Journal of Applied Botany and Food Quality 93, 248 - 256 (2020), DOI:10.5073/JABFQ.2020.093.031 1Lübecker Marzipan Fabrik v. Minden & Bruhns GmbH & Co. KG, Stockelsdorf, Germany 2Institute of Plant Science and Microbiology, University of Hamburg, Hamburg, Germany Histological and cytological comparison of seed structures of Theobroma cacao, Theobroma grandiforum and Theobroma bicolor relevant to post-harvest processing and product quality Silke Elwers1*, Reinhard Lieberei2 (Submitted: July 9, 2020; Accepted: September 27, 2020) Summary For the present study, anatomical details of seeds of Theobroma cacao L., Theobroma grandiforum (Willd. ex Spreng.) Schum. and Theobroma bicolor Humb. & Bonpl. were investigated using light and transmission electron microscopy (LM and TEM). The focus was on features with possible signifcance for the course of fermentation and drying to generate cocoa-like aroma. It was found that the seed coat of T. cacao has the smallest diameter and contains only a thin sclereid layer, whereas T. grandiforum and especially T. bicolor exhibit much more massive palisade layers. The thicker seed coats of T. grandiforum and T. bicolor could delay the passage of liquids during fermentation and seed deshelling may require more effort than with T. cacao. In addition, existing differences in the amount of plant mucilages and phenolic substances in the testa may affect the composition and activity of the microfora during fermentation. Differences were also evident in the cotyledon tissue, which is processed to obtain cocoa-like products. Besides a higher degree Fig. 1: Opened fruits of T. bicolor (left), T. grandiforum (middle) and of maceration of the parenchyma of T. grandiforum and T. bicolor, T. cacao (right). Pictures kindly provided by C. Rohsius the main distinction was the different number and distribution of polyphenol cells. There were also differences in the species-specifc Same as T. grandiforum, the macambo tree T. bicolor (Fig. 1a) is fat and protein content. commonly found in cultivation. In the case of this species, the pro- duction area comprises the entire humid tropical America, from southern Mexico to Bolivia and Brazil (CUATRECASAS, 1964). Introduction Similar to the use of T. grandiforum, the sweet-aromatic fruit pulp is All species of the genus Theobroma L. (Malvaceae) produce fruits frequently eaten while the seeds are used in many places for home- with medium to large, recalcitrant seeds which are rich in fat, made chocolate-like products like those of T. grandiforum. Also in proteins and other nutrients. They are surrounded by a fbrous seed the case of T. bicolor, there are attempts to create brands that attract coat, which in turn is embedded in a mucilagenous pulp (LOPEZ international gourmets. et al., 1987). As the essential raw material for chocolate, the cocoa Cocoa-specifc aroma precursors are generated during fermentation tree T. cacao (Fig. 1) is cultivated almost in the entire humid tropics of cocoa seeds by an acid-induced proteolysis of the cotyledonar of the world. With a total annual production of around 4 million storage proteins by endogenous proteases (VOIGT et al., 1995). The tonnes of raw cocoa, the fermented and dried cocoa seeds, it is one of seed acidifcation during the fermentation process is caused by the most important world economic plants. microbial production of lactic and acetic acid as a consequence of The other species of the genus Theobroma L. are almost unknown the pulp components (SCHWAN and FLEET, 2015). outside their natural distribution areas in South America, although For both T. bicolor and T. grandiforum, REISDORff et al. (2004) the seeds of at least 12 other Theobroma species are used locally for demonstrated the potential for the generation of a chocolate-like the manufacture of chocolate-like products (CUATRECASAS, 1964). aroma based on the storage proteins and enzymes present in the Regarding the cupuaçu tree T. grandiforum (Fig. 1b), there exists cotyledons. The majority of investigations aimed at elucidating the a widespread cultivation in the Amazon Basin (VENTURIERI, 1993). nature of chocolate favour have been directed at the biochemistry of Actually, mainly the pulp of the cupuaçu fruits is used as a popular post-harvest processing. However, the fermentation process is also ingredient of juices and sweets, but especially in recent years, there infuenced signifcantly by the histological and cytological structure have been various initiatives to generate chocolate-like products of the seeds. The permeability of the seed coat to the microbial from the seeds of T. grandiforum that are seeking their market niche metabolites produced in the pulp, which are necessary for the curing in the high-end chocolate sector. As documented in the Cocoa Atlas of the seed, determine the rate of fermentation and therefore the (ROHSIUS et al., 2010), occasional trade batches of dried cupuaçu quality of the product (LOPEZ et al., 1987). The seed coat also protects seeds arrive in Europe as supposed “white cocoa”. The further the cotyledons from the microfora during fermentation (LOPEZ et al., establishment and professionalization of a „cupulate“ (chocolate 1987). Chemical reactions in cocoa seeds during fermentation and from cupuaçu seeds) industry would increase the market value of roasting depend on the composition and post-mortem structural cupuaçu fruits signifcantly. changes of the mesophyll cells (BIEHL et al., 1981). To date, few studies exist that focus on the histological and cyto- * Corresponding author logical characteristics of T. bicolor and T. grandiforum, although Studies on the seed structure of different Theobroma species relevant for processing and favour expression 249 these features have a signifcant infuence on the course of the post- of a glass knife. The feed rate of the specimen was adjusted to 2 to harvest processing, which is essential for the generation of cocoa-like 2.5 μm. favour. Already in 1998, a comparison of the seed structures of The collecting basin attached to the glass knife was flled with T. grandiforum, T. bicolor and T. cacao was carried out as part of a fltered aqua bidest. in which the semi-thin sections obtained were diploma thesis under the supervision of Prof. Dr. Reinhard Lieberei foating. From here, they were transferred to a drop of water on the (MÜllER, 1998). The intention was to to provide a comparative slide placed on a heating plate and fxed at a temperature of about description of the microstructure of the seeds suitable for the 50 °C. production of chocolate-like products as an important information For semi-thin sections (2 μm) of the seed coats and the cotyledon for developing adjusted post-harvest processes for the different tissue a staining with Toluidine blue O according to GUTMANN Theobroma species. Although the work was completed a considerable (1995), slightly modifed was applied. The semi-thin sections fxed time ago, the results are still up to date. The present publication on a microscope slide were incubated for 30 s in a solution of sodium is intended to make them available to a wider public for the frst hypochlorite (ca. 13% Cl, Fluka, Buchs, Switzerland). After rinsing time. The data were completely revised and placed in relation to with aqua dest. and drying the slides, the samples were incubated relevant scientifc fndings that have been published since then. The for 5 min in a solution of 0.05% (w/v) Toluidine Blue O in aqua dest. universal approach that characterised all studies published under the Then the slide was rinsed with aqua dest. again and subsequently supervision of R. Lieberei was retained. dried. The treatment causes a greenish-black or (due to oxidation) brownish colour of phenolic compounds, whilst proteins and cell walls are stained blue and lipids remain colourless. Material and Methods Some semi-thin sections of the seed coat of T. cacao (thickness Material approx. 2.5 μm) were stained with Fast Green FCF and Safranin Five ripe fruits of T. grandiforum and four ripe fruits of T. bi- using a modifed procedure according to GERLACH (1984). For this color were obtained in 1998 from the experimental feld and the the samples were incubated overnight in Fast Green FCF (Serva, surroundings of the German-Brazilian scientifc cooperation project 0.1% solution in ethanol, 92%) and the following day they were SHIFT (Studies On Human Impact on Forests and Floodplains in rinsed thoroughly with tap water and dried. Afterwards, the semi thin the Tropics) near Manaus, Brazil. Five ripe fruits of T. cacao were sections were stained for several minutes in a solution of 1% Safranin bought at a fruit market in Manaus, two further cocoa fruits were (Sigma) in 50% undenatured ethanol. After rinsing again with tap kindly provided by the Technical University of Braunschweig at a water, the preparations were dried, enclosed in Styrax (Sigma) and later point of time. All cacao fruits visually corresponded to the covered with a cleaned cover glass. The Safranin stains all cell walls 'Cacao Comum' type that is frequently cultivated in Brazil, with bright red, while cytoplasm and proteins are stained green by the yellow fruit husk, as shown in Fig. 1. The export of all Theobroma Fast Green. All semi-thin sections were analyzed with an Olympus fruits and corresponding preparations was carried out with the BH-2 light microscope. Pictures were taken with an Olympus OM-2 approval of the Brazilian project partners and in accordance with the camera. then valid Brazilian guidelines. Preparation for transmission electron microscopy Preparation for light microscopy The electron microscopic preparations carried out for this work were Whole Theobroma seeds were cut into 1 mm thick sections and largely based on a method developed by SPURR (1969), modifed by fxed for at least 24 h in 4% formaldehyde in water, according to H. Quader (Institute of Plant Sciences and Microbiology, former the instructions of GERLACH (1984).