Mikrobiota autohtonog fermentisanog proizvoda sjenička ovčija stelja
Microbiota of autochthonous fermented product Sjenica sheepʼs ham
Докторанд
Žugić-Petrović, TanjaМентор
Grujović, Мirjana Ž.Чланови комисије
Mladenović, KatarinaTomović, Vladimir M.
Stefanović, Olgica
Joković, Nataša
Метаподаци
Приказ свих података о дисертацијиСажетак
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. Ispitiv...anje 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 pa...rameters 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.