Electron Microscopic Investigations of the Cell Structure in Fresh and Processed Vegetables (Carrots and Green Bean Pods)

Food Structure

Volume 3 Number 1 Article 8

1984

Monika Grote

Hans Georg Fromme

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Recommended Citation Grote, Monika and Fromme, Hans Georg (1984) "Electron Microscopic Investigations of the Cell Structure in Fresh and Processed Vegetables (Carrots and Green Bean Pods)," Food Structure: Vol. 3 : No. 1 , Article 8. Available at: https://digitalcommons.usu.edu/foodmicrostructure/vol3/iss1/8

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ELECTRQ~ MI CROSCOP I C I~VESTIGATIONS OF 1l1E CELL STRUCTURE I N FRESH AND PROCESSED VEGETAB LES (CARROTS AND GREEN BEAN PODS)

t-lonika Gr ote and Hans Georg Fromme

I nstitute of Medical Physics, ~ lu enster University Huefferstrasse 68 , D-4400 Muenster, W. Germany

The cell structure of fresh, blanched, boiled. dried and Dried carrots and green beans are traditional ingredients of rehydrated tissues from ca rrot roots and green bean pods was bag soups which have become more and more popular on the examined in the scanning and/o r transm ission electron mi cro European ma rket. In comparison with genuine instant prod ucts scope. The secondal)' phloem of carrot roots represen ts a these bag soups are characterized by comparatively long typical plant storage parenchyma characterized by a high starch cooking times which can he inOuenced - within ce rtain limits and lipid content. Gree n bean pods show many characteristics by vary ing process conditions during the production of such of assimilation tissue (e.g. chloroplasts). but they also contain a dried vege tables. The aim of indust rial research now is to considerable amount of starch. Blanching, boiling, de- and find su itable process condit ions in order to produce dried rehydration affec t the tex ture of both vegetables in a simil:u vegetables wh ich need extremely short cooking times and arc way: swe llin g of ce ll wa lls. maceration of tissue during blan similar to well cooked fresh vegetables as regard s taste and ching and boiling COlJpled with a granular denaturatio n of consistency. ln this se nse we tried to evaluat e the relati onship of cytoplasm. Drying leads to :J shrinkage and rwis ting o f th e process conditions to cel l ultr-J.stru cturl.! during the proJuction ce lls and clumping of the cy toplasm. Rch yd r.~t cd tissue is of dried vegetables and the ultrastrucrural alterations which characterized by strong cell wall swelling, ma ce ration, and occur during the rehydration process (i.e. cooking) by compa clumping of cy to plasm. t-.·lorphometric measurements of cell ring them with th e ultrastructural mo rphology of well cooked wall thick nesses after re hyd ration showed that various food fresh products. It is hoped by understanding these in terrelati on technological process parameters may strongly in nuence the ships that process conditions can be optimized to obtain d ried appearance of the rehydrated product. and reh}'d ral ed vege tables of high qualit y. There exbts large li terature on the morphological structure o f both vegetables. carrots and green beans. Most of th em were carricll out with the optical microscope, but in recent times the improved resolution power of the electron mil.:roscope was also utilized. Thus opti ca l microscopic studies in the developmental an3tomy of the root of D:1ucus ca rota we re carried out by !Iav is Initial paper received April 6, 1983 . (14), Esau (9) and Drake and Selstam (7, 8). Reck (18) Final manuscript received November 25, 1983. Direct inquiries to M. Grote. published a detailed paper on the optical microscopical Telephone nu mber : (0251) 835125 . struct urc of the plastids in the root. Scanning electron micrographs of raw and cooked ca rro t tissue arc found in the studies by Davis et al. (3 - 6). Trans miss ion electron microscopical papers on th e roots of Daucus ca rota chietl y deal wi th some cytological problems such :1s the structure of chromopla sts (II), nuclei ( 15) or the in vitro culture of ca rrot cells and tissues (28). (For a more detailed list Ke y words: Carro ts, green bean pods. elect ron microscopy, of lit erature sec the above memioned papers). cell structure, fresh and processed vegetables, morphome try. Most of the morphological studies in green bean pods that have so far been carried out were also done with the o ptical microscope. The first to examine the histological structure of th e legumen we re Kraus ( 16) and Stei nbrink (25). Like many of the following authors, some o f whom applied modern tec hniques such as polarisation mi croscopy or X-ray diffraction. they put their emphasis on the morphology of mature pericarps, especially on the structure of th e vascular bundles and fibres in

55 Grote, M. and Fromme, 1-l.G. order to investigate the dehiscence mechanism of the pods ( 10, by two different methods: by a sensory test of donenes; and by 13, 17). measuring the shear strengths of the sam ples mechanicolly (For Roth (22) treats the anatomy of the legumen as part of her further details cf. Bielig and Schwaiger ll J). comprehensive work on the anatomy and histology of the fmi ts For transmiss ion electron micoscopy (TEM), fron fresh, of angiospenns. Gassner (1 2), who deals wi th green beans blanched and rehydrated tissues (rehydrat ion by boilirg) cubes within the sco pe of a general optical microscopical in vestigation of approx. I x I mm size (from the secondary phloer.1 of the of vegetable foodstuffs, also gives some histological carrot root and the outer and inner part , respect information. ively of the green bean pod) were cut ~o.ith a sharp razor blade and flxed in 2% phosphat e buff This is even moTe the case with the detailed studies by ered glutaraldehyde (pH 7. 3) for two houn at room Reeve and Brown (2 1), who provide a survey of the develop temperature. After a short washing with the buffer solu:ion, the mental sequence and di fferentiati on of pod tissues and their samples were postfixcd fo r two hours in buffered ! Po Os04 structure and composition at edible ma turity. solution. They were dehydrated in a graded series of ettanol. In Optica l microscopic studies of the morphological cha nges 70% ethanol, block staining with phosphotungstic acil/uranyl occurring in plant tissues during preparation processes, such as acetate was carried out. After embedding the samples in Epon blanching, cooking, and dehydration/rehydration, have been resin, ultrathin sections were cut with a diamond kniff using a published by a number of authors. Reichert ultramicrotome OmU3. Sections were s1ai ned wi th Thus Simpson and Halliday (24) studied the disintegration lead citrate and examined in a Siemens Elmiskop I. of membrane material in vegetables during the cooking The specimens dried by hot air were briefl y imrrersed in procedure. Reeve and Leinbach (20) and Sterling (26) examined ethanol and then embedded in Epon resin, becwse it had the effect of mOisture and high temperature on carrot, potato, proved impossible to obtain sections from specimms directl y and apple tissues. Reeve (19) published a microscopical study embedded in the resin. o n te xtural structural changes in fresh and processed fruits and For scanning electron microscopy (SEM), fixation and vegetables. dehydration were carried out as described above After the The effects of drying and rehydration on cellulose 100% ethanol stage, the samples were critical-po.nt-dried in crystallinity of carrots were investigated by Sterling and Freon 13 using Freon 11 as a transitional fluid . The dried Shimazu (27). (For more detailed literature see the above specimens we re mounted on specimen holders wit.l "Leit-C", mentioned papers). coated with gold by sp uttering and observed in ! STE REO Transmission electron microscopy has so far not been SCAN Mk 1. employed in a systematic fine-structural investigation of For morphometric measurements of cell wall diameters in prepared and processed vegetables. TEM the semi-automatic image analysis system MOP AM /03 Likewise, the applica tion of morphometrical techniques in (Kontron, Germany) was used. In each test 10 - 15 separate combination with transmission electron mi croscopy of measurements were made on at least three parallel samples from vegetables has so far not been reported in literature. three different ex perim en ts, and the mean Vl lues were In the present study we firs t want to present the basic calculated. For obtaining comparable resu lt s, the po ints of morphologica l changes involved in the blanching, cooking, de measurement on cell walls were defin ed as follovs: midway hydration and rehydratio n of ca rrots and green beans without between two intercell ular spaces taking only thos:! ce ll walls correlating them to speciaJ food technological process condi into considcmtion which were not (yet) separated along their tions. The ultrastructural appearance of the fresh, unprocessed mid dle lamell a. cell s and tissues serves as a baseline for structure. Since the morphological appearance of the boiled raw In the second part of the present paper, we want to an a material was considered to be the " ideal" for a rehydrated lyse systemati call y the influence of special process conditions vegetable, the measured cell wall thickness of the boiled raw on the degree of cell wall swelling, which - in our opinion- is tissue was pu t at 100. Cell wa ll alterations in dried and the most conspicuous feature of dried and rehydrated ca rrots rehydrated samples were expressed as percentages of th is value. and green beans.

Mate rials and Methods Ultrastructural aspects of fresh and processed vegetal·les Two varieties of green beans ("Koralle", "Cascade") and SEM of fresh ca rrot tissues. The critical-point-tried carrot of carrots ("Bauer's Kieler Rote", "Zino") were analysed. exhibi ts a very good preserva tion of its histologicil structure Blanching, drying and storing of the vegetables were carried out (Fig. I). The tissue co nsis ts of polygonal parenchynatous cell s at the Institute of Food Technology - Fruit and Vege tables and shows many intercellular spaces. Technology - in Berl in as part of a correlated research project At higher magnifica tion, details of the cytopllsmic layer (AIF project No. 3664). Some of the results of this project were become visible. The rather homoge neous cytoplas11 includes published by Bielig and Schwaiger (I). For drying under defined particles of variable shape and size, wh ich by compuison with and reproducible conditions, a hot-air drying system previously transmission electron microscopic analyses ca n be ilen tified as designed was used (Bielig et al., 2). The d ried samples were di ffe rent cell organelles. Within the cel ls of fre;h carrots, rehydrated by cooking to "doneness" according to reports from roundish-shaped inclusions of approximately S11 n diameter the Berlin Institute of Food Technology where the optimum are seen, which have to be interpreted as starch g·ains or big cooking time for each sample had been previously detennined chromoplasts. Round particles of much smaller siz1 ( I - 2 11m

56 Electron Microscopy of Vegetables

Fig. I ~ 4 : Electron mi crographs of fresh carrot tissue

Fig. I: Phloem parenchyma cell s at low magnification in SEM (i.sp= intercellular space)

Fig. 2: Cytoplasmic layer of a singl e cell in SEM (sph=sphero some, st=starch grain)

Fig. 3: Survey of phloem paren chyma tissue in TEM (sph=sphero some, chr=chromoplast, v=vacuole, cw=cell wall)

Fig. 4: Detail from cell wall and cytoplasm in TEM (v=vacuole, m=mitochondrium, g=golgi ap paratus, chr=chromoplast, c=caro tin pigment , cw=-cell wall, pl=plas modesm)

Fig. 5 ~ 8: Electron mi crographs of fresh green bean pod ti ssue

Fig. 5: Cross section through one entire valve of the pod in SE M (o.ep=-outer epidermis, i. ep=inner epidermis, op=outcr parenchyma, ip=inner parenchyma , f=-fibre layer)

Fig. 6: Outer epid ermis of the valve with trichomes and stomata in SEM (o.ep=outer epidermis, t=trichome, s=-stoma ta)

Fig. 7: Fracture through the outer parenchyma showing the cytoplas mic layer of a single ce ll i11 SEM (n=nucleus, m=-mitochondrium, st= starch granule)

Fig. 8: Detail of parenchyma cells in TEM (v=vacuole, st=starch gra nule, i.sp=intcrccllular space, cw= cell wall)

57 Grote, M. and Fromme, I-I .G. diameter) represent lipid containing spherosomes (Fig. 2). Whereas these types of plastids occur i11 the outer parenchyma TEM of fresh carrot tissues. A survey of carrot ph loem tissue, the inner parenchyma possesses only small lens-shaped parenchyma ti ssue is shown in Figure 3. Each cell possesses a chloroplasts wi th apparently little or no starch grains. big ce ntral vacuole, whereas the cytoplasm appears as a thi11 TEM of processed carrot tissues. After blanching, carrot layer along the walls. Numerous round particles heavily stained pa renchyma ce lls show a sligh t swelling of their cell walls. They with osmium represent lipid droplets (sphcrosomes). tend to separate along their midd le lamellae. The cytoplasmic At higher magnification (Fig. 4). numerous plasmodesmata compounds still adhere to the inner side of the walls in the form as plasmic connections between neighbouring ce lls become of small granules (Fig. 9). Boiled tissue (Fig. I 0) exhibits still visible. Within the cytoplasm itself we find parts of the endo· thicker cell walls. The cytopla smic granules are coarser, but on plasmic reticulum, mitochondria of the tubuli type and golgi the whole they still retain their marginal position. Always lipid apparatuses. droplets of various sizes ca n be observed in blanched and The most remarkable morphological featllre of the carrot boiled tissues. Blanched and dried carrot root ti ssue is characte phloem parenchyma cell, however, is represented by the rized by an enormous shrinkage and twisting of the ce lls, but no ch romoplasts bearing the typical red or yellow pigment. On rupture occurs. The cytoplasm fil ls th e remaining lumina of the cross sections they show manifold shapes and structures. Within cel ls as a dense, clumpy materi al enclosing large lipid droplets the fresh carrot cells, chromoplasts in most cases are round or (sphcrosomes) which seem to stem from th e fusion of several oval and include starch grains. They often contain numerous small ones (Fig. II). plastoglobuli . irregularly shaped thylacoids and th e outlines o f The morphological appearance of blanched, dried and pigment inclusions, whereas most of the carotin p1gment itself is rehydmted cells and tissues (Fig. 11) st rongly depends on extracted during preparation procedures. Besides these typical the drying conditions (temperature, atmospheric moisture, chromoplasts amyloplasts also occur. drying time etc .. [sec below!) but, as a rule. cell walls arc uclei in ge neral show an irregular shape, wit h many thicker after blanching, drying and rehydration than they arc protrusions, and two nuclear bodies within the nu cleoplasm. after cooking alone. Intercellular sp:1ces seem to be enlarged and Closely associated with the nucleolus (1.5 - 2 p.m) there is increased in number after drying and rehydration. The cy to another nuclear body with a size of approximately 0.6 pm. plasm is more strongly condensed and clumped after re SEM of fresh green bean tissues. Cross sections through hydration and often docs not regain its o riginal marginal site, one entire valve of the pod show an outer and inner surface but remains in the centre of the ce ll s. The degree of cytoplasm ti ssue (epidermis) and a bisection of the enclosed parenchyma clumping and the localization of the cy toplasm within the ce ll ti ssue into an outer and inner parenchyma ("seed-cushion") by can also be strongly infll•enced by th e kind of dehydration a middle layer of cells with very small lumina (Fig. 5). The out em ployed. ward cpidennis reveals a pattern of trichomes. stomata and a TEM of processed green bean_!issues Ul!!.1cr_Q_<~.!_e n ch:r:~ characteristi cally ridged cuticle (Fig. 6), whilst the inner surface Blanched pod cells show ce ll walls which are slightly thickent:d. ti ssue adjoining the seed consists of small papillate cells. Cell wall junctions are slightly enlarged. their electron density Fracturing of the parenchyma tissue allows the observat ion being decreased at the same time. The cytoplasm being de of the cytoplasmic layer inside the single cc lb. Especially natured by heat fonns a marginal layer o f fin e granules still conspicuous arc round particles of approximately 1Opm dia closely lining the cell walls (Fig. 13). meter, whi<.:h arc preferably located within the cells of the outer After boiling the tissue, cell walls appear increased in parenchyma. These organelles may occur in large numbers thickness. Sometimes larger intercellular ~paces are fo rmed . The wi thin th e cells. cy toplasm consists of gra nules coarser than those in blanched As ,. comparison with transmission electron microscopical cells. The strictly marginal arrangement of the cytoplasm ic layer observations shows. they represent starch grains or chloroplasts is disturbed but, on the whole, the cytoplasmic granules are still with extensive starch inclusions. Flat-roundish bodies of assembled along the cell walls (Fig. 14). approximately l5p.m diameter might represent nuclei whert:as As in carrot tissue. blanche<.! and dehydrated pod tissue is the numerous very small particles within the cytopi:.J sm built up of shrunk and twisted, but still intact and clearly (approximately 0.6 x 0.8 pm) must be regarded :•s mitochondria distinguishable individual cells. The sma ll lumina of the cells are (Fig. 7). more or less filled up with cl umped cytoplasmic material (Fig. TEM of fresh green bean tissues. In con trast to th e inner 15). p:1renchyma. the outer parenchyma tissue of the immature pod As to the morphological picture of the bl:mched, dried and (Fig. 8) shows many intercellular spaces. The single cells possess rehydrated ti ssue (Fig. 16). many of the characteristi cs observed big central vacuoles and parietal cytoplasmic layers of varied in the rehydrated carrots arc found in the rehydrated beans as thicknesses. The cell walls themselves arc frequently traversed we ll. !! ere, too, we observe a striking dependence of the mor by plasmodesmata. A detailed analysis of the cytoplasm shows phological appearance on the influence of the preceding food many small vacuoles, flatshaped nuclei, mitochondria of the technological processing (see below). tubuli-type, golgi apparatus and frequently a richly developed rough endoplasmi c reticulum Morphometric measuret.nents of cell wall swe lling in moccsscd The morphological appeamnce of the plastids is vegetables remarkably heterogeneous. Besides we ll developed starch grains Since swelling of the cell walls seemed to be one of the (amyloplasts) we find chloroplasts with small starch grJnulcs most striking phenomena occurring in rehydrated tissues (see and chloro-amyloplasts with considemble starch inclusions, but above) , we carried out systematic measurements of cell wall there are still clearly visible stroma and grana thylacoids. thicknesses in rehydrated carrots and green beans in order to

58 Electron Mi croscopy of Vegetables

Fig. 9 - 12: TEM mi crographs of .... I • processed carrot ph loem pare n chyma tissue

Fig. 9: Blan ched ti ssue (v=vacuole, cy=cytoplasm, cw=cc ll wall, sph = spherosome,i .sp=intercell u lar space)

Fig. I 0: Boiled tissue (v=vacuole, cw=cell wall, i.sp=in tercellular space, sph=s pheroso me, cy=cy to plasm) 10 Fig. II : Dried tissue (cw=cell wall, sph=sp herosome, cy=cytoplasm)

Fig. 12: Rehydrat ed tissue (v=va v cuole, cy=cyto plasm, i.sp=intcr ccllular space, cw=ccll wall, sp h= spherosome) i ~ sp

S ph 91 Fig. 13 - 16: TEM micrographs of - C W processed green bean pod tissue 12 Fig. 13: Blanched ti ssue (cy=cy to plasm, v=va(.;uole , cw=cell wall)

Fig. 14 : Boiled tissue (v=vacuol c, cw=ccll w:.tll. cy=cy toplasm, i.sp= intercellular space) J Fig. 15: Dried ti ssue (cw=cell wall , cy=cytoplasm)

Fig. 16: Rehydrated tissue (v= vacuole, cy=cytoplasm, i.sp=inter cellular spa ce, cw= cell wall)

59 Grote, M. and Fromme, H.G.

Table I . Influence of different blanching periods upon the Table 5. Influence of the residual wa ter content of dried carrots degree of cell wall swelling in rehydrated green beans (va r. (va r. " Bauer's Kiele r Rote") upon cell wa ll swelling after "Cascade") rehydration

Sample Blanching time Cell wa ll swelling Sample Residual water Cell wall swelling No. min % No. cont ent % %

B1 135 C7 122 82 6 167 C8 13 200 83 9 190 C9 18 173 84 12 192 C10 22 184

------~~------C 11 126

Table 2. Influence of different blanchin g periods upon the Table 6. Influence of the drying temperature upon cell wall degree of cell wall swelling in rehydrated carrots (va r. "Zino") swelling in rehydrated green beans (var. "Cascade")

Sample Blan ching time Ce ll wa ll swelling Sample Blanching Drying Cell wall No. min % No. time temperature swelling min oc % cs 99 C6 10 11 9 8 7 50 160 82 70 167 B8 90 178

Table 3. lnO ucnce of blanching in different blanching media upon ce ll wall swelling in carrots (var. "Bauer's Kieler Rote") Table 7. Influence of the drying temperature upon cell wall after drying and rehydration swelling in rehydrated carrots (var. "Zino")

Sample Bl anching Blanching Ce ll wall Sampl e Bl anching Drying Cell wa ll No. time medium swellin g No. time temperature swelling min % min oc %

C1 H20 2 12 C12 70 99 C2 H20+3%NaC1 287 C13 90 90 C3 10 H20 244 C14 10 80 \04 C4 10 H20+S%NaC1 293 C15 10 90 119

Table 8. Influence of the storing temperature upon cell wall Table 4. lnOucnce of the relative humidity of the drying air swelling in carrots (var. "Bauer's Kieler Rote") after rehydra upon ce ll wall swelling in rehydrated green beans tion (var. "Korallc") Sample Storing temperature Cell wa ll swelling Sample Blanching Relative Ce ll wall No. oc % No. time humidity swelling min % % C 16 4 2 17 C17 20 273 85 2 1 189 C18 4 198 B6 3 125 C19 20 26 1

60 Electron Microscopy of Vegetables

more closely analyze the influence of so me process parameters All samples were blanched before drying and rehydration (The rehydration periods are not shown in the tables) . The SEM and TEM micrographs show that fresh carrot Influence of different blanching times. Blanching in water root parenchyma is a typical plant storage parenchyma, for different periods led to different degrees of cell wall swelling characterized by a high starch content and numerous lipid after rehydration (Tables I and 2). The values in these tables droplets. Green bean pod parenchyma, on the other hand, show that cell wall swe lling in the rehydrated tissue is correlated represents another type of plant tissue, i.e. that of assimilating with different blanching times: the longer the blanching time, tissue although the relatively high starch content within th e the greater the cell wall swelling. Different rehydration periods chloroplasts and the occurrence of intermediate forms between do not seem to influence much the alterations caused by typical chloroplasts and typical amyloplasts point out that it different blanching periods. This means that samples with short has storage functions as well. blanching periods wh ich require long rehydration times for As expected, structural changes involved in the processing reaching the point of doneness do not show the same morpho of both vegetables affect cytoplasm and cell wall in a specific logical appearance (expressed by the degree of cell wall way. swe lling) as those with long blan ching times and -consequently Cytoplasmic changes are expressed by a variable degree of - shorter rehydration periods. clumping and by translocation of cytoplasmic substance from Influence of the addition of NaCl to the blanching its marginal site into the vacuole space. medium. Blanching with variable concentrations of NaCiled to After being transfonned into a fine (blanched) or coarse a general increase in cel l waU thickness and maceration of (boiled) granular substance the cytoplasm keeps its place even the ti ssue in the rehydrated carrots as compared with blanching in boiled tissue. The strong clumping and translocation of in pure water (Table 3). It is remarkable that the addition of 3% cytoplasmic substance into the cell lumen which can be ob NaCI causes almost the same amount of cell wall swelling in 5 served in dried specimens is in general not compensated by min as the addition of 5% in 10 min. Blanching with NaCl water absorption during rehydration. So within the cytoplasm, reduced the cooking times of the samples considerably. dehydration seems to bring with it physical and chemica l Dehydration in dried air. If the relative humidity of the changes that can only partially be made reversible during drying air was reduced to 3%, the samples exhibited kss ce ll rehydration. wall swe lling than parallel samples dried in air with a relative As far as the cell wall is concerned two aspects seem humidity of 21 % (l able 4). Lowering the relative humidity al so important: (I) the degree of swelling and (::!) the separation of led to a reduction of the cooking times and a better conserva the single cells along the middle lamellae. tion of chlorophyll As the electron micrographs of raw. blanched and boiled Residual water content of the dried vegetables. As Table 5 tissues show. the effects of moisture and heat lead to a swelling shows, the morphological structure of the rehydrated carrots is of th e walls and to a beginning of separation of the individual adversely affected by a comparatively high water content (> cells. This "thermal maceration" ha s long been observed in 10%) of the dried product. Reducing the residual water content prepared tissues by optical microscopists, and has been attri to 6% by prolonged drying reduces cell wall swelling con buted to the extraction of protopectin from the middle lamella. siderably. There were no significant differences with re gard to In the present results, however, it is remarkable th:H not the cooking times. only short blanching, but also boiling to ''doneness" preserves Drying temperature. If the blanching time is kept constant. the histological structure of the ti ssue so well. Apart from whereas the drying temperature is varied, morphological moderate cell wall swelling, only occasional cell separa tion and differences in the rehydrated sample were observed (Tables 6 genuine "maceration" take place. and 7). As the figures from Table 6 show. in green beans less Like Davis and Gordon (3) we found no submicroscopic cell wall swelling was observed at low diying temperatures. In evidence that any rupture of cell walls ("holes") occurs during carrots, however, (Table 7), this is only true for samples cooking or dehydration and rehydration. as some authors blanched for I 0 min. The short blanching time (6 min) gives reported (23, 24). This would indicat e :1 degradation of the better results with the high drying temperature. In green beans. cellulose skeletal network. The observed swelling of the ce ll drying at 900 C reduced the cooking time considerably. walls, which is accompanied by a Joss of electron density, su rely Reduction of cooking time was also observed in carrots after indicates a loosening of the cellulose fibril network. If this blanching for 6 min and drying at 90o C. Carrots after loosening is caused by a breakdown of matrix material (pectin, blanching for I 0 min, however. showed shorter cooking times at hemicel!uloses, etc.) and/or by a loosening of chemical bonds low temperatures. between the cellulose fibrils or by some other factor must be Influence of storing. Dried ca rrots were stored at different decided by cytochemical analysis. temperatures (40 C, 20o C) for 5 weeks. As the figures in Table The observed shrin kage of Uehydrated ce ll is compensated 8 show there is less cell wall swelling in rehydrated samples after by water absorption during rehydration by boiling. Rehydrated storing the dried vegetables at the lower temperature. Apart samples often show more voluminous cell walls than boiled raw from a general prolongation of the cooking time due to the tissue. As other experiments demonstrated, this swelling may storing process itself, no difference between the two storing lead to cell walls being five to ten times thicker than those in temperatures as regards the cooking time could be observed. raw tissue. In most cases these tissue samples also exhibited a However, th ere was a tendency to a better conservation of higher degree of maceration. It is obvious that such enormous pigments in carrots stored at 4° C. structural deviations from the appearance of the boiled raw tissue must be regarded as undesirable from the morphologist's

61 Grote, M. and Fromme, I-I .G. point of view. Acknowledgement Another structural alteration occurring during dehydration which was observed by Sterling and Shi.mazu (27) is the increase This study was supported by AlF grants No. 3668 and in cellulose crystallinity in dehydrated samples. According 4518 as well as by the Forschungskrcis der Ernahrungsindustrie. to these authors this phenomenon may account for the oft.en observed toughness of rehydrated vegetables and the prolon gation of their cooking times, because a high amount of crystalline cellulose would prevent the cell wall from taking up I. Bielig HJ , Schwaiger M. ( 1980). Verfahrcnstechn ische water during swelling. Aspekte bei der \Varmlufttrocknung von Gcmi.ise (Pro The present results clearly demonstrated the tendency of cessing parameters i11 tl1e ilot-air dryi11g of vegetables). Z. an increased swelling of cell walls during dehydration/ rehy Lebensm. Tcchn. Verfahrenstechnik ]..!, 78-80. dration so that cellulose crystallinity might seem to be rather 2. Ui clig I-IJ , Wolff J , Schwaiger M. (1977). Entwicklung von decreased. Methoden zur Untersuchung und Herstellung von Trok The results of our morphometrical measurements of cell kengerni.isen mit optimalem Eignungswert (Dellelopmellt of wall swelling in processed vegetables show that in the pro methods for the i1111estigatio11 and prod11ctio11 of dried high duction of dried food the process parameters may exert a quality vegetables). lndustr. Obst Gemiiscverwertung §1, decisive influence on the ultrastructural appearance of the 433-435. rehydrated product. 3. Davis EA, Gordon J. (198 1). Structural studies of carrots Some of these parameters (short blanching without NaCI , by SEM, in: Studies of Food Microstructure. SEM, In c., low relative humidity of the drying air, low residual water AMF O'Hare, IL, 323-332,282. content of the dried product and storage at low temperatures) 4. Davis EA , Gordon J, Hutchinson TE . (1976a). Specimen produced samples that after rehydration showed a morpho preparation of raw and cooked carrot phloem and xylem logical status more like that of the boiled raw tissue than for the scanning electron microscope. Home Econ. samples treated in the opposite way. Whereas the influence of Research J . .±, 163-166 blanching upon cell wall swelling was remarkably distinct, even 5. Davis EA, Gordon J , Hutchinson TE. (1976b). Scanning in the rehydrated product, the ch oice of the drying temperature electron microscope studies on ca rrots: Effects of cooking did not seem to be very critical within the above limits. on the xylem and phloem. Home Econ. Res . J._1, 2 14-224. In some cases (e.g. relative humidity of the drying air) 6. Davis EA , Gordon J , Hutchinson TE. (1977). Morpho favourable morphological and favourable food technology logi cal comparison of two varieties of carrots during results (e.g. short cooking times) agreed. In other cases (most growth and storage: Scanning electron microscopy. Home prominent: long blanching times) a discrepancy was observed Econ. Res. J.Q.. 15-23. between the morphologist's claim for a good preservation of 7. Drake B, Sclstam E. ( 1971 ). Struktur hos morOtter. I structural features and the food technologist's claim for short (Stm ctHre of carrots I). Svcnska lnstitutet fOr Konser cooking times. veringsforskning. GOteborg, No. 287. Some parameters (e.g. storage temperature) did not have 8. Drake B, Selstam E. (1971). Struktur hos mor6tler. II any influence upon the cook ing times of the dried samples, (StmctHre of ca rro ts II). Svcnska Institute! fOr Konser whereas - from the morphological point of view - distinct veringsforskning. G6tcborg. No. 288. differences in cell wall swelling were observed. There was, 9. Esau K. ( 1940). Developmental anatomy of the fl eshy however, a better conservation of pigments in ca rrots at low storage organ of Daucus carota. Hilgard ia 1]. 175-226. storage temperatures. 10. Fahn A, Zohary M. ( 1955). On the pericarpial structure of the legumen, its evolution and relation to dehiscence. Phytomorphology j , 99-1 11. 11. Frey-Wyssling A, Schwegler F. (1965). Ultrastructure of Although the present investigation answers some questions the chrornoplasts in the carrot root. J. Ultrastruct. Res. dealing with the morphological consequences of food techno u. 543-559. logica l procedures, the goal pursued by both the morphologist 12. Gassner G. (1973). Mikroskopische Untersuchung pflanz and the food technologist ("The rehydrated product should be licher Lebensmittel (Microscopic investigario11 of plam similar in all aspects to the boiled raw tissue") has not yet been food). Stuttgart: G. Fischer, \49-150 and 158-159. fully reached. For the food scientist this means further im 13. Guttenberg H v. (1971). Bewegungsgewebe und Per provement of process engineering in close correlation with zcptionsgewebe (Locomotory tissue (11/d perception tissue) morphological investigations. In: Hanclbuch dcr Pflanzenanatomie, Vol. V, 5. Stuttgart: Beside cell wall swelling, these future morphological Uornrrage r, 148-176. studies should examine further characteristics of cell wall 14. Havis L. (1939). Anatomy of the hypocotyl and roots of alterations, e.g. changes in the degree of crystallinity of the Dau cus carota. J. Agr. Res.l§., 557-564. cellulose fibres (Sterling and Shimazu (27)),whichseems to be 15. Jordan EG. (1976). Nuclear structure in Dau cus carotu L. quite significant for a deeper understanding of the process Cytobiologic !.1, 171-177. involved in the drying and rehydration of plant tissues. 16. Kraus G. ( 1866). Ober den Bau trockencr Pcricarpien (On the str11cture of dry pericarps). Jahrb.wiss.Bot. 1_, 83-126.

62 Electron Microscopy of Vege tables

17 . Monsi M. (1942/1943). Untersuchungen Uber den Mecha S. Jones: What are the processing parame ters? nismu s der Schleudcrbewegung der Sojabohnenhiilsc (/11- Authors: Detailed infonnation of the processing parameters arc vestigatious 0 11 the catapult mechanism of the soy bea11 given in literature references I ,2. pod). Jap. J. Bot.ll, 437 4 74. 18. Re ck J. (1962). Untersuchunge n zur Plastidenontogcnese S. Jones: How reproducible arc your swe lling data, given th at a und Ca rotinbildung bei Daucus carota (Jn vest igtltion of subjective es timation of doneness is (probably) used? Arc your plastid ontogetty a11d caroti11 synthesis in Daucus carota). data from single or multiple runs? What are the standard errors? Di ssertation, Braunschweig. · Aut hors: Since sensory as well as shearing tests were used for 19. Reeve RM . (1970). Relationsh ips of histological structure the estimation of doneness, the reproducibility of our morpho to texture o f fresh and processed fruits and vegetables. J. metric results must be considered as good. Our da ta arc from Texture Studies I , 247-253. multiple runs extending over a total period of three yea rs. This 20. Reeve RM , Leinbach LR . (1953). Histological investi means th at the influences of three different annual growth con gations of texture in apples. I. Composition and influence ditions on the structure of the raw material are in cluded. Never of heat on structure. Food. Res. !...§., 592-603. theless, standard deviations ranged only be tween I 0 - IS %. 21. Reeve RM , Brown MS. (1968). Histological development of the green bean pod as related to culinary structure. 2. S. Jones: Are all the dried, rehydrated samples fi rst blanched? Structure and composition at edible maturity. J. Food What is blanching, specifically? If the vegetable is dried andre Sci.ll, 32 1-33 1. hydrated without blanching, is the only effect a longer cooking 22. Roth J. (1977). Fruits of angiosperms. in: Handbuch der time? Pfl anzenanatomie, Vol. V, 5. Stuttgart: Borntrage r, Authors: All samples were blanched before drying, only blan 203-225. chi ng periods and blanching media were varied (s. Tables I - 3). 23. Sherman HC. (1948). Food Products. Macmillan Co.: New Drying of vcgct.ablcs without previous blanching is - from the York , 107- 151. view of food conservation - undesirable, because blanching inhi 24. Simpson Jl , Halliday EG. (194 1). Chemical and histo bi ts enzyme activity and therefore leads to a better conservation logical studies of the disintegration of cell -membrane of the colour, taste, and chemical composit ion of vegetables. We materials ln vege tables during cooking. Food Res. §., did not check whether drying without previous blanching leads 189-206. to a prolongation of the cooking time although thi s might well 25. Steinbrink C. ( I g83). Ober den (Jffnungsmechanismus be the case. der Hi.ilsen (On th e upwing mechal"lism of pods). Ber. Deu t. Bot. Gcs. l , 27 1-276. K. Saio: Many starch grains are observed in fresh green bean 26. Sterling C. ( 1955). Effect of moisture and high tissue (Fig. 8). but not in processed green bean ti ss ues (Figs. 13 tempera ture on ce ll walls in plant tissue. Food Res. lQ, to 16). The authors do not refer to the changes in starch grains 474479. during processing. It is also considerab le in carrot ti ssue. at 27. Sterling C, Shimazu F. (1961). Cellulose crystallinit y and though the number of starch grai ns are fewer than in green the reconstitution of dehydrated carrots. J. Food Sci.l.§.. beans. Please ex pl ain . 4794 84. Au th ors: Micrograph No. 8 was taken from th e ou ter part of 28. Wi lson HJ , Israel HW , Steward FC. (1974). Morphogenesis the green bean pod (see tex t) wh ich is characterized by a high and the fi ne structure of cultured carrot ce ll s. J . Cell . Sci. starch con tent within the cells. All micrographs of processed u. 57-74. gree n beans were taken from the inner pa rt where there are only few amyloplasts occurring in the cy toplasm. We did not parti Discuss ion with Reviewers cularly focus on the fate of starch grai ns during processing in this stu dy since the degradation of starch seems to be a complex S. Jones: One of your basic assumpti ons is that the dried, re phenomenon which surely requires separate investiga ti ons. But hydrated product should resemble as closely as possible the as far as we noticed there did not seem to be big differences fresh cooked vegetable with respect to morphology of the cell between carrots and green beans with regard to these organelles. wall. Has this assumption been tested'! Authors: In most cases foodstuffs during preservation (drying, K. Saio: To resolve the "hard-to-cook"' phenomenon of dried canning, freezing) lose so me of their qualities which they had beans, soaking in salt solutions prior to cooking (quick-cooking) in the fresh or fresh cooked state. In this sense preservation can have been reported by many researches (Rockland and Jones. J. be rega rded as a kind of expedient. which is dictated by seasonal Food Sci. 3.2 , 342, 1974, Sefa-Dedeh et a!. , J. Food Sci., :L} , or commercial needs. The aim of food scienti sts is to keep these 1832, 1978, Jackson and Variano-Mortston, J. Food Sci. , .1Q, negative influences of preservation as minimal as possible as 799, 198 1 etc.). They pointed out that th e quick-cooking was regards taste, consistency and chemical composition of the pre rendered by the mechanism of ion exchange and possibly by served food . Our morphological investigations must be regarded chelation, as the middle lamella which cements the individual as part of these efforts in minimizing deviations from the original cell s together consists of a calcium sa lt of polyme rs of galactu material which occur during preservation. In this sense the mor ronic acid . The cell wall swelling in these experiments seems phology of fresh or freshl y cooked, i.e., unpreserved and un to be essentially the same as the phenomenon above. in view altered tissue, seems to be the reasonable standard for estimating of the acceleration of swelling with NaCI. The TEM micrographs the influences of processing conditions. shO\v that after processing the microstructure is largely unchanged

63 Grote, M. and Fromme, H.G .

except for enlarged intercell ular spaces and lcru'er density. But, what happens to the actual texture of the vegetables on processing? Aren't the lowering of the crispness and hardness dependent on the treatments? And how is the degree of cell wall swelling related to such tex tural properties? Au thors: We agree with you that cell wall swe ll ing and mace ration of tissue during processing is surely connected with reactio ns taking place within the pectin/protopectin compo nent of the cell wall, especially the middle lamella. Dissolu tio n of pectin/protopectin is not o nly promoted by salt addition, but also by the action of hot water which might explain the swelli ng and macera ti on of cell wall material in the experiments without the addition of salts. We suppose that the lowering o f the den sity o f cell walls which is visib le in our TEM micrographs is at least partially due to the extraction of matrix material such as pectin resulting in a loosening and broadening of the remaining cell ulose fibre network . T he actual composition and texture of the wa lls (e.g. crystallinity of cellulose fibres) cannot be elu ci dated by purely morphological methods, but their analysis seems to be the next step towards a deepened understanding of the effects of processing.