RESUMO
AIM: To study Weissella cibaria and Weissella confusa strains, lactic acid bacteria (LAB) members naturally present in food products, but not yet included in Qualified Presumption of Safety (QPS) list of European Food Safety Authority (EFSA). METHODS AND RESULTS: We carried out a comparative genome analysis of 23 sequenced W. cibaria and 7 W. confusa genomes, in parallel with a physiological and functional characterization of several strains previously isolated from sourdough-like maize bran fermentation. The genome analysis revealed the absence of dedicated pathogenicity factors. Some putative virulence genes found in Weissella genomes were also present in other LAB strains, considered safe by EFSA and commonly used as probiotics. The physiological tests carried out on our strains corroborated the genomic results. Moreover, the following functional traits of interest to application in the food sector were identified: the majority of tested strains displayed high acidification rate, high reducing ability, production of exopolysaccharides (EPS), arabinoxylan degradation ability, growth in the presence of fructo-oligosaccharides (FOS), bile and gastric juice tolerance, and antifungal activity. CONCLUSIONS: These results provide evidence for the possible use of selected strains of W. cibaria and W. confusa in the food sector. SIGNIFICANCE AND IMPACT OF THE STUDY: This polyphasic study adds to the body of knowledge on the functional and applicable characteristics of these controversial species of LAB. This knowledge contributes to design new selected cultures included in the QPS list required for food applications.
Assuntos
Weissella/genética , Weissella/metabolismo , Fermentação , Genoma Bacteriano , Genômica , Fenótipo , Polissacarídeos/biossíntese , Probióticos/análise , Probióticos/metabolismo , Weissella/classificação , Weissella/crescimento & desenvolvimento , Xilanos/metabolismoRESUMO
This study aims to describe the native microbiota of fermented spelt, taking into consideration both lactic acid bacteria (LAB) and yeasts, for which little data are available. Five samples of commercial spelt flour were subjected to spontaneous fermentation to obtain a type I sourdough. A total of 186 LAB and 174 yeast isolates were selected at different refreshment steps and subjected to further analyses. Within LAB, coccal isolates constituted 78·5% of the total LAB, with the dominance of Pediococcus pentosaceus. Although documented before as a component, this is the first report of a spelt sourdough fermentation dominated by this homofermentative LAB, characterized by a high acidification rate, ability to utilize a wide range of carbon sources and to grow in high osmolarity conditions. Yeast communities resulted in four dominant species, Saccharomyces cerevisiae, Wickerhamomyces anomalus, Pichia fermentans and Clavispora lusitaniae. This study highlights for the first time the biodiversity and dynamics of yeast communities involved in sourdough fermentation of spelt. Compared to commercial baker's yeast, autochthonous W. anomalus, P. fermentans and S. cerevisiae isolates show a good performance, and their use could be an advantage for their acquired adaptation to the environment, providing stability to the fermentation process. SIGNIFICANCE AND IMPACT OF THE STUDY: Nowadays, there is a renewed interest in products based on spelt. This 'ancient grain' is a highly nutritional grain; however, its use is limited to bread-making processes, which are not standardized. The low baking and sensory quality of spelt can be overcome through fermentation processes. However, the autochthonous microbiota of spelt sourdough is poorly known. This study highlights the dynamics of microbial communities involved in sourdough fermentation of spelt and provides the basis for the selection of autochthonous cultures, with the aim of improving the nutritional potential of spelt and its rheology and bread-making properties.
Assuntos
Pão/microbiologia , Lactobacillales/metabolismo , Consórcios Microbianos , Triticum/microbiologia , Leveduras/metabolismo , Biodiversidade , Pão/análise , Fermentação , Farinha/microbiologia , Microbiologia de Alimentos , Lactobacillales/classificação , Lactobacillales/genética , Lactobacillales/isolamento & purificação , Triticum/metabolismo , Leveduras/classificação , Leveduras/genética , Leveduras/isolamento & purificaçãoRESUMO
AIMS: To develop a SYBR Green quantitative PCR assay (qPCR) for the specific detection of Morganella morganii, a fish pathogen responsible for the Histamine Fish Poisoning. METHODS AND RESULTS: A new primer set, amplifying a 179-bp fragment of the 16S rRNA gene, was selected for specificity, and 14 M. morganii strains and 32 non-Morganella strains were evaluated. The melting temperature of 84°C was consistently specific for the amplicon. Two standard curves were constructed: the minimum detection sensitivity was 0·563 pg of pure DNA, corresponding to DNA extracted from nine cells of M. morganii. The qPCR assay was evaluated in experiments with seeded fish samples, and the regression coefficient values were calculated. CONCLUSIONS: A highly specific and rapid assay was developed for the detection of M. morganii in tuna fish samples. SIGNIFICANCE AND IMPACT OF THE STUDY: This method represents the first study about the quantification of pathogenic M. morganii in fish products. This approach can be utilized to prevent the presence of this undesirable species in the food chain.
Assuntos
Contaminação de Alimentos/análise , Morganella morganii/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Alimentos Marinhos/microbiologia , Atum/microbiologia , Animais , Primers do DNA/genética , Morganella morganii/genética , Especificidade da EspécieRESUMO
The biotechnological and safety properties of the novel enterococcal species of dairy origin, Enterococcus italicus, were investigated. The strains of the species showed technological characteristics related to their use as adjunct cultures in the production of artisanal cheeses. They were susceptible or poorly resistant to several clinical relevant antibiotics. Moreover, E. italicus strains were associated with low virulence profiles, as verified by screening for the presence of 33 different genes encoding antibiotic resistance and known virulence factors in the genus Enterococcus. From the data obtained, we deduce that the presence of E. italicus strains in cheeses results in a low health risk and that within the species new safe adjunct cultures for the dairy industry could be found.
Assuntos
Antibacterianos/farmacologia , Queijo/microbiologia , Qualidade de Produtos para o Consumidor , Enterococcus/fisiologia , Microbiologia de Alimentos , Sequência de Bases , Contagem de Colônia Microbiana , DNA Bacteriano/análise , Farmacorresistência Bacteriana , Enterococcus/efeitos dos fármacos , Enterococcus/patogenicidade , Humanos , Testes de Sensibilidade Microbiana , VirulênciaRESUMO
From Bacillus licheniformis a site-specific restriction endonuclease, named BliI, has been purified and characterized. BliI was able to digest lambda DNA at pH 9.1 over a wide temperature range (25-65 degrees C). Digestion of lambda and psi X174 DNAs with BliI produced banding patterns identical to those seen with HaeIII. Therefore, BliI and HaeIII endonculeases are isoschizomers.
Assuntos
Bacillus/enzimologia , Enzimas de Restrição do DNA/isolamento & purificação , Desoxirribonucleases de Sítio Específico do Tipo II , DNA Viral , Especificidade por SubstratoRESUMO
We isolated and identified four spore-forming bacteria from activated sludges and soil, three of which were able to degrade acifluorfen. Biochemical characteristics, DNA base composition and DNA-DNA homology indicated that the degrading strains belonged to the species Bacillus thuringiensis, Clostridium perfringens and Clostridium sphenoides. The fourth strain, identified as C. sphenoides and showing the same characteristics of the corresponding degrading strain, was unable to metabolize acifluorfen. Thus, the plasmid content of these strains was analysed to study the possible correlation between the presence of extrachromosomal elements and the ability to degrade this herbicide.
Assuntos
Bacillus thuringiensis/isolamento & purificação , Clostridium perfringens/isolamento & purificação , Clostridium/isolamento & purificação , Herbicidas/metabolismo , Nitrobenzoatos/metabolismo , Bacillus thuringiensis/genética , Bacillus thuringiensis/metabolismo , Bacillus thuringiensis/fisiologia , Clostridium/genética , Clostridium/metabolismo , Clostridium/fisiologia , Clostridium perfringens/genética , Clostridium perfringens/metabolismo , Clostridium perfringens/fisiologia , DNA Bacteriano/análise , Eletroforese em Gel de Ágar , Técnicas In Vitro , Plasmídeos/análise , Microbiologia do Solo , Esporos Bacterianos/isolamento & purificação , Esporos Bacterianos/metabolismoRESUMO
A genotypic study using amplified ribosomal DNA restriction analysis (ARDRA), random amplified polymorphic DNA fingerprinting (RAPD) and ribosomal spacer analysis (RSA) in comparison with DNA-DNA reassociation experiments was carried out with 85 thermophilic Bacillus isolates from uncultivated soil of 14 different geographical areas and seventeen reference strains representing defined thermophilic Bacillus species. This approach permitted the attribution of 51% of the new isolates to the Bacillus thermoleovorans group and the identification of 40% of the new isolates as B. "thermodenitrificans". Moreover, 2 strains were assigned to B. pallidus species and 1 isolate to B. thermosphaericus species. The remaining 6% of our thermophilic isolates from soil, constituting 2 DNA-DNA homology groups, are still unidentified. A detailed genotypic characterization of the heterogeneous species of B. thermoleovorans and B. stearothermophilus was also presented.
Assuntos
Bacillus/classificação , Bacillus/genética , Técnicas de Tipagem Bacteriana , Microbiologia do Solo , Bacillus/isolamento & purificação , Impressões Digitais de DNA , DNA Bacteriano/análise , DNA Ribossômico/análise , Temperatura Alta , Hibridização de Ácido Nucleico , Técnica de Amplificação ao Acaso de DNA Polimórfico , Mapeamento por RestriçãoRESUMO
Twenty-two Bacillus licheniformis strains, freshly isolated from pasture-land, were studied for the presence of plasmid DNA. Among these strains, 14 were shown to harbor one or more plasmids of different size. Southern-hybridization experiments showed a high homology between all plasmids investigated and a 2.2-kb PvuII/HindIII fragment of pBL1, a B. licheniformis plasmid previously isolated. Three fragments of pBL1, including the 2.2-kb PvuII/HindIII region, were cloned into pJH101 vector. The resulting chimeras were able to transform Bacillus subtilis. The fragment with high homology probably contains the region with the replicative functions of plasmids from B. licheniformis species.
Assuntos
Bacillus/genética , Plasmídeos/genética , Bacillus/efeitos dos fármacos , Southern Blotting , Quimera/genética , Clonagem Molecular , Enzimas de Restrição do DNA/metabolismo , DNA Bacteriano/genética , Resistência Microbiana a Medicamentos/genética , Eletroforese em Gel de Ágar , Vetores Genéticos/genética , Metais/farmacologia , Homologia de Sequência do Ácido Nucleico , Transformação BacterianaRESUMO
Specific regions in three genes coding for aminopeptidases C and N, and a trypsin-like serine protease were selected as species-specific primer sequences for rapid and reliable identification of Lactobacillus helveticus strains. The PCR procedures carried out gave specific 524-, 726- and 918-bp amplificates, with DNA isolated from L. helveticus. No PCR product was generated for closely related bacteria. The amplification products were also screened for their species specificity in dot blot hybridization with representatives of the most closely related genera and species and a number of other bacterial species.
Assuntos
Aminopeptidases , Proteínas de Bactérias/genética , Proteínas de Choque Térmico , Lactobacillus/classificação , Lactobacillus/genética , Proteínas Periplásmicas , Reação em Cadeia da Polimerase , Serina Endopeptidases/genética , Primers do DNA , Sondas de DNA , DNA Bacteriano/genética , Genes Bacterianos , Especificidade da EspécieRESUMO
Pediococcus acidilactici, Pediococcus parvulus and Lactobacillus plantarum pediocin AcH/PA-1 produces strains were studied with the aim to investigate their common genetic pediocin determinant using pedA, pedB, pedC and pedD gene-targeted PCR assay. Single Strand Conformation Polymorphism and restriction analysis of pedA and pedC amplified fragments from the three different species did not show any differences in sequence while these analysis carried out on pedB and pedD amplified fragments highlighted differences related to the three species analyzed harboring these plasmid encoded genes. Furthermore different multiplex PCR assay using IdhD, pedA and pedD as target genes were developed to clearly identify the pediocin AcH/PA-1 producer strains and to obtain the simultaneous identification of the P. acidilactici strains.
Assuntos
Bacteriocinas/genética , Genes Bacterianos , Lactobacillus/genética , Pediococcus/genética , Bacteriocinas/biossíntese , Southern Blotting , Amplificação de Genes , Lactobacillus/classificação , Lactobacillus/metabolismo , Óperon/genética , Pediocinas , Pediococcus/classificação , Pediococcus/crescimento & desenvolvimento , Pediococcus/metabolismo , Plasmídeos/genética , Reação em Cadeia da Polimerase , Polimorfismo Conformacional de Fita Simples , Mapeamento por Restrição , Análise de Sequência de DNARESUMO
A RAPD analysis performed using a single primer targeted to the pediocin AcH/PA-1 gene was carried out on several P. acidilactici strains and on some related species of lactic acid bacteria. The high degree of genetic variability detected in P. acidilactici strains did not allow the selection of a common RAPD fragment that could be chosen as a potential species-specific DNA marker. Nevertheless a 700 bp fragment, that was found to be peculiar of all potential pediocin producer strains analyzed, was cloned and sequenced with the aim to develop a species specific PCR marker. Sequence analysis of the cloned 700 bp fragment showed one putative small open reading frame (ORF1), with no significant homology with known genes, and a partial putative second coding region (ORF2) with a high degree of similarity with several methionyl tRNA synthesis (metS) genes. The two coding regions were separated by a short spacer region. Primers targeted to ORF2 plus part of the spacer region and primers designed for the amplification of the entire cloned RAPD fragment were found to be species-specific for the detection of P. acidilactici strains. Furthermore primers designed on the ORF1 sequence allowed the amplification of a 439 bp fragment only in some P. acidilactici strains, including pediocin producing strains.
Assuntos
Bacteriocinas/genética , Pediococcus/classificação , Técnica de Amplificação ao Acaso de DNA Polimórfico/métodos , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Marcadores Genéticos , Dados de Sequência Molecular , Pediocinas , Pediococcus/genética , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
A study of phenotypic and genotypic characteristics was carried out on 182 strains isolated from soil of different geographical areas; the type strains were B. licheniformis, B. subtilis, B. pumilus, B. cereus and B. coagulans. The results showed, primarily on the basis of phenotypic features, that all the isolates belonged to the B. licheniformis species, however DNA relatedness studies revealed only 161 to be genetically related to B. licheniformis, the DNA relatedness levels ranging from 66 to 100%. The other 21 isolates appeared to be genetically distinct not only from B. licheniformis but also from B. subtilis and B. pumilus, where there were low levels of DNA relatedness (from 4 to 37%). Nevertheless the ARDRA results indicate that the 21 atypical isolates were phylogenetically related to B. licheniformis. Our data and the phenotypic homogeneity found suggest the presence of three different genomovars.
Assuntos
Bacillus/genética , Microbiologia do Solo , Bacillus/classificação , Bacillus/fisiologia , Bacillus cereus/classificação , Bacillus cereus/genética , Bacillus cereus/fisiologia , Bacillus subtilis/classificação , Bacillus subtilis/genética , Bacillus subtilis/fisiologia , Metabolismo dos Carboidratos , DNA Bacteriano/genética , Genótipo , Fenótipo , Plasmídeos/genética , Reação em Cadeia da Polimerase , RNA Bacteriano/análise , RNA Ribossômico 16S/análise , Especificidade da EspécieRESUMO
AIMS: To develop a species-specific PCR assay with primers targeted to 16S rRNA gene for the identification of Enterococcus italicus, a new species of Enterococcus, involved in the production of Italian cheeses. METHODS AND RESULTS: The type strain of E. italicus (DSM 15952(T) - 16S rRNA gene accession no. AJ582753) and other strains of the species were subjected to a rapid identification by PCR using primer pairs located within the 16S rRNA gene. A species-specific PCR product of approximately 323 bp was obtained after amplification of all E. italicus strains tested. The specificity of the primers was validated with representatives of the most closely related genera and species and a number of other bacterial species. In addition, the technique enabled the recognition of E. italicus from cheeses. CONCLUSIONS: The protocol was highly efficient and sensitive, enabling the identification of E. italicus from cheeses. SIGNIFICANCE AND IMPACT OF THE STUDY: The species-specific PCR offers a reliable and rapid alternative to conventional phenotypic methods for the identification of E. italicus within the heterogeneous genus Enterococcus.
Assuntos
Queijo/microbiologia , Enterococcus/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Primers do DNA , DNA Bacteriano/genética , DNA Ribossômico/genética , Enterococcus/classificação , Enterococcus/genética , RNA Ribossômico 16S/genética , Sensibilidade e Especificidade , Especificidade da EspécieRESUMO
AIMS: To characterize Lactococcus garvieae strains of dairy origin and to determine their technological properties and safety for their possible use in starter culture preparation. METHODS AND RESULTS: Forty-seven L. garvieae isolates, recovered from two artisanal Italian cheeses were studied, in comparison with 12 fish isolates and the type strain of the species. Phenotypic typing revealed that the strains could be differentiated on the basis of their ecological niche of origin in lactose positive strains (all isolated from dairy sources) and lactose negative strains (all isolated from fish). Furthermore, the strains exhibited a high degree of physiological variability, showing the presence of 26 different biotypes. The strains possessed moderate acidifying and proteolytic activities and did not produce bacteriocins. A safety investigation revealed that all strains were sensitive to vancomycin and moderately resistant to kanamycin; some biotypes were tetracycline resistant. Production of biogenic amines or presence of genes encoding virulence determinants occurred in some isolates. CONCLUSIONS: The prevalence of L. garvieae in some artisanal Italian cheeses can be linked to the typicity of the products. Although in a few cases an antimicrobial resistance or a presence of virulence determinants may imply a potential hygienic risk, most of the strains showed positive properties for their possible adjunction in a starter culture preparation, to preserve the natural bacterial population responsible for the typical sensorial characteristics of the traditional raw milk cheeses. SIGNIFICANCE AND IMPACT OF THE STUDY: L. garvieae strains can be considered an important part of the microbial population associated with the natural fermentation of artisanal Italian cheeses. A deepened characterization of the strains may aid in understanding the functional and ecological significance of their presence in dairy products and in selecting new strains for the dairy industry.
Assuntos
Laticínios/microbiologia , Microbiologia de Alimentos , Lactococcus/isolamento & purificação , Animais , Antibacterianos/farmacologia , Técnicas de Tipagem Bacteriana/métodos , Aminas Biogênicas/biossíntese , Bovinos , Queijo/microbiologia , Farmacorresistência Bacteriana , Ecossistema , Genes Bacterianos/genética , Cabras , Canamicina/farmacologia , Lactococcus/efeitos dos fármacos , Lactococcus/metabolismo , Lactose/metabolismo , Oncorhynchus mykiss/microbiologia , Fenótipo , Tetraciclina/farmacologia , Vancomicina/farmacologia , VirulênciaRESUMO
AIMS: To investigate the distribution and the level of diversity of extrachromosomal molecules in Lactobacillus helveticus strains in relation to their different ecological niches. METHODS AND RESULTS: The plasmid profile of 22 Lact. helveticus strains, isolated from five different Italian cheeses, was determined. Among the tested strains, there was a variable presence of plasmids: eight plasmid-free strains and the remaining with several plasmids that could be differentiated on the basis of number and molecular weight. The profiles showed between one and five plasmid bands, which size ranged between 2.3 and 31 kb. Four of these plasmids were further analyzed by restriction digestion and compared with the plasmids from Lact. helveticus ATCC 15009(T). Analyses and comparison of their primary structures and hybridization experiments revealed the presence of different DNA homology groups. CONCLUSIONS: This study indicates that within Lact. helveticus species, there is a high degree of variability in relation to the presence of plasmid molecules. Moreover, the structural diversity found among some of these plasmids allows to hypothesize the presence of different evolutionary lineages. SIGNIFICANCE AND IMPACT OF THE STUDY: Studies on plasmid distribution and diversity should be considered as an essential component in a continuing effort to explore microbial diversity as well as to understand the real role of plasmids in the flow of genetic information in natural bacterial communities.
Assuntos
Queijo/microbiologia , Microbiologia de Alimentos , Lactobacillus helveticus/genética , Plasmídeos/genética , Variação Genética , Itália , Peso Molecular , Plasmídeos/químicaRESUMO
A total of 23 strains of Lactobacillus helveticus isolated from natural whey starter cultures for Italian hard cheeses and three reference strains were characterized by plasmid profiling, ribotyping and random amplified polymorphic DNA (RAPD) fingerprinting. The data showed an interesting strain heterogeneity in natural cheese starters, that seemed not only strain-dependent, but also related to the source of isolates. Nineteen of the strains tested harboured extrachromosomal elements, whilst 11 different plasmid profiles were detected. Ribotyping with a variety of restriction enzymes differentiated 11 strains and in a few cases, RAPD fingerprinting allowed differentiation amongst strains that were not distinguished by the other two techniques.
Assuntos
Heterogeneidade Genética , Lactobacillus/genética , Queijo/microbiologia , Impressões Digitais de DNA , DNA Bacteriano/análise , Genótipo , Lactobacillus/classificação , Lactobacillus/isolamento & purificação , Plasmídeos , RNA Bacteriano/análise , Técnica de Amplificação ao Acaso de DNA PolimórficoRESUMO
Three plasmids isolated from Lactobacillus helveticus ATCC 15009 were analysed by restriction digestion. A restriction map of the 22.0, 6.0 and 3.5 kb plasmids is presented. The existence of genetic markers such as carbohydrate fermentation, lactose metabolism, antibiotics and heavy metal ions resistance was also investigated but the plasmids remain cryptic.
Assuntos
Lactobacillus/genética , Plasmídeos/genética , Mapeamento por RestriçãoRESUMO
A polyphasic study was performed on five thermophilic strains belonging to the genus Bacillus, isolated from soil of different geographical areas. 16S rRNA gene sequence analysis placed these isolates in RNA group 5, with Saccharococcus caldoxylosilyticus and [Bacillus] thermoglucosidasius being the closest phylogenetic neighbours. The type species of Saccharococcus, Saccharococcus thermophilus, was only moderately related to these two species and the novel isolates. DNA-DNA hybridization studies and comparison of morphological, chemotaxonomic and phenotypic features supported the close relationship between the novel isolates and Saccharococcus caldoxylosilyticus. These data justify the reclassification of Saccharococcus caldoxylosilyticus. Following the transfer of the validly described Bacillus species of group 5 into the genus Geobacillus, the reclassification of Saccharococcus caldoxylosilyticus as Geobacillus caldoxylosilyticus comb. nov. is proposed. This species can be distinguished genomically from Geobacillus thermoglucosidasius, Geobacillus stearothermophilus, Geobacillus thermodenitrificans and Saccharococcus thermophilus by a specific PCR-RFLP assay targeting the 16S rDNA.
Assuntos
Bacillaceae/classificação , Bacillaceae/genética , Temperatura Alta , Microbiologia do Solo , Bacillaceae/química , Bacillaceae/fisiologia , Composição de Bases , DNA Ribossômico , Ácidos Graxos/análise , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Fenótipo , Filogenia , Reação em Cadeia da Polimerase , RNA Ribossômico 16S , Mapeamento por RestriçãoRESUMO
The study of wild strains from natural habitats is a useful means of understanding better the heterogeneity within a species of biotechnological importance, and of obtaining atypical isolates with unknown capabilities. In the present research carried out on different Lactobacillus helveticus strains isolated from natural cheese starters, it was observed that several biotechnologically important characteristics can differ greatly between strains. Biotypes were found which differ in terms of fructose, maltose and trehalose fermentation, acidifying activity, proteolytic and peptidase activity, and antibiotic and lysozyme resistance. The possibility of choosing Lact. heleveticus strains with specific biotechnological profiles will influence the quality and the variety of dairy products.