The Combined Use of Whole-cell Protein Extracts for the Identification (SDS-PAGE) and Enzyme Activity Screening of Lactic Acid Bacteria Isolated from Traditional Greek Dairy Products

A former subgenus of the genus Lactobacillus, "Streptobacterium," comprises a wide range of species, including the so-called "atypical streptobacteria," which includes the Lactobacillus sake-Lactobacillus curvatus group. Various identification systems and differentiation criteria for L. sake and L. curvatus have been described previously and are well established. The phenotypic diversity within these two species was the reason for comparing phenotypic variations with DNA homology data. Previously described biotypes of L. sake (Katagiri, Kitahara, and Fukami) and L. curvatus (Abo-Elnaga and Kandler) were the basis for selecting strains. Strains of all known biotypes of these species were examined to determine their biochemical reactions, their physiological growth characteristics, their total soluble protein contents as determined by a pattern analysis in which native polyacrylamide gel electrophoresis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis with colloidal Coomassie blue and silver diamine staining were used, and their levels of DNA homology as determined by DNA-DNA hybridization experiments. All of the phenotypic analyses revealed a diversity within the taxa, whereas the DNA-DNA hybridization analysis revealed that the level of genomic homogeneity within each species was relatively high. The phenotypic diversity and genomic homogeneity which we observed allowed us to describe subgroups of L. sake and L. curvatus. The descriptions of L. sake and L. curvatus are emended accordingly. These subgroups which we describe may be the basis for defining subspecies within these two species.The facultatively heterofermentative lactobacilli comprise a wide range of species, including species and subspecies of technological and clinical interest, such as Lactobacillus casei, Lactobacillus plantarum, Lactobacillus rhamnosus, and Lactobacillus paracasei subspecies. The so-called "atypical streptobacteria" (13, 18, 19, 34,44), which are significant spoilage bacteria, are of special interest and occur in naturally or artificially fermented meat. The habitats of Lactobacillus sake and Lactobacillus cuwatus are very diverse. These organisms are important in the production of fermented plant material (silage, sauerkraut, mixed pickles, etc.) and in the spoilage of plant material as well. They also occur in beverages (e.g., beer) and even in manure (14, 22). L. sake was first isolated from sake (rice wine) starter (22). Our strain collection also includes L. sake strains of human origin, a habitat not described previously for this species (Table 1). These bacteria have been called atypical streptobacteria compared with the "streptobacteria," a term used in classical and descriptive taxonomy (31) which is no longer used in genomically based taxonomy (46). The ability to grow at 2°C and pH 3.9 was recognized as a main criterion for differentiation. Table 2 shows other distinguishing characteristics that are based on morphological and physiological data (31, 36, 37 two species are well established, the phenotypic d...

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“…5). The usefulness of protein fingerprinting with Coomassie blue staining has been described frequently previously (17,49,50).…”

Section: Discussionmentioning

confidence: 95%

Emended Descriptions of Lactobacillus sake (Katagiri, Kitahara, and Fukami) and Lactobacillus curvatus (Abo-Elnaga and Kandler): Numerical Classification Revealed by Protein Figerprinting and Identification Based on Biochemical Patterns and DNA-DNA Hybridizations

Klein

Dicks

Pack

et al. 1996

International Journal of Systematic Bacteriology

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“…Differences in electrophoretic mobility may result from differences in charge and/or size. Multilocus enzyme analysis has been shown to be of great potential [161] in the differentiation of LAB species [182]. The electrophoretic mobility of LDH (lactate dehydrogenase) in starch gels [77] and in polyacrylamide gels [95] [146], or clinical isolates and biotechnologically-used strains of Lactobacillus rhamnosus [112], or strains of Lactobacillus casei [55] isolated from ensiled high-moisture corn grain.…”

Section: Multilocus Enzyme Electrophoresismentioning

confidence: 99%

Article

Coeuret

Dubernet

Bernardeau

et al. 2003

Lait

153

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-During the last fifteen years, the Lactobacillus genus has evolved and contains to date more than 80 species. They are present in raw milk and dairy products such as cheeses, yoghurts and fermented milks. Quality assurance programmes associated with research, development, production and validation of the health or technological benefits of these bacteria require their relevant isolation, counting and identification. This review presents the different selective media to isolate lactobacilli, and the numerous different available tools to characterise lactobacilli at genus, species or strain level using either culture-dependent methods: phenotypical, molecular or global methods, or using new culture-independent advanced molecular methods. Enzymes used for PFGE, hybridisation probes and PCR-based method primers are listed in seven tables. In conclusion, the main advantages and disadvantages associated with these techniques are presented.Lactobacillus / media / PFGE restriction enzyme / probe / primer / cheese / dairy product Résumé -Isolement, caractérisation et identification des lactobacilles des produits laitiers. Durant les quinze dernières années, le genre Lactobacillus a subi de nombreux remaniements et compte actuellement plus de 80 espèces. Les lactobacilles sont présents dans le lait cru, les produits laitiers tels que les fromages, les yaourts et les laits fermentés. Pour s'inscrire dans une démarche qualité visant au développement de lactobacilles à effet santé ou d'intérêt technologique, l'isolement, le comptage et la caractérisation parfaite de ces bactéries sont nécessaires. Cette analyse bibliographique présente les différents milieux sélectifs pour isoler les lactobacilles, ainsi que les outils disponibles à ce jour pour caractériser les lactobacilles au niveau du genre, de l'espèce ou de la souche, aussi bien par des méthodes culture dépendantes : phénotypique, moléculaire, globale, que par les nouvelles méthodes se réalisant à partir d'échantillons bruts. Les enzymes utilisées en PFGE, les sondes d'hybridation et les oligonucléotides utilisés pour les différentes techniques de PCR sont répertoriés en sept tableaux. En conclusion, les avantages et inconvénients de ces techniques sont présentés.Lactobacillus / milieu sélectif / enzyme PFGE / sonde moléculaire / amorce PCR / fromage / produit laitier

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“…equinus complex (De Vuyst and Tsakalidou, 2008). The first isolation of this species was during a survey of the LAB microbiota of naturally fermented Greek Kasseri cheese (Tsakalidou et al, 1994) and it has been also detected in several Italian raw milk samples (Franciosi et al, 2009a) and raw milk cheese products (Mora et al, 2003;Pacini et al, 2006;Lombardi et al, 2004;Poznanski et al, 2004). The characterisation of S. macedonicus (Anastasiou et al, 2007) demonstrated its food-grade and nonpathogenic status.…”

Section: Discussionmentioning

confidence: 95%

Extension of Tosèla cheese shelf-life using non-starter lactic acid bacteria

et al. 2011

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a b s t r a c tSix strains of non-starter lactic acid bacteria (NSLAB) were used to extend the shelf-life of the fresh cheese Tosèla manufactured with pasteurised cows' milk. The acidification kinetics of three Lactobacillus paracasei, one Lactobacillus rhamnosus and two Streptococcus macedonicus were studied in synthetic milk medium. Lb. paracasei NdP78 and NdP88 and S. macedonicus NdP1 and PB14-1 showed an interesting acidifying capacity and were further characterised for growth in UHT milk and production of antimicrobial compounds. Lb. paracasei NdP78 and S. macedonicus NdP1 grew more than 2 log cycles in 6 h. Lb. paracasei NdP78 was also found to produce a bacteriocin-like inhibitory substance (BLIS) active against Listeria monocytogenes. The four NSLAB strains (singly or in combination) were used to produce experimental pilotscale cheeses which were compared by a panel. The cheese manufactured with the mixed culture Lb. paracasei NdP78 -S. macedonicus NdP1 was the most appreciated for its sensory properties. The cheeses produced at factory-scale showed higher concentrations of lactobacilli (7.90 log CFU/g) and streptococci (6.10 log CFU/g), but a lower development of coliforms (3.10 log CFU/g) and staphylococci (2.78 log CFU/g) than control cheese (4.86, 4.89, 4.93 and 5.00 log CFU/g of lactobacilli, streptococci, coliforms and staphylococci, respectively) processed without NSLAB addition. The food pathogens Salmonella spp. and Listeria monocytogenes were never detected. The dominance of the species inoculated was demonstrated by denaturing gradient gel electrophoresis (DGGE), whereas strain recognition was evaluated by randomly amplified polymorphic DNA (RAPD)-PCR. From the results obtained, Lb. paracasei NdP78 and S. macedonicus NdP1 were able to persist during the storage of Tosèla cheese and their combination influenced positively the sensory characteristics and shelf-life of the final product.

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The Combined Use of Whole-cell Protein Extracts for the Identification (SDS-PAGE) and Enzyme Activity Screening of Lactic Acid Bacteria Isolated from Traditional Greek Dairy Products

Cited by 71 publications

References 14 publications

Emended Descriptions of Lactobacillus sake (Katagiri, Kitahara, and Fukami) and Lactobacillus curvatus (Abo-Elnaga and Kandler): Numerical Classification Revealed by Protein Figerprinting and Identification Based on Biochemical Patterns and DNA-DNA Hybridizations

Emended Descriptions of Lactobacillus sake (Katagiri, Kitahara, and Fukami) and Lactobacillus curvatus (Abo-Elnaga and Kandler): Numerical Classification Revealed by Protein Figerprinting and Identification Based on Biochemical Patterns and DNA-DNA Hybridizations

Article

Extension of Tosèla cheese shelf-life using non-starter lactic acid bacteria

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