Dominant lactic acid bacteria in artisanal Pirot cheeses of different ripening period

U ovom radu su ispitivana dva sira od svežeg kravljeg mleka različitog perioda zrenja. Sirevi su uzeti iz seoskog domaćinstva u regionu Stare Planine, a proizvedeni su bez dodatka starter kulture. Iz oba sira je izolovano ukupno 106 sojeva bakterija mlečne kiseline. Sojevi su testirani klasičnim fiziološkim i API 50 CH testovima. Takođe je ispitivana proteolitička i antimikrobna aktivnost. Identifikacija bakterija mlečne kiseline je rađena rep-PCR analizom sa (GTG)5 prajmerom. Osam vrsta bakterije mlečne kiseline je izolavano iz sira BGPT9 starog četiri dana (Lactobacillus plantarum, Lactobacillus paracasei subsp. paracasei, Lactobacillus delbrueckii, Lactobacillus brevis, Enterococcus faecium, Enterococcus faecalis, Enterococcus durans i Leuconostoc garlicum), dok su u siru BGPT10 starom osam meseci bile prisutne samo dve vrste (Lactobacillus plantarum i Enterococcus faecium). Proteolitičku aktivnost je pokazalo 30 izolata iz sira BGPT9, uglavnom enterokoke. Samo jedan izolat iz sira BGPT10 (koji je pripadao vrsti Lactobacillus plantarum) je posedovao delimičnu sposobnost da hidrolizuje β- kazein. Sedam enterokoka iz sira BGPT9 i četiri enterokoke iz sira BGPT10 je proizvodilo antimikrobne substance.In this study two raw cow's milk cheeses of a different ripening period were examined. The cheeses were taken from a country household in the region of mountain Stara Planina and manufactured without adding of starter culture. A total 106 lactic acid bacteria (LAB) strains were isolated from both cheeses. They are tested by classical physiological tests as well as by API 50 CH tests. Proteolytic and antimicrobial activities were done too. Identification of LAB isolates was done by repetitive extragenic palindromic-polimerase chain reaction (rep-PCR) with (GTG)5 primer. The LAB isolates from cheese BGPT9 (four days old) belonged to the eight species of LAB (Lactobacillus plantarum, Lactobacillus paracasei subsp. paracasei, Lactobacillus delbrueckii, Lactobacillus brevis, Enterococcus faecium, Enterococcus faecalis, Enterococcus durans and Leuconostoc garlicum), while in the BGPT10 cheese (eight months old) only two species were present (Lactobacillus plantarum and Enterococcus faecium). Proteolytic activity showed 30 LAB from BGPT9 cheese, mainly enterococci. From BGPT10 cheese only one isolate (which belonged to the Lactobacillus plantarum species) possessed partial ability to hydrolyze β-casein. Seven enterococci from BGPT9 cheese and four enterococci from BGPT10 cheese produced antimicrobial compounds

Influence of carbohydrates on cell properties of Lactobacillus rhamnosus

Lactobacilli represent normal commensals of the human body, particularly in the gut and vagina where they protect these environments from incoming pathogens via a variety of mechanisms. The influence of the carbohydrate source present in reconstituted MRS growth medium on the different cell properties of two Lactobacillus rhamnosus strains were examined. Two human vaginal isolates, BGHV719 and exopolysaccharide producer strain BGHV954 were analyzed. The results demonstrated that unlike in reconstituted MRS with glucose as a carbon source, the presence of fructose, mannose, or rhamnose, significantly reduced cell surface hydrophobicity of both strains. In addition, differences in cell wall protein composition of L. rhamnosus BGHV719 and alterations in colony mucoidity of L. rhamnosus BGHV954 were also demonstrated. Light and SEM microscopy revealed differences on the cellular level when BGHV719 was cultivated in the presence of different sugars. The results of this study point out the importance of complex relationships between growth medium composition and the different aspects of bacterial behavior, and call for more detailed analyses of versatile bacterial responses to the changes in the environment, including vaginal ecosystem. This is especially important since lactobacilli are amongst the most widely used of probiotics

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

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

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

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

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

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

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