The general challenge of this project is to improve the understanding of natural and man-made ecosystems as bases for the development of sustainable land use practices in the Central Amazon

LIEBEREI, R.

MÜLLER, S.

REISDORFF, C.

ROHSIUS, C.

SOUZA, A. das G. C. de

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Gerrnan-Brazilian Workshop on Neotropical Ecosystems - Achievements and Prospects of Cooperative ResearchHamburg. September 3-8, 2000Living Recources Management: Approaches. Techniques. Variability - Summaries of Lectures. Session 4Utilization of Cupuaçu Seeds as Raw Material for Chocolate-Iike Products:Variability of Morphological Traits and Biochemistry of Aroma PotentialReisdorff, C. \ Rohsius, C. \ Müller, S. \ Claret de Souza, A. G. 2 and Lieberei, R. II Universitat Hamburg. Harnburg, Germany.2 EMBRAPA Amazônia Ocidental. Manaus. Brazil.1 IntroductionThe studies on the utilization of cupuaçu seeds as rawmaterial for chocolate-like products has been done as sub-project of the SHIFr project ENV 23, which is one of thecooperational SHIFr projects located at the EMBRAPAAmazonia Ocidental near Manaus. The general challengeof this project is to improve the understanding of naturaland man-rnade ecosystems as bases for the development ofsustainable land use practices in the Central Amazon. Oneof the particular questions of our sub-project was, whethercupuaçu seeds can be used as raw material for chocolate-like products. In this summary some results of therespective investigations regarding seed inherent factorswhich are decisive for the generation of chocolate likearoma are presented.Why do we consider, that this additional use option willcontribute to concepts of sustainable production systems?The answer can be deduced from the advantages for smallholders: They will have an valuable product in addition tothe pulp, which is storable and can be sold independently ofharvesting seasons. In addition, the small holder can supp-Iya new market with price dynamics separate from those offresh products. These factors are capable to contribute tothe economic stabilization of production systems, whichhave been designed for ecological stability.The general idea to produce chocolate from cupuaçu seedscomes from the fact that the cupuaçu plant (Theobromagrandiflorum) is a close relative of the cocoa tree,Theobrorna cacao. The cupuaçu fruit contains an aromaticpulp surrounding about forty seeds. The pulp is sold at highmarket values of about I to 3 dollars per kilograrn. Theseeds are commonly not yet commercialized. Someattempts have been made to produce types of chocolatewares, however, these attempts were not very successful,mostly due to problems regarding the generation of a repro-ducible aroma quality.The typical aroma of cocoa is formed in the course of fer-mentation, drying and roasting. The most important bio-chemical reactions happen during the fermentation processoDuring the 2 to 4 days of fermentation the sugars of thepulp are degraded to acetic acid by microorganisms. Thistakes place in an anaerobic and a subsequent aerobic step.The acetic acid pene trates the cotyledons and triggers com-plex processes. These processes lead to cell death, disinte-gration of cell compartments, forrnation of an aqueousreaction phase and lowering of pH values of the seed stor-age tissue. These alterations are the pre-conditions for theforrnation of aroma precursors from seed storage proteins,namely from globulins. The biochemical reactions takeplace in the following sequence: The globulins are splicedto hydrophobic oligo-peptides by an endo-protease. In thenext step an exo-peptidase degrades these oligo-peptides tohydrophilic peptides and hydrophobic arnino-acids. Thismixture represents the precursors of cocoa aroma. The finalaroma is formed during drying and roasting. The aminogroups of the aroma precursors react with reducing sugars.These reactions, the so-called Mallard reactions, result inthe cocoa aroma.In our study we tackled the question: to which extendcupuaçu seeds possess the potential to develop a valuableand reproducible aroma. The first step was to investigatethe seed inherent factors being the pre-requisites for theforrnation of aroma. Thus, we compared the biochernicalcomponents and the morphological traits of cupuaçu seedswith the respective properties of cocoa seeds in order toprovide basic knowledge for the adaptation of seedprocessing technologies.2 ResultsConcerning the seed storage proteins we found quite simi-lar patterns in both species, T cacao and T grandiflorum.The globulins we have found in cupuaçu seeds have nearlythe same molecular weight as those of cocoa. However,cupuaçu seeds contain a little bit less of these globulins.Our results indicate that there is a high degree of qualitati-ve similarity between the globulins of cupuaçu and cocoa,whilst there are clear differences regarding the quantities.As next biochemical component of the aroma potential westudied the endo-proteases, which is responsi ble for thefirst degradative step of the reactions towards aroma pre-cursors. The comparison of the respective activities inseeds of T cacao with those of T grandiflorum revealedReisdorff. C.. Rohsius. c.. Müller: S.. Claret de Sou:a. A. G. and Lieberei. R.:Utitizution of Cupuaçu Seeds as Raw Material for Chocolate-like Products: variability of Morphological Traits and Biochemistry of Aroma Potential155Gerrnan-Brazilian Workshop on NeotropicaJ Ecosystems - Achievements and Prospects or Cooperative ResearchHamburg. September 3-8. 2000Living Recources Management: Approaches. Techniques. Variability - Summaries of Lectures. Ses ion 4that in both cases the endo-protease activities are very high,However, the highest activities were found in cocoa. ThepH dependence of the endo-protease is very similar bet-ween the two species with a maximum activity at about pH3.5. The catalytic types are identical, which was indicatedby the strong inhibition through the pepstatin A, which actsspecifically on proteases of the aspartic type.The carboxy-exo-peptidase catalyzes the cut-off of hydro-phobic amino acids. The proper result of this degradationdepends on the substrate specificity of the enzyme. Thus,we tested the substrate specificity of the cocoa andcupuaçu-enzyme by means of artificial substrates with car-boxy-terminal amino acids of different hydrophobicity. Theexperiments reveaJed that the exo-peptidase of cupuaçuseeds prefers cutting hydrophobic amino acids in the samemanner as the enzyme of cocoa does. In both species wefound considerable activities of this enzyme, however, theactivitie were higher in cocoa. In addition, the pH depen-dence shows certain differences with optimum activitiesnear pH 5.2 for cupuaçu and near pH 5.7 for cocoa.Inhibition experiments revealed that the catalytic type ofthe exo-peptidases of both species is to be classified asseryl peptidase.In addition to the biochemical parameters the followingmorphological attributes have been studied:I. The sub-cellular organization of organelles (Iiposomesand protein storage vacuoles), which is decisive for theformation of a continuous aqueous reaction phase withinthe cells.2. The seed size and shape, which determine the diffusiondistances for acetic acid and consequently determine thetime course of protein degradation.3. The morphology of the seed shells, which represent abarrier for the diffusion of acetic acid.Our respective morphological studies of the cotyledonti sue revealed that the sub-cellular structures of the stora-ge cells are nearly identical in ize and distribution of theorganelles. On the other hand, considering size and shapeof the entire seeds, one would expect differences relevantfor lhe fermentation processes, because the seeds ofcupuaçu are considerably bigger (3 - 5 g fresh weight) thanthose of cocoa (I - 2 g fresh weight). But the decisivedimension of a seed which determines the minimum diffu-sion distance of acetic acid is the seed thickness. Cocoaseeds are JO to 12 mm thick, Most of the seeds of thestudied cupuaçu plants are tlat with a thickness of J2 to14 mm. This means an additional diffusion distance ofabout I mm. which can be considered not relevant for thetime course of imbibition of acetic acid. Regarding the seedshells we observed the most evident morphologicaJ diffe-rences between the two species: The testa of cupuaçu isbigger and it is characterized by a layer of thick-walledpalisade cells. These distinctive properties of the cupuaçuseed shells might delay the diffusion rate of acetic into thecotyledons.Additional important differences between the two speciesare related to the adhering pulp: Firstly, the pulp of cupuaçuseeds is quite more fibrous and it is attached to the seedshell more tightly in comparison to cocoa. Hence, if onethinks about additional use, it has to be considered thatmost of the fruit flesh i removed for pulp production.These distinctive feature lead to problems regarding thesequence of anaerobic and aerobic fermentation, and theremight be less acetic acid for triggering the biochemical pro-ce es of aroma formation.3 ConclusionsSummarizing the results concerning the biochemicalcomponents of the aroma potential we can state thatcupuaçu seeds generally possess the biochemical com-ponents necessary for the formation of aroma precursors.However, some distinctive features might lead to a slightlyreduced yield of aroma precursors in comparison to cocoa.But the high genetic variability within the species T gran-diflorum would enable sclecting plants with higher bio-chemical aroma potential. With regard to the structuralattributes we found in both species a very similar sub-cellular organization of organelles, which allows thegeneration of an aqueous reaction phase necessary for thebiochemical reactions leading to aroma precursors. Thesize and shape of cupuaçu seed guarantee short diffusiondistances for acetic acid. But concerning the morphology ofseed shells. there is probably an evidently higher diffusionresistance for acetic acid into the torage tissue of cupuaçuseeds.The distinctive features of the seed shells and of the fruittlesh would lead to unsatisfactory results if one simplyapplies the procedures practiced in the processing of cocoaseeds. Consequently, further research is needed in order todevelop fermentation procedures which are adapted to therespective "adverse" properties of cupuaçu seeds.Reisdorff. C.. Rohsius. C.. Miiller. 5.. Claret de Sou:a. A. G. and Lieberei. R.:Utitization of Cupuaçu Seeds as Raw Material for Chocolate-like Products: variabilitv of Morphological Traits and Biochemistry of AIV/I1a Potential156

Utilization of cupuaçu seeds as raw material for chocolate-like products: variability of morphological traits and biochemistry of aroma potential.

http://www.alice.cnptia.embrapa.br/bitstream/doc/673189/1/Neotropicalpag.155156.pdf

Utilization of cupuaçu seeds as raw material for chocolate-like products: variability of morphological traits and biochemistry of aroma potential.

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