Antibiotic Susceptibility of Probiotic Bacteria

Lactic acid bacteria (LAB) are a heterogeneous group of bacteria widely distributed in nature. These bacteria are found in gastrointestinal (GI) and urogenital tract of humans and animals; they are present on plant material, in milk and meat, and numerous fermented foods. Lactic acid bacteria have been associated with traditional dairy products, cereals, vegetable and meat fermented foods, due to their natural presence leading to spontaneous fermentation. They are also used as starter cultures in industrial food production, as well as in the production of probiotic products due to their potential health benefits to consumer. Milk and dairy products are the most examined food system for the delivery of probiotic bacteria to the human gut. The probiotic concept has progressed and is now in the focus of different research. Significant improvements have been made in selection and characterization of new cultures and their application in food production. The food products, which are produced by traditional methods, exhibit a rich biodiversity with the respect to bacterial contents. From these products, new probiotic strains with the potential functional properties can been isolated and selected. The selected strains have to be further characterized in order to be used in the food industry. Before the probiotics can benefit human health, they must fulfill several criteria including: a) scientifically validated health properties; b) good technological properties meaning that they can be manufactured and incorporated into food products without loosing viability, functionality and technological performance; c) high survival through the upper gastrointestinal tract and high viability at its site of action; d) antagonistic activity to pathogens; e) antibiotic susceptibility; and f) to be able to function in the gut environment. Bearing in mind importance of antibiotic resistance of LAB in food chain, antibiotic susceptibility of potential probiotic strains is a very important criteria for their selection. In the recent decade, releasing of antibiotics in biosphere seriously increased, leading to a strong selective pressure for the emergence and persistence of resistant LAB strains. Since LAB are naturally present in traditionally made fermented food and GI tract and are also added as starter culture or probiotic bacteria in industrial food production, concerns have been raised about the antibiotic resistance of these beneficial bacteria strains. Probiotic bacteria can help maintaining balance in gastrointestinal tract in cases of diarrhea caused by antibiotic treatment. However, there is high risk associated with the ability of these resistant strains to transmit the resistance gene to pathogenic bacteria in gut microbiota. This can complicate the treatment of a patient with an antibiotic resistant bacterial infection or disease. The circulation of genes coding for antibiotic resistance from beneficial LAB in the food chain via animals to humans is a complex problem. Therefore, there is a need to evaluate the safety of potential probitic strains regarding their ability to acquire and disseminate antibiotic resistance determinants in selection of LAB. In this study, importance of LAB in the food chain will be reviewed. Morphological and biochemical characteristics of lactobacilli, bifidobactera and enterococci, as well as criteria for probiotic selection and role of probiotics in health benefit will be discussed. Antibiotic susceptibility as criteria for potential probiotic bacteria selection and mechanisms of gene transfers will be considered

Izolacija i karakterizacija autohtonih sojeva bakterija mlečne kiseline izdvojenih iz sjeničkog sira

Lactic acid bacteria strains isolated from autochtonous cheeses of Sjenica were researched. In those products, where starters were not added fermentation occurs as a result of natural flora present in the surrounding environment. Forty thermophilic, mesophilic and citrat+ LAB strains were isolated by selective mediums (MRS agar, LM17 agar and LDC agar) from 5 samples of Sjenica-cheese. These strains were exposed to the further analysis and indetification. After examination by Gram test and catalase test, 23 strains of LAB, were selected for further analysis. On the basis of lactose fermentation manner and acid production in 1% reconstituted skim milk, only 6 strains were selected. Determination of theese strains by API 50 CHL and Rapid ID 32 Strep tests, showed that 2 strains belong to the Lactococcus lactis ssp. lactis, 1 strain to the Lactococcus lactis ssp. cremoris, 1 strain to the Lactococcus lactis ssp. lactis bivar. diacetylactis and 2 strains to the Lactobacillus para. paracasei.U radu su izolovani i karakterisani sojevi bakterija mlečne kiseline izdvojeni iz sjeničkog sira, proizvedenog autohtonom tehnologijom, bez dodavanja starter kultura. Iz pet uzoraka sjeničkog sira izvršena je izolacija 40 termofilnih, mezofilnih i citrat+ sojeva BMK na odgovarajućim selektivnim podlogama (MRS agar, LM17 agar i LDC agar). Ovi sojevi su dalje podvrgnuti ispitivanju na Gram reakciju i katalaza test, posle čega je izabrano 23 soja za dalja ispitivanja. Na osnovu načina fermentacije laktoze i acidogene sposonosti praćene u 1% rekonstituisanom obranom mleku selekcionisano je samo šest sojeva. Determinacijom ovih sojeva pomoću API CHL 50 i Rapid ID 32 Strep testa, pokazalo se da dva soja pripadaju Lactococcus lactis ssp. lactis, jedan soj Lactococcus lactis ssp. cremoris jedan soj Lactococcus lactis ssp. lactis bivar. diacetylactis i dva soja Lactobacillus para. paracasei vrsti

Izolacija i karakterizacija bakterijske flore iz autohtonih fermentisanih proizvoda Srbije i Crne Gore

The natural community of lactic acid bacteria isolated from farmhouse fermented milk products has been investigated. In such products, where no starters are added, fermentation occurs as a result of natural flora present in the surrounding environment. A total of 200 isolated strains were examined for their acidification activity and EPS formation. Out of the 200 isolated strains, 27 strains were selected for further investigation. They were further identified by 16S rRNA sequencing in order to obtain the identification at species level, which showed that 14 strains belong to Lactococcus lactis subsp. lactis, 10 of them to Lactobacillus sp and 3 strains belongs to Leuconostoc sp. To differentiate between strains the pulsed-field gel electrophoresis (PFGE) patterns, of 15 isolated lactococcal strains, was generated using Smal or Ascl. Unrelated strains yielded different patterns of digestion products. The plasmid isolation of these strains has also been conducted in order to compare these results to patterns of PFGE. Phage typing of the Lactococcus sp. strains has been conducted. All lactococcal strains were resistant against 41 phages (Chr. Hansen phage collection) representing the major phage groups known for Lactococcus. These results indicate that the strains represent a possible tool for cultures that have not been exposed to any industrial selection.U ovom radu su proučavani sojevi bakterija mlečne kiseline izolovani iz autohtonih fermentisanih mlečnih proizvoda. Kod ovih proizvoda, kojima se ne dodaju starter kulture, fermentacija se odigrava kao rezultat aktivnosti prirodne mikroflore koja je prisutna u okolnoj sredini. Kod ukupno 200 sojeva proučavana je njihova sposobnost acidifikacije i sposobnost stvaranja egzopolisaharida (EPS). Od ukupno 200 izolovanih sojeva selektovano je njih 27 koji su podvrgnuti daljem proučavanju. Nakon ove selekcije, sojevi su identifikovani 16S rRNA sekvenciranjem u cilju njihove identifikacije na nivou vrste. Od 27 selektovanih sojeva, 14 je pripadalo vrsti Lactococcus lactis subsp. Lactis, 3 soja je pripadalo vrsti Leuconostoc spp., dok je 10 sojeva pripadalo vrsti Lactobacillus sp. Da bi se utvrdile razlike između različitih sojeva urađena je analiza uz korišćenje elektroforeze u pulsirajućem električnom polju (PFGE), a matrice su dobijene uz korišćenje Smal i AscI restrikcionih enzima. Sojevi koji nisu u srodstvu dali su različite matrice digestionih produkata. Izvršena je i izolacija plazmida iz ovih sojeva kako bi se potvrdili rezultati dobijeni PFGE tehnikom. Takođe je izvršena i fagotipizacija sojeva za sve sojeve za koje je dokazano da pripadaju vrsti Lactococcus sp. Svi laktokokni sojevi su bili rezistentni na 41 fag (kolekcija faga kompanije Chr. Hansen) koji predstavljaju grupu bakteriofaga koji su najčešći za vrstu Lactococcus. Dobijeni rezultati ukazuju na to da proučavani sojevi predstavljaju mogući izvor kultura koji do sada nisu bili izloženi bilo kakvoj industrijskoj selekciji

Expression of bacteriocin LsbB is dependent on a transcription terminator

The production of LsbB, leaderless class II bacteriocin, is encoded by genes (lsbB and lmrB) located on plasmid pMN5 in Lactococcus lactis BGMN1-5. Heterologous expression of the lsbB gene using the pAZIL vector (pAZIL-lsbB) in L. lactis subsp. cremoris MG7284 resulted in a significant reduction (more than 30 times) of bacteriocin LsbB expression. Subcloning and deletion experiments with plasmid pMN5 revealed that full expression of LsbB requires the presence of a complete transcription terminator located downstream of the lsbB gene. RNA stability analysis revealed that the presence of a transcription terminator increased the RNA stability by three times and the expression of LsbB by 30 times. The study of the influence of transcription terminator on the expression of other bacteriocin genes (lcnB, for lactococcin B production) indicated that this translational terminator likely functions in a lsbB-specific manner rather than in a general manner

Autohtona mikroflora sjeničkog sira

Autochthonous isolates of lactic acid bacteria and enterococci, from Sjenica cheese had been isolated and identified. From 10 samples of Sjenica cheese that were traditionally produced without starter cultures, 53 thermophile mesophile and citrate (+) isolates were isolated using selective media (MRS agar, M17 agar, LDCagar i LDV agar). According to Gram staining, cell morphology and catalase test, it was determined that all isolates were pure culture of G (+) and catalase (-) bacteria, 21 bacilli and 35 cocci. For the purpose of diferentiation enterococci and lactococci, growth of all cocci bacteria was examined on 6,5% NaCI, pH 9,6 and heat treatment 60°C 20 min. Out of 35 cocci isolates, 7 isolates were able to grow at these conditions. Identification by API CHL 50 test for lactobacilli and RAPID ID 32 Strep for cocci, showed that isolates were Lactococcus lactis ssp. lactis Lactobacillus paracasei ssp. paracasei, Lactobacillus plantarum, Lactococcus lactis ssp. lactis boiv. dicetylactis, Lactobacillus curvatus, Lactobacillus brevis, Leuconostoc sp. i Enterococcus sp. Dominant lactococci strain was Lactococcus lactis ssp. lactis (35,85%) and for lactobacilli Lactobacillus paracasei ssp. paracasei (18,86%). Strains from genus Enterococcus sp. had the rate of 13,20%.U radu su izdvojeni i identifikovani izolati bakterija mlečne kiseline i enterokoka iz sjeničkog sira, proizvedenog na tradicionalan način, bez dodavanja starter kulture. Iz 10 uzoraka sjeničkog sira izdvojeno je 53 termofilna, mezofilna i citrat+ izolata na odgovarajućim selektivnim podlogama (MRS agar, M17 agar, LDCagar i LDV agar). Postupkom bojenja po Gramu, mikroskopskim pregledom i katalaza testom je utvrđeno da svi izolati predstavljaju čiste kulture Gram-pozitivnih i katalaza-negativnih bakterija pri čemu je utvrđeno da je 21 izolat štapićastog, a 35 okruglastog oblika. Radi razdvajanja enterokoka od laktokoka, za sve okruglaste forme je ispita-na sposobnost rasta pri 6,5% NaCI, pH 9,6 i nakon termičkog tretmana na 60°C 20 minuta je utvrđeno da od 35 izolata okruglastog oblika, 7 izolata podnosi navedene uslove. Determinacijom dobijenih izolata primenom API sistema i to za štapičaste oblike API CHL50testa, a za okruglaste oblike RAPID ID 32 Strep sistema, utvrđeno je da izolati pripadaju vrstama Lac-tococcus lactis ssp. lactis, Lactobacillus paracasei ssp. paracasei Lactobacillus plantarum, Lactococcus lactis ssp. lactis boiv. dicetylactis Lactobacillus curvatus, Lactobacillus brevis, Leuconostoc sp. i Enterococcus sp. Ubedljivo najveću zastupljenost je imala okruglasta vrsta Lactococcus lactis ssp. lactis (35,85%), a od štapićastih, vrsta Lactobacillus paracasei ssp. Paracasei (18,86%). Vrste roda Enterococcus sp. bile su zastupljene sa 13,20 %

Poboljšanje metaboličke aktivnosti bakterije Lactobacillus plantarum 564 pomoću elektroporacije

The exposure of bacterial cells to pulsed electric fields (PEF) leads to the reversible formation of pores in the cell membrane if an applied energy is below the critical level. Therefore, the effect of electric field pulses with amplitudes below 14 kV/cm and the applied energy up to 12.2 J/cm3 on the growth of Lactobacillus plantarum 564 cells was investigated. After PEF treatments, the growth of lactobacilli in De Man-Rogosa-Sharpe broth at 37 °C was monitored by isothermal calorimetry, absorbance and plate counts. All the applied treatments resulted in a higher growth rate of PEF-treated cells during early and mid-log phase, especially bacterial samples treated with lower field intensities (1.3–5.5 J/cm3). The transport of ions and molecules through the cell membrane (which facilitates the growth of electroporated lactobacilli) was particularly evident in the mid-exponential growth phase, where the doubling time was reduced more than 3 times after the exposure to electric pulses of 5.5 J/cm3. The heat production rate during the growth of electroporated cells was also higher, indicating the enhanced metabolic activity of PEF-treated cells. Moreover, the electroporated cells had a better acidification ability than the untreated ones. It can be summarized that the applied PEF treatments with an energy input of below 12 J/cm3 potentially induce reversible electroporation of the cell membrane, which has a positive impact on the growth and metabolic activity of the cells of lactobacilli.Izlaganje bakterija pulsirajućim električnim poljima uzrokuje reverzibilno stvaranje pora na staničnoj membrani, ako je energija električnog polja ispod kritične razine. Stoga je istražen utjecaj pulsirajućih električnih polja na rast stanica bakterije Lactobacillus plantarum 564 primjenom polja jačine do 12.2 J/cm3, tj. amplituda manjih od 14 kV/cm. Rast laktobacila u De Man-Rogosa-Sharpe bujonu na 37 °C nakon tretmana praćen je pomoću izotermalne kalorimetrije, te mjerenjem apsorbancije i ukupnog broja stanica. Utvrđeno je da su stanice izložene pulsirajućem električnom polju brže rasle tijekom rane i srednje logaritamske faze, što je naročito bilo izraženo kod stanica tretiranih poljima niskog intenziteta (1.3-5.5 J/cm3). Transport iona i molekula kroz staničnu membranu (što olakšava rast elektroporiranih laktobacila) bio je posebno izražen tijekom eksponencijalne faze rasta, kad je generacijsko vrijeme stanica tretiranih pulsevima energije od 5.5 J/cm3 bilo trostruko kraće. Tijekom rasta tretiranih stanica oslobođena je veća količina topline, što znači da se metabolička aktivnost stanica povećala nakon tretmana. Osim toga, elektroporirane su stanice jače zakiseljavale sredinu. Iz dobivenih se rezultata može zaključiti da pulsirajuća električna polja jačine manje od 12.2 J/cm3 uzrokuju reverzibilnu elektroporaciju stanične membrane, što ima pozitivan učinak na rast i metaboličku aktivnost stanica laktobacila

Karakteristike rasta i aktivnosti laktobacila u UF mleku

The application of ultrafiltration has an increasing importance in the manufacture of dairy products, primarily of soft cheeses. However, in semi-hard and hard cheeses, manufac­ture ultrafiltration has not been applied at industrial level yet. Considering that UF milk is characterised by increased buffer capacity, lactic acid bacteria, that could be used as starter cultures, must have an expressed acidogenic ability. Also, practice has shown that cheeses manufactured of UF milk ripen slowly, because it is necessary that starter cultures have an expressed and proteolytic activity. The buffer capacity of milk equivalent (CF=1) and retentates (CF=3 and CF=4) were studied. Furthermore, the influence of milk equivalent and these retentates on the growth and activity of two strains of lactic acid bacteria: Lactobacillus lielveticus HD-50 and Lactobacillus acidophilus MC. The growth and production of lactic acid of the tested strains in UF milk were being observed during eight hours of fermentation. Proteolytic activity of these strains was examined by determination of tyrosine value and electrophoretic detection prt+ strains which were capable to hydrolyse p casein. In strain iMctobacillus helveticus HD-50 was detected better acidogenic ability, but in strain Lactobacillus acidophilus MC was detected better proteolytic activity.Primena ultrafiltracije ima sve veći značaj u proizvodnji mlečnih proizvoda, u prvom redu mekih sireva, dok je u proizvodnji polutvrdih i tvrdih sireva zastupljena u manjem obimu. S obzirom da se UF mleko karakteriše povećanim puferskim kapacitetom, bakterije mlečne kiseline koje bi se mogle koristiti kao starter kulture, moraju se odlikovati izraženom acidogenom sposobnošću. Takodje, praksa je pokazala da sirevi dobijeni iz UF mleka sporije sazrevaju, zbog čega je neophodno da starter kulture poseduju izražena i proteolitička svojstva. U radu je prikazan puferski kapacitet UF mleka različitog stepena koncentrovanja i to mlečnog ekvivalenta (CF=1) i retentata CF=3 i CF=4. Zatim je ispitivan uticaj UF mleka navedenih koncentracija na aktivnost i rast dva soja bakterija mlečne kiseline i to : Lactobacillus helveticus HD-50 i Lactobacillus acidophilus MC. Rast i acidogena sposobnost ispitivanih sojeva je praćena tokom osam sati fermentacije u mlečnom ekvivalentu i retenlatima. Proteolitička aktivnost ispitivanih sojeva je determinisana određivanjem tirozinske vrednosti i elektroforetskom detekcijom prt+ sojeva koji hidrolizuju P kazein. Ispitivanja su pokazala da soj Lactobacillus helveticus HD-50 ima bolju acidogenu sposobnost, a soj Lactobacillus acidophilus MC je pokazao bolju proteolitičku aktivnost

Expression of bacteriocin LsbB is dependent on a transcription terminator

The production of LsbB, leaderless class II bacteriocin, is encoded by genes (lsbB and lmrB) located on plasmid pMN5 in Lactococcus lactis BGMN1-5. Heterologous expression of the lsbB gene using the pAZIL vector (pAZIL-lsbB) in L. lactis subsp. cremoris MG7284 resulted in a significant reduction (more than 30 times) of bacteriocin LsbB expression. Subcloning and deletion experiments with plasmid pMN5 revealed that full expression of LsbB requires the presence of a complete transcription terminator located downstream of the lsbB gene. RNA stability analysis revealed that the presence of a transcription terminator increased the RNA stability by three times and the expression of LsbB by 30 times. The study of the influence of transcription terminator on the expression of other bacteriocin genes (lcnB, for lactococcin B production) indicated that this translational terminator likely functions in a lsbB-specific manner rather than in a general manner

Determinacija autohtonih bakterija mlečne kiseline iz kačkavalja

Autochthonous isolates of lactic acid bacteria and enterococci from Kashkaval cheese had been isolated and identified. From 6 samples of Kashkaval cheese that were traditionally produced without starter cultures, 27 isolates were isolated using selective media (MRS agar, M17 agar) and two incubation temperatures (37ºC and 43ºC). According to Gram staining, cell morophology and catalase test, it was determined that all isolates were pure culture of G (+) and catalase (-) bacteria, 12 bacilli and 15 cocci. For the purpose of diferentiation between enterococci and lactococci, growth of all cocci bacteria was examined on 6,5% NaCl, pH 9,6 and heat treatment 60ºC 20 min. Out of 15 cocci isolates, 6 isolates were able to grow at these conditions. Acidogenic activity was determinated by measuring pH values of 1% inoculated reconstituted skim milk during 24 hours. Researched isolates showed significant difference in lactic acid production. Ten isolates had good acidogenic activity (pH 4,12-4,90) and seventeen isolates were weak producers (pH 5,10 - 6,48). Identification by API CHL 50 test for lactobacilli and RAPID ID 32 Strep for cocci, showed that isolates were Lactobacillus delbrueckii ssp. delbrueckii, Lactobacillus casei, Lactobacillus plantarum Lactobacillus fermentum, Lactobacillus curvatus, Streptococcus salivarius ssp. thermophilus, Lactococcus lactis ssp. lactis, Lactococcus lactis ssp. cremoris, Enterococcus faecium i Enterococcus durans.U radu su izdvojeni i okarakterisani izolati bakterija mlečne kiseline i enterokoka iz kačkavalja, proizvedenog bez dodavanja starter kulture. Iz 6 uzoraka kačkavalja različite starosti izdvojeno je 27 izolata na odgovarajućim selektivnim podlogama MRS i M17 agaru i temperaturama rasta od 37ºC i 43ºC. Postupkom bojenja po Gramu, mikroskopskim pregledom i katalaza testom je utvrđeno da svi izolati predstavljaju čiste kulture Gram-pozitivnih i katalaza-negativnih bakterija, pri čemu je utvrđeno da je 12 izolata štapićastog, a 15 okruglastog oblika.. Radi razdvajanja enterokoka od bakterija mlečne kiseline, za sve okruglaste forme je ispitana sposobnost rasta pri 6,5% NaCl, pH 9,6 i nakon termičkog tretmana na 60ºC 20 minuta utvrđeno je da od 15 izolata okruglastog oblika, 6 izolata podnosi navedene uslove. Acidogena aktivnost je određena merenjem pH vrednosti rekonstituisanog obranog mlaka sa 1 % inokuluma u toku 24 sata, pri čemu je utvrđeno da se izolati značajno razlikuju u sposobnosti stvaranja kiseline. Deset izolata je pokazalo dobru acidogenu sposonost (pH 4,12-4,90), dok je 17 izolata slabo produkovalo kiselinu (pH 5,10 - 6,48). Determinacijom dobijenih izolata primenom API sistema i to za štapićaste oblike API CHL 50 testa, a za okruglaste oblike RAPID ID 32 Strep sistema, utvrđeno je da izolati pripadaju vrstama: Lactobacillus delbrueckii ssp. delbrueckii, Lactobacillus casei Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus curvatus Streptococcus salivarius ssp. thermophilus, Lactococcus lactis ssp. lactis Lactococcus lactis ssp. cremoris, Enterococcus faecium i Enterococcus durans

Saccharomyces boulardii jedini kvasac probiotik

Yeasts as eukaryotes do have a more diverse enzymatic profile than lactic acid bacteria. They provide protection against pathogenic bacteria and toxic compounds by surface binding. Saccharomyces boulardii, a non-pathogenic yeast, has been used both as a preventative and therapeutic agent for the treatment of a variety of diarrheal diseases. Experimental studies clearly demonstrate that S. boulardii has specific probiotic properties, and recent data has opened the door for new therapeutic uses of this yeast. Today, a considerable number of pharmaceutical preparations (capsules, powders, tablets, pellets) containing probiotic yeast Saccharomyces boulardii cells are commercially available, and are marketed mainly via pharmacies. Also, the application of the Saccharomyces boulardii as a probiotic microorganism seems promising especially in milk and yogurts, the production of amounts of alcohol and gas formation, however, are for the moment major problem. .Probiotici danas predstavljaju pravi hit u proizvodnji funkcionalne hrane i parafarmaceutskih proizvoda. Po pravilu, svi ovi proizvodi zasnivaju se na bakterijama mlečne kiseline (BMK) i bifidobakterijama, a rezultati velikog broja istraživanja opravdavaju njihovu sve veću primenu. Međutim, kod parafarmaceutskih proizvoda često je prisutan kvasac Saccharommyces boulardii koji je intenzivno testiran u pogledu probiotskih svojstava, a koji je prema najnovijim molekularnim ispitivanjima veoma blizak vrsti Saccharomyces cerevisiae. Prema nekim autorima, radi se o varijetetu navedene vrste. Saccharommyces boulardii pokazuje terapeutska svojstva kod dijareja izazvanih delovanjem antibiotika, prisustvom Clostridium dificile, pojave iritabilnog kolona, kod osoba obolelih od AIDSa, itd. Kako je navedena vrsta nađena u kulturama čajne gljive kombuhe i kefira, učinjeni su prvi pokušaji u pogledu primene ove vrste u proizvodnji fermentisanih mlečnih napitaka u zajednici sa BMK. Iako nije u stanju da koristi laktozu, Saccharommyces boulardii je koristio prisutne organske kiseline, galaktozu i glukozu nastalu u toku metabolizma BMK. Pojava gasa i alkohola i dalje predstavlja glavni problem za dalju primenu ove vrste, ali istraživanja treba nastaviti jer se potencijalno mogu dobiti proizvodi sa visokom nutritivnom vrednošću.