Investigating the fermentation of cocoa by correlating Denaturing Gradient Gel Electrophoresis profiles and Near Infrared spectra Dennis S. Nielsen ⁎, Pia Snitkjaer, Frans van den Berg Department of Food Science, Faculty of Life Sciences, Centre for Advanced Food Studies (LMC), University of Copenhagen, Denmark ABSTRACT ARTICLE INFO Article history: Received 28 September 2007 Received in revised form 6 March 2008 Accepted 24 March 2008 Keywords: DGGE NIR spectroscopy Multivariate data analysis Cocoa Raw cocoa has an astringent, unpleasant taste and flavour, and has to be fermented, dried and roasted in order to obtain the characteristic cocoa flavour and taste. During the fermentation microbial activity outside the cocoa beans induces biochemical and physical changes inside the beans. The process is complex involving activity of several different groups of microorganisms which bring about numerous biochemical and physical changes inside the beans. Due to the complexity of these processes no thorough investigations of the interactions between the microbial activities on the outside of the beans and the chemical processes inside the beans have been carried out previously. Recently it has been shown that Denaturing Gradient Gel Electrophoresis (DGGE) offers an efficient tool for monitoring the microbiological changes taking place during the fermentation of cocoa. Near Infrared (NIR) spectroscopy has previously been used to determine various components in cocoa beans, offering a rapid alternative compared to traditional analytical methods for obtaining knowledge about changes in the chemical composition of the cocoa beans during fermentation. During a number of cocoa fermentations bean samples were taken with 24 h intervals to be dried and analysed by NIR. Cocoa pulp samples taken simultaneously during the same fermentations have previously been characterised using DGGE [Nielsen, D.S., Teniola, O.D., Ban-Koffi, L., Owusu, M., Andersson, T., Holzapfel, W.H. (2007). The microbiology of Ghanaian cocoa fermentations analysed using culture dependent and culture-independent methods. International Journal of Food Microbiology 114,168–186.]. Here we report the first study where microbiological changes during the fermentation determined using DGGE are correlated to changes inside the beans determined by NIR using multivariate data analysis. Following data pre-processing (baseline correction followed by Co-shift correction or Correlation Optimised Warping) the DGGE spectra were analysed using Principal Component Analysis (PCA). A clear grouping according to fermentation time was seen demonstrating the microbial succession taking place during the fermentation. Subsequently the DGGE spectra were correlated to the NIR spectra using Partial Least Squares regression models (PLS2). Correlations of 0.87 (bacterial derived DGGE spectra) and 0.81 (yeast derived DGGE spectra) were obtained indicating the relationship between the microbial activities in the pulp and the (bio) chemical changes inside the beans. By comparing the X-block loadings of the PLS2 models and the DGGE spectra it was possible to directly link several microbial species with changes in the NIR spectra and consequently also with changes inside the beans. © 2008 Elsevier B.V. All rights reserved. 1. Introduction Cocoa beans are the principal raw material of chocolate. West Africa produces more than two-third of the World's cocoa with Cote d'Ivorie and Ghana alone accounting for 40 and 20% of the World production, respectively (Anon., 2005). Cocoa beans originate as seeds in fruit pods of the tree Theobroma cacao, where each fruit pod contains 30 to 40 beans embedded in a mucilaginous pulp. Raw cocoa has an astringent, unpleasant taste and flavour, and has to be fermented, dried and roasted in order to obtain the characteristic cocoa flavour and taste (Thompson et al., 2001). The fermentation of cocoa is a spontaneous process. Following opening of the pods the mucilaginous, acidic and sugar rich pulp surrounding the cocoa beans is contaminated with a variety of microorganisms originating from workers hands, containers used for transport, knives, pod surfaces, etc. (Roelofsen, 1958; Thompson et al., 2001; Jespersen et al., 2005). During the fermentation various yeasts, lactic acid bacteria (LAB), acetic acid bacteria (AAB) and possibly Ba- cillus spp. develop in a form of succession carrying out the fermentation (Roelofsen, 1958; Schwan et al., 1995; Thompson et al., International Journal of Food Microbiology 125 (2008) 133–140 ⁎ Corresponding author. Department of Food Science, Faculty of Life Sciences, University of Copenhagen, Rolighedsvej 30,1958 Frederiksberg C, Denmark. Tel.: +45 35 33 32 87; fax: +45 35 33 32 14. E-mail address: dn@life.ku.dk (D.S. Nielsen). 0168-1605/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2008.03.040 Contents lists available at ScienceDirect International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro