hemijskom analizom sjeničke ovčije stelje utrđeno je da uzorci ispunjavaju zahteve koji definišu propisi o kvalitetu proizvoda od mesa, kao i elaborata o zaštiti geografskog porekla sjenička očija stelja u pogledu sadržaja vode, masti, proteina mesa, soli i pepela. Promene pH vrednosti tokom tehnološkog procesa su odgovarale vrsti proizvoda. Na kraju zrenja pH vrednosti su se kretale u intervalima od 5,15 do 5,6, što ukazuje na odgovarajuću zrelost proizvoda. Tokom zrenja sjeničke ovčije stelje došlo je do promene aw vrednosti u proizvodu koje su ukazivale na mikrobiološku stabilnost tradicionalnog proivoda. Senzornom analizom nisu zabeležena oštećenja, mrlje i diskoloracije proizvoda, pri čemu je istaknuta specifičnost proizvoda po svim istraživanim senzornim parametima Rezultati ispitivanja mikrobiote istakli su dominaciju BMK, koje je pratilo prisustvo KNS i plesni. Prisusto bakterija iz familije Enterobacteriaceae i Pseudomonadaceae je potrđeno u toku procesa proizvodnje, dok na kraju procesa zrenja nisu detektovane. Prisustvo vrsta iz roda Salmonella i Listeria monocytogenes, nije detektovano ni u jednoj fazi proizvodnog procesa ovčije stelje u svim ispitivanim uzorcima iz svih sezona. Od ukupno 432 BMK izolata, identifikovano je ukupno 6 vrsta, od kojih je 3,83% pripadalo rodu Leuconostoc, 8,35% rodu Enterococcus, 2,03% rodu Lactiplantibacillus, a najveći procenat (87,58%) je pripadao rodu Lactobacillus. Od ukupno 376 izolata KNS identifikovano je 152 izolata (40,42%) kao Staphylococcus equorum, 103 izolata (27,39%), kao Staphylococcus xylosus, 64 izolata (17,2%) kao Staphylococcus carnosus i 46 izolata (12,23%) je identifikovano kao Staphylococcus saprophyticus. Značajan deo mikrobiote sjeničke očije stelje su činile plesni, ukupno 221 izolata, pri čemu je karakterizacijom i identifikacijom determinisano ukupno 4 roda: Aspergillus (2,71%), Eurotium (10,86%), Penicillium (83,71%) i Mucor (2,71%). Rast plesni iz ovčije stelje je bio pod direktnim uticajem aktivnosti vode u supstratu. Tehnološkom karakterizacijom izolata je utvrđeno da izolati iz roda Lactobacillus pokazuju sposobnost rasta na temperaturi do 45ºC kao i na svim testiranim koncentracijama soli i različitim pH vrednostima, bez proteolitičke i lipolitičke aktivnosti. Izolati L. plantarum i L. mesenteroides su uspešno rasli na temperaturi do 15ºC, koncentracijama soli do 6,5% i pri pH vrednostima iznad 5. Izolati L. mesenteroides su pokazali proteolitičku aktivnost. Izolati koji su pripadali vrstama E. faecium i E. faecalis su rasli na temperaturama do 15ºC i na svim koncentracijama soli, i pokazali su proteolitičku i lipolitičku aktivnost. E. faecium izolati su rasli na svim testiranim pH vrednostima, dok su izolati E. faecalis rasli na pH vrednostima većim od 6. Antimikrobnu aktivnost prema testiranim indikatorskim vrstama pokazalo je 47,61% testiranih BMK izolata, sa najvećom zonom inhibicije prema E. coli ATCC 25922. Najveću antilisterijsku aktivnost je pokazao E. faecium Ios4 sa zonom inhibicije od 25 mm. KNS izolati nisu pokazali antimikrobnu sposobnost prema odabranim indikatorskim vrstama. Antimikrobna aktivnost poluprečišćenih bakteriocina izolata L. curvatus IIos4, L. curvatus IIos6, L. sakei IIb11, L. sakei IIa13, L. mesenteroides IIos4i i E. faecium Ios4 se potpuno izgubila pod delovanjem proteolitičkih enzima, dok se dobra antimikrobna aktivnost polipeptida zadržala na pH 7. Prisustvo hemijskih jedinjenja nije uticalo na antimikrobnu aktivnost poluprečišćeih bakteriocina. Proizvodnja antimikrobnih jedinjenja je zapažena u eksponencijalnoj fazi rasta. Tokom produžene inkubacije u stacionarnoj fazi (nakon 30 h) aktivnost supernatanta je značajno opala, da bi nakon inkubacije od 48 h antimikrobna aktivnost prečišćenih bakteriocina nestala. Testirani izolati BMK su pokazali dobar probiotski potencijal. Izolati Lactobacillus spp. su pokazali visok nivo tolerancije na nisku pH (2 i 3), kao i sposobnost rasta u prisustvu žučnih soli. Izolati L. plantarum i E. faecalis su pokazali slabu toleranciju na nisku pH (2 i 3) i prisustvo žučnih soli. Odabrani izolati su pokazali dobru sposobnost preživljavanja u simuliranim uslovima želudca i tankog creva i visok nivo sposobnost rasta u prisustvu fenola. Hidrofobnost prema hloroformu, n-heksadekanu i ksilolu se kretala u rasponu od 5,2% do 80,9%. Sposobnost autoagregacije i koagregacije sa E. coli ATCC 25922, testiranih izolata BMK izolata je bila izolat specifična. Testirani izolati nisu pokazali sposobnost sinteze biogenih amina kao ni hemolizu na krvnom agaru. Apsolutnu rezistenciju na antibiotike nije pokazao ni jedan testirani izolat. Osetljivost na istraživane antibiotike zabeležena je kod 69,04% testiranih izolata. Rezultati dobijeni ovom disertacijom ukazuju na činjenicu da se autohtoni fermentisani proizvod sjenička očija stelja odlikuje svim specifičnostima kvaliteta suvomesnatog proizvoda, koji u mnogome zavisi od mikrobiote koja se u toku fermentacije razvija u proizvodu. Rezultati probiotskih i bezbedonosnih svojstava BMK izolata iz sjenčike ovčije stelje otvaraju mogućnost daljih istraživanja potencijalne primene određenih sojeva u mesnoj industriji kao startera za proizvodnju suvomesnatih proizvoda.Sjenica’s sheep ham is an autochthonous fermented sheep meat product that is traditionally produced in rural households in the municipalities of Sjenica and Tutin. The product is prepared according to a unique recipe and technology, from boneless and shaped sheep carcasses of the indigenous breed of sheep called Sjenička pramenka which were raised on the Sjenica-cave plateau, without the addition of starter cultures and additives. For the purposes of the experiment, Sjenica’s sheep ham produced in selected households in the villages of Blato (A), Krajinoviće (B), and Rasno (V) from the Pešter area in three production seasons was used (2016/17 - 1; 2017/18 - 2; 2018/19 - 3), at the end of autumn and the beginning of spring, during 120 days of ripening. This dissertation defines the quality parameters of Sjenica’s sheep ham by examining the chemical composition and sensory characteristics of the product as well as by examining the indigenous microbiota of the product and comparing all parameters through different seasons, maturation periods, and households. Examination of the microbiota composition of the autochthonous product Sjenica’s sheep ham included examination of the presence and abundance of aerobic mesophilic bacteria, lactic acid bacteria (LAB), coagulase-negative staphylococci (CNS), bacteria from the families Enterobacteriaceae and Pseudomonadaceae, species of the genera Salmonella and Listeria monocytogenes as well as molds. Identification of LAB and CNS was performed using biochemical tests and MALDI-TOF mass spectrophotometry. Mold isolation was performed on Dichloran 18% glycerol agar (DG18) from the surface of sheep litter samples, while identification was performed using identification keys. The technological properties of LAB and CNS isolates and molds were examined. The antimicrobial potential of LAB and CNS isolates against the growth of Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, Listeria monocytogenes ATCC 19115, Pseudomonas aeruginosa ATCC 27853 and Bacillus cereus ATCC 14579 was examined by well diffusion method. Isolates with the best antimicrobial potential were selected to analyze the influence of enzymes, pH, temperature, and chemical compounds on the antimicrobial activity of semi-purified bacteriocins and the kinetics of growth and biosynthesis of antimicrobial compounds. To assess the probiotic potential, 42 LAB isolates were selected. Isolates were tested for their ability to tolerate GIT conditions (tolerance to low pH, presence of pepsin, pancreatin, and bile salts, presence of phenols), hydrophobicity, ability to autoaggregation and co-aggregation, and safety assessment of isolates was performed by testing blood agar hemolysis and resistance on antibiotics. Chemical analysis of Sjenica’s sheep ham determined that the samples meet the requirements defined by the regulations on the quality of meat products, as well as the study on the protection of geographical origin of Sjenica’s sheep ham in terms of water, fat, meat protein, salt, and ash. Changes in pH values during the technological process correspond to the type of product. At the end of ripening, the pH values ranged from 5.15 to 5.6, which indicates the appropriate maturity of the product. During the ripening of the Sjenica’s sheep ham, there was a change in aw value of the product, which indicated the microbiological stability of the traditional product. The sensory analysis did not show damage, stains, and discoloration of the product, emphasizing the specificity of the product for all examined sensory parameters. The results of microbiota testing highlighted the dominance of LAB, which was accompanied by the presence of CNS and mold. The presence of bacteria from the families Enterobacteriaceae and Pseudomonadaceae was confirmed during the production process, while at the end of the maturation process, they were not detected. The presence of species from the genera Salmonella and Listeria monocytogenes was not detected at any stage of the production process of sheep ham in all tested samples from all seasons. Out of a total of 432 LAB isolates, a total of 6 species were identified, of which 3.83% belonged to the genus Leuconostoc, 8.35% to the genus Enterococcus, 2.03% to the genus Lactiplantibacillus and the largest percentage (87.58%) belonged to the genus Lactobacillus. Out of a total of 376 CNS isolates, 152 isolates (40.42%) were identified as Staphylococcus equorum, 103 isolates (27.39%) as Staphylococcus xylosus, 64 isolates (17.2%) as Staphylococcus carnosus and 46 isolates (12.23 %) was identified as Staphylococcus saprophyticus. A significant part of the microbiota of the Sjenica’s sheep ham consisted of molds, a total of 221 mold isolates, with the characterization and identification of a total of 4 genera: Aspergillus (2.71%), Eurotium (10.86%), Penicillium (83.71%) and Mucor (2.71%). The growth of mold from sheep ham was directly affected by water activity in the substrate. Technological characterization of isolates has determined that isolates of the genus Lactobacillus show the ability to grow at temperatures up to 45ºC as well as at all tested salt concentrations and different pH values, without proteolytic and lipolytic activity. Isolates of L. plantarum and L. mesenteroides successfully grew at temperatures up to 15ºC, salt concentrations up to 6.5%, and pH values above 5. Isolates of L. mesenteroides showed proteolytic activity. Isolates belonging to E. faecium and E. faecalis species grew at temperatures up to 15ºC and at all salt concentrations and showed proteolytic and lipolytic activity. E. faecium isolates grew at all tested pH values, while E. faecalis isolates grew at pH values greater than 6. Antimicrobial activity according to the tested indicator species was shown by 47.61% of tested LAB isolates, with the highest zone of inhibition according to E. coli ATCC 25922. The highest antilisterial activity was shown by E. faecium Ios4 with a zone of inhibition of 25 mm. CNS isolates did not show antimicrobial ability according to the selected indicator species. The antimicrobial activity of semi-purified bacteriocins of L. curvatus IIos4, L. curvatus IIos6, L. sakei IIb11, L. sakei IIa13, L. mesenteroides IIos4i, and E. faecium Ios4 isolates was completely lost by proteolytic enzymes, while the good antimicrobial activity of the polypeptide was maintained at pH 7. The presence of chemical compounds did not affect the antimicrobial activity of semi-purifying bacteriocins. The production of antimicrobial compounds was observed in the exponential growth phase. During the prolonged incubation in the stationary phase (after 30 h) the activity of the supernatant decreased significantly, and after the incubation of 48 h, the antimicrobial activity of the purified bacteriocins disappeared. LAB isolates testing showed good probiotic potential. Isolates of Lactobacillus spp. showed a high level of tolerance to low pH (2 and 3), as well as the ability to grow in the presence of bile salts. Isolates of L. plantarum and E. faecalis showed poor tolerance to low pH (2 and 3) and the presence of bile salts. Selected isolates showed good survival ability in simulated gastric and small intestine conditions and a high level of growth ability in the presence of phenol. Hydrophobicity towards chloroform, n-hexadecane, and xylene ranged from 5.2% to 80.9%. The ability of auto-aggregation and coaggregation with E. coli ATCC 25922, tested isolates of LAB isolates was isolate specific. The tested isolates did not show the ability to synthesize biogenic amines or hemolysis on blood agar. None of the tested isolates showed absolute antibiotic resistance. Susceptibility to the studied antibiotics was observed in 69.04% of the tested isolates. The results obtained by this dissertation indicate the fact that the autochthonous fermented product Sjenica’s sheep ham is characterized by all the specifics of the quality of cured meat product, which largely depends on the microbiota that develops in the product during fermentation. The results of probiotics and safety properties of LAB isolates from the shade of sheep litter open the possibility of further research into the potential application of certain strains in the meat industry as a starter to produce meat products

The evolution of lactic acid bacteria community during the development of mature sourdough

In order to follow the composition and changes in lactic acid bacteria (LAB) population of rye flour sourdough that was continuously propagated by a repeated inoculation, sixty-two strains of LAB were isolated and characterized. The LAB were the only bacteria detected, both at the end of the second propagation step and in the stage of mature sourdough (after two weeks of continuous daily refreshment). The stable ecological system in rye sourdough could be established from the second propagation step onward. The predominant genera of LAB during the development of sourdough were lactobacilli, which were grouped in eight clusters. Heterofermentative lactobacilli were in majority in both propagation step two and a mature sourdough participating 56% and 70% of total bacterial count, respectively. The identification based on a phenotypic characterization that was carried out by using a set of 36 tests, showed that the lactobacilli contained in the two sourdough steps did not clearly belong to any known species of the genus Lactobacillus. In addition, the structure of the bacterial population were monitored by two statistical techniques (Hierachical Cluster Analysis and Principal Component Analysis), being applied to phenotypical characteristics of the isolates