Microbiota of cocoa powder with particular reference to aerobic thermoresistant spore-formers Lídia J.R. Lima a , Henri J. Kamphuis b , M.J. Rob Nout a, * , Marcel H. Zwietering a a Laboratory of Food Microbiology, Wageningen University, P.O. box 8129, 6700 EV Wageningen, The Netherlands b Cargill Cocoa & Chocolate, P.O. Box 82,1530 AB Wormer, The Netherlands article info Article history: Received 17 September 2010 Received in revised form 17 November 2010 Accepted 18 November 2010 Available online 24 November 2010 Keywords: Cocoa Bacillus subtilis Salmonella Spore heat-resistance Thermotolerance 16S rDNA phylogeny Amplified fragment length polymorphism abstract The microbiological criteria of commercial cocoa powder are defined in guidelines instituted by the cocoa industry. Twenty-five commercial samples were collected with the aim of assessing the compli- ance with the microbiological quality guidelines and investigating the occurrence and properties of aerobic Thermoresistant Spores (ThrS). Seventeen samples complied with the guidelines, but one was positive for Salmonella, five for Enterobacteriaceae and two had mould levels just exceeding the maximum admissible level. The treatment of the cocoa powder suspensions from 100  C to 170  C for 10 min, revealed the presence of ThrS in 36% of the samples. In total 61 ThrS strains were isolated, of which the majority belonged to the Bacillus subtilis complex (65.6%). Strains resporulation and spore crops inactivation at 110  C for 5 min showed a wide diversity of heat- resistance capacities. Amplified fragment length polymorphism analysis revealed not only a large intraspecies diversity, but also different clusters of heat-resistant spore-forming strains. The heat- resistance of spores of six B. subtilis complex strains was further examined by determination of their D and z-values. We concluded that B. subtilis complex spores, in particular those from strain M112, were the most heat-resistant and these may survive subsequent preservation treatments, being potentially problematic in food products, such as chocolate milk. Ó 2010 Elsevier Ltd. All rights reserved. 1. Introduction The processing chain of cocoa beans (Theobroma cacao L. fruit seeds) for cocoa powder production starts in tropical countries, where farmers, after harvesting the cocoa pods, submit the beans to a process of natural fermentation, the “sine qua non” for the distinctive cocoa flavour development ensued by roasting (Lima et al., in press; Schwan and Wheals, 2004). Following the fermen- tation, the cocoa beans are dried and shipped to industrial plants, where intermediate or final products are manufactured. For the production of cocoa powder, cocoa beans from different origins are blended, roasted and processed into a mass, which is subsequently partially defatted to minimum levels of 20% or between 10 and 20% (dry weight matter) and pulverised (Anon., 1981; Kamphuis, 2009). Prior to roasting, alkalisation of cocoa beans is generally applied to improve organoleptic and technological attributes of cocoa powder (Kamphuis, 2009). Whereas diverse microorganisms are present at high numbers during cocoa beans fermentation, subsequent post-harvest and industrial processing operations allow only the survival of a micro- biota dominated by the genus Bacillus and relatives (Barrile et al., 1971). The ability of members of this group to form endospores, in some cases of extreme heat-resistance, implies that they may survive industrial processes and pose spoilage and safety problems (Huemer et al., 1998; Oh and Cox, 2009; Oomes et al., 2007). The general microbiological composition of commercial cocoa powder was investigated previously, revealing total aerobic micro- bial levels between 2 and 4.4 log cfu/g, with Bacillus licheniformis, Bacillus cereus, Bacillus megaterium and Bacillus subtilis constituting, respectively, 45, 20, 10 and 8% of the isolates (Gabis et al., 1970). In another study, in addition to total aerobic microorganisms, spores populations surviving heat-treatments of 80  C and 100  C during 1 and 5 min were analysed, and this showed B. subtilis and B. lichen- iformis to represent 83% of the isolates (Mossel et al., 1974). Although the aforementioned reports are of interest in compar- ative studies of cocoa microbiota, they offer less insight into the occurrence and properties of highly heat-resistant spores in cocoa * Corresponding author. Tel.: þ31 317 48 2834; fax: þ31 317 48 4978. E-mail address: rob.nout@wur.nl (M.J.R. Nout). Contents lists available at ScienceDirect Food Microbiology journal homepage: www.elsevier.com/locate/fm 0740-0020/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.fm.2010.11.011 Food Microbiology 28 (2011) 573e582