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
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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
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. The generally running in the direction of the long epidermal cells (Figs. 4-5}, form a uni-seri ate axis of the seed (Figs. 1-2). The parenchyma laye r of tubular cells which completely surround cells of this layer nearest the cotyledon are the cotyledons and are continuous with the compressed (Fig. 3). Between the testa and the epidermis of the embryonic axis. These cells are cotyledons is a thin 'bees wing' or endosperm not uniform and those on the abax; a 1 surfaces (Duncan and Todd, 1972) which is also referred are larger than those of the adaxial surfaces. to as the perispen11 (Roelofsen, 1958; Vaughan, Cuticle and starch granules are also evident and 1970). This is a film of thin -walled, flattened some of the cells have multi-cellular trichomes cells that completely surround the cotyledons, arising from their surfaces (Fig . 5). The and also protrude into the cotyledonary folds surface formed by the cotyledon's epidermal ce lis (Figs. 4 and 14). was smooth but undulating and interrupted only by the occasional presence of trichomes.
Fig. 3. Portion of the testa parenchyma con taining the vascular bundles (V) and flattened parenchyma layer at the inner surface (PA). Fig. 5. Multicellular trichomes (TR) arising Bar = 10 ~ m. from the s urface of epidermal cells in the cotyledon folds. The epidermal cells ( E) fon11 an undulating surface; no superficial wax-like secretions were noted . Bar= IOO u m.
The ground parenchyma (Figs. 4 and 6), which constitutes the major part of the cotyledon tissue, consists of two types of thin walled cells. These are closely packed in an irregular fashion with virtually no spaces between them. The most abundant cell types are the smaller lipid/protein storage cells (Fig. 6). They contain a large number of 1 ipid vacuol es or globules embedded in cytoplasm which also contains starch granules and protein bodies. Scattered among these cells and often occurring in groups are larger cells whose interiors are filled almost entirely by a large vacuole (Fig. 6) . These cells are recognized as polyphenol cells and contain virtually all of the seed's polyphenolic material and alkaloids (Roelofsen, 1958; Va ughan, 1970) . In the Fig . 4 . Unicellular sheet of endosperm or peri Forestero cocoa, under l ow power of the l ight sperm ( ES) which 1 i es between the testa and the microscope, they appear as violet streaks due to cotyledon (Cot) of the seed. Lipid/ protein the presence of anthocyanin pigments which give stora9e cells (LP), polypheno l cells (PP) and the cotyledons their characteristic viol et or muci l age filled cells (M) are also evident. purp l e color. Vascular tissue of composite The smaller, uniseriate epidermal cells in bundles of xylem and ph l oem is a I so present contact with the endosperm do not bear within the cotyledons (Fig. 7). trichomes. Bar = 10 ~m.
11 A. S. Lope z, P. S. Dim ic k and R. M. Wa ls h
Fig. 6 . Cotyledon section showing 1 ipid/protein storage cells (LP) and polyphenol cells (PP) containing remnants of cytoplasm and starch and other embedded inc 1 us ions . Bar ;: 10 \.1 m.
Fig. 8 . LS, and Figure 9 CS of t he embryonic axi~ showing the root-~ap (RC) and hypocotyl (H) reg10n. (E) o epiderm1S, (C) o cortex, (PC) Fig. 7. Vascular tissue within the cotyledon procambium, (Pi)"' pith and (V) =vascular (Y). Bar "' 100 \.l m. reg10n. Bar = 100 1.1m .
In longitudinal sections of the embryonic In the fermented cacao seed structural axis, the morpho logical differentiation in the c hanges resulting from the treatment are minimal. epicotyl described by Duncan and Todd ( 1972) The most noticeable change is in the testa, pre was not evident. This may be due to the plane cisely in the pulp layer where most of the tubu of the fracture during sample preparation. lar cells have flaked away . Those few remaining However, epidermis, cortex, procambi urn and pith cells show masses of bacilli and yeasts occupy were well defined in the hypocotyl and root cap ing intercellular spaces (Figs. 11-12). Exami regions (Figs. 8-9). No stoma were seen nation of the testa of freeze dried beans showed a 1though they have been reported (Duncan and the presenc~ of polyhedral crystals similar to Todd, 1972). Starch granu !es and cuticle were those of calcium oxalate seen in the parenchyma observed in the epidermis . Vascular tissue was of many plant tissues {Fig. 13). also evident in the procambium region. The The peri sperm or endosperm t hat 1 i es be innermost cells of the pith show extensive tween the testa and the cotyledon did not appear mucilage (Fig. 10). to undergo structura 1 changes. In the unfer mented seeds, however, it closely adheres to the cotyledon surface while in the fermented
12 SEM of Cocoa Bea ns
Fig . 10 . CS of embryoni c axis show i ng mucilage Fi g . 12 . CS of testa showing detail of com cells (M) in t he pith . Ba r = 10 " m. pressed pul p cells with bacilli and yeast embedded between cells. Bar= 10 JJ ffi.
Fig. 11. CS ot the testa of the fermented seed showing the remnants of the pu lp (P), the vas Fig. 13. Microflora embedded in the pulp of the cu l ar ti ssue (V), the flattened parenchyma (PA) ferme nted testa shows yeast cells (Y), bacilli and the mucilage cells (S) . Bar= IOO " m. (B) and crystalline bodies (X). Bar= I" m.
seeds it is often fou nd loose or attached to the for the fat. changes in the other components are testa. It still remains attached to the no t manifested as obvious structural modifi cotyledons however, where it dips ; nto the cations detectable with the SEM. cotyledonary folds (Fig. 14). As me ntioned above, the lipids and pro Since the testa protects the cotyledons teins are stored in cells apart from the poly- from the microflora during fermentation, changes phenols. In general, the appearance of the such as cell lysis that might be expected to be sec tions of the fermented seed appear denser than provoked by microbial action, are not noted. thos e of unfermented seeds. This apparent dif Nor i s there evidence in the 1 iterature that ference in density mainly invol ves the 1 ipid/ sugge sts that microbia l enzymes produced in the protein cells whose cytop l asm in the fermented testa and capable of lysing cellular componen ts seed appears to be reticulate and riddled with are actually transported into the cotyledon. holes ( Figs. 15-16 ). This is most l i kely due to The enzymatic changes in the seed during fermen the r emova l of fat during the dehydration step. tation are intracell ular and involve the hydro Other discernible changes are in the poly lys is ar.d mo bi I ization of proteins and poly phenol cells . In the sections from the unfer phenols, and the redeposition of fat within mented seeds, they were generally void except the cell. The starch is believed to remain for part of the cytoplasm 1 ining of the vacuole unchanged ( Schmei der and Keeney, 1980). Except (Fig. 6). In the fermented seeds however, the
13 A. S. Lopez, P. S. Dimick and R. M. Walsh
Fig. 14. The fermented seed endosperm (ES) at a point where it enter s the cotyl edon fold. Bar = 100 u rn. cell ular contents appear to have soli dified (Fig . 14) and when not fractured, the solidified mass gave t he appearance of having remnants of the cytoplasm and other spherical bodies embedded in it. This suggests that some fat protein and starch may also be conta i ned in the polyphenol cells (Fig. 16). However, empty polypheno l cells were most frequently observed. Di spersed t hroug hout the cotyledon tissue, there were cell s filled with what appeared to be dehy drated mucilage . No obvious changes in the structure of the embryonic axis of the fermented samples were not i ced but sharper secti ons were obtained . This could be the result nf the intP.raction between protei ns and polyphenols (fig . 17). Figs . 15, 16. Fermen t ed cacao seed coty ledon . (E)= epidermal layer, (LP) = lipid/ protein cells and (PP) = po l y phenol cell s with cytoplasm During the in itial phase of ferme ntati on , remnants or solidified interi or . t he seed undergoes changes typi ca l ly assoc iated Ba r = 10 llm . with germination. However these orderl y bio chemical reactions £ive way to s pontaneous enzymatic and chemical changes on the death of the seed . Polyphenols diffuse throughout t he tissue and combine with protei ns and other compounds in complex chemical react i ons. Th e cotyledon an d the enzymatic transfor matifJOS t hat they undergo during fermentation are of interest to chocolate processors . However, the othe r components of the seed , though often ignored , play important, though indirect, roles in the curing process . The testa , for example , serves not on l y in providing substrates fo r the mi crobial fennentation and a barrier against t he entry of microorganisms and insect pests, bu t also has a direct bearing on the fermentation process . The permeabi 1 i ty of the testa to the microbi al metabolites produced in the pulp, which are necessary for the curing of the seed , df:'!ermine the rate of fermentation and t herefore t he quality of the product (lopez, 1986). The observati ons in this study confirm Fig . 17 . CS of the embryoni c axis of the that t he microfl ora of fermentation do not pene fermented seed. (TR) = trichomes, (E) = trate the testa to invade the seed but are con epidermis, (C) =- cortex, (PC) = procambium, fined t o the periphery. The ro l e of t he (V) = vascular t i ssue . Bar = 100 j..lm.
14 SEM of Cocoa Beans
embryonic axis in flavor precursor development Duncan, F. J. and Todd , A. W. ( 1972). is not clear although incipient germination of Structure of t he mature embryo of Theobroma the seed is believed necessary for flavor cacao L. Ann Bot. 16_:939-945. --- development. There is however, some evidence that suggests that germination determines the Esau, K. (1977). Embryo and seedl ing. In: texture of the cured seed as well as influences Anatomy of Seed Plants. John Wi 1 ey and Sons the oxidative phase dunng the latter stages at Inc., N.Y. 475- 476 p. fermentation. It has been observed for example ( Lopez, 1984), that in fermentations where Hoskin, J. M. , Dimick, P. S. and Daniel, R. R. conditions that encourage germination prevai 1, (1980). Scanning electron microscopy of the t he cured seeds genera 1 ly have an open and Theobroma cacao seed . J . Food Sci. 45: 1538-154D plump appearance that chocolate manufacturers aiidT54"5." -- - prefer (Anon., 1968). On the other hand, when gennination is inhibited or halted by the rapid Humphreys, W. J ., Spurlock, B. D. and Johnson, development of heat and acetic acid of ferme nta J. S. (1974) . Crit ical point drying of ethanol tion, the seeds are more likely to be compact infiltrated, cryofractured b iological specimens and cheesy (lopez, 1986). Plump beans are pre for scanning electron microscopy. Scanning ferred because they are eas i er to de hull and Electron Microsc. 1974: 275- 282. there is less carry- over of shell into the product. Furthermore, the open structure of the Jaenick, J. ( 197 3) . Elektronenmikroskopische seed allows for better oxidation of the coty untersuchungen an embryonen von Theobroma cacao ledon polyphenols and therefore a greater L. ( Ka kao) wah rend der embri ogeneseuna- - reduction in the astringency and bitterness . Semen ke i mung. Des serta t ion , Tie ra z t 1 i c he The reason for t he occurrence of compact seeds Hochschule Hannover. was not obvious from the samples examined in t h is study. Changes in the coty 1 edons resulting Lopez, A. S . (1984). Difusao de substancia from fermentation and res pons i b 1 e for flavor qu imi ca da testa de amendoas de cacau . precursor format i on a re still not clear ( Lopez , Jnforme Tecni co , CEPEC/CEPLAC. llheus 8a , 1986). However, it is well established that Brasil. 168-171 p. among the more important of these are the changes in the proteins and the polyphenols. Lop ez , A. S. (1986). Chemical changes occur Both are fragmented into numerous smallf'r units ring during the processing of cacao . Proceedings which themselves interact to form a large number of the Biotechnology Symposium. (Ed) . P. S. of camp lex substances. Evidence from SEM Dimick. The Pennsylvania State University. suggesting that protein bodies undergo changes 19-54 p. was not conclusive mainly due to thf' interfer ence of the lipid fraction. Biehl (1973) Roelofsen, P. A. (1958). Fe r mentation, drying bel ieves that under certain circumstances the and s t orc1ge of cacao beans. Adv . Food Res. fat isolates hydrophi lie constituents in the 1!. : 225-296. seed, preventing them from mix ing and reacting. Fat coating of cellular material was observed in Schmeider, R. L. and Keeney, P. G. (1980). some samp l es but it was not conclusive that this Characterization and quantification of starch had _occurred during fermentation and that enzy in cacao beans a nd chocolate products. J . Food matlc changes had been prevented . Confirmation Sci . ~:555 - 557. of this as well as other c hemical transformations \'lill be possible during the next phase of the Vaughan, J. G. (1970). Ster culiaceae-cacao . study which aims at investigating intracellul ar In: Structure and Utilization of Oil Seeds . reactions with differential stains a nd light Chapman and Hall Ltd. London . 241 - 243 p . microscopy, in conjunction with SEM.
Acknow 1 ed~ements
Authorized for publication as Paper No. 7JY9 in the journal series of the Pennsylvania Discussion with Reviewers Agricultural Experimentational Station. J. F. Heathcock: Hi'.ve the authors made any observations on the differences in structure References between the inner and outer port i ons of the cotyledons? 1 Anon. {1968). Raw cacao: Manufacturers Authors : Yes. The cotyledons are the quality requirements, Al liance, Vale, Surry . ccnvo luted laminae of the first pair of "leaves" 12 p. wh ich functicn as storage organs during growth . They do develop some chlorophyll too,but later Biehl, B. ( 1973). Veranderungen der sub fall off when the radicle has emerged and the cellularen struktur in keimblattern von 11 11 first three true leavf'~ have formed. (The kakaosamen (T heobroma cacao) wahrend der fermen cellular composit i on of the surface of the tation und trocknung. T.lebensm. Unters. cotyl edon and the interior are similar in that Frosch. ill: 137 - 150. t hey are composed of the same type of ce 11 s.)
15 A. S. Lopez , P. S. Dimick and R. M. Walsh However, the l ayers are different in t ha t t he re is a di sti nct cuticular l ayer which carries the multi cellu lar trichomes . These are more numer ous on the unders ide of the cotyledons.
J. F. Heathcock: Cou ld you comment further on the a ppea ranee of fat c rys tal s on the prep a r ations? This may suggest inadequate fixation. What diffe rencf's would you expect to see be tween well fixed tissue and tissue in which the fat has been solvent extracted? Authors : It is possible that ine~dequate fixation was responsible for some of the abe rrations seen in the micrographs. In the unfermented cacao seed two types of cel ls have been descri bed: the protein storage cells and the polyphenolic cells. The former contain s tarch grains and agglomerates, protei n vacuoles and nucl eic ma terial surrounded by fat gl obul es ( l-6 "min di ame ter ) , which form the bulk of the cell contents . Fermentation produces changes that cause the fat globules to coalesce a nd fonn l arger bodies, surrounded by a hydrop hilic protein phase (Biehl, personal comm.). The starch is believed to be unchanged. The poly pheno l ic cells contain a single large polyphenol vacuole surrounded by a thin cytoplasmic layer which contai ns starch grains, protein bodi es and the nucleus. The polyphenol cells would appear as empty cells because of the extraction of the polyphenols by acetone during preparation.
J. F. Heathcock: What changes do you think ilre ~oing on in the polyphenol cells to produce the "solidified" contents that you describe ? Authors: During fermentation, hydrolysis of prote1ns ard polyphenols occur. Besi des these e nzymati c degradative processes , r ecombination and conde nsation reactions between t ne poly phenolic fragments and also between them and the proteins occur. It is believed that these reactions will produce insoluble pol yp he nol-pro t e in complexes which are probably those observed in the polyp henol-protein cells of fe rmented cotyledon tissue.
J. F. Heathcock: Coul d you describe fur t her what you th1nk are the structural similarities and differences between germination and fermentation and do you have any evidence that some cocoa beans may s tart germinating prematurely before they are even fermented? Authors: The fermentation of cocoa may be ~as encompassing all the changes that occ ur in the seed during the curing process. Th ese are both the external microbial and the intercellular erzymatic fermentations as well as the pregermination changes that would occur prior to the death of the embryo . This germi na t ion may be expected to begin even in the mature fruit on the t ree and is governed by an inhibitor contained i n the pulp of the seed. Physiological stress is believed to cause a des truction of the inhibitor and provoke premature germination in the case of drought a nd frost damage.
16