Improvements of regional compared to global forecasts in long term integrations. Umnoženo za odbranu

U okviru disertacije ispituje se zaključak bojnih autora koji su predstavili eksperimente u kojima se beleži slabljenje cirkulacije velikih razmera u integracijama klimatskim regionalnim modelom kada se ne primenjuje spektralno forsiranje velikih razmera (npr. VON STORCH i dr., 2000; BINER i dr., 2000; ROCKEL i dr., 2008; SANCHEZ-GOMEZ i dr., 2008; ALEXANDRU i dr., 2009; između ostalih). Porede se uspesi prognoziranja velikih razmera regionalnim Eta modelom i globalnim ECMWF modelom u slučaju ansambla koji čini 26 32-dnevnih prognoza. Dalje, ispituje se šema za bočne granične uslove. Pitanje na koje se traži odgovor je, da li je uveliko zastupljena, ali računski zahtevnija, relaksaciona šema za bočne granične uslove neophodna za uspešno prognoziranje/simuliranje velikih razmera regionalnim klimatskim modelom. Odgovor će se dobiti poređenjem rezultata dobijenih integracijama Eta modela u dve verzije, koje se razlikuju samo po šemi za bočne granične uslove. Jedna šema je, uveliko zastupljena, relaksaciona šema, a druga je originalna Etina šema kod koje se informacija iz globalnog modela dobija samo u jednom spoljašnjem redu tačaka i na izlaznoj granici se ne definišu sve promenljive. Uspesi ova dva skupa prognoza regionalnog Eta modela se porede kako međusobno, tako i sa uspehom globalnog modela od kojeg se uzimaju granični uslovi. Novina u eksperimentu je metod za verifikaciju. U cilju ocene uspeha prognoziranja velikih razmera, prati se tačnost prognoze položaja najjačih vetrova na nivou mlazne struje, za koji se uzima da odgovara nivou pritiska od 250 hPa. Računaju se dva skora: nepristrasna mera šanse da događaj potpadne pod određenu kategoriju (da brzina vetra na nivou 250 hPa potpada pod kategoriju vetra većeg od 45 m s–1) podešena odnosom čestina prognoziranog i osmotrenog događaja (MESINGER, 2008) i odnos čestina prognoziranog i osmotrenog događaja. Za verifikaciju se koriste ECMWF analize. Takođe se računaju u verifikaciji uobičajeni skorovi – RMS razlike kao i odstupanja između prognoziranog i analiziranog vetra na istom nivou tj. standardni bias. Rezultati eksperimenta pokazuju da je uspeh prognoze velikih razmera Eta modelom, bez dodatnog forsiranja, gotovo isti ili čak bolji nego globalnim modelom od koga dolaze granični uslovi. Što se tiče šeme za bočne granične uslove, nije uočljiva prednost relaksacione šeme u odnosu na originalnu Etinu šemu, uprkos tome što originalna šema koristi informacije iz globalnog modela u samo jednom spoljašnjem redu tačaka. Naprotiv, rezultati Ete sa Etinim LBC su bili mnogo češće bolji od onih sa relaksacionim LBC.A considerable number of authors presented experiments in which degradation of large scale circulation occurred in regional climate integrations when large-scale nudging was not used (e.g., VON STORCH et al., 2000; BINER et al., 2000; ROCKEL et al., 2008; SANCHEZ-GOMEZ et al., 2008; ALEXANDRU et al., 2009). We here revisit this issue by comparing the large scale skill of the Eta RCM against that of the global ECMWF forecasts used as its driver. Another issue we are looking into is that of the lateral boundary condition (LBC) scheme. Specifically, the question we ask is whether the almost universally used but somewhat costly relaxation scheme is necessary for a desirable RCM performance? We address this by running the Eta model in two versions differing in the lateral boundary scheme used. One of these is the traditional relaxation scheme and the other is the Eta model scheme in which information is used at the outermost boundary only and not all variables are prescribed at the outflow boundary. The skills of these two RCM forecasts are compared against each other and also against that of their driver. Our global data dynamically downscaled to address these issues is an ECMWF 32-day ensemble forecast. A novelty in our experiments is the verification used. In order to test the large scale skill we are looking at the forecast position accuracy of the strongest winds at the jet stream level, which we have taken as 250 hPa. We do this by calculating bias adjusted equitable threat scores (MESINGER, 2008) and frequency bias scores for wind speeds greater than a chosen wind speed threshold, with the ECMWF analyses used as truth. For greater confidence we also calculate a traditional RMS difference between the forecast and analyzed winds at this same level. Our results show the Eta RCM skill in forecasting large scales with no interior nudging to be just about the same as that of the driver model. As to the LBC impact no disadvantage compared to relaxation was seen from using the Eta LBC scheme, in spite of its requiring information from the outermost RCM boundary only. Moreover, results of the Eta with standard were more times better than those with relaxation LBC scheme

Molecular analysis of enterolysin A and entL gene cluster from natural isolate Enterococcus faecalis BGPT1-10P

Soj Enterococcus faecalis BGPT1-10P je izolovan iz domaćeg polutvrdog sira, poreklom sa Stare Planine. Rezultati pokazuju da soj BGPT1-10P sintetiše termolabilan bakteriocin, enterolizin A, sa širokim spektrom delovanja, uključujući patogene bakterije roda Listeria i Candida. EntL gen, odgovoran za sintezu ovog bakteriocina, je hromozomalno lokalizovan. Analiza nukleotidne sekvence entL gena kod prirodnog izolata En. faecalis BGPT1-10P je identična sa entL genom soja En. faecalis LMG 2333, koji je prethodno okarakterisan. Pokazana je jedinstvena sekvenca entL gena i njegove okoline, koju čine orf1, orf2 i orf3 geni, kao i scpE gen. Prvi put je kod prirodnog izolata okarakterisan scpE gen, koji kodira virulentni faktor stafopain peptidazu. Funkcionalna analiza entL gena je pokazala da je kompletna genetička informacija, neophodna za sintezu i aktivnost enterolizina A, sadržana u entL genu. Soj BGPT1-10P osim enterolizina, sintetiše i želatinazu i citolizin i sadrži set različitih virulentnih faktora. Pored toga, BGPT1-10P nosi ermB i tetM gene, odgovorne za rezistenciju na eritromicin i tetraciklin.Strain Enterococcus faecalis BGPT1-10P was isolated from artisanal semi-hard homemade cheese from Stara Planina, Serbia. Results showed that BGPT1-10P synthesized a heat labile bacteriocin with a broad spectrum of activity, including Listeria and Candida species. Further analysis revealed that synthesized bacteriocin is enterolysin A. Moreover, the entL gene encoding enterolysin A was found to be located on the chromosome. The entL gene was cloned and sequenced. Analysis of nucleotide sequence showed that the entL gene in natural isolate En. faecalis BGPT1-10P is identical to that of the entL gene described previously in En. faecalis LMG 2333. Within the cloned DNA fragment containing the entL gene, four ORFs were detected. One of them was identified as the scpE gene, which encodes a virulent factor staphopain peptidase. Functional analysis of the entL gene showed that the complete genetic information necessary for the synthesis of enterolysin A were directly linked solely to it. Strain BGPT1-10P also synthesized gelatinase and citolysin, and contained a set of virulent factors. In addition, BGPT1-10P carries the ermB and tetM genes conferring the resistance to erythromycin and tetracycline, respectively

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

Molecular analysis of enterolysin a and entL gene cluster from natural isolate Enterococcus faecalis BGPT1-10P

Strain Enterococcus faecalis BGPT1-10P was isolated from artisanal semi-hard homemade cheese from Stara Planina, Serbia. Results showed that BGPT1-10P synthesized a heat labile bacteriocin with a broad spectrum of activity, including Listeria and Candida species. Further analysis revealed that synthesized bacteriocin is enterolysin A. Moreover, the entL gene encoding enterolysin A was found to be located on the chromosome. The entL gene was cloned and sequenced. Analysis of nucleotide sequence showed that the entL gene in natural isolate En. faecalis BGPT1-10P is identical to that of the entL gene described previously in En. faecalis LMG 2333. Within the cloned DNA fragment containing the entL gene, four ORFs were detected. One of them was identified as the scpE gene, which encodes a virulent factor staphopain peptidase. Functional analysis of the entL gene showed that the complete genetic information necessary for the synthesis of enterolysin A were directly linked solely to it. Strain BGPT1-10P also synthesized gelatinase and citolysin, and contained a set of virulent factors. In addition, BGPT1-10P carries the ermB and tetM genes conferring the resistance to erythromycin and tetracycline, respectively. [Projekat Ministarstva nauke Republike Srbije, br. 173019

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

Examination of antimicrobial potential in natural isolates of lactobacillus casei/paracasei group

Cilj ove studije je izučavanje antimikrobnog potencijala 52 prirodna izolata vrste L. casei/paracasei. Učestalost gena koji kodiraju BacSJ (bacSJ2-8/bacSJ2-8i genski klaster), acidocin 8912 (acdT), ABC-transporter (abcT) i pomoćni protein (acc) su takođe izučavani. Genski klaster bacSJ2-8/bacSJ2-8i prisutan je kod 49 (94.23%), a acdT kod 41 (78.85%) od 52 testirana soja. Četrdeset sojeva (76.92%) poseduje oba analizirana gena. Interesantno je da samo 17 sojeva (32.69%) koji poseduju bacSJ2-8/bacSJ2-8i genski klaster i/ili acdT gen proizvode bakteriocine. Soj L. paracasei BGNK1-62 poseduje bacSJ2-8/bacSJ2-8i genski klaster, ali ne proizvodi bakteriocin BacSJ što je verovatno posledica nedostatka abcT i acc gena. Nakon transformacije soja BGNK1-62 konstruktom pA2A koji poseduje abcT i acc gene ostvarena je proizvodnja bakteriocina BacSJ. Osim toga, utvrđeno je da soj L. paracasei BGGR2-66 proizvodi nov bakteriocin označen kao BacGR, koji je biohemijski okarakterisan, a određena je i njegova N-terminalna sekvenca.The aim of this study was to investigate the antimicrobial potential of 52 natural isolates of Lactobacillus casei/paracasei. The incidence of relevant genes encoding BacSJ (bacSJ2-8/bacSJ2-8i gene cluster), acidocin 8912 (acdT), ABC-transporter (abcT) and accessory protein (acc) was also studied. These genes were found to be widespread amongst the analyzed L. casei/paracasei strains. The bacSJ2-8/bacSJ2-8i gene cluster was present in 49 (94.23%) and acdT in 41 (78.85%) of the 52 tested strains. Forty of these strains (76.92%) harbored both analyzed genes. Interestingly, only 17 strains (32.69%) with the bacSJ2-8/bacSJ2-8i gene cluster and/or the acdT gene showed bacteriocin production. Strain L. paracasei BGNK1-62 contained the bacSJ2-8/bacSJ2-8i gene cluster, but did not produce bacteriocin BacSJ possibly due to absence of the abcT and acc genes. Hence, these genes were introduced into BGNK1-62 by transformation with constructed plasmid pA2A, after which BacSJ was produced. In addition, it was found that L. paracasei BGGR2-66 produced new bacteriocin designated as BacGR that was biochemically characterized and its N- terminal sequence was determined

Analysis of the lactic acid bacteria microflora in traditional Caucasus cow's milk cheeses

A total of 157 lactic acid bacteria (LAB) were isolated from three hand-made cheeses taken from different households in the region of the Caucasus Mountains. The cheeses were manufactured from cow's milk without the addition of a starter culture. The isolates of LAB were characterized by subjecting them to phenotypic and genotypic tests. The results of identification of LAB indicate that the examined cheeses contained 10 species, viz., Lactobacillus plantarum, Lactobacillus paraplantarum, Lactobacillus arizonensis, Lactobacillus farciminis, Lactobacillus brevis, Lactococcus lactis subsp. lactis, Leuconostoc mesenteroides subsp. mesenteroides, Leuconostoc pseudomesenteroides, Enterococcus faecium, and Enterococcus faecalis. The strains within the species L. plantarum, L. arizonensis, L. paraplantarum, L. farciminis, and L. pseudomesenteroides showed good proteolytic activity

Analysis of the lactic acid bacteria microflora in traditional Caucasus cow's milk cheeses

Iz tri ručno pravljena sira uzeta iz različitih domaćinstava smeštenih u regionu planine Kavkaz, izolovano je ukupno 157 bakterija mleč ne kiseline (LAB). Sirevi su pravljeni od kravljeg mleka bez dodatka starter kulture. Izolati LAB su okarakterisani fenotipskim i genotipskim testovima. Rezultati identifikacije LAB pokazuju da je u ispitivanim sirevima prisutno deset vrsta, kao što su: Lactobacillus plantarum, Lactobacillus paraplantarum, Lactobacillus arizonensis, Lactobacillus farciminis, Lactobacillus brevis, Lactococcus lactis subsp. lactis, Leuconostoc mesenteroides subsp. mesenteroides, Leuconostoc pseudomesenteroides, Enterococcus faecium i Enterococcus faecalis. Sojevi u okviru vrsta L. plantarum, L. arizonensis, L. paraplantarum, L. farciminis i L. pseudomesenteroides su pokazivali dobru proteolitičku aktivnost.A total of 157 lactic acid bacteria (LAB) were isolated from three hand-made cheeses taken from different households in the region of the Caucasus Mountains. The cheeses were manufactured from cow's milk without the addition of a starter culture. The isolates of LAB were characterized by subjecting them to phenotypic and genotypic tests. The results of identification of LAB indicate that the examined cheeses contained 10 species, viz., Lactobacillus plantarum, Lactobacillus paraplantarum, Lactobacillus arizonensis, Lactobacillus farciminis, Lactobacillus brevis, Lactococcus lactis subsp. lactis, Leuconostoc mesenteroides subsp. mesenteroides, Leuconostoc pseudomesenteroides, Enterococcus faecium, and Enterococcus faecalis. The strains within the species L. plantarum, L. arizonensis, L. paraplantarum, L. farciminis, and L. pseudomesenteroides showed good proteolytic activity

Novel Aggregation Promoting Factor AggE Contributes to the Probiotic Properties of Enterococcus faecium BGGO9-28

The understanding of mechanisms of interactions between various bacterial cell surface proteins and host receptors has become imperative for the study of the health promoting features of probiotic enterococci. This study, for the first time, describes a novel enterococcal aggregation protein, AggE, from Enterococcus faeciurn BGGO9-28, selected from a laboratory collection of enterococcal isolates with auto aggregation phenotypes. Among them, En. faecium BGG09-28 showed the strongest auto -aggregation, adhesion to components of ECM and biofilm formation. Novel aggregation promoting factor AggE, a protein of 178.1 kDa, belongs to the collagen -binding superfamily of proteins and shares similar architecture with previously discovered aggregation factors from lactic acid bacteria (LAB). Its expression in heterologous enterococcal and lactococcal hosts demonstrates that the aggE gene is sufficient for cell aggregation. The derivatives carrying aggE exhibited the ten times higher adhesion ability to collagen and fibronectin, possess about two times higher adhesion to mucin and contribute to the increase of biofilm formation, comparing to the control strains. Analysis for the presence of virulence factors (cytolysin and gelatinase production), antibiotic resistance (antibiotic susceptibility) and genes (cylA, egg, gelE, esp, hyiN, ace, efaks, and efagn) showed that BGG09-28 was sensitive to all tested antibiotics, without hemolytic or gelatinase activity. This strain does not carry any of the tested genes encoding for known virulence factors. Results showed that BGGO9-28 was resistant to low pH and high concentrations of bile salts. Also, it adhered strongly to the Caco-2 human epithelial cell line. In conclusion, the results of this study indicate that the presence of AggE protein on the cell surface in enterococci is a desirable probiotic feature

Characterization of lactic acid bacteria isolated from artisanal Zlatar cheeses produced at two different geographical location

Iz belog polutvrdog zlatarskog sira, označenog kao BGNV, izolovan je osamdeset jedan soj bakterija mlečne kiseline (BMK). Sir je uzet iz jednog seoskog domaćinstva smeštenog na severnoj strani planine Zlatar. Zlatarski BGNV sir je napravljen od svežeg kravljeg mleka bez dodatka starter kulture. Svi izolati BMK su okarakterisani fenotipskim i genotipskim testovima. Identifikacija sojeva je rađena rep-PCR analizom sa (GTG)5 prajmerom i 16S rDNK sekvenciranjem. Najzastupljenije vrste u zlatarskom BGNV siru su bile Lactobacillus casei/paracasei (65.43%) i Enterococcus faecalis (29.63%. Dva fakultetivno anaerobna štapića su identifikovana kao Lactobacillus plantarum (2.47%), a dva obligatna heterofermentativna izolata BMK su identifikovani kao Lactobacillus parabuchneri (2.47%). Od svih 81 testiranih izolata, samo osam eneterokoka su bili proizvođači antimikrobnih komponenti. Četrnaest od 16 testiranih izolata laktobacila je pokazivalo srednju do vrlo dobru proteolitičku aktivnost. Svih 57 laktobacila iz zlatarskog BGNV sira veoma sporo grušaju mleko, ili ga uopšte ne grušaju. Međutim, tri izolata enterokoka, BGNV1-63, BGNV1-76 i BGNV1-80, su pokazivala vrlo dobru aktivnost u mleku i grušala su ga za 5 h. Ove enterokoke su pokazivale vrlo visoku proteolitičku aktivnost potpuno hidrolizirajući αs1- i κ- kazein nakon 3 h, a β-kazein nakon 30 min inkubacije. Pored toga, ova tri izolata enterokoka degradovala su želatin. Upoređujući dobijene rezultate sa onima prethodno dobijenim ispitivanjem BMK u drugom zlatarskom BGZLS siru, napravljenom takođe, od svežeg kravljeg mleka, može se zaključiti da je mikroflora BMK zlatraskog BGNV sira manje raznovrsna.Eighty-one strains of lactic acid bacteria (LAB) were isolated from white semi-hard homemade cheese, designated Zlatar BGNV, which was taken from household settled on Northern side of mountain Zlatar. The Zlatar BGNV cheese was manufactured from raw cow's milk without addition of the starter culture. All isolates of LAB were characterized by phenotypic and genotypic tests. Identification of strains was done by the repetitive extragenic palindromic-polymerase chain reaction (rep-PCR) with (GTG)5 primer and 16S rDNA sequence analysis. The most present species in Zlatar BGNV cheese were Lactobacillus casei/paracasei (65.43%) and Enterococcus faecalis (29.63%). Two facultative heterofermentative rods were identified as Lactobacillus plantarum (2.47%), and two obligate hetrofermentative LAB isolates as Lactobacillus parabuchneri (2.47%). Among all 81 tested isolates, only eight enterococci were producers of antimicrobial compounds. Fourteen of 16 tested lactobacilli isolates showed medium to very good proteolytic activity. All 57 lactobacilli from the Zlatar BGNV cheese curdled milk very slowly or did not curdle milk at all. However, three isolates of enterococci, BGNV1-63, BGNV1-76 and BGNV1-80, showed very good activity in milk and curdled milk within 5 h. They showed very high proteolytic activity hydrolyzing completely αs1- and κ-casein after 3 h, and β-casein after 30 min of incubation. In addition, those three enterococcal isolates degraded gelatin. Comparing obtained results with those previously achieved in examination of LAB microflora in another Zlatar BGZLS cheese made also from raw cow's milk, it can be concluded that LAB microflora in the Zlatar BGNV cheese is less diverse