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ABSTRACT: AIMS: The aim of this study was to analyse the bacterial microbiota of water kefir using culture-independent methods. METHODS AND RESULTS: We compared four water kefirs of different origins using 16S rDNA amplicon sequencing and ARDRA. The microbiota consisted of different proportions of the genera Lactobacillus (Lb.), Leuconostoc (Lc.), Acetobacter (Ac.) and Gluconobacter. Surprisingly, varying but consistently high numbers of sequences representing members of the genus Bifidobacterium (B.) were found in all kefirs. Whereas part of the bifidobacterial sequences could be assigned to B. psychraerophilum, a majority of sequences identical to each other could not be assigned to any known species. A nearly full-length sequence of the latter exhibited a beyond-species similarity (96.4%) with the sequence from the closest relative species B. psychraerophilum. A Bifidobacterium-specific ARDRA analysis reflected the abundance of the novel Bifidobacterium species by revealing its unique MboI restriction profile. Attempts to isolate the bifidobacteria were successful for B. psychraerophilum only. CONCLUSIONS: The complexity of the water kefir microbiota has been underestimated in previously studies. The occurrence of Bifidobacteria as part of the consortium is novel. SIGNIFICANCE AND IMPACT OF STUDY: These data give new insights in the understanding of the complexity of food fermentations and underlines the need for approaches detecting non-cultivable organisms. © 2013 The Authors Journal of Applied Microbiology © 2013 The Society for Applied Microbiology.
Journal of Applied Microbiology 01/2013; · 2.34 Impact Factor
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ABSTRACT: Enterococcus (E.) faecalis is found as commensal in healthy humans, in a variety of fermented foods. It can serve as probiotic but also as pathogen causing endocarditis, bacteremia and urinary tract infections. We have employed a proteomic study with E. faecalis strain OG1RF under different growth conditions and in contact to mouse intestinal cells to identify novel latent and adaptive fitness determinants. These relate to changes in catabolic pathways (BudA), protein biosynthesis (AsnS), cellular surface biosynthesis (RmlA) and regulatory mechanisms (OmpR). This knowledge can be used to derive novel evidence-based targets, which can be used to further elucidate gene expression changes enhancing pathogenicity or fitness in a commensal strain and possibly delineate this species into groups of higher and lower risk for applications in a food or a medical context versus improved treatment strategies of the so far hard to cure diseases.
Archives of Microbiology 12/2012; · 1.43 Impact Factor
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ABSTRACT: Branched chain aminotransferases (IlvE/BcaT), specific for leucine, isoleucine, and valine initialize the formation of methyl-branched volatiles, which are strongly linked to the typical aroma of cured meat products. Lactobacillus sakei, one of the dominating lactic acid bacteria species for meat fermentations and commonly used as starter lacks this enzyme, whereas the presence of IlvE has been reported for Lactobacillus paracasei, a non-starter lactic acid bacterium occurring in meat products and with probiotic properties, in Staphylococcus carnosus, a catalase positive cocci also used as starter for meat products, and in Enterococcus faecalis belonging to the natural microbiota of meat and that may impact on the aroma of fermented meat products. The genes for branched-chain aminotransferases of these three bacterial species were used to complement the IlvE negative strain L. sakei TMW1.1322. For that purpose, ilvE genes were heterologously expressed in L. sakei TMW1.1322 under the control of the constitutive L. sakei promotor pldhL via replicative plasmids and chromosomal integration. To examine effective expression the constructs were transcriptionally coupled to mCherry, a red fluorescent protein. Aminotransferase activities and formation of volatile compounds were compared. Activities of L. sakei ArcT and AspD purified enzymes were also measured. Conversion of branched chain amino acids to the corresponding α-keto-acids was significantly increased in all transformants expressing the ilvE genes. The activity of IlvE obtained from L. paracasei was highest. Substrate specificities of IlvEs towards leucine, isoleucine and valine were similar. However, enhanced transaminase activities did not increase formation of the respective methyl-branched volatiles by recombinant L. sakei strains. This indicates that presence of ilvE cannot be the only bottleneck in aroma formation from amino acids. Amino acid or peptide uptake into the cell via specific transport systems and the conversion of α-keto acids to the corresponding aldehydes, alcohols and carboxylic acids must be considered as further limiting steps.
Food Microbiology 04/2012; 29(2):205-14. · 3.28 Impact Factor
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ABSTRACT: The microbial diversity of water kefir, made from a mixture of water, dried figs, a slice of lemon and sucrose was studied. The microbial consortia residing in the granules of three water kefirs of different origins were analyzed. A collection of 453 bacterial isolates was obtained on different selective/differential media. Bacterial isolates were grouped with randomly amplified polymorphic DNA (RAPD)-PCR analyses. One representative of each RAPD genotype was identified by comparative 16S rDNA gene sequencing. The predominant genus in water kefirs I and II was Lactobacillus, which accounted for 82.1% in water kefir I and 72.1% in water kefir II of the bacterial isolates. The most abundant species in water kefirs I and II were Lactobacillus hordei and Lb. nagelii followed by considerably lower numbers of Lb. casei. Other lactic acid bacteria (LAB) were identified as Leuconostoc mesenteroides and Lc. citreum in all three water kefirs. The most abundant species in water kefir III was Lc. mesenteroides (28%) and Lc. citreum (24.3%). A total of 57 LAB belonging to the species of Lb. casei, Lb. hordei, Lb. nagelii, Lb. hilgardii and Lc. mesenteroides were able to produce exopolysacchrides from sucrose. Non LABs were identified as Acetobacter fabarum and Ac. orientalis. The Acetobacter species were more prevalent in consortium III. Cluster analyses of RAPD-PCR patterns revealed an interspecies diversity among the Lactobacillus and Acetobacter strains. Aditionally, Saccharomyces cerevisiae, Lachancea fermentati, Hanseniaospora valbyensis and Zygotorulaspora florentina were isolated and identified by comparison of partial 26S rDNA sequences and FTIR spectroscopy.
International journal of food microbiology 12/2011; 151(3):284-8. · 3.01 Impact Factor
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ABSTRACT: Enterococcus faecalis and Enterococcus faecium are human commensals frequently found in fermented foods or used as probiotics, but also recognized as opportunistic pathogens. We investigated 62 Enterococcus strains isolated from clinical, food and environmental origins towards a rationale for safety evaluation of strains in food or probiotic applications. All isolates were characterised with respect to the presence of the virulence determinants fsrB, sprE, gelE, ace, efaAfs/fm, as, esp, cob and the cytolysin operon. In addition RAPD-PCR was used to obtain genomic fingerprints that were clustered and compared to phenotypic profiles generated by MALDI-TOF-MS. The gelatinase phenotype (GelE) and the haemolytic activity (β-haemolysis) were analysed. E. faecium strains contained esp and efaAfm only, and none of them contained any CRISPR elements. The amenability of E. faecalis strains to acquisition of virulence factors was investigated along the occurrence of CRISPR associated (cas) genes. While distribution of most virulence factors, and RAPD versus MALDI-TOF-MS typing patterns were unrelated, 2 out of 5 RAPD clusters almost exclusively contained clinical E. faecalis isolates, and an occurrence of CRISPR elements versus reduced number of virulence factors was observed. The presence of the cytolysin operon, cob and as encoding pheromone and aggregation substance, respectively, significantly corresponded to absence of cas. As their production promote genetic exchange, their absence limits further gene acquisition and distribution. Thus, absence of the cytolysin operon, cob and as in a cas positive environment suggests itself as promising candidate for E. faecalis evaluation towards their occurrence in food fermentation or use as probiotics.
Systematic and Applied Microbiology 09/2011; 34(8):553-60. · 3.37 Impact Factor
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ABSTRACT: A total of 51 Lactobacillus sakei and 28 Lactobacillus curvatus strains from different origins were screened for their potential to produce biogenic amines (BAs), and for their diversity of peptidolytic systems and specific aminotransferases (AraT, BcaT) that initiate amino acid conversion to volatiles relevant for aroma formation in meat products. The profiles of volatiles formed (volatilomes) were analysed in the headspace of fermentations by solid phase microextraction followed by GC-MS analysis. Tyramine-forming potential was detected only within L. curvatus and was strain-dependent. Histamine decarboxylase (HDC) activity could only be detected in one L. sakei strain, previously described as histidine decarboxylase positive (HDC(+)). Peptide transporters and peptidases were nearly ubiquitous in L. sakei and only a few strains lacked single peptidases. In L. curvatus, differences were detected in the occurrence of peptidase genes detected with PCR primers derived from L. sakei. All strains lacked known aminotransferases specific for branched-chain amino acids (BCAAs) and aromatic amino acids (ACAAs). Although L. sakei is suggested as a genetically very heterogenous species, and relatedness between L. curvatus and L. sakei at the genomic level is rather low, they appeared to be nearly uniform in the genes forming the peptidolytic system. The volatilomes of L. sakei and L. curvatus strains were qualitatively nearly identical. However, slight differences in the formation of single volatile compounds and the interaction with staphylococci may impact upon sausage fermentation which occurs over a period of many weeks. Among the compounds expected to contribute to the aroma were dimethyldisulphide, 3-methyl-1-butanol, acetic acid, 1-butanol and butanoic acid.
Systematic and Applied Microbiology 07/2011; 34(5):311-20. · 3.37 Impact Factor
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ABSTRACT: Lactic acid bacteria (LAB) are generally accepted as beneficial to the host and their presence is directly influenced by ingestion of fermented food or probiotics. While the intestinal lactic microbiota is well-described knowledge on its routes of inoculation and competitiveness towards selective pressure shaping the intestinal microbiota is limited. In this study, LAB were isolated from faecal samples of breast feeding mothers living in Syria, from faeces of their infants, from breast milk as well as from fermented food, typically consumed in Syria. A total of 700 isolates were characterized by genetic fingerprinting with random amplified polymorphic DNA (RAPD) and identified by comparative 16S rDNA sequencing and Matrix Assisted Laser Desorption Ionization-Time-Of-Flight Mass Spectrometry (MALDI-TOF-MS) analyses. Thirty six different species of Lactobacillus, Enterococcus, Streptococcus, Weissella and Pediococcus were identified. RAPD and MALDI-TOF-MS patterns allowed comparison of the lactic microbiota on species and strain level. Whereas some species were unique for one source, Lactobacillus plantarum, Lactobacillus fermentum, Pediococcus pentosaceus and Lactobacillus brevis were found in all sources. Interestingly, identical RAPD genotypes of L. plantarum, L. fermentum, L. brevis, Enterococcus faecium, Enterococcus faecalis and P. pentosaceus were found in the faeces of mothers, her milk and in faeces of her babies. Diversity of RAPD types found in food versus human samples suggests the importance of host factors in colonization and individual host specificity, and support the hypothesis that there is a vertical transfer of intestinal LAB from the mother's gut to her milk and through the milk to the infant's gut.
Systematic and Applied Microbiology 02/2011; 34(2):148-55. · 3.37 Impact Factor
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01/2010: pages 221ff; , ISBN: 978-3642107153
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ABSTRACT: A Gram-stain-positive, catalase-negative and rod-shaped bacterium was isolated from a brewery environment. Its phylogenetic affiliation was determined by using 16S rRNA gene sequence analysis. It was found that strain TMW 1.1424(T) belongs to the genus Lactobacillus, with the three nearest neighbours Lactobacillus parabrevis LMG 11984(T) (97 %), Lactobacillus brevis DSM 20054(T) (95.9 %) and Lactobacillus hammesii DSM 16381(T) (96.2 %). Comparative sequencing of additional phylogenetic marker genes tuf and pheS confirmed the 16S rRNA gene sequence tree topology. The DNA G+C content of strain TMW 1.1424(T) is 46.6 mol%. Genomic DNA-DNA relatedness values with L. brevis DSM 20054(T), L. parabrevis LMG 11984(T) and L. hammesii DSM 16381(T) do not exceed 52.8 %, revealing that the novel isolate represents a separate genomic species. The strain can be distinguished from other related species of the genus Lactobacillus by physiological and biochemical tests. Based on biochemical, physiological and phylogenetic data, it is proposed that the new isolate be classified as a novel species of the genus Lactobacillus, Lactobacillus paucivorans sp. nov. The type strain is TMW 1.1424(T) (=DSM 22467(T) =LMG 25291(T)).
INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY 11/2009; 60(Pt 10):2353-7. · 2.11 Impact Factor
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ABSTRACT: A novel lactic acid bacterium, strain TMW 2.694(T), was isolated among other lactic acid bacteria from palm wine, an alcoholic beverage produced from the sap of various palm tree species. Strain TMW 2.694(T) is a Gram-positive, facultatively anaerobic, catalase-negative, non-spore-forming coccus, occurring in long chains. Phylogenetic analysis based on 16S rRNA gene sequencing positioned strain TMW 2.694(T) in a distinct line of descent within the genus Leuconostoc, with the closest neighbours being Leuconostoc lactis JCM 6123(T) (98.7 % sequence similarity) and Leuconostoc citreum DSM 5577(T) (98.8 % sequence similarity). Comparative sequencing of the additional phylogenetic markers dnaK and recA confirmed the 16S rRNA gene tree topology. Genomic DNA-DNA similarities of strain TMW 2.694(T) to L. lactis DSM 20202(T) and L. citreum DSM 5577(T) were 45.1 and 17.7 %, respectively. The DNA G+C content is 36.4 mol%. Thus, we propose a novel species within the genus Leuconostoc, with the name Leuconostoc palmae sp. nov. and the type strain TMW 2.694(T) (=DSM 21144(T) =LMG 24510(T)).
International journal of systematic and evolutionary microbiology 06/2009; 59(Pt 5):943-7. · 2.27 Impact Factor
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04/2008: pages 95 - 114; , ISBN: 9780470376409
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ABSTRACT: The fermentative biota of the majority of different sourdough types consists of lactic acid bacteria (LAB), most of which
belong to the genus Lactobacillus, and yeasts. The exploitation of these organisms emerging from a long history in sourdough fermentations forms an emerging
field for baking applications and design of added-value food. The deliberate use of functional traits within these bacteria
is strongly supported by knowledge of their diversity, phylogenetic and environmental status, the characterization of their
genome structure, gene regulation and metabolic potential. This knowledge base is required to achieve a status of qualified
presumption of safety (QPS) as recently proposed by the European Authority for Food Safety (EFSA). Molecular methodology in
taxonomy and genetics are the major contributors to gain these insights in biodiversity, behavior and functionality of sourdough
microorganisms.
12/2007: pages 119-144;
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ABSTRACT: Two strains of Gram-positive, catalase-negative, lactic acid bacteria, strains TMW 1.1309(T) and TMW 1.1313, were isolated at an interval of several years from an industrial type II sourdough. They occurred at cell numbers of 8x10(8) c.f.u. g(-1) and therefore were considered to be one of the dominant members of the microbiota in this type of fermentation. Cells of both strains grow exclusively on modified MRS containing trypsin-digested rye-bran extract. Both strains possessed identical 16S rRNA gene sequences, but could be discriminated by RAPD fingerprints. Comparative 16S rRNA and tuf gene sequence analyses positioned strain TMW 1.1309(T) as part of the Lactobacillus reuteri phylogenetic group within the genus Lactobacillus. The 16S rRNA gene sequence similarities to the closest related species, Lactobacillus coleohominis and Lactobacillus ingluviei were 97.1 and 95.4 %, respectively. The DNA G+C content of strain TMW 1.1309(T) was 48 mol%. Growth characteristics, biochemical features and DNA-DNA hybridization values below 70 % with all the nearest neighbours demonstrated that the isolates represent a novel Lactobacillus species. The name Lactobacillus secaliphilus sp. nov. is proposed for the novel isolates, with the type strain TMW 1.1309(T) (=DSM 17896(T)=CCUG 53218(T)).
International journal of systematic and evolutionary microbiology 04/2007; 57(Pt 4):745-50. · 2.27 Impact Factor
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ABSTRACT: Endospores of proteolytic type B Clostridium botulinum TMW 2.357 and Bacillus amyloliquefaciens TMW 2.479 are currently described as the most high-pressure-resistant bacterial spores relevant to food intoxication and spoilage in combined pressure-temperature applications. The effects of combined pressure (0.1 to 1,400 MPa) and temperature (70 to 120 degrees C) treatments were determined for these spores. A process employing isothermal holding times was established to distinguish pressure from temperature effects. An increase in pressure (600 to 1,400 MPa) and an increase in temperature (90 to 110 degrees C) accelerated the inactivation of C. botulinum spores. However, incubation at 100 degrees C, 110 degrees C, or 120 degrees C with ambient pressure resulted in faster spore reduction than treatment with 600 or 800 MPa at the same temperature. This pressure-mediated spore protection was also observed at 120 degrees C and 800, 1,000, or 1,200 MPa with the more heat-tolerant B. amyloliquefaciens TMW 2.479 spores. Inactivation curves for both strains showed a pronounced pressure-dependent tailing, which indicates that a small fraction of the spore populations survives conditions of up to 120 degrees C and 1.4 GPa in isothermal treatments. Because of this tailing and the fact that pressure-temperature combinations stabilizing bacterial endospores vary from strain to strain, food safety must be ensured in case-by-case studies demonstrating inactivation or nongrowth of C. botulinum with realistic contamination rates in the respective pressurized food and equipment.
Applied and Environmental Microbiology 06/2006; 72(5):3476-81. · 3.83 Impact Factor
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ABSTRACT: The effect of sublethal hydrostatic pressure on the transcriptome of Lactobacillus sanfranciscensis was determined using a shot-gun-microarray. Among the 750 spots that passed quality analysis 42 genes were induced, while six were repressed when cells were incubated at 45 MPa for 30 min. The nature of genes and their differential expression clearly indicate cellular efforts to counteract a decrease in translational capacity. The majority of high pressure affected genes were found to encode either translation factors (EF-G, EF-TU), ribosomal proteins (S2, L6, L11), genes changing translational accuracy or molecular chaperones (GroEL, ClpL). These data agree with previously reported effects observed in in vitro studies as well as with physiological and proteomic data. This study provides in vivo evidence to identify ribosomes and impaired translation among primary targets for high pressure treatment. The observed induction of heat as well as cold shock genes (e.g. hsp60, gyrA) may be explained as a result of high pressure affected protein synthesis.
Archives of Microbiology 11/2005; 184(1):11-7. · 1.43 Impact Factor
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ABSTRACT: Protein hydrolysis and amino acid metabolism contribute to the beneficial effects of sourdough fermentation on bread quality. In this work, genes of Lactobacillus sanfranciscensis strain DSM 20451 involved in peptide uptake and hydrolysis were identified and their expression during growth in sourdough was determined. Screening of the L. sanfranciscensis genome with degenerate primers targeting prt and analysis of proteolytic activity in vitro provided no indication for proteolytic activity. Proteolysis in aseptic doughs and sourdoughs fermented with L. sanfranciscensis was inhibited upon the addition of an aspartic protease inhibitor. These results indicate that proteolysis was not linked to the presence of L. sanfranciscensis DSM 20451 and that this strain does not harbor a proteinase. Genes encoding the peptide transport systems Opp and DtpT and the intracellular peptidases PepT, PepR, PepC, PepN, and PepX were identified. Both peptide uptake systems and the genes pepN, pepX, pepC, and pepT were expressed by L. sanfranciscensis growing exponentially in sourdough, whereas pepX was not transcribed. The regulation of the expression of Opp, DtpT, and PepT during growth of L. sanfranciscensis in sourdough was investigated. Expression of Opp and DtpT was reduced approximately 17-fold when the peptide supply in dough was increased. The expression of PepT was dependent on the peptide supply to a lesser extent. Thus, the accumulation of amino nitrogen by L. sanfranciscensis in dough is attributable to peptide hydrolysis rather than proteolysis and amino acid metabolism by L. sanfranciscensis during growth in sourdough is limited by the peptide availability.
Applied and Environmental Microbiology 11/2005; 71(10):6260-6. · 3.83 Impact Factor
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ABSTRACT: Twenty morphologically different strains were chosen from French wheat sourdough isolates. Cells were Gram-positive, non-spore-forming, non-motile rods. The isolates were identified using amplified-fragment length polymorphism, randomly amplified polymorphic DNA and 16S rRNA gene sequence analysis. All isolates were members of the genus Lactobacillus. They were identified as representing Lactobacillus plantarum, Lactobacillus paralimentarius, Lactobacillus sanfranciscensis, Lactobacillus spicheri and Lactobacillus sakei. However, two isolates (LP38(T) and LP39) could be clearly discriminated from recognized Lactobacillus species on the basis of genotyping methods. 16S rRNA gene sequence similarity and DNA-DNA relatedness data indicate that the two strains belong to a novel Lactobacillus species, for which the name Lactobacillus hammesii is proposed. The type strain is LP38(T) (=DSM 16381(T)=CIP 108387(T)=TMW 1.1236(T)).
International journal of systematic and evolutionary microbiology 04/2005; 55(Pt 2):763-7. · 2.27 Impact Factor
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ABSTRACT: Exopolysaccharides (EPS) produced in situ by sourdough lactobacilli affect rheological properties of dough as well as bread quality and may serve as prebiotics. The aim of this study was to characterize EPS-formation by Lactobacillus sanfranciscensis TMW 1.392 at the molecular level. A levansucrase gene from L. sanfranciscensis TMW 1.392 encompassing 2,300 bp was sequenced. This levansucrase is predicted to be a cell-wall associated protein of 879 amino acids with a relative molecular weight (M(R)) of 90,000. The levansucrase gene was heterologously expressed in Escherichia coli and purified to homogeneity. The recombinant enzyme exhibited transferase and hydrolase activities and produced glucose, fructose, 1-kestose and levan from sucrose; truncation of the N-terminal domain did not affect catalytic activity. Kestose formation was enhanced relative to fructose and levan formation by low temperature or high sucrose levels. During growth in wheat doughs, strain TMW 1.392 utilized sucrose to form fructose, 1-kestose, and fructan, whereas a levansucrase deletion mutant, L. sanfranciscensis TMW 1392Deltalev, lost the ability to hydrolyze sucrose, and did not produce fructan or 1-kestose. These results indicate that, in L. sanfranciscensis TMW 1.392, sucrose metabolism and formation of fructan and 1-kestose is dependent on the activity of a single enzyme, levansucrase.
Applied Microbiology and Biotechnology 04/2005; 66(6):655-63. · 3.42 Impact Factor
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ABSTRACT: "Lactobacillus pastorianus" (Van Laer, 1892) is not a validly described species and is not included in the Approved List of Bacterial Names. The strain is available in multiple culture collections as Lactobacillus sp. DSM 20197, L. brevis ATCC 8291, "L. pastorianus" CECT 5926, L. brevis JCM 1113, and "L. pastorianus" LMG 11990. Nearly identical 16S rRNA sequences and protein encoding genes for 6-phosphogluconate dehydrogenase (99.9%) revealed this strain as L. paracollinoides. A 16S-23S rRNA intergenic spacer region-based PCR assay did not differentiate "L. pastorianus" DSM 20197 from L. paracollinoides DSM 15502(T). Highly similar RAPD profiles differentiated both strains below species level.
Systematic and Applied Microbiology 02/2005; 28(1):54-6. · 3.37 Impact Factor
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ABSTRACT: The inactivation of bacterial endospores by hydrostatic pressure requires the combined application of heat and pressure. We have determined the resistance of spores of 14 food isolates and 5 laboratory strains of Bacillus subtilis, B. amyloliquefaciens, and B. licheniformis to treatments with pressure and temperature (200 to 800 MPa and 60 to 80 degrees C) in mashed carrots. A large variation in the pressure resistance of spores was observed, and their reduction by treatments with 800 MPa and 70 degrees C for 4 min ranged from more than 6 log units to no reduction. The sporulation conditions further influenced their pressure resistance. The loss of dipicolinic acid (DPA) from spores that varied in their pressure resistance was determined, and spore sublethal injury was assessed by determination of the detection times for individual spores. Treatment of spores with pressure and temperature resulted in DPA-free, phase-bright spores. These spores were sensitive to moderate heat and exhibited strongly increased detection times as judged by the time required for single spores to grow to visible turbidity of the growth medium. The role of DPA in heat and pressure resistance was further substantiated by the use of the DPA-deficient mutant strain B. subtilis CIP 76.26. Taken together, these results indicate that inactivation of spores by combined pressure and temperature processing is achieved by a two-stage mechanism that does not involve germination. At a pressure between 600 and 800 MPa and a temperature greater than 60 degrees C, DPA is released predominantly by a physicochemical rather than a physiological process, and the DPA-free spores are inactivated by moderate heat independent of the pressure level. Relevant target organisms for pressure and temperature treatment of foods are proposed, namely, strains of B. amyloliquefaciens, which form highly pressure-resistant spores.
Applied and Environmental Microbiology 01/2005; 70(12):7321-8. · 3.83 Impact Factor
