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Isolation and Characterisation of New Spore-Forming Lactic Acid Bacteria with Prospects of Use in Food Fermentations and Probiotic Preparations

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Isolation and Characterisation of New Spore-Forming Lactic Acid Bacteria with Prospects of Use in Food Fermentations and Probiotic Preparations African Journal of Microbiology Research Vol. 4 (11), pp. 1016-1025, 4 June, 2010 Available online http://www.academicjournals.org/ajmr ISSN 1996-0808 © 2010 Academic Journals Full Length Research Paper Isolation and characterisation of new spore-forming lactic acid bacteria with prospects of use in food fermentations and probiotic preparations Ali Bayane1*, Bréhima Diawara2, Robin Dauphin Dubois3, Jacqueline Destain3, Dominique Roblain3 and Philippe Thonart3 1 Biotechnology Research Institute, National Council of Research Canada, 6100 Royalmount, Montréal, Québec H4P 2R2 Canada. 2Département de Technologie Alimentaire, IRSAT/CNRST. 03 BP 7047 Ouagadougou 03 Burkina Faso. 3FUSAGx, Unité de Bio-industrie, Centre Wallon de Biologie Industrielle (CWBI), Passage des Déportés 2, 5030 Gembloux, Belgique. Accepted 25 January, 2010 Five spore-forming bacteria producer of lactic acid were isolated from soils sampled in the vicinity of poultry farms in Burkina Faso. All isolates were Gram-positive, motile, mesophilic, facultative anaerobic, catalase positive rods, and with L(+) lactic acid production. The isolates have been characterized and identified by a polyphasic approach, combining various phenotypic and genetic characteristics. The 16S-rDNA-sequence analyses revealed the membership of two isolates to the genus Bacillus and the three other to the genus Paenibacillus. The physiological and biochemical analyses showed that the isolates were quite different from known spore forming lactic acid bacteria. Several relevant technological properties were observed, particularly the resistance of the isolates to bile salts and acidic conditions, even the productions of amylolytic and proteolytic enzymes, which could make them good candidates for certain technological applications such as food fermentations and probiotic formulations. Furthermore, the isolation of these microorganisms in the vicinity of farms reinforces the feasibility of their involvement in animal feedstuffs preparations. In conclusion, this work shows an important diversity within the spore-forming lactic acid bacteria and confirms the conclusions of previous works, which have already shown that the SFLAB (Spore Forming Lactic Acid Bacteria) were good candidates for food fermentation and the probiotic formulations. Key words: Lactic acid, spore-forming bacteria, biodiversity, bacillus, 16S rDNA. INTRODUCTION Lactic acid bacteria are the most used microbial groups in eria are exploited in vitro for food preservation (Mellefont fermentation industries (Harlander, 1989). Lactic acid and Ross, 2007) and in vivo through probiotic formu- bacteria are involved in several aspects of human life, lations (Saulnier et al., 2009). In the lactic acid bacteria from food processing to health care (Mombelli and group the genus Lactobacillus is the best known and the Gismondo, 2000; Coleman et al., 2003). The anti- most used in food fermentations (Collins et al, 1998; bacterial properties of lactic acid produced by this bact- Gomez and Malcata, 1999; Kandler and Weiss, 1986). However, the majority of the species belonging to this genus have specific nutritional needs (Elvira et al., 2004) and lower resistance from the environmental conditions *Corresponding author. E-mail: [email protected]. using in biomass production at industrial scale, that is, Tel: +1/514-283-7212. Fax: +1/514-496-6265. drying and storage processes (Zamora et al., 2006; Kets et al., 1996; Hood and Zoitola, 1988). On the other hand, Abbreviations: SFLAB, Spore forming lactic acid bacteria. their use in vivo is often affected by the physico-chemical Bayane et al. 1017 TM TM properties of the digestive tract (Buckenhüskes, 1993). then incubated under anaerobic condition (BBL GasPak , H2 + This situation leads regularly the industries to develop in CO2) at 30°C for two days. After incubation, 100 µl of the turbid collaboration with the researchers new programmes in broth was spread onto GYP Agar plates prepared with 0,005% (w/v) bromocresol purple. The plates were incubated under aerobic order to improve the functional properties and main- condition at 30°C for two days. Thus, all acid-producing bacteria tenance condition of the strain already used or to find were recognized by the appearance of yellow zones around colo- new techniques of production, which can be less restrict- nies. The acidifying bacteria were isolated and purified by repeated tive or finally to isolate new bacteria, which could better isolation and the purity was checked by microscope examination. resist to the conditions of production, storage and formu- All positive isolates for the screening criteria were maintained as frozen stocks at -80°C in the presence of 30% glycerol as lation without losing their functional properties (Berner cryoprotective agent. and O' Donnell, 1998; Klein et al., 1998; Schillinger, 1999). Within this framework our work aims at isolating new acidifying bacteria producing lactic acid selected Identification and phenotypical characterization from extreme environmental conditions by the means of their ability to sporulate (Sow et al., 2005). The principal Cell morphology was examined by phase-contrast microscopy. Gram determination was performed using the KOH method, spore-forming lactic acid bacteria (SFLAB) are according to Gregersen (1978). All growth tests were performed in recognized for their ability to produce lactic acid, while a modified GYP medium described by Bayane et al. (2006). All they can also produce endospores. The most known cultures were performed at 30°C according to general methods of SFLAB belong to the genera Bacillus, including B. bacteriology (Gerhardt, 1994), including the examination of coagulans, B. racemilacticus et B. laevolacticus, and tolerance to different NaCl concentrations. Catalase production test Sporolactobacillus, including S. inulinus, S. racemicus, S. were determined by transferring fresh colonies from Agar to a slide glass and adding 5% H2O2 reagent. Cell motility was detected by lactosus, etc (Yanagida et al., 1997; Cavazzoni and cultivation in a semi-solid medium. Production of gas from glucose Adami, 1993; Nakayama and Yanoshi, 1967a). Beside was assayed in roll tubes supplemented with Durham tubes in GYP the Lactobacillus species, the SFLAB (Bacillus and broth. The quantity and the enantiomeric of lactic acid produced, Sporolactobacillus) have the advantage to have lower was determined by enzymatic tests (Roche Molecular Biochemi- nutritional requirements (Ohara and Yahata, 1996; cals, Boehringer Mannheim GmbH, Germany). The ability of the isolates to produce acid from various carbon sources was deter- Holzapfel and Botha, 1988), a good resistance to mined by API 50 CHB test (BioMérieux sa, Lyon, Marcy l’étoile, different environmental stress (Hyronimus et al., 2000). In France) according to manufacturer’s instructions. addition the SFLAB can provide the major required functional properties wanted among species of the genus Lactobacillus (Cavazzoni and Adami, 1993). Hence, the Determination of the technological properties of the isolates SFLAB are good candidates for industrial using, both in The ability of the isolates to produce technological enzymes, lactic acid production and probiotic preparations. Why it is including lipases, proteases and amylases were examined on better to know that certain SFLAB, such as B. coagulans modified GYP agar according to Larpent and Larpent (1985). The and Sp. inulinus are already used as probiotic for human tolerance to acidic conditions was tested in both sterile phosphate and animal nutrition (Cavazzoni and Adami., 1993; buffer saline and GYP broth according to Hyronimus et al. (2000). Hyronimus et al., 2000). Researches presented here The tolerance to bile salts was performed on GYP agar medium were aimed at one part of the isolation and character- containing oxgall bile (0.1 - 1% w/v) (Sigma Chemical Co., St Louis, MO, USA) according to Hyronimus et al. (2000). The minimal inhi- rization of new SFLABs and on the other hand to bitory concentration (MIC) of bile salts for a strain was determined highlight the technological properties of the new isolates as the lowest concentration totally inhibiting the growth of cells as with a view on their using as probiotics. judged from visual examination of the agar plates. MATERIALS AND METHODS Total cellular fatty acids analysis Samples enrichment and isolation procedures Isolates were grown on tryptone soya broth medium for fatty acid extraction. Fatty acid methyl esters (FAME) were obtained from wet Soil samples were collected in the vicinity of poultry farm in Burkina biomass (approx. 40 mg) by saponification, methylation and Faso with sterile spoons, and kept into clean bags. Six soil samples extraction as described by Osterhout et al. (1991). FAME mixtures were collected in three farms i.e., two samples by farm. The were separated using a model 5898A microbial identification sys- microbial enrichment and bacterial isolation were performed on the tem (Microbial ID, Newark, DE, USA) which consisted of a Hewlett- GYP (Glucose-Yeast-Peptone) medium described by Nakayama Packard model 3392 gas chromatograph fitted with a 5% phenyl- and Yanoshi (1967a). The GYP medium consists of 1% (w/v) glu- methyl silicone capillary column (0.2 mm x 25 m), a flame ionization cose, 1% (w/v) yeast extract, 1% (w/v) peptone, 0.5% (w/v) CaCO3 detector, a Hewlett-Packard model 3392 integrator, Hewlett- and 2,5% (v/v) mineral solution. The medium was adjusted to pH Packard model 7673A automatic sampler
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