Food Structure Volume 6 Number 1 Article 3 1987 Scanning Electron Microscopy Studies of the Cellular Changes in Raw, Fermented and Dried Cocoa Beans A. S. Lopez P. S. Dimick R. M. Walsh Follow this and additional works at: https://digitalcommons.usu.edu/foodmicrostructure Part of the Food Science Commons Recommended Citation Lopez, A. S.; Dimick, P. S.; and Walsh, R. M. (1987) "Scanning Electron Microscopy Studies of the Cellular Changes in Raw, Fermented and Dried Cocoa Beans," Food Structure: Vol. 6 : No. 1 , Article 3. Available at: https://digitalcommons.usu.edu/foodmicrostructure/vol6/iss1/3 This Article is brought to you for free and open access by the Western Dairy Center at DigitalCommons@USU. It has been accepted for inclusion in Food Structure by an authorized administrator of DigitalCommons@USU. For more information, please contact [email protected]. FOO D MICROSTRUCTURE, Vol. 6 (1987), pp. 9- 16 0730-54 19/87$ 3. 00+. 00 Scanning Mic roscopy In ternational, Chicago (AMF O ' Hare) , I L 60666 USA SCANNIN G ELECTRON MICROSCOPY STUDIES OF THE CELLULAR CHANGES IN RAW, FERM ENTED AND DRIED COCOA BEANS A. S. Lopez*, P. S. Dimick and R. M. Walsh Department of Food Science, The Pennsylvania State University, 116 Borland Laboratory , University Park, PA 16802 *Current Address: Cacao Research Center, CEPLAC/CEPEC, 45600 ltabuna Brazil Abstract Introduction Cocoa beans are submitted to a curing pro­ Chocolate, which is manufactured from the cess of fermentation and drying to develop flavor seeds of Theobroma cacao owes its pop ularity and precursors . The beans must have reached maturi ­ wor ld- wi deappeartOl"'tS unique and character­ ty; otherwi se, no amount of processing can pro­ i s tic flavor . Prior to being processed into duce the desired flavor . Early work with cacao chocolate, the seeds from the ripe fruits , cell cultures showed that only when the cells irm1ediately follow i ng harvesting , are subjected have "matured" could a chocolate or cocoa flavor to a fermentation and drying process during result from further processing. Fermentation is whi c h the flavor precursors are developed. Only therefore require d because unfermented beans may after undergoing such a process do the seeds develop littl e chocolate flavor when roasted. (common l y refer red to as beans) possess those l i kewise , the outcome of excessive fermentation attributes necessa ry and des i rabl e for the ma nu­ 1 1 may also r esult in unwanted flavor . Thus , the factu re of c hocolate. The term, fermentation , first major post- harvesting phase t o have an is misleading when a pplied to the curi ng of impact on flavor development is that of fer­ cocoa because , a lthough typiccJl a l cohol, l act i c mentation. During this phase of curing, the and acetic fermentati ons occur in the pulp that mucilaginous pulp s urrounding the beans undergoes su r rounds the seed , the reactions that are an ethanol, acetic and lactic fermentation. The responsible for the formation of t he flavor pre­ acid and heat ge nerated kill the beans with a cursors are reactions between the seed enzymes resulting c hange in cell memb ranes . This facil­ and their substretes ( Lopez , 19B6). In the itates e nz yme and substrate movement with notable intact bean these s ubstrates are separated and swelling of the bean. Changes induced in the compa rtme nta l ized by biological barriP.rs whi ch beans during the process affect the texture and brea k down during the fermentation treatment. flavo r qucJlity . This paper rel<ltes texture of The liberation cJnd intermixing of enzymes and the bean to cellular and subcellular transforma ­ sub strates lead to spontaneous reacti ons wh i ch tions observed by sca nning electron microscopy. are governed by the physicochemical conditions of fermentation. They are infl uenced by the external microflora of fermentation and the Initial paper received April 18, 1986 method used. Thus the fermentation process and Man uscript received April 06, 198 7 the biochemica l transformations that it provokes Direct inquiries t o P . S. Di mic k inside the bean are of major importance in the Te lephone nu mber: 814 863 2962 development of c hocolate f l avor . The majority of investigati ons a i me d a t e l ucidating the nature of c hocolate f l a vor have bee n directed at the chemistry of fe r mentat i on a nd the manufacturing process in contrast to the few reports on the micro- structure of t he seed (Roelofsen, 1958; Duncan and Todd, 1972 ; Vaughan 1970) and that provoked by fermentati on (Biehl, 1973 ; Hoskin et al. , 1980) . The present study attempts to KEY WORDS: Theobroma cacao, scanning electron address this subject utilizing scanning electron microscopy of cocoa seeds, ce 11u 1ar changes, mi croscopy (SE M) techn i ques. fermentati on. Mate r ials a nd Methods Ripe fruit of Theobroma cacao of the Forestero variety were obta 1necrTrom the Cacao Research Center of CEPLAC (Com i ssao Executive do Plano da Lavoura Cacaueiro) . Immediately after removal from the fruits, samples of the seeds A. S. Lopez, P. S . Dimick and R. M. Wa l sh including the testa cotyleCon and the embyronic parenchyma c~lls (Figs. l, 2) . These, together ax i s, were prefixed in 3% 9lutaraldehyde in O.lM with the underlying spongy parenchyma contain the Sorensen's buffer, ( pH 7.1 } and post-fi xed in 1% substrates for tne microorganisms during fermen ­ tation. The pulp is separated tram the rest of ~~~~ 1!~ e~;: ~~;~n~:~~~r~~:~e~h~~:g~ ~- qr!~~ent the testa by a s ingle layer of smaller epidermal ethanol series , frozen in liquid nitrogen and cells ( Fig. 2) beneath which are found the large c ryofractured to expose a surface unaffected by slime or mucilaqe cells whi ch measure from 100 to the initial cutting (Humphreys et al., 1974) . 400 fl ffi , These Cell types are embedded in a mass Samples were critical point dried in a Polaron E3000 with C02 as a transitional fluid, mounted on aluminum stubs with silver adhesive and gold ­ coated in a Polaron PS-2 sputter coater on a cold stage with 280 to 420A of gold. When fat, which wa s liberated from the sample, recrystallized on the surface it obscured the field. Th ese samp l es were then refractured , immersed 0ver- night in acetone, air-dried and recoated with gol d. Fer­ me nted beans (seeds ) of mi xed Trinitario-Forestero crosses, were obtained from the Hurrmingbird He r s hey farm in Bel ize, Central Ame ri ca. These fe rmentations were carried out i n a seri es of s hallow and deep boxes currently bei ng used on the farm . Th e fermenting seeds were ~erated after 24, 48, 96 and 120 h during the 6-day fer­ mentation. Bean samples were obtained after 3 and 6 days of fermentation and were prepared as in the case of the fresh control beans, except that vacuum infiltration was appli ed at the initial fixing step to insure thorough penetra­ tion throughout the sample. Prepared sample s of unfermented and fermented beans were viewed us ing Fig. 1. SE micrograph of a cross section of the an ISI-60 SE M at lOkV. Photomi crographs were unfermented cacao seed tes ta s howing the tubu lar taken with a Polaroid 545 land Camera and pulp cells (P) and the spongy parenchyma below Polaroid 52 film (AS A-400 ) or with a 50mm it, the large slime or mucilage cells (5) Tahkumar lens and Plus X 35mm film (ASA 125) . embedded in the parenc hyma of the testa (PA) which also contains the vascular bundles (V) . Observations IS = i nterna 1 surface of the testa. B"r = 100 IJ ffi. In this study , the identification of ce ll types and the cellular inclusions relied upon the de scripti ons of cocoa seed t i ssue prov ided by Roe l ofsen (1958), Vaughan(1970), Dun can a nd Todd ( 1972) and Jaenick ( 1973). No staining for identification at t he light microscope was undertaken in this phase of the study. There is a considerable amount of ambiguity with respect to the terminology used to describe the cocoa seed or bean. For our purpose, the interpreta­ tion used by Duncan and Todd (1972), Jaenick (1973) and Esau ( 1977) whe reby the embryo is regarded as consisting of the embryonic axis and the two cotyledons was adopted. The embryo of the cocoa seed is somewhat oval in shape and flattened. It is comprised of two irregularly formed cotyledons and the embryonic axis to which tney are attached . These are enclosed in a f ibrous testa, the seed coat or shell wnich it­ self is enveloped in a mucilagenous pulp. Only the coty l edons are utilized in the ma nufacture of chocolate and although at this s tep, both the embryonic axis and the testa with remnants of Fig. 2. Details of the slime cell s (S) separ­ the pulp are di scarded , they serve a very ated by a layer of e pidermal cells (E) from the important ro 1 e in the fermentation process whi ch spongy parenchyma (PA) above . Be low the slime i s a prerequ i site for the manufacture of cells are vascular bundl es (V) embedded in t he chocolate . parenchyma. Bar =100 lJm . The muci lagenous pulp or endocarp of the fres h intact cocoa seed comprises large tubular 10 SEM of Cocoa Beans of thin-walled parenchyma cells which also con­ Fat globules and crystals (Hoskin et al., 1980) tain scatter ed bundles of vascular tissue were not observed on the en dosperm surface.