Lactic acid bacteria isolated from canine faeces

AIMS: Lactic acid bacteria (LAB) were isolated and sequenced from the faeces of healthy dogs. Five of these strains were selected and further characterized to clarify the potential of these strains as probiotics for canine. METHODS AND RESULTS: LAB were found in 67% (14/21) of the canine faeces samples when plated on Lactobacilli Selective Media without acetic acid. Out of 13 species identified with partial 16S rRNA gene sequencing, Lactobacillus fermentum LAB8, L. mucosae LAB12, L. rhamnosus LAB11, L. salivarius LAB9 and Weissella confusa LAB10 were selected as candidate probiotic strains based on their frequency, quantity in faeces, growth density, acid tolerance and antimicrobial activity. The minimal inhibitory concentration values of these isolates were determined for 14 antibiotics. L. salivarius LAB9, W. confusa LAB10 and L. mucosae LAB12 were viable in pH 2 for 4 h (mLBS), indicating tolerance to acidity and thus the potential to survive in gastrointestinal tract of the canine. The LAB8-LAB12 strains showed antimicrobial activity against Micrococcus luteus A1 NCIMB86166. CONCLUSIONS: Thirteen different LAB species were found from the faecal microbiota of the healthy canines. Five acid tolerant and antimicrobially active LAB strains with the capacity to grow to high densities both aerobically and anaerobically were chosen to serve as candidate probiotics. SIGNIFICANCE AND IMPACT OF THE STUDY: The selected LAB strains are among the first host-specific LAB with antimicrobial activity isolated from canines that could serve as potential probiotics for canine use.     

A genus-specific PCR method for differentiation between Leuconostoc and Weissella and its application in identification of heterofermentative lactic acid bacteria from coffee fermentation

A genus-specific PCR analysis method was developed for a rapid and reliable differentiation between the two heterofermentative lactic acid bacteria genera Leuconostoc and Weissella. Primer sets specific for target regions of the 16S rRNA genes were designed and the specificity of the PCR was evaluated using the type strains of 13 species of Leuconostoc and 11 species of Weissella. In addition, the newly developed genus-specific PCR analysis was applied to characterize 72 lactic acid bacteria (LAB) strains isolated from coffee fermentation and which were presumptively classified as Leuconostoc or Weissella species. Additionally, a total of 34 LAB isolates from various other fermented foods were included. The investigations of these strains were conducted to test the effectiveness of correct characterization of field isolates using the genus-specific PCR approach. The correct assignment to one of these two genera by the application of the genus-specific primers was confirmed by further identifying the strains using repetitive extragenic palindromic-PCR and 16S rRNA gene sequencing.     

Species diversity and relative abundance of vaginal lactic acid bacteria from women in Uganda and Korea

AIMS: Lactobacilli play an important role in maintaining vaginal health of women. The aim of this study was to compare the species richness and relative abundance of Lactobacillus and other lactic acid bacteria in women of two geographically distant countries, Uganda and Korea. METHODS AND RESULTS: Vaginal samples were obtained from two women populations in Uganda and Korea. The Lactobacillus Rogosa SL agar was used for initial isolation of lactic acid bacteria. After phenotypic analyses, the 16S rRNA gene was amplified by polymerase-chain reaction and analysed by the BLAST program and phylogenetic tree construction. A total of 338 (128 Korean and 210 Ugandan) vaginal lactic acid bacterial strains were isolated, including five genera: Lactobacillus, Leuconostoc, Pediococcus, Streptococcus and Weissella. While Lactobacillus crispatus was common in both populations, Lactobacillus fermentum was common only in Korean women, and Lactobacillus gasseri, Lactobacillus reuteri and Lactobacillus vaginalis only in Ugandan women. Among other lactic acid bacteria, Weissella was more common in Ugandan, and Pediococcus in Korean women. All Weissella strains produced hydrogen peroxide, and all Pediococcus strains inhibited Candida species. CONCLUSION: Although many lactic acid bacteria colonize women, their species distributions may be different in women of geographically separated communities. SIGNIFICANCE AND IMPACT OF THE STUDY: The knowledge of species richness and relative abundance of vaginal lactic acid bacteria, including Lactobacillus, Pediococcus and Weissella, may lead to the design of better probiotic products as bacterial replacement therapy.     

Identification of lactic acid bacteria isolated from human vaginal secretions

A total of 57 lactic acid bacteria were isolated from the vaginal secretions of 259 patients. Of these strains, 37 were isolated from patients attending pre-natal clinics and the remaining strains from patients attending post-natal clinics. The strains were identified by using simple physiological and biochemical tests and their phenotypic relatedness determined by numerical analysis of total soluble cell protein patterns. The genotypic relatedness of representative strains selected from each of the protein profile clusters was determined by numerical analysis of the DNA banding patterns obtained from RAPD-PCR. The majority of lactobacilli isolated belonged to the species Lactobacillus pentosus, Lactobacillus fermentum and Enterococcus faecium. A few strains of Lactobacillus plantarum and Weissella viridescens were also isolated. One strain, TV 1029, grouped into the same protein profile cluster as E. faecium, but revealed a DNA banding pattern closer related to Enterococcus faecalis. This is the first report of W. viridescens associated with the human vagina. This revised version was published online in June 2006 with corrections to the Cover Date.     

Interactions between Saccharomyces cerevisiae and lactic acid bacteria in sourdough

The aim of this study was to assess the interactions between Saccharomyces cerevisiae and lactic acid bacteria that either form a stable consortium in Greek wheat sourdoughs (i.e. Lactobacillus sanfranciscensis and L. brevis) or occasionally constitute the secondary microbiota (i.e. Weissella cibaria, L. paralimentarius, Pediococcus pentosaceus and Enterococcus faecium). For this purpose, wheat dough was prepared by using strains of the above mentioned species either as single starters, or in combination of the yeast with each of the lactic acid bacteria strains. The determination of the metabolic products in sourdough samples was performed by HPLC analysis. Presence of lactic acid bacteria had no effect on S. cerevisiae final cell yield but affected negatively the maximum specific growth rate. Ethanol production was primarily affected negatively while the co-culture had a variable effect on glycerol production. On the other hand, the presence of S. cerevisiae favoured mannitol and acetic acid production, had a species-dependent effect on maximum specific growth rate and had no effect on final cfu/g sourdough and lactic acid production by the lactic acid bacteria and at the same time caused the depletion of glucose, fructose and maltose.     

Characterization of sourdough lactic acid bacteria based on genotypic and cell-wall protein analyses

AIMS: To evaluate the effectiveness of two independent methods in differentiating a large population of lactic acid bacteria (LAB) isolated from wheat flours and sourdoughs and to correlate eventual differences/similarities among strains with their geographical origin and/or process parameters. METHODS AND RESULTS: One hundred fifty strains belonging to Lactobacillus spp. and Weissella spp., plus eight type strains, one for each species, and two unidentified isolates, were characterized by randomly amplified polymorphic DNA (RAPD) and SDS-PAGE of cell-wall proteins. The RAPD analysis separated the eight type strains but did not always assign all the strains of a species to the same group, while SDS-PAGE cell-wall protein profiles were species-specific. Frequently, strains isolated from sourdoughs of the same geographical origin or produced by similar raw material/process parameters showed similar RAPD and/or cell-wall profiles. CONCLUSIONS: The combined use of the RAPD and cell-wall protein analysis represents a useful tool to classify large adventitious microbial populations and to discriminate the diversity of the strains. SIGNIFICANCE AND IMPACT OF THE STUDY: This study represents a typing of a large collection of flour/sourdough LAB and provides evidence of the advantage of using two independent methods in the classification and traceability of microorganisms.     

Discovering lactic acid bacteria by genomics

This review summarizes a collection of lactic acid bacteria that are now undergoing genomic sequencing and analysis. Summaries are presented on twenty different species, with each overview discussing the organisms fundamental and practical significance, nvironmental habitat, and its role in fermentation, bioprocessing, or probiotics. For those projects where genome sequence data were available by March 2002, summaries include a listing of key statistics and interesting genomic features. These efforts will revolutionize our molecular view of Gram–positive bacteria, as up to 15 genomes from the low GC content lactic acid bacteria are expected to be available in the public domain by the end of 2003. Our collective view of the lactic acid bacteria will be fundamentally changed as we rediscover the relationships and capabilities of these organisms through genomics.     

Preliminary characterization of lactic acid bacteria isolated from Zlatar cheese

AIMS: Isolation, characterization and identification of lactic acid bacteria (LAB) from artisanal Zlatar cheese during the ripening process and selection of strains with good technological characteristics. METHODS AND RESULTS: Characterization of LAB was performed based on morphological, physiological and biochemical assays, as well as, by determining proteolytic activity and plasmid profile. rep-polymerase chain reaction (PCR) analysis and 16S rDNA sequencing were used for the identification of LAB. PCR analysis was performed with specific primers for detection of the gene encoding nisin production. Strains Lactobacillus paracasei subsp. paracasei, Lactobacillus plantarum, Lactobacillus brevis, Lactococcus lactis subsp. lactis, Enterococcus faecium and Enterococcus faecalis were the main groups present in the Zlatar cheese during ripening. CONCLUSIONS: Temporal changes in the species were observed during the Zlatar cheese ripening. Mesophilic lactobacilli are predominant microflora in Zlatar cheese. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study we determined that Zlatar cheese up to 30 days old could be used as a source of strains for the preparation of potential starter cultures in the process of industrial cheese production. As the Serbian food market is adjusting to European Union regulations, the standardization of Zlatar cheese production by using starter culture(s) based on autochtonous well-characterized LAB will enable the industrial production of this popular cheese in the future.     

Acquired antibiotic resistance in lactic acid bacteria from food

Acquired antibiotic resistance, i.e. resistance genes located on conjugative or mobilizable plasmids and transposons can be found in species living in habitats (e.g. human and animal intestines) which are regularly challenged with antibiotics. Most data are available for enterococci and enteric lactobacilli. Raw material from animals (milk and meat) which are inadvertantly contaminated with fecal matters during production will carry antibiotic resistant lactic acid bacteria into the final fermented products such as raw milk cheeses and raw sausages. The discovered conjugative genetic elements of LAB isolated from animals and food are very similar to elements studied previously in pathogenic streptococci and enterococci, e.g. -type replicating plasmids of the pAM1, pIP501-family, and transposons of the Tn916-type. Observed resistance genes include known genes like tetM, ermAM, cat, sat and vanA. A composite 29'871 bp resistance plasmid detected in Lactococcus lacti s subsp. lactis isolated from a raw milk soft cheese contains tetS previously described in Listeria monocytogenes, cat and str from Staphylococcus aureus. Three out of five IS elements on the plasmid are almost or completely identical to IS1216 present in the vanA resistance transposon Tn1546. These data support the view that in antibiotic challenged habitats lactic acid bacteria like other bacteria participate in the communication systems which transfer resistance traits over species and genus borders. The prevalence of such bacteria with acquired resistances like enterococci is high in animals (and humans) which are regularly treated with antibiotics. The transfer of antibiotic resistant bacteria from animals into fermented and other food can be avoided if the raw substrate milk or meat is pasteurized or heat treated. Antibiotic resistance traits as selectable markers in genetic modification of lactic acid bacteria for different purposes are presently being replaced, e.g. by metabo lic traits to generate food-grade vectors.     

Characterization of lactic acid bacteria isolated from seafood

Lactic acid bacteria were isolated from various samples of seafood: fresh pollock, brine shrimp, gravad fish, vacuum-packed seafood (surimi, smoked tuna, salted cod), and fish stored under 100% CO₂ at 5°C (smoked tuna, fresh and salted cod, salmon). Eighty-six independent isolates were obtained and were grouped according to cell morphology, presence or absence of diaminopimelic acid in the cell wall, and lactate configuration. Fifty-four isolates were identified as belonging to the genus Lactococcus and most of them exhibited DNA homologies with L. lactis subsp. lactis. Four strains were identified as Lactobacillus plantarum , eight strains as genus Leuconostoc and 16 belonged to the genus Carnobacterium. One facultative heterofermentative Lactobacillus and three other isolates were not identified. Of the strains 47% showed similar patterns of carbohydrate fermentations especially among strains belonging to the genera Lactococcus and Carnobacterium. Most of the strains (64%) grew at 5°C, in salted media and in fish extract medium without added sugar. Carnobacterium piscicola and Carn. divergens were the only reference strains able to grow in the same conditions as well as psychrotroph strains isolated from seafood. A numerical analysis could not be used because of the divergent properties of isolates of the same genus and strong similarities between different genera.     

Identification of lactic acid bacteria isolated from Portuguese wines and musts by SDS-PAGE

Thirty-two strains were isolated from spoiled port wines, from musts and from various styles of young, Northeastern Portuguese red table wines that had undergone spontaneous malolactic fermentation. Comparison of their SDS-PAGE whole-cell protein patterns with an SDS-PAGE database of lactic acid bacteria indicated that the isolates were members of the species Leuconostoc oenos or Lactobacillus paracasei subsp. paracasei. The latter were found in low acidity table wines and in port wine. The isolation of Lactobacillus paracasei strains from wines indicates the importance of using known strains for wine deacidification because spontaneous malolactic fermentation of table wines can occur from an indigenous flora, adapted to the particular composition of the wine.     

Characterization and antimicrobial spectrum of bacteriocins produced by lactic acid bacteria isolated from traditional Bulgarian dairy products

Aims:  To isolate bacteriocin-producing lactic acid bacteria (LAB) with high wide spectrum antibacterial activity and to characterize their inhibitory peptides.
Method and Results:  Seven LAB strains [ Lactobacillus casei ssp. rhamnosus (PC5), Lactobacillus delbrueckii ssp. bulgaricus (BB18), Lactococcus lactis ssp. lactis (BCM5, BK15), Enterococcus faecium (MH3), Lactobacillus plantarum (BR12), Lactobacillus casei ssp. casei (BCZ2)], isolated from authentic Bulgarian dairy products were capable of producing bacteriocins, inhibiting the widest range of pathogenic bacteria. The bacteriocins were resistant to heating at 121°C for 15 min, stable at pH 2–10, sensitive to protease, insensitive to α-amylase and lipase. Two of bacteriocins produced by Lact. bulgaricus BB18 (bulgaricin BB18) and E. faecium MH3 (enterocin MH3) were purified and the molecular masses were determined. The N -terminal amino acid sequence of bulgaricin BB18 did not show strong homology to other known bacteriocins.
Conclusions:  Lactobacillus bulgaricus BB18 and E. faecium MH3 produce two novel bacteriocins highly similar to the pediocin-like nonlantibiotics.
Significance and Impact of the Study:  The two bacteriocins are potential antimicrobial agents and, in conjunction with their producers, may have use in applications to contribute a positive effect on the balance of intestinal microflora. Furthermore, bulgaricin BB18 strongly inhibits Helicobacter pylori .     

Glutamine deamidation by cereal-associated lactic acid bacteria

AIMS: It was the aim of our work to investigate glutamine deamidation by lactic acid bacteria isolated from cereal fermentations and to elucidate the ecological and technological relevance in baking of the conversion of glutamine to glutamate. METHODS AND RESULTS: Lactobacillus sanfranciscensis and Lact. reuteri were found to display glutaminase activity. The addition of glutamine to modified Man, Rogosa and Sharp medium increased the cell yields of Lact. sanfranciscensis, as well as the production of lactic and acetic acid. The final pH; however, was increased in the glutamine-containing medium. The addition of 47 mmol kg(-1) glutamate to chemically acidified doughs significantly changed the bread flavour. In sourdoughs with enhanced proteolytic activity, strain-dependent production of 27-120 mmol glutamate per kilogram sourdough was observed. CONCLUSIONS: Lactobacillus sanfranciscensis and Lact. reuteri converted glutamine into glutamate; this conversion improves the acid tolerance of lactobacilli and significantly influences wheat bread flavour. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper illustrates the complex interaction of sourdough-lactobacilli with their environment: the flour provides substrates for metabolic activities that enable the lactobacilli to reach higher cell counts, and the produced metabolite may be one of the reasons why the flavour of fermented breads is different to the flavour of chemically acidified breads.     

Characterization of starter lactic acid bacteria from the Finnish fermented milk product viili

Aims: Phenotypic and molecular methods were used to identify and compare the strain composition of three industrial dairy starters used for the manufacture of viili. Methods and Results: Preliminary differentiation was made by phenotypic methods. Genotypic differentiation was carried out using polymerase chain reaction (PCR) and further characterization at strain level by pulsed‐field gel electrophoresis (PFGE). The isolates could be assigned as acid‐producing Lactococcus lactis strains of both lactis and cremoris subspecies, and aroma producers, identified as L. lactis subsp. lactis biovar diacetylactis and Leuconostoc mesenteroides. PCR analysis discriminated between the lactococcal subspecies, and cluster analysis of the digestion patterns of PFGE analysis revealed different genotypes in each subspecies. Each Leuconostoc‐genotype seemed to be specific to only a single starter mix. Conclusions: The work proved that in addition to L. lactis subsp. lactis biovar diacetylactis and Leuc. mesenteroides subsp. cremoris, commercial viili starters of traditional origin may contain (i) only L. lactis subsp. cremoris, (ii) both L. lactis subsp. cremoris and L. lactis subsp. lactis as a minority, and – as a new discovery – (iii) only L. lactis subsp. lactis. Significance and Impact of the Study: The results obtained give an overview of the microbial population of viili starters and can be exploited in the development of optimized starter cultures for industrial‐scale manufacture of viili.     

Influence of carbohydrates on the isolation of lactic acid bacteria

Aims: To determine the influence of carbohydrates on enrichment isolation of lactic acid bacteria from different niches. Methods and Results: Lactic acid bacteria in three traditional fermented products in southern Africa (amasi, mahewu and tshwala) and in three fresh samples (two flowers and a fruit) were enrichment cultured in media supplemented with 13 different carbohydrates. Diversity of lactic acid bacteria was determined by PCR‐denaturing‐gradient gel electrophoresis. Carbohydrates used in enrichment media had a big impact on the isolation of lactic acid bacteria from fermented products. Depending on the carbohydrates tested, the number of species detected ranged from one to four in amasi, one to five in mahewu and one to three in tshwala. Fructose and mannitol selected for relatively higher numbers of lactic acid bacteria in fermented products. Specific relationships between substrates and lactic acid bacteria have been noted. On the other hand, small influences were found among carbohydrates tested in flowers and fruit. Conclusion: Carbohydrates have a big impact on the isolation of a variety of lactic acid bacteria in fermented food. Significance and Impact of the Study: This is the first study that reports the influence of carbohydrates on the enrichment of lactic acid bacteria.     

Production of natural folates by lactic acid bacteria starter cultures isolated from artisanal Argentinean yogurts

Folate is a B-group vitamin that cannot be synthesized by humans and must be obtained exogenously. Although some species of lactic acid bacteria (LAB) can produce folates, little is known about the production of this vitamin by yogurt starter cultures. Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus strains were isolated from artisanal Argentinean yogurts and were grown in folate-free culture medium (FACM) and nonfat milk after which intracellular and extracellular folate production were evaluated. From the initial 92 isolated LAB strains, 4 L. delbrueckii subsp. bulgaricus and 32 S. thermophilus were able to grow in the absence of folate. Lactobacillus delbrueckii subsp. bulgaricus CRL 863 and S.?thermophilus CRL 415 and CRL 803 produced the highest extracellular folate levels (from 22.3 to 135?μg/L) in FACM. In nonfat milk, these strains were able to increase the initial folate concentrations by almost 190%. This is the first report where native strains of L. delbrueckii subsp. bulgaricus were shown to produce natural folate. The LAB strains identified in this study could be used in developing novel fermented products bio-enriched in natural folates that could in turn be used as an alternative to fortification with the controversial synthetic chemical folic acid.     

Construction of theta-type shuttle vector for Leuconostoc and other lactic acid bacteria using pCB42 isolated from kimchi

The pCB42 plasmid from Leuconostoc citreum CB2567, a strain isolated from kimchi, was characterized, and a shuttle vector for Escherichia coli and lactic acid bacteria (LAB) was constructed. The pCB42 plasmid has a circular structure of 4312 bp, a low G + C content, and no single-stranded DNA intermediates during replication, which indicates that pCB42 replicates via the theta-type replication mechanism. In silico analysis of this plasmid revealed 6 open reading frames: 1 transposase gene, 1 DNA-binding gene, 2 putative replication genes, and 2 unknown genes. The fragment encompassing ORF5 contains a functional plasmid replicon. This plasmid was capable of replicating in various LAB, including L. citreum, L. mesenteroides, Lactobacillus plantarum, Lb. reuteri, Lactococcus lactis, Streptococcus thermophilus, Weissella confusa, and Oenococcus oeni. The LAB-E. coli shuttle vector was constructed by ligating pCB42 and pEK104, and the resulting shuttle vector, pLeuCM42, showed a high segregational stability in L. citreum CB2567 after 100 generations of cell division. By using this shuttle vector, the β-gal gene from Lb. plantarum was successfully expressed in the host strain, L. citreum CB2567. The pLeuCM42 shuttle vector can serve as a useful gene-delivery and expression tool for the genetic study or metabolic engineering of various strains of LAB.     

Exocellular polysaccharides produced by lactic acid bacteria

Abstract The production of homopolysaccharides (dextrans, mutans) and heteropolysaccharides by lactic acid bacteria, their chemical composition, their structure and their synthesis are outlined. Mutans streptococci, which include Streptococcus mutans and S. sobrinus produce soluble and insoluble α-glucans. The latter may contain as much as 90%α-1–3 linkages and possess a marked ability to promote adherence to the smooth tooth surface causing dental plaque. Dextrans produced by Leuconostoc mesenteroides are high molecular weight α-glucans having 1–6, 1–4 and 1–3 linkages, varying from slightly to highly branched; 1–6 linkages are predominant. Emphasis is put on exopolysaccharide producing thermophilic and mesophilic lactic acid bacteria, which are important in the dairy industry. The produced polymers play a key role in the rheological behaviour and the texture of fermented milks. One of the main problems in this field is the transitory nature of the thickening trait. This instability is not yet completely understood. Controversial results exist on the sugar composition of the slime produced, but galactose and glucose have always been identified with galactose predominating in most cases.