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Occurrence, Isolation and DNA Identification of Streptococcus Thermophilus Involved in Algerian Traditional Butter 'Smen'

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Occurrence, Isolation and DNA Identification of Streptococcus Thermophilus Involved in Algerian Traditional Butter 'Smen' Occurrence, isolation and DNA identification of streptococcus thermophilus involved in algerian traditional butter ’Smen’ Farid Bensalah, Asmaa Labtar, Christine Delorme, Pierre Renault To cite this version: Farid Bensalah, Asmaa Labtar, Christine Delorme, Pierre Renault. Occurrence, isolation and DNA identification of streptococcus thermophilus involved in algerian traditional butter ’Smen’. African Journal of Biotechnology, Academic Journals, 2011, 10 (75), pp.17251 - 17257. 10.5897/AJB11.2462. hal-01000016 HAL Id: hal-01000016 https://hal.archives-ouvertes.fr/hal-01000016 Submitted on 29 May 2020 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. African Journal of Biotechnology Vol. 10(75), pp. 17251-17257, 28 November, 2011 Available online at http://www.academicjournals.org/AJB DOI: 10.5897/AJB11.2462 ISSN 1684–5315 © 2011 Academic Journals Full Length Research Paper Occurrence, isolation and DNA identification of Streptococcus thermophilus involved in Algerian traditional butter ‘Smen’ Farid Bensalah 1*, Asmaa Labtar 1, Christine Delorme 2 and Pierre Renault 2 1Faculté des Sciences, Département de Biologie, LGM (Laboratoire de Génétique Microbienne), Université Es-Sénia, Oran 31000, Algérie. 2INRA-CRJ, Institut National de la Recherche Agronomique, Micalis Institute, Département de Microbiologie et Chaîne Alimentaire. 78352 Jouy-En- Josas, cedex, France. Accepted 19 October, 2011 Streptococcus thermophilus isolates from traditional butter ‘Smen’, a fermented product from cow’s and ewe’s milk in arid area was subjected to taxonomical investigations. The identification procedure included phenotypic approaches, molecular characterization by using genus polymerase chain reaction (PCR) amplifications for sodA gene encoding the manganese-dependant superoxide dismutase A, and species-specific primers from gene encoding glucose kinase (glcK ), gene encoding DNA polymerase III (dnaE ) and gene encoding threonyl-tRNA synthetase (thrS ) housekeeping genes in order to distinguish among reference, and wild strains of S. thermophilus and for their differentiation from Enterococcus spp. A total of 12 strains were tested by DNA identification analysis and these indigenous isolates were unambiguously characterised by their housekeeping gene profiles. Finally, four genotypes were recognised. To our knowledge, this is the first report on the isolation and molecular characterization of S. thermophilus strains from fermented milk transformed into home-production butter provided from Southern West Algeria. These results are of particular interest as they favour the selection of future cultures starters as S. thermophilus from traditional dairy products. It is, therefore, of great importance to preserve the genetic pool of the wild strains. Key words: Streptococcus thermophilus , traditional butter, PCR, Enterococcus , DNA, species-specific primer. INTRODUCTION The microbial composition of traditional dairy products ‘Smen’ is a traditional butter, which, so far has not been and, particularly, of European artisanal cow’s and ewe’s investigated for microbiological genotypic strains fermented milk and cheeses, have been subjected to characterization. The nature of this fermented product is microbiological investigation at genus and species level different from one region to another and depends on the (Tornadjio et al., 1995; Cogan et al., 1997; Mannu et al., local indigenous microflora; which in turn reflects the 2000; Mannu and Paba, 2002; Alegría et al., 2009; climatic conditions of the area. However, there is very Secchi et al., 2011). Many indigenous fermented foods little information on strain composition within species, and from Algeria are based on lactic fermentation from cow’s on the specific contribution of each wild strain to the lactic and ewe’s milk (Bensalah et al., 2009). Among these, fermentation. Moreover, the study of strain heterogeneity in African traditional butter (El-Baradei et al., 2008; Ongol and Asano, 2009), and cheese starters is believed to be of great importance to the dairy industry (Girrafa et al., *Corresponding author. E-mail: [email protected]. 1998). Abbreviation: PCR , Polymerase chain reaction; glcK , gene Fermented milk ‘leben’ or 'rayeb' (Elotmani et al., 2002; encoding glucose kinase; dnaE , gene encoding DNA Hegazi, 1988; Bensalah et al., 2009) can be eaten fresh polymerase III; thrS , gene encoding threonyl-tRNA synthetase. or suffers a churning and creaming in goatskin called 17252 Afr. J. Biotechnol. ‘Chekoua’. Skimming is usually done in the morning; intergenic sequences 16S-23S rRNA (Tissala-Timisjarvi gourd is half filled with milk fermented, then strained by and Alatossava, 1997). swelling, and then the ‘Chekoua’ is tied and shaken Moreover, since little is known about the involvement of vigorously for half an hour. The formation of fat globules thermophilic cocci with Algerian artisanal foods, this study is considered by the sound change that occurs within the was aimed at characterizing phenotypically and geno- skin. To assist the agglomeration of particles of butter, typically, the streptococci associated with the traditional water is usually added, hot or cold depending on the fermented cow’s and ewe’s raw milk transformed into temperature of milk. The fresh butter is removed home-production butter provided from southern west manually in a single lump called ‘Zebda’ which is trans- Algeria. We applied the developed methodology of genus formed into ‘Smen’ by washing with salt water. ‘Smen’ and species-specific primers from sodA , glcK , dnaE and has a very strong smell, easily recognizable during thrS housekeeping genes in order to distinguish among ripening. Implication of Streptococcus thermophilus in reference, and wild strains of S. thermophilus and for Egyptian artisanal butter ‘zabady’ was reported recently their differentiation from Enterococcus spp. (El-Baradei et al., 2008). This strain is an essential micro- organism in the manufacture of many types of fermented dairy products, occurring in natural as well as commercial MATERIALS AND METHODS starter cultures (Coppola et al., 1998; Delorme, 2008), and the increasing number of commercial strains Sampling and bacterial strains available used as starters requires rapid and reliable Six samples of cow’s and ewe’s traditional butter ‘Smen’ were methods to accurately differentiate strains at both species obtained from individual households in rural arid area. Samples and strain levels in pure and mixed cultures in order to were collected in sterile small bottles and stored in Laboratoire de protect patent rights and eliminate risks of use confusion Génétique Microbienne, Es-Sénia, Algeria (LGM) under (Ramos and Harlander, 1990; Moschetti et al., 1998). refrigeration at 4°C until use. Reference strains representing four Enterococcus Gram-positive species were used in this study and were provided strains can harbour specific biochemical from INRA collection (Jouy-En-Josas, France). These included S. traits that are essential in the manufacture of various thermophilus CNRZ 1066, Enterococcus faecalis JH2-2, fermented milk products, and some strains are Enterococcus faecium 64/3 and Corynebacterium glutamicum technologically exploited as functional starters or ATCC 13032. probiotics (Giraffa et al., 1997; Franz et al., 1999; Bensalah et al., 2006). Enterococci has also been implicated in the spoilage of processed meats (Franz and Isolation and phenotypic characterization of selected isolates Holy, 1996) and include strains that have been recog- Serials dilutions of homogenized 10 g of ‘Smen’ samples in 90 ml nized as emerging human pathogens mostly in sterile solution saline 0.85% NaCl were used for microbial isolation nosocomial urinary tract infections and bacteremias with M17 agar (Terzaghi and Sandine, 1975) containing 1% lactose (Giraffa et al., 2000; Martinez and Baquero, 2002). (LM17). Plates were incubated anaerobically at 37, 45 or 50°C for In the last few years, the development of new methods 48 to 72 h for isolation of thermophilic streptococci. Identification involving various DNA-based typing techniques (Farber, was carried out according to previously published characterization 1996), widely used in epidemiological studies for the schemes (Devriese et al., 1993; Moschetti et al., 1998; Muller et al., 2001; Michaylova et al., 2007). Gram staining, catalase activity, identification of medically important bacterial strains ammonia production from arginine and gas production from glucose (Kuhn et al., 1995; Descheemaeker et al., 1997), has were determined. Tests for growth on Slanetz-Bartley (SB) medium, opened up new perspectives for typing strains from raw in M17 broth at 10°C, pH value adjusted to 9.6 and with NaCl 6.5% milk and from traditional cheeses (Bouton et al., 1998; were performed for all the isolates. Colonies morphology were Desmasures et al., 1998). It was previously reported that determined for overnight cultures by using phase-contrast a genus-specific PCR assay based on the use of microscopy and all isolates were stored
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