The Industrialization of Cocoa Bean Fermentations Hugh Dircks

The Industrialization of Cocoa Bean Fermentations Hugh Dircks and Graham Fleet Food Science Group School of Chemical Sciences and Engineering University of New South Wales Sydney, Australia

Cocoa beans are the raw material of chocolate production

Cocoa Statistics • Chocolate industry : $ 70 billion, globally • Cocoa beans : $ 3000/ tonne • Major producers of cocoa beans • West Africa-- -- 70% • Asia –Pacific--- 20% • South/Central Americas--- 10%

Key unit operations in the cocoa supply chain.

Beans in a Pod

Cocoa Bean Processing * Harvest pods from trees * Storage, transport of pods to fermentary * Remove beans and pulp from pod * Fermentation (3 -6 days) * Drying, roasting of beans * Processing to cocoa butter, cocoa powder

Fermentation is essential for cocoa bean quality, chocolate aroma and flavour 1. Hydrolysis of pulp 2. Metabolic end-products, enzymes diffuse into bean 3. Physico-chemical changes cause bean death. Activate endogenous degradation of bean constituents. 4. Produces precursors of chocolate flavour which develops during roasting 5. NO fermentation; NO chocolate flavour

Fermentation is a traditional, uncontrolled process Inefficient ; beans of inconsistent quality

Origin of Microorganisms 1. Beans aseptically extracted from pods are sterile 2. Implements for breaking pods and extracting beans; hands of workers 3. Containers for transport 4. Fermentation vessels 5. Air 6. Fruit flies, insects, animals, birds 7. Natural origin (totally organic!!)

The microbial ecology of cocoa bean fermentations— 100 years of research • Yeasts: Hanseniaspora guilliermondii; Saccharomyces cerevisiae; Pichia membranifaciens, P. fermentans; Issatchenkia orientalis; Candida spp • Lactic acid bacteria: Lactobacillus plantarum, L. fermentum; Leuconostoc spp; Pediococcus acidilactici • Acetic acid bacteria: Acetobacter pasteurianus; Gluconobacter oxydans • Bacillus spp • Fungal species

Complex microbial ecology • Which species are essential ? ? ? • How do individual species impact on cocoa bean quality and chocolate character? ? ? • Controlled Fermentation Studies • Sanchez J. et al (1985)--- S. cerevisiae • Schwan R. (1998)--- S. cerevisiae

The challenge of industrialized fermentations • Define microbial starter cultures • Design of fermentation vessel --- to allow uniform mixing, control of aeration, temperature, p. H, cleaning etc

Impact of yeasts on cocoa bean quality and chocolate character Innisfail, Queensland

Cocoa Bean Plantations in Queensland South Johnstone after cyclone (April 2006)

Experimental Strategy • Pilot scale fermentations (50 -100 kg beans) in North Queensland (Department Primary Industry, Queensland). Monitor : • Microbiology (yeasts, fungi, bacteria) • Chemistry of pulp and cotyledon (sugars, acids, ethanol, p. H) • Temperature (fermentation profile) • Quality (beans, chocolate)

Pilot Scale Cocoa Bean Fermentations Box Heap Barrel

Microbiological Analyses ISOLATION • Yeasts and moulds. MEA; DRBC • Lactic acid bacteria. MRS • Acetic acid bacteria. WL-Nutrient • Other bacteria. TSA IDENTIFICATION • Base morphology, biochemistry, DNA sequencing DGGE KVL University, Denmark

Microbiology of Fermentation Yeasts: Hanseniaspora uvarum, H. guillermondii, Issatchenkia orientalis, Saccharomyces cerevisiae, Pichia spp. , Kluyveryomyces marxianus Lactic acid bacteria: Lactobacillus plantarum, Pediococcus acidilactici Acetic acid bacteria: Acetobacter pasteurianus

Yeast starter culture fermentations • Various mixtures : S. cerevisiae; I. orientalis H. guilliermondii; P. membranifaciens; K. marxianus • Monitor • Fermentation kinetics • Criteria of cocoa bean quality • Cocoa bean chemistry • Chocolate character

Experimental Strategy • Controlled micro-scale fermentations (5 kg beans) at UNSW (Sydney) Monitor : • Microbiology (yeasts, fungi, bacteria) • Chemistry (GC-MS, aroma volatiles of beans) • Quality (beans, chocolate)

1 cocoa pods 2 at the beginning of fermentation 5 drying 4 end of fermentation 3 fermenter box

YEAST 1 STARTER CULTURE DEVELOPMENT PROCESS Spontaneous fermentation Screening of isolates Mass culture YEAST 2 YEAST 1 Lab fermentation Pilot-fermentation Further screening

Pilot Scale Fermentations Control - no added starter Hanseniaspora spp. Pichia spp. Saccharomyces cerevisiae Acetic acid bacteria Lactic acid bacteria Starter mixture 1

Starter yeast affects chemistry of fermentation faster ferment, more Et. OH, less acids Enhanced quality and sensory outcomes Criteria Industry standard Control With Starter Fermentation index > 30 70 80 5. 5 - 6. 0 5. 6 6. 0 5. 0 6. 9 8. 1 Cocoa bean p. H Flavour score

Quantitative sensory profiling • • Profiled using trained panel and GC-MS Starter directly affect sensory profile of beans and chocolate Control Starter Ghana

Accelerated proteolysis • Vicilin class proteins • Enzymatic breakdown leads to chocolate flavour precursors • Orange arrows: faster proteolysis with starters • Shorter fermentation possible Start (0 h) Middle (48 h) End (120 or 96 h)

Functional properties Increased retention of polyphenols during fermentation Inhibition of mould growth during drying

Conclusions • Fermentations in barrels with added cultures of yeasts gave commercially acceptable cocoa beans and chocolate • Faster fermentations ( 4 -5 days) • Chocolate flavour varied according to yeast species used • Some improved functional qualities • PLATFORM TO INDUSTRIALIZED PROCESS

Acknowledgements • Cadbury—Schweppes • Queensland Government

Team Cocoa

Chemical Changes during Fermentation PULP BEAN Before 2. 4% After 1% Before 1% After 0. 5% Lactic acid 0. 03% 0. 6% 0. 01% 0. 2% Acetic acid 0. 04% 1% 0. 09% 2. 0% Citric acid Beans: Lipid 45 -55%; lipases? Protein 1 -7%; proteases; free amino acid content increases 4 -5 fold; formation of flavour specific peptides Glycosidases -- active Polyphenoloxidases -- active

Chemical Changes during Fermentation PULP Before BEAN After Before After Fructose 6% 1% 0. 4% Glucose 5% 0. 05% 0. 01% Sucrose 3% 0% 2% 0% Ethanol 0. 1% 6% 1% 1% 0. 1% 3%

Predominant Yeasts in Cocoa Beans of Different Countries INDONESIA Saccharomyces cerevisiae; Candida tropicalis; Kloeckera apis BRAZIL MALAYSIA GHANA IVORY COAST Saccharomyces cerevisiae; Kloeckera apiculata; Kluyveromyces marxianus; Candida spp. Saccharomyces, Kloeckera, Pichia, Candida, Debaryomyces Saccharyomyces, Kloeckera, Pichia, Candida, Schizosaccharomyces Saccharomyces chevalieri, Kloeckera

Pilot-Scale Fermentations of Cocoa Beans • Box, Heap, Barrel • Barrel; with nutrient supplementation • Box; with increased turning and mixing • Box; increased fermentation time