Karakterizacija prirodnog izolata Lactobacillus sp. BGRA43 kao potencijalnog probiotika

Abstract

Prirodni izolat BGRA43, izolovan iz gastrointestinalnog trakta čoveka, determinisan je na osnovu klasičnih mikrobioloških i molekularno-bioloških metoda kao vrsta Lactobacillus helveticus. Soj poseduje jedan plazmid (pRA1), veličine 2.4 kb, koji se stabilno održava u ćeliji i nakon 10 sukcesivnih presejavanja. Za potrebe genetičkih manipulacija soj je uspešno transformisan plazmidom pA1-6, mada je efikasnost transformacije niska. Optimalna temperatura za rast ovog soja je 42oC, pri čemu je pokazano da soj dobro raste kako u MRS-u tako i u 10% obranom mleku. Nakon 6 sati rasta u 10% obranom mleku na temperaturi od 37oC dolazi do obaranja pH vrednosti u mleku na 4.53 pri čemu takodje dolazi do formiranja homogenog gruša visokog stepena viskoznosti. Soj BGRA43 pokazuje antimikrobijalnu aktivnost na veći broj Gram-pozitivnih i Gram-negativnih bakterija. Takodje je utvrdjeno da soj BGRA43 poseduje ekstracelularnu proteinazu. Cele ćelije ovog soja hidrolizuju sve tri glavne kazeinske frakcije u Na-fosfatnom puferu pH 6.5 i temperaturi od 45oC za samo 2 h. Proteolitička aktivnost soja BGRA43 inhibirana je inhibitorom serinskih proteaza (PMSF). Oslobadjanje proteinaze soja BGRA43 sa ćelijskog zida nije zavisno od Ca++-jona. Na osnovu DNK-DNK hibridizacija, PCR analize kao i sekvenciranjem kloniranog PCR produkta koji odgovara katalitičkom regionu proteinaze utvrdjen je visok stepen homologije sa ptrH genom iz soja Lactobacillus helveticus CNRZ32. Izolovan je i okarakterisan spontani Prt- derivat soja BGRA43, označen kao BGRA433. Derivat BGRA433 nije hidrolizovao ni jednu kazeinsku frakciju i pokazano je da poseduje deleciju u katalitičkom regionu proteinaznog gena. Ćelije soja BGRA43 preživljavaju u visokom procentu u uslovima niske pH vrednosti kao i u prisustvu 0.3% žučnih soli. Humano poreklo, široki spektar antimikrobijalnog delovanja i sposobnost preživljavanja u uslovima koji vladaju u gastrointestinalnom traktu čine soj BGRA43 potencijalnim probiotikom. Lactobacillus helveticus BGRA43 je uspešno iskorišćen i kao starter kultura za proizvodnju jogurta i kiselog mleka.

Natural isolate BGRA43 was isolated from the human intestinal tract. Using the microbiology and molecular biology tests this thermophilic human isolate was determined as Lactobacillus helveticus. The strain BGRA43 was transformed with plasmid pA1-6, but efficiency of transformation was very low. It was found that BGRA43 contains only one small plasmid pRA1 about 2.4 kb. The strain BGRA43 grow very fast in milk (6h, pH 4.5) as well as in MRS (A600nm, 0.858, 10h). Growth of Lactobacillus helveticus BGRA43 in non-fat skim milk after 6 h at 37oC resulted in a lowering of the pH value to 4.53. Besides the fast acidification, the strain generated a high viscosity of skim milk. The strain BGRA43 produced antagonistic substances against both Gram-positive and Gram-negative bacteria and also exhibited an inhibitory effect on the growth of Clostridium sporogenes. Determination of caseinolytic activity done under optimal conditions (pH 6.5 and 45oC), revealed that proteinase from this strain completely hydrolysed all three casein fractions. The proteolytic activity of whole cells was inhibited by serine proteinase inhibitor (PMSF), suggesting that BGRA43 strain produce serine-type of proteinase. In addition, release of the proteinase from the cell envelope of strain BGRA43 is not Ca++-dependent. DNA-DNA hybridisation, PCR analysis and analysis of subcloning PCR fragments from catalytic region of proteinase showed high similarity (98.9% identity) with prtH gene isolated from Lactobacillus helveticus CNRZ32. Spontaneous Prt- derivative BGRA433 was isolated. This derivative is not able to hydrolyse any of casein fractions and hybridisation and PCR analysis showed deletion in catalytic region of proteinase gene. Human origin, antimicrobial activity and surviving at low pH and in presence of bile salts are all properties that consider this strain to be a potential probiotic. Finally, it was demonstrated that the strain BGRA43 can be very attractive as starter culture for production of fermented milk product.