The microbiology of Ghanaian cocoa fermentations analysed using culture-dependent and culture-independent methods D.S. Nielsen a, ⁎ , O.D. Teniola b , L. Ban-Koffi c , M. Owusu d , T.S. Andersson a , W.H. Holzapfel e a Department of Food Science, Food Microbiology, Center for Advanced Food Studies (LMC), Royal Veterinary and Agricultural University, Frederiksberg, Denmark b Federal Institute of Industrial Research, Oshodi, Lagos, Nigeria c Centre National de Recherche Agronomique, Abidjan, Cote d'Ivoire d CSIR-Food Research Institute, Accra, Ghana e Institute für Hygeiene und Toxikologie, Bundesforschungsanstalt für Ernährung und Lebensmittel, Karlsruhe, Germany Received 27 March 2006; received in revised form 14 July 2006; accepted 15 September 2006 Abstract Export of cocoa beans is of great economic importance in Ghana and several other tropical countries. Raw cocoa has an astringent unpleasant taste and a spontaneous fermentation is the first step in a process leading to cocoa beans with the characteristic cocoa flavour and taste. The microbiology of Ghanaian cocoa fermentations was investigated using culture-dependent and culture-independent methods. Samples were collected at 12 hour intervals during 96–144 hour tray and traditional heap fermentations. Yeast, Lactic Acid Bacteria (LAB), Acetic Acid Bacteria (AAB) and Bacillus spp. were enumerated on suitable substrates and identified using phenotypic and molecular methods. The yeast and bacterial micro-populations involved in the cocoa fermentation were further investigated using the culture-independent method Denaturing Gradient Gel Electrophopresis (DGGE). A microbiological succession was observed during the fermentations. At the onset of fermentation yeasts were the dominating microorganisms. Lactic Acid Bacteria became dominant after 12–24 h of fermentation and remained predominant throughout the fermentations with AAB reaching high counts in the mid phase of fermentation. Bacillus spp. were only detected during heap fermentations where they reached high numbers during the later stages of fermentation. Hanseniaspora guilliermondii was the predominant yeast during the initial phase and Pichia membranifaciens during the later phases of fermentation. A number of other yeast species including three putatively undescribed species were isolated during the fermentations. Lactobacillus fermentum was the dominant LAB in most samples. Several other LAB including Lactobacillus plantarum, Leu- conostoc pseudomesenteroides, Leuconostoc pseudoficulneum, Pediocococcus acidilactici and a putatively undescribed LAB species were detected during the fermentations. Acetobacter syzygii, Acetobacter pasteurianus and Acetobacter tropicalis were the predominant AAB in all investigated fermentations. During the later stages of heap fermentation Bacillus licheniformis and occasionally other Bacillus spp. were detected in high numbers. In general the culture-based findings were confirmed using DGGE. However, DGGE indicated that Lc. pseudoficulneum plays a more important role during the fermentation of cocoa than expected from the culture-based findings as it yielded a strong band in most DGGE fingerprints. Cluster analysis of the DGGE fingerprints revealed that the DGGE fingerprints clustered according to fermentation site. Within each fermentation site the profiles clustered according to fermentation time. The DGGE method seems to offer a relatively fast and reliable tool for studying yeast and bacterial dynamics during cocoa fermentations. © 2006 Elsevier B.V. All rights reserved. Keywords: Cocoa; Yeast; Lactic Acid Bacteria; Acetic Acid Bacteria; Bacillus; DGGE 1. Introduction Ghana is the world's second largest producer of cocoa beans producing approximately 20% of the world's cocoa (Anon, 2005). Being the largest export commodity cocoa is of great International Journal of Food Microbiology 114 (2007) 168 – 186 www.elsevier.com/locate/ijfoodmicro ⁎ Corresponding author. Department of Food Science, Royal Veterinary and Agricultural University, Rolighedsvej 30, 1958 Frederiksberg C, Denmark. Tel.: +45 35 28 32 87. E-mail address: dn@kvl.dk (D.S. Nielsen). 0168-1605/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2006.09.010