Mikrobiota autohtonog fermentisanog proizvoda sjenička ovčija stelja

Abstract

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.