Fiziološka karakterizacija bakterija mlečne kiseline izolovanih iz autohtonog sira jugoistočne Srbije i evaluacija njihovih biotičkih potencijala

Abstract

U ovom rukopisu, ispitano je i utvrđeno prisustvo i fiziološke karakteristike bakterija mlečne kiseline (BMK) izolovanih iz tradicionalno proizvedenog sira iz jugoistočne Srbije (područje Sokobanje). Tri uzorka, iz različitih seoskih domaćinstava, uzorkovana su tokom proleća, leta i jeseni. Ispitane su hemijske karakteristike uzoraka sira. Identifikacija BMK je izvršena korišćenjem biohemijskih testova i MALDI-TOF masene spektrofotometrije. Utvrđen je antimikrobni potencijal u odnosu na rast Escherichia coli ATCC 25922 i Proteus mirabilis ATCC 12453, kao i na rast nekih Gram-negativnih bakterija, izolovanih iz istog sira (Klebsiella oxytoca KGPMF1, Klebsiella ornithinolytica KGPMF8 i Aeromonas hydrophila KGPMF64), korišćenjem metode difuzije iz bunarića u agar. Za dalje istraživanje, odabrano je 16 izolata, na osnovu prethodno istraženih i utvrđenih biohemijskih karakteristika i antimikrobnog potencijala u odnosu na izabrane enterobakterije. Izvršena je procena probiotskog potencijala odabranih BMK (tolerancija na različite gastrointestinalne uslove i osetljivost na klinički relevantne antimikrobne agense). Ispitan je uticaj različitih vrsta šećera na rast izolovanih BMK, sposobnost formiranja biofilma, sposobnost bakterija da se vezuju za rastvarače i epitelne ćelije tankog creva svinje, kao i sposobnost auto- i koagregacije između njih i kliničkog izolata Escherichia coli. Aktivnost enzima (kisela i alkalna invertaza, alkalna fosfataza, alkalna proteaza, invertaza) kao i ukupna koncentracija proteina u fermentacionoj tečnosti je izmerena spektrofotometrijskom metodom. Procenjen je uticaj različitih temperatura u sinergizmu sa različitim pH i koncentracijama soli na planktonski rast izolata BMK. Prema procentu masnoće u suvoj materiji, sokobanjski sir pripada punomasnoj grupi sireva, dok prema sadržaju vlage u materiji bez masnoće, sokobanjski sir pripada kiseloj grupi sireva. Brojnost bakterija je zavisila od sezone, domaćinstva i vrste podloge koja se koristila za izolaciju i brojanje. Identifikovana su četiri različita roda BMK (Lactobacillus, Lactococcus, Enterococcus i Streptococcus). U okviru roda Lactobacillus identifikovani su Lb. fermentum, Lb. brevis i Lb. plantarum. U okviru roda Lactococcus identifikovanini su L. lactis subsp. lactis i L. lactis subsp. lactis biovar. diacetylactis. U okviru roda Enterococcus identifikovani su E. faecium, E. faecalis, E.II hirae i E. durans. U okviru roda Streptococcus identifikovani su S. uberis i S. agalactiae. Svi testirani izolati, osim izolata iz roda Streptococcus, pokazali su dobru acidifkacionu sposobnost u standardnom i obogaćenom mleku. Testirani izolati su inhibirali rast bar jedne od pet indikatorskih vrsta, sa prečnikom zone inhibicije od 10 do 26 mm. S. uberis (7 izolata) i S. agalactiae (1 izolat) su izolovani iz sira uzorkovanog tokom leta. Testirani Streptococcus izolati su pokazali osetljivost na tetraciklin, hloramfenikol, novobiocin i rifampicin, sa prečnikom zone inhibicije od 36 do 48 mm. Zbog specifičnosti tradicinalnog načina proizvodnje sokobanjskog sira, smatra se da su ovi izolati dospeli u mleko, a kasnije u sir sa vimena krava. Izabrani izolati BMK su pokazali toleranciju na simulirane gastrointestinalne uslove i osetljivost na antibiotike, posebno na ampicilin (MIK na 0,195 μg mL-1 za laktobacile; od 0,195 – 3,125 μg mL-1 za laktokoke; od 0,19 – 2,5 μg mL-1 za enterokoke). Sposobnost formiranja biofilma izolata BMK je bila izolat specifična. Najveći procenat adhezije je detektovan sa hloroformom, dok je adheziona sposobnost odabranih izolata na epitel tankog creva svinje u korelaciji sa rezultatima adhezione sposobnosti prema rastvaračima. Sposobnost autoagregacije izolata je bila jaka. Lactobacillus spp. su pokazali snažnu koagregaciju sa E. coli, dok je koagregacija izolata iz rodova Lactococcus i Enterococcus bila izolat specifična. Svi izolati iz roda Lactobacillus su pokazali aktivnost proteaze, amilaze i alkalne fosfataze, dok aktivnost kiselih i alkalnih invertaza nije uočena. Izolati iz roda Lactococcus su pokazali aktivnost proteaze, kisele invertaze i alkalne fosfataze, osim izolata KGPMF50, koji nije pokazao aktivnost alkalne fosfataze. Izolati iz roda Enterococcus su pokazali slabu i izolat specifičnu enzimsku aktivnost. Proteini su detektovani u fermentacionoj tečnosti svih izolata. Izolati iz rodova Lactobacillus i Lactococcus su pokazali veću toleranciju na kiselu pH, dok su izolati iz roda Enterococcus bili tolerantniji na baznu pH, na 37°S. Temperatura na 4°S je bila limitirajući faktor za rast svih testiranih izolata. Ograničavajući faktori za planktonski rast izolata iz rodova Lactobacillus i Lactococcus su bili bazna pH, koncentracija soli iznad 6,5% i temperatura na 20°S. Ograničavajući faktori za rast izolata iz roda Enterococcus su bili kisela pH, koncentracija soli oko 8% i temperatura na 20°S. U kombinaciji sa 8% soli i temperaturom od 20°S, samo su izolati iz roda Lactococcus pokazali minimalanu sposobnost rasta, dok izolati iz roda Lactobacillus nisu pokazali sposobnost rasta.III Rezultati su ukazali na potencijalna probiotska svojstva BMK izolovanih iz sokobanjskog sira i otvorila mogućnost za dalja istraživanja i potencijalnu primenu u mlečnoj industriji. Izolati su uglavnom pokazali visoku toleranciju na ekstremne uslove koji vladaju u gastrointestinalnom traktu, osetljivost na antibiotike, kao i sposobnost preživljavanja u prisustvu fenola. Izolati nisu pokazali sposobnost da sintetišu histamin i tiramin, što je poželjna karakteristika pri izboru mogućih probiotika ili starter kultura. Na osnovu rezultata sposobnosti adhezije i agregacije, izdvojili su se izolati Lb. fermentum KGPMF28, Lb. fermentum KGPMF29, Lb. brevis KGPMF35 i L. lactis subsp. lactis biovar. diacetylactic KGPMF57, koji su pokazali potencijal za upotrebu i buduća istraživanja.

In this paper, the presence and physiological characteristics of lactic acid bacteria (LAB) isolated from traditionally made cheese from Southeastern Serbia (Sokobanja area) were examined. Three samples, from different households, were obtained during the spring, summer and autumn. Chemical characteristics of cheese samples were examined. Identification of LABs was done by using the biochemical tests and MALDI-TOF mass spectrometry. The antimicrobial potential on the growth of Escherichia coli ATCC 25922 and Proteus mirabilis ATCC12453 as well as on the growth of some Gram‐negative bacteria, isolated from the same cheese (Klebsiella oxytoca KGPMF1, Klebsiella ornithinolytica KGPMF8 and Aeromonas hydrophila KGPMF64) was examined by using agar-well diffusion method. The 16 isolates were chosen for further investigation, according to the previously investigated biochemical characteristics and antagonistic potential against enterobacteria isolated from the same cheese. The evaluation of the probiotic potential of chosen LABs (tolerance to different gastrointestinal conditions and sensitivity to clinically relevant antimicrobial agents) was evaluated. The effect of different type of sugars on the growth of isolated LAB, was examined. The ability of biofilm formation of LAB, the ability of bacteria to adhere to solvents and to pig intestinal epithelium, as well as the ability of auto- and co-aggregation between them and Escherichia coli clinical isolate, were investigated. The enzymatic activities (acid and alkaline invertase, alkaline phosphatase, alkaline protease, invertase) as well as the total concentration of protein in fermentation liquid were measured by using the spectrophotometry method. The effect of different temperature in synergism with different pH and salt concentration, on the ability of planktonic growth of selected LAB, was also evaluated. According to the fat in dry matter, the cheese belong to full-fat, while according to moisture in nonfat substance in acid-curd soft cheese group. The number of viable bacterial cells showed seasonal, household and type of substrate dependence. Briefly, four different genera of LAB (Lactobacillus, Lactococcus, Enterococcus and Streptococcus) were identified. The members of the genus Lactobacillus were Lb. fermentum, Lb. brevis and Lb. plantarum. The members of the genus Lactococcus were L. lactis subsp. lactis and L. lactis subsp. lactis biovar. diacetylactis. The members of the genus Enterococcus were E. faecium, E. faecalis, E. hirae and E. durans. The members of genus Streptococcus were S. uberis and S. agalactiae. All tested isolates, except the members of genus Streptococcus, showed good acidification ability in pure and enriched milk. Tested isolates inhibited the growth of at least one indicator strain. Diameter of the growth inhibition zone was from 10 to 26 mm.V d S. agalactiae (1 isolate) were isolated from the cheese samples taken in summer. All isolates were susceptible to tetracycline, hloramphenicol, novobiocin and rifampicin, with a growth inhibition zone from 36 – 48 mm. Due to the specific making of cheese from Sokobanja, these isolates are, probably, originating from cows’ udders. Chosen isolates were tolerant to the simulated gastrointestinal condition and showed sensitivity to antibiotics, especially to ampicillin (MIC at 0.195 μg mL-1 for lactobacilli; from 0.195 – 3.125 μg mL-1 for lactococci; from 0.19 – 2.5 μg mL-1 for enterococci). The sugar metabolism and the ability of biofilm formation of LABs were strain specific. The highest percentage of adhesion was detected with chloroform, while the adhesion ability of selected isolates to pig intestinal epithelium was in the correlation with the results of adhesion ability to solvents. The auto-aggregation ability of isolates was strong. Lactobacillus spp. demonstrated hight co-aggregation with E. coli, while co-aggregation of Lactococcus spp. and Enterococcus spp. was strain specific. All Lactobacillus isolates showed the protease, amylase and alkaline phosphatase activity, while the activity of acid and alkaline invertase was not observed. The Lactococcus isolates showed the protease, acid invertase and alkaline phosphatase activity, except KGPMF 50 isolate, which showed no alkaline phosphatase activity. Tested Enterococcus isolates showed weak and strain specific enzymatic activity. The proteins were detected in the fermentation liquid of all isolates. Tested Lactobacillus and Lactococcus isolates showed tolerance to acidic pH, while Enterococcus isolates showed tolerance to basic pH, at 37°C. Temperature at 4°C was limiting factor for the growth of all tested bacteria. The limiting factors for the planktonic growth of Lactobacillus and Lactococcus isolates were basic pH, salt concentration above 6.5% and temperature at 20°C. The limiting factors for the growth of Enterococcus isolates were acidic pH, salt concentration around 8% and temperature at 20°C. In combination of 8% of salt and 20°C, only Lactococcus isolates showed minimal growth, while Lactobacillus isolates showed no growth at all. These results indicated the potential probiotic properties of the LABs isolated from Sokobanja cheese and give evidence for further potential application in the dairy industry. Isolates mostly showed a high tolerance to extremes of gastrointestinal conditions, sensitivity to antibiotics, as well as the ability of surviving in the presence of phenol. They showed no ability to synthesize histamine and tyramine, which is desirable characteristic when selecting possible probiotics or starter cultures. Based on the results of adhesion and aggregation ability, Lb. fermentum KGPMF28, Lb. fermentum KGPMF29, Lb. brevis KGPMF35 and L. lactis subsp. lactis biovar. diacetylactic KGPMF57 isolates showed the potential for usage and future investigation.