ORIGINAL ARTICLE Bacillus amyloliquefaciens ssp. plantarum strains as potential protective starter cultures for the production of Bikalga, an alkaline fermented food C.S. Compaor e 1,2,3 , D.S. Nielsen 2 , H. Sawadogo-Lingani 1 , T.S. Berner 2 , K.F. Nielsen 4 , D.B. Adimpong 2 , B. Diawara 1 , G.A. Ou edraogo 3 , M. Jakobsen 2 and L. Thorsen 2 1D epartement Technologie Alimentaire (DTA/IRSAT/CNRST), Ouagadougou, Burkina Faso 2 Department of Food Science, Faculty of Science, University of Copenhagen, Frederiksberg C, Denmark 3 Institut du D eveloppement Rural/Universit e Polytechnique de Bobo, BAMSB, Bobo-Dioulasso, Burkina Faso 4 DTU Systems Biology, Department of Systems Biology, Center for Microbial Biotechnology, Technical University of Denmark, Lyngby, Denmark Keywords antimicrobial activity, Bacillus amyloliquefaciens ssp. plantarum, bikalga, lipopeptides, polyketides, starter cultures. Correspondence Clarisse S. Compaor e, D epartement Technologie Alimentaire (DTA/IRSAT/CNRST), Ouagadougou 03 BP 7047, Burkina Faso. E-mail: compaclara@yahoo.fr 2013/2241: received 15 December 2012, revised 4 March 2013 and accepted 2 April 2013 doi:10.1111/jam.12214 Abstract Aims: To identify and screen dominant Bacillus spp. strains isolated from Bikalga, fermented seeds of Hibiscus sabdariffa for their antimicrobial activities in brain heart infusion (BHI) medium and in a H. sabdariffa seed-based medium. Further, to characterize the antimicrobial substances produced. Methods and Results: The strains were identified by gyrB gene sequencing and phenotypic tests as B. amyloliquefaciens ssp. plantarum. Their antimicrobial activity was determined by the agar spot and well assay, being inhibitory to a wide range of Gram-positive and Gram-negative pathogenic bacteria and fungi. Antimicrobial activity against Bacillus cereus was produced in H. sabdariffa seed-based medium. PCR results revealed that the isolates have potential for the lipopeptides iturin, fengycin, surfactin, the polyketides difficidin, macrolactin, bacillaene and the dipeptide bacilysin production. Ultra-high- performance liquid chromatography-time of flight mass spectrometry analysis of antimicrobial substance produced in BHI broth allowed identification of iturin, fengycin and surfactin. Conclusions: The Bacillus amyloliquefaciens ssp. plantarum exhibited broad- spectrum antifungal and antibacterial properties. They produced several lipopeptide antibiotics and showed good potential for biological control of Bikalga. Significance and Impact of the Study: Pathogenic bacteria often occur in spontaneous food fermentations. This is the first report to identify indigenous B. amyloliquefaciens ssp. plantarum strains as potential protective starter cultures for safeguarding Bikalga. Introduction There is an increased interest in searching for bacteria with new antimicrobial properties that can be used as: (i) protective starter cultures for foods; (ii) as new probiotics or as producers of alternatives to existing antibiotics; (iii) against antibiotic resistant pathogenic bacteria (Guo et al. 2012). Bikalga is a traditional food condiment obtained by a spontaneous and alkaline fermentation of the plant H. sabdariffa seeds. It is widely used in Burkina Faso as a flavouring agent in soups and sauces and constitutes an affordable source of proteins (22–30%), lipids, carbohy- drates, essential amino, fatty acids and vitamins (Bengaly et al. 2006). Bacillus subtilis group species are the main micro-organisms involved in the fermentation of H. sab- dariffa seeds into Bikalga (Ouoba et al. 2008a). The spon- taneous nature of the fermentation processes sometimes results in the occurrence of pathogenic and spoilage micro-organisms, leading to products that are unsafe and of varying quality (Ouoba et al. 2008b; Agbobatinkpo Journal of Applied Microbiology 115, 133--146 © 2013 The Society for Applied Microbiology 133 Journal of Applied Microbiology ISSN 1364-5072