Microbial consortia involved in fermented spelt sourdoughs: dynamics and characterization of yeasts and lactic acid bacteria.

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.

A polyphasic approach to characterize Weissella cibaria and Weissella confusa strains.

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.

Species-specific DNA probe and development of a quantitative PCR assay for the detection of Morganella morganii.

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.

A survey on biotechnological potential and safety of the novel Enterococcus species of dairy origin, E. italicus.

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.

Phenotypic typing, technological properties and safety aspects of Lactococcus garvieae strains from dairy environments.

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.

Rapid identification of Enterococcus italicus by PCR with primers targeted to 16S rRNA gene.

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.

Plasmids from Lactobacillus helveticus: distribution and diversity among natural isolates.

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.

Autolytic activity and pediocin-induced lysis in Pediococcus acidilactici and Pediococcus pentosaceus strains.

AIMS: To evaluate the autolytic phenotype of Pediococcus acidilactici and P. pentosaceus, the peptidoglycan hydrolases content and the effect of pediocin AcH/PA-1 and autolysins on cell lysis. METHODS AND RESULTS: The autolytic phenotype of Pediococcus strains was evaluated under starvation conditions in potassium phosphate buffer. The strains tested showed an extent of autolysis ranging between 40 and 90% after 48 h of starvation at 37 degrees C. Peptidoglycan hydrolase content was evaluated by renaturing sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) using cells of Micrococcus lysodeikticus as a target for the enzymatic activity and a major activity band migrating at about 116 kDa was detected. Additional secondary lytic bands migrating in a range of molecular weight between 45 and 110 kDa were also detected. The lytic activity, evaluated in the presence of different chemicals, was retained in 15 mM CaCl2 and in a range of pH between 5 and 9.5 but was strongly reduced in presence of 8% NaCl and in the presence of protease inhibitors. The substrate specificity of peptidoglycan hydrolases of Pediococcus strains was evaluated in renaturing SDS-PAGE incorporating cells of different bacterial species. Lytic activity was detected against cells of Lactococcus lactis subsp. lactis, L. delbrueckii subsp. bulgaricus, Lactobacillus helveticus and Listeria monocytogenes. The interaction between pediocin AcH/PA-1 and autolysis was evaluated and a relevant effect of bacteriocin in cell-induced lysis was observed. CONCLUSIONS: The autolytic phenotype is widely distributed among P. acidilactici and P. pentosaceus and the rate of autolysis is high in the majority of the analysed strains. Several autolytic bands, detected by renaturing SDS-PAGE, retained their activity against several lactic acid bacteria and L. monocytogenes. SIGNIFICANCE AND IMPACT OF THE STUDY: The characterization of the autolytic phenotype of Pediococcus acidilactici and P. pentosaceus strains should expand the knowledge of their role in fermentation processes where these species occur as primary or secondary bacterial population.

Genetic diversity and technological properties of Streptococcus thermophilus strains isolated from dairy products.

AIMS: To evaluate the genetic diversity and the technological properties of 44 strains of Streptococcus thermophilus isolated from dairy products. Methods METHODS AND RESULTS: Strains were analysed for some relevant technological properties, i.e. exopolysaccharide (EPS) production, growth kinetic in skim milk medium, urease activity and galactose fermentation. The EPS production, determined by evaluating the colour of the colonies grown in ruthenium red milk agar, was observed in 50% of the analysed strains. Urease activity, determined by colorimetric and conductimetric methods, showed that 91% of the isolates, all except four, could hydrolyse urea. A conductimetric approach was also used for the evaluation of the overall metabolic behaviour in milk of Strep. thermophilus strains and the differences observed allowed grouping of the strains in seven different clusters. A total of 11 strains were able to produce acid in presence of galactose. Genetic diversity of Streptococcus thermophilus strains, evaluated by Random Amplified Polymorphic DNA fingerprinting (RAPD) and amplified epsC-D restriction analysis, allowed the identification of 21 different genotypes. CONCLUSIONS: Comparison between the genotypic and phenotypic data highlights an interesting correlation between some important technological properties and well-defined genotypes. SIGNIFICANCE AND IMPACT OF THE STUDY: The genetic and technological characterization carried out on several Strep. thermophilus strains of dairy origin should expand the knowledge on this important lactic acid bacteria species and lead to a simple, rapid, and reliable identification of strains on the basis of well-defined biotechnological properties.

Comparison between Streptococcus macedonicus and Streptococcus waius strains and reclassification of Streptococcus waius (Flint et at. 1999) as Streptococcus macedonicus (Tsakalidou et al. 1998).

Two species of dairy streptococci, Streptococcus waius and Streptococcus macedonicus, were originally characterized by 16S-23S intergenic spacer sequence analysis, random amplified polymorphic DNA fingerprinting, PFGE analysis and DNA-DNA reassociation experiments. All genetic data suggested that S. waius strains belong to the previously described species S. macedonicus. Likewise, the phenotypic characterization showed that strains of S. macedonicus and S. waius were highly related and easily differentiated from the closest phylogenetic neighbour, Streptococcus bovis, principally by their failure to produce a blackening reaction in medium containing aesculin. The utilization of maltose and cellobiose by S. macedonicus/S. waius strains allowed their differentiation from the most studied dairy species, Streptococcus thermophilus. On the basis of genetic and phenotypic data S. macedonicus and S. waius species should be considered synonyms and S. macedonicus has the priority.

Ureibacillus gen. nov., a new genus to accommodate Bacillus thermosphaericus (Andersson et al. 1995), emendation of Ureibacillus thermosphaericus and description of Ureibacillus terrenus sp. nov.

A polyphasic taxonomic study was performed on the type strain of Bacillus thermosphaericus DSM 10633T and three related soil isolates. On the basis of phenotypic characteristics, chemotaxonomic profiles and phylogenetic data a new genus, Ureibacillus gen. nov., is proposed for the strains in the Bacillus thermosphaericus cluster. Strains of this cluster fall into two DNA-DNA similarity groups: while one group contains the type strain of Ureibacillus thermosphaericus comb. nov. and a single soil isolate, the other contains two soil isolates. The two groups differed in the composition of isoprenoid quinones and some phenotypic properties. These data support the description of a novel species of Ureibacillus for which the name Ureibacillus terrenus is proposed. The type strain of this new species is TH9AT (= DSM 12654T = LMG 19470T).

Specific identification of Lactobacillus helveticus by PCR with pepC, pepN and htrA targeted primers.

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.

Reclassification of Saccharococcus caldoxylosilyticus as Geobacillus caldoxylosilyticus (Ahmad et al. 2000) comb. nov.

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.

Development of molecular RAPD marker for the identification of Pediococcus acidilactici strains.

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.

Bacillus thermodenitrificans sp. nov., nom. rev.

A polyphasic study was performed on 10 soil isolates of thermophilic denitrifying Bacillus strains from different geographical areas. The presence of two main characteristic bands following amplification of the internal transcribed spacer (ITS) region of rrn operons suggests a close relatedness to 'Bacillus thermodenitrificans'. The isolates cluster around two strains of 'B. thermodenitrificans' in riboprint and fatty acid analyses, though differences occur at the strain level. Subsequent DNA-DNA reassociation studies including the 10 isolates, 'B. thermodenitrificans' DSM 465T and DSM 466, and Bacillus stearothermophilus ATCC 12980T and Bacillus thermoleovorans ATCC 43513T revealed such a high level of genomic relatedness between the isolates and the DSM strains (> 73% similarity) that they must be considered strains of the same taxon. The degree of DNA-DNA similarity between the 12 strains of 'B. thermodenitrificans' and the type strains of the other two phylogenetically neighbouring Bacillus species was significantly lower (21-43% similarity). Based upon phylogenetic, chemotaxonomic and phenotypic evidence, the designation of B. thermodenitrificans sp. nov., nom. rev. is proposed. The type strain of B. thermodenitrificans is DSM 465T.

Discrimination among Pediocin AcH/PA-1 producer strains by comparison of pedB and pedD amplified genes and by multiplex PCR assay.

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.

Genotypic heterogeneity among Lactobacillus helveticus strains isolated from natural cheese starters.

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.

Contribution to phenotypic and genotypic characterization of Bacillus licheniformis and description of new genomovars.

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.

Genotypic characterization of thermophilic bacilli: a study on new soil isolates and several reference strains.

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.

Lactobacillus helveticus heterogeneity in natural cheese starters: the diversity in phenotypic characteristics.

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.