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

Ubiquity and diversity of multidrug resistance genes in Lactococcus lactis strains isolated between 1936 and 1995

The presence and the nucleotide sequence of four multidrug resistance genes, lmrA, lmrP, lmrC, and lmrD, were investigated in 13 strains of Lactococcus lactis ssp. lactis, four strains of Lactococcus lactis ssp. cremoris, two strains of Lactococcus plantarum, and two strains of Lactococcus raffinolactis. Multidrug resistance genes were present in all L. lactis isolates tested. However, none of them could be detected in the strains belonging to the species L. raffinolactis and L. plantarum, suggesting a different set of multidrug resistance genes in these species. The analysis of the four deduced amino acid sequences established two different variants depending on the subspecies of L. lactis. Either lmrA, or lmrP, or both were found naturally disrupted in five strains, while full-length lmrD was present in all strainsThis work was financed by the European Union STREP project ACE-ART (FP6-506214).Peer reviewe

Regulatory and Safety Requirements for Food Cultures

The increased use of food cultures to ferment perishable raw materials has potentiated the need for regulations to assess and assure the safety of food cultures and their uses. These regulations differ from country to country, all aimed at assuring the safe use of food cultures which has to be guaranteed by the food culture supplier. Here we highlight national differences in regulations and review a list of methods and methodologies to assess the safety of food cultures at strain level, at production, and in the final product

Evaluation of Reuterin Production in Urogenital Probiotic Lactobacillus reuteri RC-14▿

Classified as a distinct species in 1980, Lactobacillus reuteri strains have been used in probiotic formulations for intestinal and urogenital applications. In the former, the primary mechanism of action of L. reuteri SD2112 (ATCC 55730) has been purported to be its ability to produce the antibiotic 3-hydroxypropionaldehyde (3-HPA), also known as reuterin. In the vagina, it has been postulated that probiotic Lactobacillus reuteri RC-14 does not require reuterin production but mediates a restoration of the normal microbiota via hydrogen peroxide, biosurfactant, lactic acid production, and immune modulation. The aim of the present study was to determine whether strain RC-14 produced reuterin. Using PCR and DNA dot blot analyses, numerous Lactobacillus species, including RC-14, were screened for the presence of the gene encoding the large subunit of glycerol dehydratase (gldC), the enzyme responsible for reuterin production. In addition, lactobacilli were grown in glycerol-based media and both high-performance liquid chromatography and a colorimetric assay were used to test for the presence of reuterin. L. reuteri RC-14 was determined to be negative for gldC sequences, as well as for the production of reuterin when cultured in the presence of glycerol. These findings support that the probiotic effects of L. reuteri RC-14, repeatedly demonstrated during numerous studies of the intestine and vagina, are independent of reuterin production

Development of a freeze substitution technique to examine the structure of Lactobacillus casei GR-1 grown in agar and under batch and chemostat culture conditions

Classified as a distinct species in 1980, Lactobacillus reuteri strains have been used in probiotic formulations for intestinal and urogenital applications. In the former, the primary mechanism of action of L. reuteri SD2112 (ATCC 55730) has been purported to be its ability to produce the antibiotic 3-hydroxypropionaldehyde (3-HPA), also known as reuterin. In the vagina, it has been postulated that probiotic Lactobacillus reuteri RC-14 does not require reuterin production but mediates a restoration of the normal microbiota via hydrogen peroxide, biosurfactant, lactic acid production, and immune modulation. The aim of the present study was to determine whether strain RC-14 produced reuterin. Using PCR and DNA dot blot analyses, numerous Lactobacillus species, including RC-14, were screened for the presence of the gene encoding the large subunit of glycerol dehydratase (gldC), the enzyme responsible for reuterin production. In addition, lactobacilli were grown in glycerol-based media and both high-performance liquid chromatography and a colorimetric assay were used to test for the presence of reuterin. L. reuteri RC-14 was determined to be negative for gldC sequences, as well as for the production of reuterin when cultured in the presence of glycerol. These findings support that the probiotic effects of L. reuteri RC-14, repeatedly demonstrated during numerous studies of the intestine and vagina, are independent of reuterin production. Copyright © 2008, American Society for Microbiology. All Rights Reserved

Ubiquity and diversity of multidrug resistance genes in Lactococcus lactis

The presence and the nucleotide sequence of four multidrug resistance genes, lmrA, lmrP, lmrC, and lmrD, were investigated in 13 strains of Lactococcus lactis ssp. lactis, four strains of Lactococcus lactis ssp. cremoris, two strains of Lactococcus plantarum, and two strains of Lactococcus raffinolactis. Multidrug resistance genes were present in all L. lactis isolates tested. However, none of them could be detected in the strains belonging to the species L. raffinolactis and L. plantarum, suggesting a different set of multidrug resistance genes in these species. The analysis of the four deduced amino acid sequences established two different variants depending on the subspecies of L. lactis. Either lmrA, or lmrP, or both were found naturally disrupted in five strains, while full-length lmrD was present in all strainsThis work was financed by the European Union STREP project ACE-ART (FP6-506214).Peer reviewe