Karakterizacija prirodnog izolata Lactobacillus sp. BGRA43 kao potencijalnog probiotika

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

Analysis of natural isolates of Lactobacilli resistant to bacteriocin nisin

Kolekcija bakterija mlečne kiseline (BMK) je napravljena od mikroorganizama izolovanih iz fermentisanih mlečno-kiselinskih proizvoda dobijenih na tradicionalan način. Iz kolekcije 51 izolat je identifikovan kao Lactobacillus sp. Svi izolovani laktobacili pripadaju grupi mezofilnih sojeva koji dobro rastu na temperaturama od 15°C i 30°C, a ne rastu na temperaturi od 45°C. Testiranje sposobnosti rasta u prisustvu nizina pokazalo je da su izolati BGCGK4, BGHN40, BGBUK2-8, BGBUK2-7 i BGBUK2-16 rezistentni na bakteriocin nizin. U eksperimentu određivanja minimalne inhibitorne koncentracije (MIK) za nizin pokazano je da je najrezistentniji izolat Lactobacillus sp. BGCGK4. Izolat BGBUK2-16, determinisan kao Lactobacillus paracasei subsp. paracasei, produkuje bakteriocin označen kao Bac217 i pokazuje rezistenciju na 8000 IU/ml. Čišćenjem plazmida iz soja BGBUK2-16 dobijena su 2 derivata označena kao BGBUK2-16/K2 i BGBUK2-16/K4. Derivat BGBUK2-16/K2 zadržao je rezistenciju na Bac217 i nizin, ali je izgubio sposobnost sinteze Bac217, dok je derivat BGBUK2-16/K4 pored gubitka sposobnosti sinteze Bac217 postao senzitivan na Bac217 i nizin. Prirodno rezistentni laktobacili se mogu iskoristiti za pripremanje starter kultura u kombinaciji sa nizinom kao konzervansom u cilju kontrolisane mlečno-kiselinske fermentacije.The collection of lactic acid bacteria (LAB) was made by isolation of microorganisms from fermented products traditionally manufactured in different geographical regions (high mountains, river valleys, seaside, etc). Among collected LAB, 51 isolates were identified as Lactobacillus sp. Results showed that all isolated lactobacilli were mesophilic strains, since they grew at 15°C and 30°C but not at 45°C. Testing the ability of isolated lactobacilli to grow in the presence of nisin revealed that Lactobacillus sp. isolates designed BGCGK4, BGHN40, BGBUK2-8, BGBUK2-7 and BGBUK2-16 were resistant to nisin. Determination of the minimal inhibitory concentrations (MIC) for nisin revealed that the most resistant isolate was Lactobacillus sp. BGCGK4. Isolate BGBUK2-16, determined as Lactobacillus paracasei subsp. paracasei, produces bacteriocin, named Bac217 and showed a resistance to 8000 IU/ml of nisin. Plasmid curing of BGBUK2-16 resulted in derivatives BGBUK2-16/K2 and BGBUK2-16/K4. Derivative BGBUK2-16/K2 retained resistance to Bac217 and nisin, but lost the ability to synthesise Bac217. Derivative BGBUK2-16/K4 lost concomitantly the resistance to both Bac217 and nisin

The presence of prtP proteinase gene in natural isolate Lactobacillus plantarum BGSJ3-18

Aims: The study of proteolytic activity and examination of proteinase gene region organization in proteolytically active Lactobacillus plantarum strains from different natural sources. Methods and Results: A set of 37 lactobacilli was distinguished by using multiplex PCR assay. Results showed that 34 strains were Lact. plantarum and three of them were Lact. paraplantarum. The examination of proteolytic activity revealed that 28 Lact. plantarum and two Lact. paraplantarum hydrolyse beta-casein. Further analyses of all proteolytically active Lact. plantarum with primers specific for different types of CEPs demonstrated that strain BGSJ3-18 has prtP catalytic domain as well as prtP-prtM intergenic region showing more than 95% sequence identity with the same regions present in Lact. paracasei, Lact. casei and L. lactis. No presence of prtB, prtH or prtR proteinase genes was detected in any of tested Lact. plantarum strains. Conclusions: One out of 28 analysed Lact. plantarum strains harbours the prtP-like gene. The other proteolytically active Lact. plantarum probably possesses a different type of extracellular proteinase(s). Significance and Impact of the Study: It is the first report about the presence of the prtP-like gene in Lact. plantarum, which illustrates the mobility of this gene and its presence in different species

Characterization and antimicrobial activity of vaginal lactobacillus isolate

The aim of this study was to investigate the probiotic potential of bacteriocin-producing lactobacilli strain Lactobacillus plantarum G2 isolated from the vaginal mucus of healthy women. The antimicrobial effect of G2 was confirmed in the mixed culture with pathogenic Escherichia coli, Staphylococcus aureus, Salmonella abony and Pseudomonas aeruginosa, while bacteriocine activity was detected against S. aureus and S. abony only. The strain showed an excellent survival rate in low pH and in the presence of bile salts. The percentage of adhered cells of L. plantarum G2 to hexadecane was 63.85±2.0 indicating the intermediate hydrophobicity

Comparative analysis of antimicrobial and proteolytic activity of lactic acid bacteria isolated from Zlatar cheese

Tradicionalni zlatarski sir pripada grupi belih, polutvrdih sireva proizvedenih u domaćinstvu. Sir se proizvodi od nepasterizovanog kravljeg mleka bez dodavanja bilo kakvih poznatih starter kultura. Ukupno je izolovano 253 Gram pozitivnih i katalaza negativnih bakterija mlečne kiseline (BMK). Rezultati su pokazali da 70 od 253 analiziranih izolata proizvodi antimikrobna jedinjenja poznatih kao bakteriocini. Većina izolata koji pripadaju rodovima Lactococcus i Enterococcus, kao i izolati vrsta Lactobacillus plantarum i Lb. brevis ne sintetišu ekstracelularne proteinaze. Nasuprot njima, izolati prodvrste Lb. paracasei subsp. paracasei pokazuju veoma dobru proteolitičku aktivnoist. Pokazano je da ne postoji korelacija između dobre proteolitičke i antimikrobne aktivnosti u većini izolata.Traditional artisan Zlatar cheese belongs to the group of white, semi hard home-made cheeses, which are produced from no pasteurized cow's milk, without addition of any known bacterial starter culture. In total, 253 Gram-positive and catalase negative lactic acid bacteria (LAB) were isolated. Results showed that 70 out of 253 analyzed isolates produced antimicrobial compounds known as bacteriocins. Most isolates from genera Lactococcus and Enterococcus, and isolates belonging to species Lactobacillus plantarum and Lb. brevis, do not synthesize extracellular proteinase. In contrast, isolates from subspecies Lb. paracasei subsp. paracasei showed very good proteolytic activity. It was observed that good proteolytic activity of isolates was not in correlation with their good antimicrobial activity in the most of isolates

A successful use of a new shuttle cloning vector pA13 for the cloning of the bacteriocins BacSJ and acidocin 8912

The aim of this paper was to research the molecular cloning of genes encoding the novel bacteriocin BacSJ from Lactobacillus paracasei subsp. paracasei BGSJ2-8 by using a newly constructed shuttle cloning vector pA13. A new shuttle-cloning vector, pA13, was constructed and successfully introduced into Escherichia coli, Lactobacillus and Lactococcus strains, showing a high segregational and structural stability in all three hosts. The natural plasmid pSJ2-8 from L. paracasei subsp. paracasei BGSJ2-8 was cloned in the pA13 using BamHI, obtaining the construct pB5. Sequencing and in silico analysis of the pB5 revealed 15 open reading frames (ORF). Plasmid pSJ2-8 harbors the genes encoding the production of two bacteriocins, BacSJ and acidocin 8912. The combined N-terminal amino acid sequencing of BacSJ in combination with DNA sequencing of the bacSJ2-8 gene enabled the determination of the primary structure of a bacteriocin BacSJ. The production and functional expression of BacSJ in homologous and heterologous hosts suggest that bacSJ2-8 and bacSJ2-8i together with the genes encoding the ABC transporter and accessory protein are the minimal requirement for the production of BacSJ. Biochemical and genetic analyses showed that BacSJ belongs to the class II bacteriocins. The shuttle cloning vector pA13 could be used as a tool for genetic manipulations in lactobacilli and lactococci

Natural isolate Enterococcus faecalis BGPM3 produces an inducible extracellular proteinase

Enterococcus faecalis BGPM3 proizvodi proteinazu sposobnu da hidrolizuje ukupan kazein kao i frakcije αs1-, β-, i k-kazeina. Ova proteinaza, takođe, hidrolizuje želatin, ali ne deluje na denaturisani goveđi serum albumin ili hemoglobin. Ustanovljeno je da se optimalna hidroliza kazeina u prisuetvu BGPM3 proteinaze postiže na pH 6.5, a njegova maksimalna hidroliza na 37°C. Prisustvo proteolitičke aktivnosti u supernatantu, koji ne sadrži žive ćelije, ukazuje da izolat E. faecalis BGPM3 proizvodi ekstracelularnu proteinazu. Sinteza ove proteinaze se odigrava tokom celokupnog ciklusa rasta bakterije, pri čemu se maksimum proizvodnje postiže u stacionarnoj fazi. Trstman BGPM3 proteinaze sa helatorima metalnih jona dovodi do potpunog gubitka proteolitičke aktivnosti. Međutim, moguće je povratiti proteolitičku aktivnost (do 75%) ako se tretiranom enzimu dodaju joni Zn2+ što ukazuje da je BGPM3 proteinaza metaloenzim. Proteolitička aktivnost ovog enzima je inhibirana jonima Cu2+, čak i u prisustvu jona Zn2+. Eksperimentalni rezultati ukazuju da je proizvodnja BGPM3 proteinaze indudibilna, tj., dolazi do povećanja njene sinteze kada bakterija raste u prisustvu smeše oligopeptida (kazitona). Tako se dobija desetostruko povećanje proteolitičke aktivnosti u bezćelijskom supernatantu kada se pripremi iz kulture koja sadrži kaziton. Određivanje molekulske mase BGPM3 proteinaze je pokazalo da je to protein od oko 29 kDa.Enterococcus faecalis BGPM3 produces a proteinase that hydrolyzes total casein as well as α s1-, β-, and k-casein fractions. This proteinase was also able to hydrolyse gelatine, but not denatured bovine serum albumin and haemoglobin. The optimal pH of casein hydrolysis was 6.5 (determined at 30°C). Maximum caseinolytic activity was obtained at 37°C. The presence of proteolytic activity in cell-free supernatant strongly indicated that E. faecalis BGPM3 produces strictly extracellular proteinase. Proteinase production occurred through the growth cycle reaching a maximum at stationary phase. Pretreatment of the BGPM3 proteinase with metal ion chelators resulted in a total loss of proteolytic activity. Restoration of activity (75%) was obtained only with Zn2+ suggesting that the BGPM3 proteinase is zinc-metalloenzyme. Cu2+ even in the presence of Zn2+ inhibited proteolytic activity. It seems that production of proteinase is induced by oligopeptides (casitone), since 10-fold higher proteolytic activity was obtained in the cell-free supernatant prepared from the cultures containing casitone. The molecular mass determination revealed that extracellular BGPM3 proteinase has a molecular mass of about 29 kDa

Lactobacillus fermentum Postbiotic-induced Autophagy as Potential Approach for Treatment of Acetaminophen Hepatotoxicity

The aim of this study was to investigate the potential of postbiotics originated from Lactobacillus fermentum BGHV110 strain (HV110) to counteract acetaminophen (APAP)-induced hepatotoxicity in HepG2 cells. This strain was selected according to its autophagy inducing potential, based on previous studies reporting protective role of autophagy in APAP caused cellular damage. Cell viability was assessed using MTT and LDH assays, while autophagy was monitored by qPCR analysis of BECN1, Atg5, p62/SQSTM1, and PINK1 mRNA expression and by Western blot analysis of p62/SQSTM1 and lipidated LC3 accumulation. Our results showed that detrimental effect of APAP on cell viability was suppressed in the presence of HV110 which was linked with increased conversion of LC3 protein and p62/SQSTM1 protein degradation. Additionally, higher p62/SQSTM1 and PINK1 mRNA transcription were noticed in cells co-treated with APAP/HV110, simultaneously. In conclusion, this study suggests that HV110 enhances activation of PINK1-dependent autophagy in HepG2 cells and its eventual co-supplementation with APAP could be potentially used for alleviation of hepatotoxic side effects caused by APAP overdose

Lactobacilli hydrolysis of cows' milk proteins abrogates their humoral immunoreactivity in patients with immune-mediated diseases

The level of humoral immunoreactivity to total cows' milk proteins (TCMP) in sera of patients suffering from recurrent oral ulcerations, gastrointestinal diseases or haematological malignancies was investigated. TCMP were also hydrolysed with two different species of lactobacilli and dramatic changes in the levels of specific IgG and IgE were found with statistically significant decreases in the levels of specific antibodies in sera from all patient groups. The levels below cut-off values of IgG specific for TCMP hydrolysates were detected in sera from all patients, while values of IgE for hydrolysates obtained with Lactobacillus helveticus BGRA43 and Lactobacillus zeae LMG17315were below cut-off in 85% and 97% of patients, respectively. Competitive ELISA confirmed the specificity of antibodies for immunogenic TCMP epitopes, demonstrating that lactobacilli hydrolyse TCMP by degrading immunogenic epitopes, and could therefore be used in processing of milk proteins to obtain products suitable for patients with altered immune response on TCMP.This is the peer reviewed version of the paper: Vukotic, G., Matic, I., Begovic, J., Besu, I., Kojic, M., Djokic, J., Juranic, Z., & Strahinic, I. (2016). Lactobacilli hydrolysis of cows’ milk proteins abrogates their humoral immunoreactivity in patients with immune-mediated diseases. International Dairy Journal, 63, 1–7. [https://doi.org/10.1016/j.idairyj.2016.07.009

Effects of soybean carbohydrates and lactobacillus helveticus bgra43 on metabolic processes in rat colon

Aim of this work was to assess the metabolic and physiological changes that occurred in the hind gut of rats after feeding with soybean carbohydrates alone and in combination with Lactobacillus helveticus BGRA43. Wistar rats were gavaged with soybean flour for 28 days. The parameters assessed included fecal volatile organic compounds, and L-lactate, reducing sugars, proteins, ammonia and water levels in the colonic lumen. The presence of lactic acid (LAB), sulfate reducing (SRB) and methanogenic bacteria was assessed by semi-quantitative PCR. Malondialdehyde levels as well as lymphoid tissue size in ileal and colonic mucosa were also evaluated. On the basics of the results obtained, correlation network was created, setting the parameters tested in research in two metabolic groups: saccharolytic and proteolytic fermentation group. The principal finding of the study is a negative correlation between oral administration of BGRA43 and increase of parameters related to carbohydrate fermentation in the gut, and a positive correlation to factors related to proteolytic fermentation. On the contrary, soybean carbohydrates were correlated with increased values of factors related to carbohydrate catabolism. Different effects of BGRA43 and soybean carbohydrates on metabolic processes in colonic lumen indicate the possibility of applying the BGRA43 in alleviating the gastrointestinal symptoms occurring after consuming hardly digestible carbohydrates