Raw Cocoa (Theobroma cacao L.) Quality Parameters - with special Reference to West Africa. Dissertation submitted for the award of the Doctorate Degree -Dr. rer. nat.- at the Biology Department, Faculty of Mathematics, Informatics and Natural Sciences, University of Hamburg Presented by Isaac Yaw Berbiye, From Ghana Hamburg, 2014 2 "Nine out of ten people like chocolate. The tenth person always lies." - John Q. Tullius This work is dedicated to all the Berbiye, Boachie and Asemiah-Yeboah Families 3 4 Table of Contents General Summary ............................................................................................................................... 8 Zusammenfassung.............................................................................................................................. 9 1. Introduction .................................................................................................................................. 10 2. Background information .............................................................................................................. 12 2.1 The cacao tree (Theobroma cacao L.) ........................................................................................ 12 2.1.1 History and origin .................................................................................................................... 12 2.1.2 Classification and types of cacao ............................................................................................ 13 2.2 Cacao cultivation: ecology .......................................................................................................... 14 2.3 Flowering, fertilization and pod development ........................................................................... 15 2.4 Harvesting .................................................................................................................................. 16 2.5 Cacao diseases and pests ........................................................................................................... 16 2.6 Processing and utilization of cacao ............................................................................................ 18 2.6.1 Fermentation ........................................................................................................................... 18 2.6.2 Drying, transport and storage ................................................................................................. 20 2.6.3 Roasting ................................................................................................................................... 21 2.7 Heterogeneity of raw cocoa ....................................................................................................... 22 3. Materials and methods ................................................................................................................ 25 3.1 Plant materials ........................................................................................................................... 25 3.1.1 Description of plant materials used in the biochemical analysis ............................................ 25 3.1.2 Description of plant materials used in the fermentation-like incubation of fresh cocoa bean seeds................................................................................................................................................. 25 3.2 Methods ..................................................................................................................................... 25 3.2.1 Defatting of raw cocoa bean samples ..................................................................................... 26 3.2.2 pH determination .................................................................................................................... 26 3.2.3 Antioxidative potential determination ................................................................................... 26 3.2.4 Phenolic compounds determination ....................................................................................... 27 3.2.4.1 Determination and extraction of polyphenols and anthocyanins using the RP-HPLC ......... 27 3.2.4.2 Determination of total phenolic substances using the Folin-Ciocalteus phenol reagent method .............................................................................................................................................. 29 3.2.5 Determination of the amount of acetic and lactic acids ......................................................... 30 3.2.6 Determination of the amount of methylxanthines (theobromine and caffeine) ................... 32 3.2.7 Determination of sugar contents ............................................................................................ 33 3.2.8 Determination of the free amino acids contents by means of HPLC ...................................... 34 3.2.9 Preparation of acetone-dried powder .................................................................................... 36 5 3.2.10 Measurement and detection of oligopeptide linkages ........................................................... 36 3.2.11 Fermentation index value ..................................................................................................... 37 3.2.12 Fermentation-like incubation of raw cocoa bean seeds ....................................................... 38 3.2.13 Characterization of individual protein by separation on the SDS-polyacrylamid-gel electrophoresis (SDS-PAGE) ............................................................................................................. 39 3.2.13.1 Preparation of the samples for gel electrophoresis ........................................................... 42 3.2.13.2 Staining of polyacrylamide gels with Coomassie Brilliant Blue R 250 ................................ 42 4. Results .......................................................................................................................................... 43 4.1 Quality assessment tests of raw cocoa seeds ............................................................................ 43 4.1.1 Sample structure of raw cocoa bean seeds ............................................................................ 43 4.1.2 Differences in the sample structure of raw cocoa bean samples ........................................... 44 4.2 Interpretation of quality parameters of different raw cocoa bean samples ............................. 45 4.2.1 Phenolic compounds ............................................................................................................... 45 4.2.1.1 Total phenolic compounds ................................................................................................... 45 4.2.1.2 Epicatechin content.............................................................................................................. 47 4.2.1.3 Catechin content .................................................................................................................. 49 4.2.2 Antioxidative capacity ............................................................................................................. 51 4.2.3 Methylxanthines ..................................................................................................................... 53 4.2.3.1 Theobromine content .......................................................................................................... 53 4.2.3.2 Caffeine content ................................................................................................................. 55 4.2.3.3 Ratio of theobromine to caffeine (T/C) value ...................................................................... 57 4.2.4 Moisture content.................................................................................................................... 59 4.2.5 Fermentation index value ....................................................................................................... 61 4.2.6 pH value .................................................................................................................................. 63 4.2.7 Acetic acid content .................................................................................................................. 65 4.2.8 Lactic acid content .................................................................................................................. 67 4.2.9 Free amino acid content ........................................................................................................ 69 4.2.9.1 Individual amounts of free amino acids ............................................................................... 71 4.2.9.2 Classification of free amino acids ......................................................................................... 72 4.2.9.3 Group 1: Hydrophobic amino acids-alanine, leucine, phenylalanine, tyrosin, valine and isoleucine. ......................................................................................................................................... 77 4.2.9.4 Group 2: Acidic free amino acids-asparagine, aspartic acid, glutamine, glutamic acid and histidine. ........................................................................................................................................... 80 4.2.9.5 Group 3: Other free amino acids-tryptophan, lysine, serine, glycine, arginine and threonine. .........................................................................................................................................................