In vitro fermentation studies for selection and evaluation of Bacillus strains as starter cultures for the production of okpehe, a traditional African fermented condiment Folarin A. Oguntoyinbo a,c , Abiodun I. Sanni b , Charles M.A.P. Franz c , Wilhelm H. Holzapfel c, ⁎ a Department of Botany and Microbiology, Faculty of Science, University of Lagos, Akoka, Lagos, Nigeria b Department of Botany and Microbiology, Faculty of Science, University of Ibadan, Nigeria c Federal Research Centre for Nutrition and Food, Institute for Hygiene and Toxicology, Haid-und-Neu-Strasse 9, D-76131 Karlsruhe, Germany Received 5 June 2005; received in revised form 3 May 2006; accepted 3 July 2006 Abstract Selected Bacillus and Enterococcus strains, isolated from traditional okpehe fermentations, were studied for their suitability as starter cultures in laboratory-scale fermentations of Prosopis africana seeds for the production of okpehe, a traditional fermented vegetable product of Nigeria. The strains were selected on the basis of highest proteolytic activity, as determined with the APIZYM (BioMerieux) test. The choice of starter strains was narrowed to Bacillus subtilis strains BFE 5301 and BFE 5372. These were determined as the best starter combination because of rapid growth, high amylolytic and proteolytic activities, high levels of polyglutamic acid production by strain BFE 5372, as well as bacteriocin production by strain BFE 5301. Other mixed culture fermentations did not yield sensorically acceptable products. Although a monoculture fermentation, using only B. subtilis strain BFE 5372, produced okpehe with very good sensory characteristics, the growth of B. cereus could be detected after 48 h fermentation, indicating that this starter did not sufficiently contribute to product safety. Mixed culture fermentation with the combination of bacteriocin-producing starter B. subtilis BFE 5301 and the non-bacteriocin-producing B. subtilis BFE 5372, produced a product with good sensory characteristics, in which growth of B. cereus was delayed. The bacteriocin produced by B. subtilis strain BFE 5301 was identified as subtilisin, using subtilisin-specific primers and PCR amplification of the subtilisin gene. The bacteriocin was heat-stable at 100 °C for 10 min and exhibited highest activity at pH values lower or equal to pH 6.0. The bacteriocin was sensitive to the proteolytic enzymes trypsin and α-chymotrypsin at concentrations of 10 mg/ml. © 2006 Elsevier B.V. All rights reserved. Keywords: Bacillus subtilis; Okpehe; Fermentation; Starters; Subtilisin 1. Introduction Okpehe is a traditional fermented soup condiment produced from Prosopis africana seeds (Guill, Perr) Taub. (Tchongdjeu et al., 1998; Sanni and Onilude, 1999). The condiment serves as a tasty, low cost protein source. P. africana seeds are inedible for humans and animals, but they can be consumed when processed and fermented. This results in hydrolysis of proteins and oligosaccharides, which is based on the metabolic activities of the fermenting microorganisms. For traditional fermentation of okpehe, seeds of P. africana are boiled 15–24 h till soft, manually dehulled by pressing them between the palms of the hands, after which the cotyledons are washed and boiled again for 3–5 h. These are then spread in wide calabash trays or earthenware pots already lined with pawpaw leaves, and the trays are then tightly wrapped with jute bags. Fermentation takes place within 3–5 days at temperatures of 30–35 °C am- bient temperature, resulting in a sticky, dark brown and pungent-smelling mash. The condiment is added generously to soups in many homes as a flavouring agent, but many urban and rural people also use it as a meat substitute on some occasions (Sanni, 1993; Obeta and Ugwuanyi, 1996). Some International Journal of Food Microbiology 113 (2007) 208 – 218 www.elsevier.com/locate/ijfoodmicro ⁎ Corresponding author. Tel.: +49 721 6625 450; fax: +49 721 6625 453. E-mail address: Wilhelm.Holzapfel@bfe.uni-karlsruhe.de (W.H.Holzapfel). 0168-1605/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2006.07.006