Molecular characterization of semi-hard homemade cheese microflora

U poslednjoj deceniji zabeležen je nagli razvoj molekularnih tehnika baziranih na 16S i 23S rRNK, koje se koriste u izučavanju biodiverziteta mikroorganizama. U ovom radu ispitivana je mikroflora polutvrdog sira pripremljenog u domaćinstvu. Zbog visokog sadržaja masti u ovom siru razvili smo novu tehniku za izolaciju totalne DNK iz sira (metod je baziran na bead beating-u). Brza izolacija DNK iz mikroflore sira omogućila nam je molekularnu identifikaciju BMK (BAKTERIJE MLEČNE KISELINE) na osnovu umnožavanja gena za 16S rRNK PCR metodom. Za umnožavanje su korišćeni prajmeri specifični za gene 16S rRNK lakto-bacila, ali su uslovi PCR reakcije bili takvi da su omogućavali i umnožavanje gena 16S rRNK laktokoka. Rezultati RFLP analize pokazali su da mikrofloru sira pripremljenog u domaćinstvu čine predominantno laktokoke.This decade has shown an impressive development in the application of molecular techniques based on 16S and 23S rRNA genes to study the microbial diversity in various ecosystems. Microflora of semi-hard homemade cheese was examined in this work. We developed a novel technique for DNA extraction (a bead beating based method) due to high fat content of this cheese. Rapid extraction of DNA from cheese microflora enabled a molecular identification of the LAB (Lactic Acid Bacteria) strains based on PCR amplification of 16S RNA coding sequences. The specific primers for 76S RNA gene of lactobacilli were used for amplification. The PCR reaction was performed at lower temperature, where the specificity of the annealing reaction was reduced and lactococcal sequences of 16S RNA genes were also amplified. The results of RFLP analysis revealed that the microflora of Doboj homemade cheese encompases mostly lactococci

Effect of methionine and cysteine deprivation on growth of different natural isolates of Lactobacillus spp. in chemically defined media

Cilj ovog rada je bio da se utvrdi sposobnost prirodnih izolata laktobacila,izolovanih iz različitih ekoloških niša da rastu u hemijski definisanom medijumu sa ili bez prisustva aminokiselina koje sadrže sumpor, metionin i/ili cistein. Dobijeni rezultati su pokazali da je esencijalna aminokiselina za rast izolata L. paracasei subsp. paracasei BGHN14 i BGSJ2-8 cistein, dok je za izolate BGHN40, BGCG31, BGHV54T, koji pripadaju vrsti L. plantarum-metionin. Metionin je esencijalna aminokiselina za rast soja L. rhamnosus BGHV58T. Ostali analizirani sojevi,kao što su L. plantarum BGSJ3-18,BGZB19,BGHV52Ta i BGHV43T za svoj rast zahtevaju prisustvo obe aminokiseline u medijumu.The purpose of this study was to determine the ability of natural isolates of lactobacilli from different ecological niches to grow in a chemically defined medium in the presence or absence of sulphur-containing amino acids, methionine and/or cysteine. The obtained results indicate that cysteine is essential for growth of L. paracasei subsp. paracasei BGHN14 and BGSJ2-8, while methionine is essential for isolates BGHN40, BGCG31, and BGHV54T of the species L. plantarum. Methionine is also essential for growth of L. rhamnosus BGHV58T. Other analyzed strains, such as L. plantarum BGSJ3-18, BGZB19, BGHV52Ta, and BGHV43T, require the presence of both amino acids for their growth

Improved sensitivity and reproducibility of the PCR method for detection of Listeria spp. and L. monocytogenes in milk

Listeria monocytogenes, prouzrokovač listerioze kod ljudi i životinja, je fakultativan intraćelijski mikroorganizam široko rasprostranjen u prirodi. U cilju izolacije i detekcije L. monocytogenes iz hrane koriste se tradicionalne mikrobiološke i nove molekularno-genetičke metode. Cilj ovog rada je bio povećanje osetljivosti i ponovljivosti PCR metode u detekciji L. monocytogenes u mleku. U tu svrhu, uzorci pasterizovanog mleka su kontaminirani serijskim razblaženjima sojeva L. monocytogenes 4b ATCC 119115 i Listeria innocua ATCC 33090. Dobijeni rezultati na veštački kontaminiranim uzorcima pasterizovanog mleka, ukazuju da osetljivost PCR metode zavisi od perioda inkubacije i izbora prajmera. Najbolji rezultati su dobijeni nakon 24 h inkubacije, pomoću prajmera za hlyA gen, kada je bilo moguće detektovati 1 ćeliju L. monocytogenes tj. 1 CFU/ml.Listeria monocytogenes is a facultative intracellular Grampositive bacterium, ubiquitous in nature and capable of causing listeriosis in humans and animals. Conventional microbiological techniques and modern molecular approaches are currently used for the isolation and detection of L. monocytogenes in food samples. The aim of this study was to improve the sensitivity and reproducibility of PCR for the detection of Listeria spp. in milk. For that purpose milk samples were artificially inoculated with serial dilutions of L. monocytogenes 4b ATCC 19115 and L. innocua ATCC 33090. The results obtained on artificially contaminated milk samples indicated that incubation time and target genes have an influence on the sensitivity of PCR detection. The best results were obtained after 24 h of preenrichment, with primers complementary to the hlyA gene, when it was possible to detect 1 CFU/mL of Listeria spp

Molecular characterization of the CmbR activator-binding site in the metC-cysK promoter region in Lactococcus lactis

The metC-cysK operon involved in sulphur metabolism in Lactococcus lactis is positively regulated by the LysR-type protein CmbR. Transcription from the metC promoter is activated when concentrations of methionine and cysteine in the growth medium are low. The metC promoter region contains two direct and three inverted repeats. Deletion analysis indicated that direct repeat 2 (DR2) is required for activation of the metC promoter by CmbR. Gel mobility shift assays confirmed that CmbR binds to a 407 bp DNA fragment containing the rnetC promoter. This binding was stimulated by O-acetyl-L-serine. Competition experiments with deletion variants of the metC promoter showed that CmbR binding only occurred with fragments containing an intact DR2, confirming that DR2 is the CmbR binding site within the metC promoter

Enterococci from Raw-Milk Cheeses: Current Knowledge on Safety, Technological, and Probiotic Concerns

The present study is focused on the safety, technological characteristics, and probiotic evaluation of Enterococcus species from different artisanal raw milk dairy products, mainly cheeses with ripening. Apart from proteolytic and lipolytic activities, most enterococci show the ability to metabolize citrate and convert it to various aromatic compounds. Long-ripened cheeses therefore have a specific flavor that makes them different from cheeses produced from thermally treated milk with commercial starter cultures. In addition, enterococci are producers of bacteriocins effective against spoilage and pathogenic bacteria, so they can be used as food preservatives. However, the use of enterococci in the dairy industry should be approached with caution. Although originating from food, enterococci strains may carry various virulence factors and antibiotic-resistance genes and can have many adverse effects on human health. Still, despite their controversial status, the use of enterococci in the food industry is not strictly regulated since the existence of these so-called desirable and undesirable traits in enterococci is a strain-dependent characteristic. To be specific, the results of many studies showed that there are some enterococci strains that are safe for use as starter cultures or as probiotics since they do not carry virulence factors and antibiotic-resistance genes. These strains even exhibit strong health-promoting effects such as stimulation of the immune response, anti-inflammatory activity, hypocholesterolemic action, and usefulness in prevention/treatment of some diseases

The presence of Listeria spp. and Listeria monocytogenes in a chosen food processing establishment in Serbia

The aim of the study was to establish a protocol to evaluate the presence of Listeria spp. in food processing environments. The presence of Listeria spp. was evaluated in a selected restaurant in Serbia on three occasions. Samples were collected from 47 sampling spots in the commercial kitchen equipment and environment. The presence of Listeria spp. and Listeria monocytogenes were detected by conventional culture methods and by the PCR method. The obtained results showed that 23 swab samples were positive for Listeria spp. Interestingly, the swabs from the bread-cutting board and meat defrosting sink were positive for L. monocytogenes

Next-generation probiotics: Health-promoting bacteria of the human gut

In recent years, a vast number of human diseases have been correlated with gut microbiota dysbiosis. The development of modern methods in molecular microbiology, such as the culturomics approach, as well as various multi-omics methods like next generation sequencing, transcriptomics and metabolomics analysis, coupled with large data sets correlation analysis, enabled the cultivation and characterization of novel anaerobic hitherto uncultivated Next-Generation Probiotics. In addition, the results of host-microbe interactions studies helped to reveal the mechanisms involved in the beneficial effects of Next-Generation Probiotics. Eventually, the obtained data on Next-Generation Probiotics will help to broaden the scientific knowledge on these bacteria, in terms of both their safety and health-promoting effects, unravel opportunities for the development of novel therapeutic strategies for prevention and treatment of tumors, metabolic, neuropsychiatric and other diseases, with the aim of relieving the symptoms of the diseases and increasing the quality of life for patients and their families. So far, the best characterized probiotics of the new generation are Akkermansia muciniphila, Faecalibacterium prauznitzii and Bacteroides fragilis

Myeloid-derived suppressor cells prevent disruption of the gut barrier, preserve microbiota composition, and potentiate immunoregulatory pathways in a rat model of experimental autoimmune encephalomyelitis

Over-activated myeloid cells and disturbance in gut microbiota composition are critical factors contributing to the pathogenesis of Multiple Sclerosis (MS). Myeloid-derived suppressor cells (MDSCs) emerged as promising regulators of chronic inflammatory diseases, including autoimmune diseases. However, it remained unclear whether MDSCs display any therapeutic potential in MS, and how this therapy modulates gut microbiota composition. Here, we assessed the potential of in vitro generated bone marrow-derived MDSCs to ameliorate experimental autoimmune encephalomyelitis (EAE) in Dark Agouti rats and investigated how their application associates with the changes in gut microbiota composition. MDSCs differentiated with prostaglandin (PG)E2 (MDSC-PGE2) and control MDSCs (differentiated without PGE2) displayed strong immunosuppressive properties in vitro, but only MDSC-PGE2 significantly ameliorated EAE symptoms. This effect correlated with a reduced infiltration of Th17 and IFN-gamma-producing NK cells, and an increased proportion of regulatory T cells in the CNS and spleen. Importantly, both MDSCs and MDSC-PGE2 prevented EAE-induced reduction of gut microbiota diversity, but only MDSC-PGE2 prevented the extensive alterations in gut microbiota composition following their early migration into Payer's patches and mesenteric lymph nodes. This phenomenon was related to the significant enrichment of gut microbial taxa with potential immunoregulatory properties, as well as higher levels of butyrate, propionate, and putrescine in feces. This study provides new insights into the host-microbiota interactions in EAE, suggesting that activated MDSCs could be potentially used as an efficient therapy for acute phases of MS. Considering a significant association between the efficacy of MDSC-PGE2 and gut microbiota composition, our findings also provide a rationale for further exploring the specific microbial metabolites in MS therapy

Capability of exopolysaccharide-producing Lactobacillus paraplantarum BGCG11 and its non-producing isogenic strain NB1, to counteract the effect of enteropathogens upon the epithelial cell line HT29-MTX

The putative protective role of the exopolysaccharide (EPS)-producing Lactobacillus paraplantarum BGCG11, and its non-EPS-producing isogenic strain NB1, was tested upon HT29-MTX monolayers challenged with seven opportunistic pathogens. The probiotic strain Lactobacillus rhamnosus LMG18243 (GG) was used as a reference bacterium. Tested lactobacilli were able to efficiently reduce the attachment to HT29-MTX of most pathogens. Lb. paraplantarum NB1 and Lb. rhamnosus GG were more efficient reducing the adhesion of Clostridium difficile or Yersinia enterocolitica than Lb. paraplantarum BGCG11, while stain BGCG11 reduced, to a greater extent, the adhesion of Escherichia coli and Listeria monocytogenes. The detachment and cell lysis of HT29-MTX monolayers in the presence of pathogens alone and co-incubated with lactobacilli or purified EPS was followed. L. monocytogenes induced the strongest cell detachment among the seven tested pathogens and this effect was prevented by addition of purified EPS-CG11. The results suggest that this EPS could be an effective macromolecule in protection of HT29-MTX cells from the pathogen-induced lysis. Regarding innate intestinal barrier, the presence of C. difficile induced the highest IL-8 production in HT29-MTX cells and this capability was reinforced by the co-incubation with Lb. paraplantarum NB1 and Lb. rhamnosus GG. However, the increase in IL-8 production was not noticed when C difficile was co-incubated with EPS-producing Lb. paraplantarum BGCG11 strain or its purified EPS-CG11 polymer, thus indicating that the polymer could hinder the contact of bacteria with the intestinal epithelium. The measurement of mucus secreted by HT29-MTX and the expression of mud, muc2, muc3B and muc5AC genes in the presence of pathogens and lactobacilli suggested that all lactobacilli strains are weak "co-adjuvants" helping some pathogens to slightly increase the secretion of mucus by HT29-MTX, while purified EPS-CG11 did not induce mucus secretion. Taking altogether, Lb. paraplantarum BGCG11 could act towards the reinforcement of the innate mucosal barrier through the synthesis of a physical-protective EPS layer which could make difficult the contact of the pathogens with the epithelial cells.This is the peer reviewed version of the paper: Zivkovic, M., Hidalgo-Cantabrana, C., Kojic, M., Gueimonde, M., Golic, N., & Ruas-Madiedo, P. (2015). Capability of exopolysaccharide-producing Lactobacillus paraplantarum BGCG11 and its non-producing isogenic strain NB1, to counteract the effect of enteropathogens upon the epithelial cell line HT29-MTX. Food Research International, 74, 199–207.[ https://doi.org/10.1016/j.foodres.2015.05.012]Published version : [https://imagine.imgge.bg.ac.rs/handle/123456789/794

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