Naslov (srp)

Mikrobiota autohtonog fermentisanog proizvoda sjenička ovčija stelja

Autor

Žugić-Petrović, Tanja, 1976-

Doprinosi

Grujović, Мirjana Ž., 1990-
Mladenović, Katarina, 1990-
Tomović, Vladimir M., 1974-
Stefanović, Olgica, 1979-
Joković, Nataša, 1972-
Kocić-Tanackov, Sunčica, 1974-

Opis (srp)

Sjenička ovčija stelja je autohtoni fermentisani proizvod od ovčijeg mesakoji se tradicionalno proizvodi u seoskim domaćinstvima na teritorijama opštinaSjenica i Tutin. Proizvod se priprema prema jedinstvenoj recepturi i tehnologiji,od iskošćenih i oblikovanih ovčijih trupova autohtone rase ovaca sjeničkapramenka koje su gajene na sjeničko-pešterskoj visoravni, bez dodataka starterkultura i aditiva. Za potrebe eksperimenta je korišćena sjenička ovčija steljaproizvedena 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 doktorskojdisertaciji definisani su parametri kvaliteta sjeničke očije stelje krozispitivanje hemijskog sastava i senzornih karakteristika proizvoda kao i krozispitivanje autohtone mikrobiote proizvoda i poređenje svih parametara krozrazličite sezone, periode zrenja i domaćinstva. Ispitivanje sastava mikrobioteautohtonog proizvoda sjenička ovčija stelja je podrazumevalo ispitivanje prisustva ibrojnosti aerobnih mezofilnih bakterija, bakterija mlečne kiseline (BMK),koagulaza-negativnih stafilokoka (KNS), bakterija iz familija Enterobacteriaceae iPseudomonadaceae, vrsta iz roda Salmonella i Listeria monocytogenes kao i plesni.Identifikacija BMK i KNS je izvršena korišćenjem biohemijskih testova iMALDI-TOF masene spektrofotometrije. Izolacija plesni je izršena na Dihloran18% glicerol agaru (DG18) sa površine uzoraka ovčije stelje, dok je identifikacijasprovedena prema ključevima za determinaciju. Ispitane su tehnološke osobineizolata BMK, KNS i plesni. Antimikrobni potencijal izolata BMK i KNS u odnosuna rast Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, Listeriamonocytogenes ATCC 19115, Pseudomonas aeruginosa ATCC 27853 i Bacillus cereusATCC 14579 je ispitan metodom difuzije iz bunarića. Izolati sa najboljimantimikrobnim potencijalom su odabrani za analizu uticaja enzima, pH, temperaturei hemijskih jedinjenja na antimikrobnu aktivnost poluprečišćenih bakteriocina ikinetike rasta i biosinteze antimikrobnih jedinjenja. Za procenu probiotskogpotencijala, odabrano je 42 BMK izolata. Izolati su ispitani na sposobnosttolerancije na uslove GIT-a (tolerancija na nisku pH, prisustvo pepsina,pankreatina i žučnih soli, prisustvo fenola), hidrofobnost, sposobnostautoagregacije i koagregacije i urađena je bezbednosna procena izolata krozispitivanje sposobnosti hemolize na krvnom agaru i rezistencije na antibiotike.Hemijskom analizom sjeničke ovčije stelje utrđeno je da uzorci ispunjavajuzahteve koji definišu propisi o kvalitetu proizvoda od mesa, kao i elaborata ozaštiti geografskog porekla sjenička očija stelja u pogledu sadržaja vode, masti,proteina mesa, soli i pepela. Promene pH vrednosti tokom tehnološkog procesa suodgovarale vrsti proizvoda. Na kraju zrenja pH vrednosti su se kretale u intervalimaod 5,15 do 5,6, što ukazuje na odgovarajuću zrelost proizvoda. Tokom zrenja sjeničkeovčije stelje došlo je do promene aw vrednosti u proizvodu koje su ukazivale namikrobiološku stabilnost tradicionalnog proivoda. Senzornom analizom nisuzabeležena oštećenja, mrlje i diskoloracije proizvoda, pri čemu je istaknutaspecifičnost proizvoda po svim istraživanim senzornim parametimaRezultati ispitivanja mikrobiote istakli su dominaciju BMK, koje je pratiloprisustvo KNS i plesni. Prisusto bakterija iz familije Enterobacteriaceae iPseudomonadaceae je potrđeno u toku procesa proizvodnje, dok na kraju procesa zrenjanisu detektovane. Prisustvo vrsta iz roda Salmonella i Listeria monocytogenes, nijedetektovano ni u jednoj fazi proizvodnog procesa ovčije stelje u svim ispitivanimuzorcima iz svih sezona. Od ukupno 432 BMK izolata, identifikovano je ukupno 6vrsta, 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. Odukupno 376 izolata KNS identifikovano je 152 izolata (40,42%) kao Staphylococcusequorum, 103 izolata (27,39%), kao Staphylococcus xylosus, 64 izolata (17,2%) kaoStaphylococcus carnosus i 46 izolata (12,23%) je identifikovano kao Staphylococcussaprophyticus. Značajan deo mikrobiote sjeničke očije stelje su činile plesni, ukupno221 izolata, pri čemu je karakterizacijom i identifikacijom determinisano ukupno4 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 usupstratu.Tehnološkom karakterizacijom izolata je utvrđeno da izolati iz rodaLactobacillus pokazuju sposobnost rasta na temperaturi do 45ºC kao i na svimtestiranim koncentracijama soli i različitim pH vrednostima, bez proteolitičke ilipolitičke aktivnosti. Izolati L. plantarum i L. mesenteroides su uspešno rasli natemperaturi do 15ºC, koncentracijama soli do 6,5% i pri pH vrednostima iznad 5.Izolati L. mesenteroides su pokazali proteolitičku aktivnost. Izolati koji supripadali vrstama E. faecium i E. faecalis su rasli na temperaturama do 15ºC i na svimkoncentracijama 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 je47,61% testiranih BMK izolata, sa najvećom zonom inhibicije prema E. coli ATCC25922. Najveću antilisterijsku aktivnost je pokazao E. faecium Ios4 sa zonominhibicije od 25 mm. KNS izolati nisu pokazali antimikrobnu sposobnost premaodabranim indikatorskim vrstama. Antimikrobna aktivnost poluprečišćenihbakteriocina izolata L. curvatus IIos4, L. curvatus IIos6, L. sakei IIb11, L. sakei IIa13, L.mesenteroides IIos4i i E. faecium Ios4 se potpuno izgubila pod delovanjemproteolitičkih enzima, dok se dobra antimikrobna aktivnost polipeptida zadržalana pH 7. Prisustvo hemijskih jedinjenja nije uticalo na antimikrobnu aktivnostpoluprečišćeih bakteriocina. Proizvodnja antimikrobnih jedinjenja je zapažena ueksponencijalnoj fazi rasta. Tokom produžene inkubacije u stacionarnoj fazi (nakon30 h) aktivnost supernatanta je značajno opala, da bi nakon inkubacije od 48 hantimikrobna aktivnost prečišćenih bakteriocina nestala.Testirani izolati BMK su pokazali dobar probiotski potencijal. IzolatiLactobacillus spp. su pokazali visok nivo tolerancije na nisku pH (2 i 3), kao isposobnost rasta u prisustvu žučnih soli. Izolati L. plantarum i E. faecalis supokazali slabu toleranciju na nisku pH (2 i 3) i prisustvo žučnih soli. Odabraniizolati su pokazali dobru sposobnost preživljavanja u simuliranim uslovimaželudca i tankog creva i visok nivo sposobnost rasta u prisustvu fenola.Hidrofobnost prema hloroformu, n-heksadekanu i ksilolu se kretala u rasponu od5,2% do 80,9%. Sposobnost autoagregacije i koagregacije sa E. coli ATCC 25922,testiranih izolata BMK izolata je bila izolat specifična. Testirani izolati nisupokazali 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% testiranihizolata.Rezultati dobijeni ovom disertacijom ukazuju na činjenicu da se autohtonifermentisani proizvod sjenička očija stelja odlikuje svim specifičnostimakvaliteta suvomesnatog proizvoda, koji u mnogome zavisi od mikrobiote koja se u tokufermentacije razvija u proizvodu. Rezultati probiotskih i bezbedonosnih svojstavaBMK izolata iz sjenčike ovčije stelje otvaraju mogućnost daljih istraživanjapotencijalne primene određenih sojeva u mesnoj industriji kao startera zaproizvodnju suvomesnatih proizvoda.

Opis (srp)

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Opis (eng)

Sjenica’s sheep ham is an autochthonous fermented sheep meat product that istraditionally produced in rural households in the municipalities of Sjenica and Tutin. Theproduct is prepared according to a unique recipe and technology, from boneless and shapedsheep carcasses of the indigenous breed of sheep called Sjenička pramenka which were raisedon the Sjenica-cave plateau, without the addition of starter cultures and additives. For thepurposes of the experiment, Sjenica’s sheep ham produced in selected households in thevillages of Blato (A), Krajinoviće (B), and Rasno (V) from the Pešter area in three productionseasons was used (2016/17 - 1; 2017/18 - 2; 2018/19 - 3), at the end of autumn and thebeginning of spring, during 120 days of ripening. This dissertation defines the qualityparameters of Sjenica’s sheep ham by examining the chemical composition and sensorycharacteristics of the product as well as by examining the indigenous microbiota of theproduct and comparing all parameters through different seasons, maturation periods, andhouseholds. Examination of the microbiota composition of the autochthonous productSjenica’s sheep ham included examination of the presence and abundance of aerobicmesophilic bacteria, lactic acid bacteria (LAB), coagulase-negative staphylococci (CNS),bacteria from the families Enterobacteriaceae and Pseudomonadaceae, species of the generaSalmonella and Listeria monocytogenes as well as molds. Identification of LAB and CNSwas performed using biochemical tests and MALDI-TOF mass spectrophotometry. Moldisolation was performed on Dichloran 18% glycerol agar (DG18) from the surface of sheeplitter samples, while identification was performed using identification keys. Thetechnological properties of LAB and CNS isolates and molds were examined. Theantimicrobial potential of LAB and CNS isolates against the growth of Staphylococcusaureus ATCC 25923, Escherichia coli ATCC 25922, Listeria monocytogenes ATCC 19115,Pseudomonas aeruginosa ATCC 27853 and Bacillus cereus ATCC 14579 was examined bywell diffusion method. Isolates with the best antimicrobial potential were selected to analyzethe influence of enzymes, pH, temperature, and chemical compounds on the antimicrobialactivity of semi-purified bacteriocins and the kinetics of growth and biosynthesis ofantimicrobial 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, presenceof pepsin, pancreatin, and bile salts, presence of phenols), hydrophobicity, ability to autoaggregation and co-aggregation, and safety assessment of isolates was performed by testingblood agar hemolysis and resistance on antibiotics.Chemical analysis of Sjenica’s sheep ham determined that the samples meet therequirements defined by the regulations on the quality of meat products, as well as the studyon the protection of geographical origin of Sjenica’s sheep ham in terms of water, fat, meatprotein, salt, and ash. Changes in pH values during the technological process correspond tothe type of product. At the end of ripening, the pH values ranged from 5.15 to 5.6, whichindicates the appropriate maturity of the product. During the ripening of the Sjenica’s sheepham, there was a change in aw value of the product, which indicated the microbiologicalstability of the traditional product. The sensory analysis did not show damage, stains, anddiscoloration of the product, emphasizing the specificity of the product for all examinedsensory parameters.The results of microbiota testing highlighted the dominance of LAB, which wasaccompanied by the presence of CNS and mold. The presence of bacteria from the familiesEnterobacteriaceae and Pseudomonadaceae was confirmed during the production process,while at the end of the maturation process, they were not detected. The presence of speciesfrom the genera Salmonella and Listeria monocytogenes was not detected at any stage of theproduction process of sheep ham in all tested samples from all seasons. Out of a total of 432LAB isolates, a total of 6 species were identified, of which 3.83% belonged to the genusLeuconostoc, 8.35% to the genus Enterococcus, 2.03% to the genus Lactiplantibacillus andthe largest percentage (87.58%) belonged to the genus Lactobacillus. Out of a total of 376CNS 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 46isolates (12.23 %) was identified as Staphylococcus saprophyticus. A significant part of themicrobiota of the Sjenica’s sheep ham consisted of molds, a total of 221 mold isolates, withthe 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 hamwas directly affected by water activity in the substrate.Technological characterization of isolates has determined that isolates of the genusLactobacillus show the ability to grow at temperatures up to 45ºC as well as at all tested saltconcentrations and different pH values, without proteolytic and lipolytic activity. Isolates ofL. plantarum and L. mesenteroides successfully grew at temperatures up to 15ºC, saltconcentrations up to 6.5%, and pH values above 5. Isolates of L. mesenteroides showedproteolytic activity. Isolates belonging to E. faecium and E. faecalis species grew attemperatures up to 15ºC and at all salt concentrations and showed proteolytic and lipolyticactivity. E. faecium isolates grew at all tested pH values, while E. faecalis isolates grew at pHvalues 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 25mm. CNS isolates did not show antimicrobial ability according to the selected indicatorspecies. 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 Ios4isolates was completely lost by proteolytic enzymes, while the good antimicrobial activity ofthe polypeptide was maintained at pH 7. The presence of chemical compounds did not affectthe antimicrobial activity of semi-purifying bacteriocins. The production of antimicrobialcompounds was observed in the exponential growth phase. During the prolonged incubationin the stationary phase (after 30 h) the activity of the supernatant decreased significantly, andafter the incubation of 48 h, the antimicrobial activity of the purified bacteriocinsdisappeared.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 thepresence of bile salts. Isolates of L. plantarum and E. faecalis showed poor tolerance to lowpH (2 and 3) and the presence of bile salts. Selected isolates showed good survival ability insimulated gastric and small intestine conditions and a high level of growth ability in thepresence of phenol. Hydrophobicity towards chloroform, n-hexadecane, and xylene rangedfrom 5.2% to 80.9%. The ability of auto-aggregation and coaggregation with E. coli ATCC25922, tested isolates of LAB isolates was isolate specific. The tested isolates did not showthe ability to synthesize biogenic amines or hemolysis on blood agar. None of the testedisolates showed absolute antibiotic resistance. Susceptibility to the studied antibiotics wasobserved in 69.04% of the tested isolates.The results obtained by this dissertation indicate the fact that the autochthonousfermented product Sjenica’s sheep ham is characterized by all the specifics of the quality ofcured meat product, which largely depends on the microbiota that develops in the productduring fermentation. The results of probiotics and safety properties of LAB isolates from theshade of sheep litter open the possibility of further research into the potential application ofcertain strains in the meat industry as a starter to produce meat products.

Jezik

srpski

Datum

2022

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