Karakterizacija Enterobacteriaceae poreklom iz autohtonog sira Srbije sa posebnim osvrtom na vrste iz rodova Klebsiella i Serratia

Abstract

Proizvodnja i konzumiranje sira datira nekoliko hiljada godina unazad tako da sir predstavlja jednu od najstarijih fermentisanih namirnica. Tradicionalni sirevi su autohtoni za neko područje i predstavljaju kulturnu baštinu tog područja. Proizvodnja autohtonih mlečnih proizvoda na teritoriji Balkanskog poluostrva je očuvana vekovima. Proces fermentacije je iskorišćen za proizvodnju mlečnih proizvoda, kiselog mleka, maslaca, sireva i dr. U siru je prisutna prirodna mikroflora koju čine bakterije mlečne kiseline koje potiču iz mleka kao sirovine. One se razvijaju u siru tokom zrenja, a nisu dodate u obliku startera (sirila). Enzimi pomenute mikroflore imaju značajnu ulogu u proteolitičkim promenama tokom zrenja i doprinose formiranju specifičnih senzornih svojstava sireva. Pomenuti deo mikroflore sireva je u najvećoj meri nekontrolisan. Mlečnu kiselinu, kao glavni proizvod fermentacije u anaerobnim uslovima, stvaraju bakterije mlečne kiseline. Zbog prisutne kiseline, prisutan je i karakteristični kiseli ukus sira. Pored bakterija mlečne kiseline, u prirodnu mikrofloru se ubrajaju i druge vrste bakterija koje mogu dospeti u mleko. Sirovo sveže, nekuvano mleko je izvor mnogih vrsta bakterija bez obzira od koje vrste životinja se dobija. Enterobakterije su činioci normalne flore nepasterizovanih sireva i jedni su od predstavnika heterofermentativnih bakterija. Varijabilnost u brojnosti i zastupljenosti vrsta je velika i varira u zavisnosti od sezone u kojoj se sir proizvodi. Enterobakterije mogu da utiču na kvalitet i senzorne karakteristike sireva. Cilj ove doktorske disertacije je utvrđivanje hemijskih karakteristika i klasifikacija do sada neistraženog autohtonog mlečnog proizvoda iz Srbije (sokobanjski sir), utvrđivanje higijensko – mikrobiološkog statusa sokobanjskog sira na osnovu prisustva vrsta iz fam. Enterobacteriaceae kao indikatora, izolacija enterobakterija, njihova identifikacija i formiranje bakterioteke u okviru Laboratorije za mikrobiologiju PMF-a. Sastavni deo ovih istraživanja je analiza dinamike bakterija sa sezonskog aspekta (proleće, leto, jesen) proizvodnje sira. Utvrđivanje biohemijskih osobina identifikovanih bakterija i poređenje biohemijskih i fizioloških osobina izolata sa standardima – ATCC sojevima, kao i proširivanje ukupnih znanja o vrstama iz prirodne bakteriobiote sokobanjskog sira saII aspekta kontrole mikroorganizama, povećanja kvaliteta sira i smanjenja higijenskog rizika, a koja uključuju: osetljivost bakterija na antibiotike zbog mogućnosti rezistencije, kao i ispitivanje sposobnosti aglutinacije E. coli, ispitivanje karakteristika planktonskog i biofilm rasta izabranih vrsta enterobakterija sa posebnim osvrtom na uticaj ekoloških faktora (temperatura, pH, koncentracija NaCl, glukoze i laktoze), ispitivanje hidrofobnosti i sposobnosti adhezije za epitel creva, ispitivanje koagregacija enterobakterija sa E. faecalis KGPMF 49, koja je izolovana iz istog sira, ispitivanje lipolitičke i proteolitičke aktivnosti bakterija i sposobnosti sinteze ekstracelularnih enzima. Sokobanjski sir je autohtoni prehrambeni proizvod koji se se pravi od nekuvanog kravljeg mleka, na tradicionalan način, u domaćinstvima okoline Sokobanje (jugoistočna Srbija). Po svojim hemijskim karakteristikama sokobanjski sir pripada mladim, kiselim sirevima, polumasnim do punomasnim i polutvrdim sirevima. Dominantnu zajednicu bakterija u sokobanjskom siru čine aerobne mezofilne bakterije čija se brojnost menja u odnosu na sezonu u kojoj je sir pravi i sirila koja se koristi u procesu dobijanja sira. Sirilo je bez bakterijskih startera. U okviru bakteriobiote sokobanjskog sira identifikovana su 32 izolata koji su pripadali vrstama iz rodova Escherichia, Klebsiella, Serratia i Enterobacter. Izolati E. coli su dominatno prisutni u prolećnim uzorcima (uzorak 3), a izolati Klebsiella spp. su dominantni u siru pravljenom tokom leta i jeseni gde je korišćeno sirilo „Sirela“. U prolećnim uzorcima 1 i 2 nema enterobakterija jer se koristilo hemijsko sirilo „Maja Rekorderka“ koje je onemogućilo pojavu enterobakterija. Vrsta E. gergoviae se pojavljuje u letnjim uzorcima i njegova pojava se povezuje sa bolešću mlečnih žlezda životinje kao i sa lošijom higijenom. Biohemijske i fiziološke osobine izolovanih enterobakterija ne odstupaju od standarnih sojeva ATCC sojeva. Bakterije poreklom iz sokobanjskog sira su pokazale različit stepen osetljivosti prema antibioticima. 28 izolata su osetljiva na hlorafenikol, streptomicin i tetraciklin, a 4 (dva soja E. coli, S. marescens, S. odorifera) izolata pokazuju rezistenciju na tetraciklin. Pojedini sojevi E. coli poseduju sposobnost aglutinacije. Poznavanje uticaja različitih ekoloških faktora na rast izolovanih vrsta je značajno sa aspekta kontrole bakterija, povećanja kvaliteta i smanjenja higijenskog rizika. Poznavanje uticaja različitih ekoloških faktora na biofilm izolovanih vrsta je značajno sa aspekta smanjenja higijenskog rizika. Uticaj svih ekoloških faktora na formiranje i formirani biofilm je zavisio od vrste. Enterobakterije pokazuju su nizak stepen hidrofobnosti zavisnu od rastvaračaIII (hloroform < etil acetat < ksilen). Utvrđena je adhezivna sposobnost K. pneumoniae, K. ornithinolytica i S. marcescens za epitel svinjskog creva. Izolovani sojevi poseduju sposobnost stupanja u interakcije sa bakterijama iz drugih rodova. Poznato je da bakterije iz fam. Enterobacteriaceae mogu uticati na organoleptičke osobine sira ali testirane bakterije u ovom doktoratu uglavnom nisu pokazale proteolitičku i lipolitičku aktivnost. Enzimska aktivnost bakterija, kao i njihov rast u siru je smanjena zbog niske pH koje stvaraju bakterije mlečne kiseline i zbog veće koncentracije NaCl. Rezultati ove disertacije doprinose boljem razumevanju neistražene mikroflore sokobanjskog sira. Iako su enterobakterije nepoželjne u mlečnim proizvodima, one čine uobičajenu mikrofloru nepasterizovanog mleka i proizvoda koji se dobijaju od takvog mleka. Njihova brojnost i aktivnost je kontrolisana niskom pH sira i visokom koncentracijom NaCl.

Production and consumption of cheese dates back several thousand years, so cheese is one of the oldest fermented foods. Traditional cheeses are autochthonous for some area and represent the cultural heritage of the area. The production of autochthonous dairy products in the territory of the Balkan area has been preserved for centuries. The fermentation process is used for production of dairy products, sour milk, butter, cheese. In the cheese, a natural microflora is consisting of lactic acid bacteria derived from milk as a raw material or from environment. They are developing in cheese during ripening, but they are not added in the form of a starter (rennet). The enzymes of these microflora play an important role in proteolytic changes during ripening and contribute to the formation of specific sensory properties of cheese. Mentioned part of cheese microflora is largely uncontrolled. Lactic acid, as the main product of fermentation in anaerobic conditions, is produces by lactic acid bacteria. Due to the presence of acid, there is a characteristic acidic taste of cheese. In addition to lactic acid bacteria, other types of bacteria that can be found in the milk, are also a part of the natural microflora. Raw fresh, uncooked milk is the source of many types of bacteria, no matter what kind of animal is obtained. Enterobacteriaceae are members of the normal flora of non-pasteurized cheeses and they are one of the representatives of heterofermentative bacteria. The variability in the number and species representation is high and varies depending on the season in which the cheese is produced. Enterobacteriaceae can affect the quality and sensory characteristics of cheese. The aims, of this doctoral dissertation is to determine the chemical characteristics and classification of the so far unexplored autochthonous dairy product from Serbia (Sokobanja cheese), to determine the hygienic - microbiological status of the Sokobanja cheese based on the presence of species from fam. Enterobacteriaceae, isolation of enterobacteria, their identification and collection of bacteria within the Laboratory for Microbiology of PMF. An integral part of this research is the analysis of the dynamics of bacteria from the seasonal aspect (spring, summer, autumn) of cheese production as well as determination of biochemical properties of identified bacteria and comparison of biochemical and physiological properties of isolates with standards - ATCC strains. Also, the aim was the expanding the knowledge of species from the natural bacteriobiota of the Sokobanja cheese from the aspect of control of microorganisms, increasing the quality of cheese and reducing hygienic risk, which include: the susceptibility of bacteria to antibiotics due to the possibility of resistance, as well as the examination of potential ability of agglutination of E. coli, examination of the characteristicsV of planktonic and biofilm growth of selected types of enterobacteria with special emphasis on the influence of ecological factors (temperature, pH, concentration of NaCl, glucose and lactose); to test hydrophobicity and the ability of adhesion to gut epithelium, coaggregation enterobacteria with E. faecalis KGPMF 49, which has been isolated from the same cheese; lipolytic and proteolytic activity of the bacteria and the ability to synthesize extracellular enzymes. Sokobanja cheese is an autochthonous product made from unwashed cow milk, in the traditional way, in the households of the Sokobanja region (southeastern Serbia). In its chemical characteristics, the Sokobanja cheese belongs to acidic, semi-fat-full-fat cheeses and semi-hard cheeses. The dominant bacterial community in the Sokobanja cheese consists of aerobic mesophilic bacteria whose number changes in comparison to the season in which the cheese is made and the cheese used in the process of cheese production. Bacterial starter was not added. 32 isolates, who belongs to the genera Escherichia, Klebsiella, Serratia and Enterobacter, were identified. Isolates of E. coli are dominantly present in spring samples (sample 3) were isolates of Klebsiella spp. are dominant in cheese made during the summer and autumn where the "Sirela" was used as a rennet. In spring samples 1 and 2 there are no enterobacteria because a chemical rennet „Maja Rekorderka“ was used, which prevented the occurrence of enterobacteria. E. gergoviae appears in summer samples and its occurrence is associated with the disease of the animal's gastric glands as well as with poorer hygiene. The biochemical and physiological properties of isolated enterobacteria (except species from the genus Serratia) do not deviate from the standard strains of ATCC strains. Bacteria originating from Sokobanja cheese showed a different degree of antibiotic sensitivity. 28 isolates are susceptible to chlorophenol, streptomycin and tetracycline, and 4 isolates (two strains of E. coli, S. marescens, S. odorifera) showed resistance to tetracycline. Certain strains of E. coli have ability of agglutination. Knowing the influence of various ecological factors on the growth of isolated species is significant from the aspect of controlling bacteria, increasing the quality and reducing hygienic risk. Knowing the impact of various ecological factors on biofilm-isolated species is significant from the aspect of reducing hygienic risk. The influence of all ecological factors on the biofilm formation and formed biofilm was species dependent. Tested enterobacteria showed a low degree of hydrophobicity which is dependent to solvent (chloroform <ethyl acetate <xylene). The adhesive ability of K. pneumoniae, K. ornithinolytica and S. marcescens for the pig epithelium was determined. Certain strains of fam. Enterobacteriaceae have ability to interacts with bacteria from another genera. It is known that bacteria from fam. Enterobacteriaceae may affect the organoleptic properties of the cheese, butVI the tested bacteria in this doctoral dissertation, have not shown proteolytic and lipolytic activity. The enzymatic activity of bacteria, as well as their growth in cheese, is reduced. Low pH made by lactic acid bacteria and higher concentration of NaCl reduced their growth and enzymatic activity. The results of this dissertation contribute to a better understanding of the unexplored microflora of Sokobanja cheese. Although enterobacteria are undesirable in dairy products, they form a common microflora of non-pasteurized milk and dairy products which are produced from them. Their number and activity are controlled by low pH of cheese and high concentration of NaCl.