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Rudi F Vogel - One of the best experts on this subject based on the ideXlab platform.
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Glutathione reductase from Lactobacillus sanfranciscensis DSM20451T: contribution to oxygen tolerance and thiol exchange reactions in wheat sourdoughs.
Applied and environmental microbiology, 2007Co-Authors: André Jänsch, Rudi F Vogel, Maher Korakli, Michael G GanzleAbstract:The effect of the glutathione reductase (GshR) activity of Lactobacillus sanfranciscensis DSM20451T on the thiol levels in fermented sourdoughs was determined, and the oxygen tolerance of the strain was also determined. The gshR gene coding for a putative GshR was sequenced and inactivated by single-crossover integration to yield strain L. sanfranciscensis DSM20451TΔgshR. The gene disruption was verified by sequencing the truncated gshR and surrounding regions on the chromosome. The gshR activity of L. sanfranciscensis DSM20451TΔgshR was strongly reduced compared to that of the wild-type strain, demonstrating that gshR indeed encodes an active GshR enzyme. The thiol levels in wheat doughs fermented with L. sanfranciscensis DSM20451 increased from 9 μM to 10.5 μM sulfhydryl/g of dough during a 24-h sourdough fermentation, but in sourdoughs fermented with L. sanfranciscensis DSM20451TΔgshR and in chemically acidified doughs, the thiol levels decreased to 6.5 to 6.8 μM sulfhydryl/g of dough. Remarkably, the GshR-negative strains Lactobacillus pontis LTH2587 and Lactobacillus reuteri BR11 exerted effects on thiol levels in dough comparable to those of L. sanfranciscensis. In addition to the effect on thiol levels in sourdough, the loss of GshR activity in L. sanfranciscensis DSM20451TΔgshR resulted in a loss of oxygen tolerance. The gshR mutant strain exhibited a strongly decreased aerobic growth rate on modified MRS medium compared to either the growth rate under anaerobic conditions or that of the wild-type strain, and aerobic growth was restored by the addition of cysteine. Moreover, the gshR mutant strain was more sensitive to the superoxide-generating agent paraquat.
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carbohydrate peptide and lipid metabolism of lactic acid bacteria in sourdough
Food Microbiology, 2007Co-Authors: Michael G Ganzle, Nicoline Vermeulen, Rudi F VogelAbstract:The metabolic pathways of lactic acid bacteria that influence bread quality are coupled to the central carbon flux by the availability of cofactors influencing the cellular and environmental redox potential. Homo- and heterofermentative metabolism differ fundamentally with respect to the requirement for regeneration of reduced cofactors, NADH or NADPH. The utilization of co-substrates such as oxygen or fructose as electron acceptors by obligate heterofermentative lactobacilli is coupled to an increased production of acetate in dough. Recently, several oxidoreductases involved in cofactor regeneration were characterized and glutathione and short-chain aldehydes derived from lipid oxidation were identified as substrates for cofactor regeneration by Lactobacillus sanfranciscensis. Based on the different metabolic requirements for cofactor regeneration, homo- and heterofermentative lactobacilli exert divergent effects on redox-reactions in sourdough that influence bread quality beyond the formation of acetate. Proteolysis, followed by peptide or amino acid metabolism by LAB is one of the key routes of flavour formation in bread flavour, and enables the strain-specific formation of antifungal metabolites. Peptide metabolism as well as the metabolism of cysteine, arginine, and phenylalanine in Lactobacillus plantarum, L. sanfranciscensis, and Lactobacillus pontis is increasingly understood and these insights provide new opportunities for the directed application of sourdough LAB for improved bread quality.
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In Situ Production of Exopolysaccharides during Sourdough Fermentation by Cereal and Intestinal Isolates of Lactic Acid Bacteria
Applied and environmental microbiology, 2003Co-Authors: Markus Tieking, Matthias A Ehrmann, Michael G Ganzle, Maher Korakli, Rudi F VogelAbstract:EPS formed by lactobacilli in situ during sourdough fermentation may replace hydrocolloids currently used as texturizing, antistaling, or prebiotic additives in bread production. In this study, a screening of >100 strains of cereal-associated and intestinal lactic acid bacteria was performed for the production of exopolysaccharides (EPS) from sucrose. Fifteen strains produced fructan, and four strains produced glucan. It was remarkable that formation of glucan and fructan was most frequently found in intestinal isolates and strains of the species Lactobacillus reuteri, Lactobacillus pontis, and Lactobacillus frumenti from type II sourdoughs. By the use of PCR primers derived from conserved amino acid sequences of bacterial levansucrase genes, it was shown that 6 of the 15 fructan-producing lactobacilli and none of 20 glucan producers or EPS-negative strains carried a levansucrase gene. In sourdough fermentations, it was determined whether those strains producing EPS in MRS medium modified as described by Stolz et al. (37) and containing 100 g of sucrose liter−1 as the sole source of carbon also produce the same EPS from sucrose during sourdough fermentation in the presence of 12% sucrose. For all six EPS-producing strains evaluated in sourdough fermentations, in situ production of EPS at levels ranging from 0.5 to 2 g/kg of flour was demonstrated. Production of EPS from sucrose is a metabolic activity that is widespread among sourdough lactic acid bacteria. Thus, the use of these organisms in bread production may allow the replacement of additives.
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contribution of sourdough lactobacilli yeast and cereal enzymes to the generation of amino acids in dough relevant for bread flavor
Cereal Chemistry, 2002Co-Authors: C Thiele, Michael G Ganzle, Rudi F VogelAbstract:ABSTRACT The amino acid release was determined in wheat doughs supplied with salt, acid, dithiothreitol, or starter cultures to evaluate the relevance of the amino acid concentration on bread flavor. Wheat flour proteinases almost linearly released amino acids and the highest activity of wheat flour proteinases was found in acidified and reduced doughs. The effects of starter cultures on amino acid concentrations depended on their composition. Yeasts exhibited a high demand for amino acids, however, the total amino acid concentrations were not markedly affected by lactic acid bacteria. The individual amino acid contents were determined by the pH during fermentation and microbial metabolism. The formation of proline was favored by values higher than pH 5.5, whereas release of phenylalanine, leucine and cysteine mainly occurred at lower pH. Ornithine was found only in doughs fermented with Lactobacillus pontis. To determine effects of the amino acid concentration on bread aroma, fermented doughs were evalua...
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Monitoring the growth of Lactobacillus species during a rye flour fermentation
Food Microbiology, 2001Co-Authors: Martin Müller, Peter Stolz, Matthias A Ehrmann, Georg Wolfrum, Rudi F VogelAbstract:Abstract The natural microbial community conducting an industrial sourdough fermentation was investigated by molecular biological methods using the following strategy: strains were isolated and subjected to RAPD (randomly-amplified polymorphic DNA) PCR. After computer-supported pattern analysis and clustering of the strains the 16S rDNA of members of each distinct cluster were partially (530 bp) or completely (1570 bp) sequenced and identified by comparative sequence analysis. The predominant strains of this fermentation could be allotted to the species Lactobacillus amylovorus, Lactobacillus pontis and a species, which phylogenetically takes an intermediate position between L. pontis and L. panis. Sporadically, strains were identified as L. reuteri. In a second step the effect of external factors was investigated under the controlled conditions of a lab-scale process. Fermentations were carried out at 34°C, 40°C and 46°C. The development of the flora was consistent in independent fermentations as proved by RAPD typing of randomly-picked colonies. The microbial community in these fermentations was identical to those found in an industrial scale. The qualitative composition of the flora was not affected by the temperature. L. amylovorus was the dominant species. With increasing fermentation time, a shift toward the predominance of heterofermentative lactobacilli was observed. This finding was underlined by metabolic studies and stoichiometric calculations of the metabolic pathways. With increasing temperature the percentage of homofermentative organisms was reduced. Furthermore, the growth rate and the metabolic activity increased, followed by an immediate decrease of the growth rate at 46°C and lower terminal values of lactate, acetate and ethanol, respectively.
Xavier Dousset - One of the best experts on this subject based on the ideXlab platform.
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a one step reaction for the rapid identification of Lactobacillus mindensis Lactobacillus panis Lactobacillus paralimentarius Lactobacillus pontis and Lactobacillus frumenti using oligonucleotide primers designed from the 16s 23s rrna intergenic sequ
Journal of Applied Microbiology, 2008Co-Authors: Mounir Ferchichi, R. Valcheva, Hervé Prévost, B. Onno, Xavier DoussetAbstract:Aims: Species-specific primers targeting the 16S–23S ribosomal DNA (rDNA) intergenic spacer region (ISR) were designed to rapidly discriminate between Lactobacillus mindensis, Lactobacillus panis, Lactobacillus paralimentarius, Lactobacillus pontis and Lactobacillus frumenti species recently isolated from French sourdough. Methods and Results: The 16S–23S ISRs were amplified using primers 16S/p2 and 23S/p7, which anneal to positions 1388–1406 of the 16S rRNA gene and to positions 207–189 of the 23S rRNA gene respectively, Escherichia coli numbering (GenBank accession number V00331). Clone libraries of the resulting amplicons were constructed using a pCR2·1 TA cloning kit and sequenced. Species-specific primers were designed based on the sequences obtained and were used to amplify the 16S–23S ISR in the Lactobacillus species considered. For all of them, two PCR amplicons, designated as small ISR (S-ISR) and large ISR (L-ISR), were obtained. The L-ISR is composed of the corresponding S-ISR, interrupted by a sequence containing tRNAIle and tRNAAla genes. Based on these sequences, species-specific primers were designed and proved to identify accurately the species considered among 30 reference Lactobacillus species tested. Conclusions: Designed species-specific primers enable a rapid and accurate identification of L. mindensis, L. paralimentarius, L. panis, L. pontis and L. frumenti species among other lactobacilli. Significance and Impact of the Study: The proposed method provides a powerful and convenient means of rapidly identifying some sourdough lactobacilli, which could be of help in large starter culture surveys.
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A one‐step reaction for the rapid identification of Lactobacillus mindensis, Lactobacillus panis, Lactobacillus paralimentarius, Lactobacillus pontis and Lactobacillus frumenti using oligonucleotide primers designed from the 16S–23S rRNA intergenic s
Journal of Applied Microbiology, 2008Co-Authors: Mounir Ferchichi, R. Valcheva, Hervé Prévost, B. Onno, Xavier DoussetAbstract:AIMS: Species-specific primers targeting the 16S-23S ribosomal DNA (rDNA) intergenic spacer region (ISR) were designed to rapidly discriminate between Lactobacillus mindensis, Lactobacillus panis, Lactobacillus paralimentarius, Lactobacillus pontis and Lactobacillus frumenti species recently isolated from French sourdough. METHODS AND RESULTS: The 16S-23S ISRs were amplified using primers 16S/p2 and 23S/p7, which anneal to positions 1388-1406 of the 16S rRNA gene and to positions 207-189 of the 23S rRNA gene respectively, Escherichia coli numbering (GenBank accession number V00331). Clone libraries of the resulting amplicons were constructed using a pCR2.1 TA cloning kit and sequenced. Species-specific primers were designed based on the sequences obtained and were used to amplify the 16S-23S ISR in the Lactobacillus species considered. For all of them, two PCR amplicons, designated as small ISR (S-ISR) and large ISR (L-ISR), were obtained. The L-ISR is composed of the corresponding S-ISR, interrupted by a sequence containing tRNA(Ile) and tRNA(Ala) genes. Based on these sequences, species-specific primers were designed and proved to identify accurately the species considered among 30 reference Lactobacillus species tested. CONCLUSIONS: Designed species-specific primers enable a rapid and accurate identification of L. mindensis, L. paralimentarius, L. panis, L. pontis and L. frumenti species among other lactobacilli. SIGNIFICANCE AND IMPACT OF THE STUDY: The proposed method provides a powerful and convenient means of rapidly identifying some sourdough lactobacilli, which could be of help in large starter culture surveys.
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Molecular identification of the microbiota of French sourdough using temporal temperature gradient gel electrophoresis
Food Microbiology, 2007Co-Authors: Mounir Ferchichi, Hervé Prévost, Rosica Valcheva, Bernard Onno, Xavier DoussetAbstract:Abstract The microbiota of four industrial French sourdoughs (BF, GO, VB and RF) was characterized by PCR temporal temperature gel electrophoresis (TTGE). The TTGE technique reveals differences in the 16S rDNA V6–V8 regions of these bacteria. DNA was extracted directly from sourdough samples. A specific TTGE fingerprint was determined for 30 bacterial species, including members of the genera Lactobacillus, Leuconostoc and Weissella, all known to be present in sourdough. These sourdoughs contain different species of lactic acid bacteria (LAB) depending on ecological conditions prevailing in the different sourdough fermentations. Only a few LAB species were found to be competitive and became dominant. Lactobacillus sanfranciscensis was observed as the most frequently found species. In sourdough GO, L. sanfranciscensis, Lactobacillus panis and two new species, Lactobacillus nantensis and Lactobacillus hammesii, were detected. Sourdough BF contain L. sanfranciscensis, Lactobacillus spicheri and Lactobacillus pontis. In sourdough VB, which differed in the process temperature, we identified exclusively L. sanfranciscensis and Leuconostoc mesenteroides subsp. mesenteroides. Lactobacillus frumenti, L. hammesii and Lacobacillus paralimentarius became the predominant species in sourdough RF. Compared with conventional bacteriological methods, the use of this new molecular approach to analyze the sourdough ecosystem should therefore allow a more complete and rapid assessment of its specific microbiota.
Stefan Roos - One of the best experts on this subject based on the ideXlab platform.
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Microbiological Characterization of Wet Wheat Distillers' Grain, with Focus on Isolation of Lactobacilli with Potential as Probiotics
Applied and environmental microbiology, 2004Co-Authors: C. Pedersen, Hans Jonsson, Jan Erik Lindberg, Stefan RoosAbstract:Wet wheat distillers' grain (WWDG), a residue from ethanol fermentation, was examined from a microbiological perspective. After storage, WWDG was characterized by a high content of lactobacilli, nondetectable levels of other bacteria, occasional occurrence of yeasts, and a pH of about 3.6 and contained a mixture of lactic acid, acetic acid, and ethanol. The composition of lactobacilli in WWDG was simple, including primarily the species Lactobacillus amylolyticus, Lactobacillus panis, and Lactobacillus pontis, as determined by 16S rRNA gene sequencing. Since the use of WWDG as pig feed has indicated a health-promoting function, some relevant characteristics of three strains of each of these species were examined together with basal physiological parameters, such as carbohydrate utilization and growth temperature. Seven of the strains were isolated from WWDG, and two strains from pig feces were included for comparison. It was clear that all three species could grow at temperatures of 45 to 50 degrees C, with L. amylolyticus being able to grow at temperatures as high as 54 degrees C. This finding could be the explanation for the simple microflora of WWDG, where a low pH together with a high temperature during storage would select for these organisms. Some strains of L. panis and L. pontis showed prolonged survival at pH 2.5 in synthetic stomach juice and good growth in the presence of porcine bile salt. In addition, members of all three species were able to bind to immobilized mucus material in vitro. Especially the isolates from pig feces but, interestingly, some isolates from WWDG as well possessed properties that might be of importance for colonization of the gastrointestinal tracts of pigs.
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Lactobacillus mucosae sp. nov., a new species with in vitro mucus-binding activity isolated from pig intestine.
International Journal of Systematic and Evolutionary Microbiology, 2000Co-Authors: Stefan Roos, Fredrik Karner, Lars Axelsson, Hans JonssonAbstract:A new Lactobacillus species from pig small intestine has been identified. In an attempt to isolate Lactobacillus reuteri strains carrying the putative colonization-factor gene (mub, for mucus binding) a mub-derived gene probe was used to screen pig intestinal material. A number of isolates were obtained and primary characterization showed that they were Gram-positive, catalase-negative, non-spore-forming, non-motile rods. Growth occurred at 45 degrees C but not at 15 degrees C and the DNA G+C content was 46 mol%. Cell wall analysis together with DNA-DNA hybridization and analysis of the 16S rRNA sequence revealed that the new isolates represent a previously undescribed Lactobacillus species closely related to L. reuteri, Lactobacillus fermentum and Lactobacillus pontis. The name Lactobacillus mucosae is proposed for this species and the type strain is S32T.
Matthias A Ehrmann - One of the best experts on this subject based on the ideXlab platform.
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In Situ Production of Exopolysaccharides during Sourdough Fermentation by Cereal and Intestinal Isolates of Lactic Acid Bacteria
Applied and environmental microbiology, 2003Co-Authors: Markus Tieking, Matthias A Ehrmann, Michael G Ganzle, Maher Korakli, Rudi F VogelAbstract:EPS formed by lactobacilli in situ during sourdough fermentation may replace hydrocolloids currently used as texturizing, antistaling, or prebiotic additives in bread production. In this study, a screening of >100 strains of cereal-associated and intestinal lactic acid bacteria was performed for the production of exopolysaccharides (EPS) from sucrose. Fifteen strains produced fructan, and four strains produced glucan. It was remarkable that formation of glucan and fructan was most frequently found in intestinal isolates and strains of the species Lactobacillus reuteri, Lactobacillus pontis, and Lactobacillus frumenti from type II sourdoughs. By the use of PCR primers derived from conserved amino acid sequences of bacterial levansucrase genes, it was shown that 6 of the 15 fructan-producing lactobacilli and none of 20 glucan producers or EPS-negative strains carried a levansucrase gene. In sourdough fermentations, it was determined whether those strains producing EPS in MRS medium modified as described by Stolz et al. (37) and containing 100 g of sucrose liter−1 as the sole source of carbon also produce the same EPS from sucrose during sourdough fermentation in the presence of 12% sucrose. For all six EPS-producing strains evaluated in sourdough fermentations, in situ production of EPS at levels ranging from 0.5 to 2 g/kg of flour was demonstrated. Production of EPS from sucrose is a metabolic activity that is widespread among sourdough lactic acid bacteria. Thus, the use of these organisms in bread production may allow the replacement of additives.
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Monitoring the growth of Lactobacillus species during a rye flour fermentation
Food Microbiology, 2001Co-Authors: Martin Müller, Peter Stolz, Matthias A Ehrmann, Georg Wolfrum, Rudi F VogelAbstract:Abstract The natural microbial community conducting an industrial sourdough fermentation was investigated by molecular biological methods using the following strategy: strains were isolated and subjected to RAPD (randomly-amplified polymorphic DNA) PCR. After computer-supported pattern analysis and clustering of the strains the 16S rDNA of members of each distinct cluster were partially (530 bp) or completely (1570 bp) sequenced and identified by comparative sequence analysis. The predominant strains of this fermentation could be allotted to the species Lactobacillus amylovorus, Lactobacillus pontis and a species, which phylogenetically takes an intermediate position between L. pontis and L. panis. Sporadically, strains were identified as L. reuteri. In a second step the effect of external factors was investigated under the controlled conditions of a lab-scale process. Fermentations were carried out at 34°C, 40°C and 46°C. The development of the flora was consistent in independent fermentations as proved by RAPD typing of randomly-picked colonies. The microbial community in these fermentations was identical to those found in an industrial scale. The qualitative composition of the flora was not affected by the temperature. L. amylovorus was the dominant species. With increasing fermentation time, a shift toward the predominance of heterofermentative lactobacilli was observed. This finding was underlined by metabolic studies and stoichiometric calculations of the metabolic pathways. With increasing temperature the percentage of homofermentative organisms was reduced. Furthermore, the growth rate and the metabolic activity increased, followed by an immediate decrease of the growth rate at 46°C and lower terminal values of lactate, acetate and ethanol, respectively.
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Lactobacillus frumenti sp nov a new lactic acid bacterium isolated from rye bran fermentations with a long fermentation period
International Journal of Systematic and Evolutionary Microbiology, 2000Co-Authors: Martin Müller, Matthias A Ehrmann, Rudi F VogelAbstract:Within the framework of the characterization of the microflora of an industrial sourdough fermentation, strains of Lactobacillus amylovorus, Lactobacillus pontis and two other strains were isolated which could not be associated with a valid species. These latter strains were Gram-positive, catalase-negative, non-spore-forming, non-motile rods that could be clearly differentiated from known species by 16S rDNA sequence analysis. For further characterization, the morphological, physiological (sugar fermentation, formation of DL-lactate, hydrolysis of arginine, growth temperature, CO2 production) and chemotaxonomic (G+C content, cell wall composition, SDS-PAGE of whole-cell proteins) properties were determined. Fitting of the complete 16S rDNA sequence into alignments of such sequences, together with the subsequent phylogenetic calculations, allowed the reconstruction of a phylogenetic tree. These data showed that the two strains were phylogenetically related but formed an independent cluster distinct from their closest neighbours, L. pontis, Lactobacillus panis, Lactobacillus oris, Lactobacillus vaginalis and Lactobacillus reuteri. The results of DNA-DNA hybridization experiments indicated that the two isolates represent a new Lactobacillus species, for which the name Lactobacillus frumenti is proposed; the type strain of this species is DSM 13145T (= LMG 19473T).
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Multiplex PCR for the Detection of Lactobacillus pontis and Two Related Species in a Sourdough Fermentation
Applied and environmental microbiology, 2000Co-Authors: Martin Müller, Matthias A Ehrmann, Rudi F VogelAbstract:A specific multiplex PCR assay based on the amplification of parts of the 16S rRNA molecule was designed. Primers derived from variable regions of the 16S rRNA provided a means of easily differentiating the species Lactobacillus pontis and Lactobacillus panis. They could be clearly discriminated from the phylogenetically related species Lactobacillus vaginalis, Lactobacillus oris, and Lactobacillus reuteri and from other lactobacilli commonly known to be present in sourdough. Other strains isolated together with L. pontis from an industrial sourdough fermentation could be clearly separated from these species by comparative sequence analysis and construction of a specific PCR primer. For a fast identification a DNA isolation protocol based on the ultrasonic lysis of cells from single colonies was developed. To demonstrate the potential of such techniques for tracking these organisms in a laboratory-scale fermentation, we combined the specific PCR assay with direct DNA extraction from the organisms in the sourdough without previous cultivation.
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Identification of lactobacilli from sourdough and description of Lactobacillus-pontis sp-nov.
International Journal of Systematic and Evolutionary Microbiology, 1994Co-Authors: Rudi F Vogel, Peter Stolz, Daniela Fanta, G. Böcker, Matthias A Ehrmann, Karl-heinz Schleifer, Wolfgang Ludwig, Karel Kersters, Walter P. HammesAbstract:The microflora of sourdough preparations was investigated by examining the physiological characteristics, whole-cell protein patterns, and 16S rRNA sequences of Lactobacillus isolates. Strains isolated from sourdough were placed in the species Lactobacillus brevis, Lactobacillus sanfrancisco, and Lactobacillus reuteri. 16S rRNA sequences were determined for L. brevis, Lactobacillus fructivorans, Lactobacillus fermentum, L. sanfrancisco, and L. reuteri, and oligonucleotide probes for fast specific identification of these sourdough lactobacilli were deduced. The physiological characteristics, protein patterns, and 16S rRNA sequences of these organisms were compared with data for other sourdough lactobacilli and additional reference strains. Strains of a Lactobacillus species were isolated from rye sourdough; these strains may account for most of the flora in sourdough made from wheat or rye. These organisms were differentiated from other sourdough lactobacilli by their protein pattern, 16S rRNA sequence, G+C content, and physiological properties. The 16S rRNA sequence of this species was determined, and we constructed a phylogenetic tree which reflected the relationships of this species to other lactobacilli. This organism is closely related to L. reuteri. A new Lactobacillus species, Lactobacillus pontis, is proposed. The type strain is L. pontis LTH 2587 (= DSM 8475 = LMG 14187). We describe a general strategy in which a polyphasic approach was used to characterize a new species.
Mounir Ferchichi - One of the best experts on this subject based on the ideXlab platform.
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a one step reaction for the rapid identification of Lactobacillus mindensis Lactobacillus panis Lactobacillus paralimentarius Lactobacillus pontis and Lactobacillus frumenti using oligonucleotide primers designed from the 16s 23s rrna intergenic sequ
Journal of Applied Microbiology, 2008Co-Authors: Mounir Ferchichi, R. Valcheva, Hervé Prévost, B. Onno, Xavier DoussetAbstract:Aims: Species-specific primers targeting the 16S–23S ribosomal DNA (rDNA) intergenic spacer region (ISR) were designed to rapidly discriminate between Lactobacillus mindensis, Lactobacillus panis, Lactobacillus paralimentarius, Lactobacillus pontis and Lactobacillus frumenti species recently isolated from French sourdough. Methods and Results: The 16S–23S ISRs were amplified using primers 16S/p2 and 23S/p7, which anneal to positions 1388–1406 of the 16S rRNA gene and to positions 207–189 of the 23S rRNA gene respectively, Escherichia coli numbering (GenBank accession number V00331). Clone libraries of the resulting amplicons were constructed using a pCR2·1 TA cloning kit and sequenced. Species-specific primers were designed based on the sequences obtained and were used to amplify the 16S–23S ISR in the Lactobacillus species considered. For all of them, two PCR amplicons, designated as small ISR (S-ISR) and large ISR (L-ISR), were obtained. The L-ISR is composed of the corresponding S-ISR, interrupted by a sequence containing tRNAIle and tRNAAla genes. Based on these sequences, species-specific primers were designed and proved to identify accurately the species considered among 30 reference Lactobacillus species tested. Conclusions: Designed species-specific primers enable a rapid and accurate identification of L. mindensis, L. paralimentarius, L. panis, L. pontis and L. frumenti species among other lactobacilli. Significance and Impact of the Study: The proposed method provides a powerful and convenient means of rapidly identifying some sourdough lactobacilli, which could be of help in large starter culture surveys.
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A one‐step reaction for the rapid identification of Lactobacillus mindensis, Lactobacillus panis, Lactobacillus paralimentarius, Lactobacillus pontis and Lactobacillus frumenti using oligonucleotide primers designed from the 16S–23S rRNA intergenic s
Journal of Applied Microbiology, 2008Co-Authors: Mounir Ferchichi, R. Valcheva, Hervé Prévost, B. Onno, Xavier DoussetAbstract:AIMS: Species-specific primers targeting the 16S-23S ribosomal DNA (rDNA) intergenic spacer region (ISR) were designed to rapidly discriminate between Lactobacillus mindensis, Lactobacillus panis, Lactobacillus paralimentarius, Lactobacillus pontis and Lactobacillus frumenti species recently isolated from French sourdough. METHODS AND RESULTS: The 16S-23S ISRs were amplified using primers 16S/p2 and 23S/p7, which anneal to positions 1388-1406 of the 16S rRNA gene and to positions 207-189 of the 23S rRNA gene respectively, Escherichia coli numbering (GenBank accession number V00331). Clone libraries of the resulting amplicons were constructed using a pCR2.1 TA cloning kit and sequenced. Species-specific primers were designed based on the sequences obtained and were used to amplify the 16S-23S ISR in the Lactobacillus species considered. For all of them, two PCR amplicons, designated as small ISR (S-ISR) and large ISR (L-ISR), were obtained. The L-ISR is composed of the corresponding S-ISR, interrupted by a sequence containing tRNA(Ile) and tRNA(Ala) genes. Based on these sequences, species-specific primers were designed and proved to identify accurately the species considered among 30 reference Lactobacillus species tested. CONCLUSIONS: Designed species-specific primers enable a rapid and accurate identification of L. mindensis, L. paralimentarius, L. panis, L. pontis and L. frumenti species among other lactobacilli. SIGNIFICANCE AND IMPACT OF THE STUDY: The proposed method provides a powerful and convenient means of rapidly identifying some sourdough lactobacilli, which could be of help in large starter culture surveys.
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Molecular identification of the microbiota of French sourdough using temporal temperature gradient gel electrophoresis
Food Microbiology, 2007Co-Authors: Mounir Ferchichi, Hervé Prévost, Rosica Valcheva, Bernard Onno, Xavier DoussetAbstract:Abstract The microbiota of four industrial French sourdoughs (BF, GO, VB and RF) was characterized by PCR temporal temperature gel electrophoresis (TTGE). The TTGE technique reveals differences in the 16S rDNA V6–V8 regions of these bacteria. DNA was extracted directly from sourdough samples. A specific TTGE fingerprint was determined for 30 bacterial species, including members of the genera Lactobacillus, Leuconostoc and Weissella, all known to be present in sourdough. These sourdoughs contain different species of lactic acid bacteria (LAB) depending on ecological conditions prevailing in the different sourdough fermentations. Only a few LAB species were found to be competitive and became dominant. Lactobacillus sanfranciscensis was observed as the most frequently found species. In sourdough GO, L. sanfranciscensis, Lactobacillus panis and two new species, Lactobacillus nantensis and Lactobacillus hammesii, were detected. Sourdough BF contain L. sanfranciscensis, Lactobacillus spicheri and Lactobacillus pontis. In sourdough VB, which differed in the process temperature, we identified exclusively L. sanfranciscensis and Leuconostoc mesenteroides subsp. mesenteroides. Lactobacillus frumenti, L. hammesii and Lacobacillus paralimentarius became the predominant species in sourdough RF. Compared with conventional bacteriological methods, the use of this new molecular approach to analyze the sourdough ecosystem should therefore allow a more complete and rapid assessment of its specific microbiota.
