Microstructural Approach to Legume Seeds for Food Uses

Food Structure

Volume 12 Number 3 Article 6

1993

Kyoko Saio

Michiko Monma

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Recommended Citation Saio, Kyoko and Monma, Michiko (1993) "Microstructural Approach to Legume Seeds for Food Uses," Food Structure: Vol. 12 : No. 3 , Article 6. Available at: https://digitalcommons.usu.edu/foodmicrostructure/vol12/iss3/6

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]. FOOD STRUCTURE, Vol. 12 (1993). pp. 333-341 I 046-705X/93$5.00+ .00 Scanning Mi c roscopy International, Chicago (AMF O'Hare), IL 60666 USA

MICROSTRUCTURAL APPROACH TO LEGUME SEEDS FOR FOOD USES

Kyoko Saio1 and Michiko Monma

National Food Research Institute 2-1 2 Kannondai Tsukuba, lbaraki, Japan 305 1Present address: National Agricu ltural Research Center, 1- 1-3 Kannondai, Tsukuba, Japan 305

Abstract Introduction

This review summarizes the microstructures of The family Leguminosae includes numerous spe several seed legumes based on previous work and some cies and they are widely distributed from the tropic to new findings. Fifteen species of tropically grown leg the arctic regions. Especially in Asian countries, sever umes, adzuki bean and soybeans (a leading variety and al legume seeds have been traditionally used as staple two local va rieties) were examined by light and trans foods and have assumed a role of protein supplements in mission electron microscopy in relation to food uses. the inhabitants' diet. Most legume seeds are starch-rich Processing of adzuki beans to fo rm Q!l bean paste is dis but also richer in protein as compared with cereals. cussed to illustrate the effects of processing on micro Some of them are starch-poor but protein-rich and/or structure of starch g rains. Differences in contents, oil-rich. Historically both types have been cooked and shape and size of starch grains are emphasized in a com manufactured into sophisticated foods in various coun parison of soybeans wi th other legumes. tries ( I , 2).

Materia ls and Methods

Mat erials Tropically grown legume seeds were coll ected by the Tropical Agricultural Research Center, Tsukuba. The kinds of legumes and countries of production are as follows: Benas (Phaseolus vulgaris, Ind ia) , black g ram (Vigna mungo, India), chickpea (Cicerarietinum , India), cowpea (Vigna unguiculata, Indonesia), c lu sterbean (Cyamopsis tetragonoloba, India) , green gram (Vigna Key \Vords: Legume, adzuki, Qll, soybean, light mi radiata, India), hyacinth bean (Lablab purpueus, Indone croscopy, transmission electron microscopy, starch. sia), khesari (Lathyrus sativus, India) , lentil (Lens culinaris, India), pea (Piswn sarivum, Indi a) , pigeonpea (Cajanus cajun, India), riccbean (Vigna umbel/ora, Thai land) , swordbean (Canavalia ensiformis, Indonesia), winged bean (Psophocarpus rerragonolohus , Thailand), and yambean (Pachyrhizus erosus, Thailand). Commercial adzuki beans (spelled azuki in Japa nese, Phaseolus radiarus, Hokkaido, Japan) were used to prepare {lJ! (a Japanese bean paste) particles . Dried fll1. was prepared by the laboratory of Kyoritsu Women ' s University, Tokyo. Fifty grams of adzuki beans were cooked carefully at IOO oc for 90 minutes with 250 ml of water. The hulls were removed after cooling and sepa Initial paper received December 20, 1992 ration of the cooking water. The cotyledons were Manusc ript received July 5, 1993 ground, filtered and freeze-d ried (10, II). Direct inquiries to K. Saio Soybeans (Glycine ma.:c) were cultivated at the Telephone number: 81-298 38 8486 Agricultural Experiment Station of Hyogo prefecture, Fax number: 81-298 38 8484 Wadayama, Japan.

333 K. Saio and M. Monma

Figure I (above). Light micrographs (at same magn ifications) of cowpea (Vigna unguiculara) stained by three meth ods. A: Coomassie Brilli ant Blue (CBB), B: Periodic acid-Schiff (PAS), C : Sudan Bl ack B (SB). Bar in B =50 J

Figure 2 (on t he facing page). Light micrographs (at identical magnifications) of seed leg um es (see F igure I legend for th e identification of staining techniques given in parenthesis). A: Benas (PAS) , B: Black gram (PAS), C: Chick pea (SB), D: Cowpea (PAS), E: Swordbean (SB), F: Green gram (CBB), G: Hyacinth bean (SB), H: Kh esari (CBB), 1: Lentil (CBB), J : Pea (CBB), K : Pigeonpea (SB) , L: Ricebean (CBB), M: Clusterbean (SB), N : Wi nged bean (PAS), 0 : Yam bean (SB), P: Soybean (CBB). Bar in H = SO J

Table I . Characteristics of soybean varieties used.

name weight of total crude crude ripening variety I 00 seeds (g) sugar(%) protein (%) fat(%) period (days) status

En rei 32 20.5 (0.6)' 45 20 75 leading Tanbaguro 55 24 (3)' 45 23 109 local Aomame 36 27 43 25 83 local

av alues in parenthesis are starch coment. Data were analyzed by the Hyogo Prefecture Ex periment Station (1992).

P repara tion of microscopic specimens periodic acid solution ( PA S); and (c) lipids were stained Small pieces (less than 1 x l x 3 mm ) of cotyle with a saturated solution of Sudan Black B in 50% eth donary tissue from each legume seed were cut with a ra anol (7). For transmission electron mi croscopy (TEM, zor blade, fixed with 0 .5 % glutaraldehyde and then I % JEOL SX- 100 or EX-1200), the blocks used for the light osmium tetroxide, dehydrated with a graded acetone ser mic roscope were sliced (about 0.2-0.3 J.Lffi thick, to re ies (40% to 100%) and embedded in Epon or Spurr's tain starch grains in the ti ssues) with a Rei chert-Jung resin . For light mi croscopy (LM), the blocks, prepared Ultratome E (the specimens shown in Figure 10 were as described above, were sliced with an Ultratome and ultrath in slices) and specimens were observed with or affixed onto a glass slide. Three staining techniques without double-staining with saturated uranyl acetate were used: (a) specimens were stained for protein with solution and saturated lead acetate. 0.5% sol ution of Coomassie Brilliant Blue in 7% acetic An. particles were suspended in melted agar at less acid-50% methanol overnight and decolorized with 7% than 40oC and cooled to gelatinize. The coagulated agar acetic acid-50% meth anol ; (b) polysaccharides were gel was cut into small pi eces, which were then fixed, stained with Schiff's reagent after oxidation with 0 .5% dehydrated and embedded in resin .

334 Legume seeds for food use

335 K. Saio and M. Monma

Figure 4 (facing page, top). Transmission electron micrographs of cotyledonary cells of tropical legume seeds. A: Benas, 8 : Cowpea, C: Hyacinth bean, D : Pi geonpea. Bars = l J.Lffi. S: starch granule, CW: cell wall, PB: protein body, arrow: pit-pair.

belong to the genus Vigna, show similar structures, hav ing a number of round , elli psoidal or kidney-shaped starch grains. Ri cebean, however, was a little different , having bigger ellipsoidal starch granules with irregul ar swelling, in spite of being in the same genus. Rather, the shape of ricebean starch granules resembled those in pigeon pea and swordbean which were bigger and fewer in number per cell. Clusterbean , winged bean , yambean and soybean are all protein-rich legumes. Globular materials in yambean and clusterbean appeared to be protein bodies, whereas winged bean and soybean definitely had protein bodies, but all four contained few , if any, starch grains, being different from the starch-rich legumes. Moreover, size and shape of starch grains, when present , in those four beans were quite different. Some of the tropically grown legume seeds had very thick cell walls with pit-pairs, as reported pre viously in winged bean (8). As an exampl e, a li ght mi crograph of green gram is shown in Figure 3. The result s ofT EM examinati on for four tropical ly grown legumes, benas, cowpea, hyacinth bean, and pigeonpea, are shown in Figure 4. T he starch grains, whi ch vary in shape and size, were observed. In the cytoplasmic matrix, fo r example in hyacinth bean and cowpea (Figures 5A and 5B) , many cellular materials, such as vacuoles, protein bodies, and lipid bodies, were F igure 3. Light micrograph of green gram. Bar = 10 observed. Protein bodies were faint in electron density J.Lm. S: starch granule, arrow: pit-pair. and lipid bodies rarely were found adjacent to cell walls, as compared with protein-rich legumes such as soybean and winged bean. The thick cell walls with pit-pairs de Results and Discussion scribed above were often found in tropically grown leg ume seeds. Pit-pairs with plasmodesmata, in benas, are Tropically grown legume seeds shown in Fi gure 6. The mi c rostru cture of tropical leg Figure 1 shows li ght micrographs of cotyledonary ume seeds were reported previously also (3). tissue in cowpea stained by the three methods. Starchy T ropicall y grown leg umes are eate n in diverse cell s of cowpea could be clearly observed by a ll meth food products prepared through processes such as soak ods, where sin gle starch grains rangin g from 10-30 J.Lm ing, cooking , roasting, mashing, milling, fe rmenting, in diameter were distributed. Starch and cell walls were frying , puffing, baking, gel-forming, germ inating, etc., stai ned with PAS . Cytoplasmic matrix was stained with or without decortication. These processing proce faintly with Sudan Black. dures have improved through history for effective use of Since the principal features of legume seed struc the various legume characteristics. The thi ck cell walls ture were observed by any of the three staining methods, often found in these seeds, are digested by fermentation , one of the clearest photographs was selected for each an d some bioactive components, such as trypsin inhibitor legume seed and they are shown in Figure 2. and hemagglutinin , are inactivated by heating. The tra Benas, black gram, chickpea, cowpea, swordbean, ditional use of Phaseolus beans for t.m (bean paste), de green gram, hyacinth bean, khesari, lentil, pea, pigeon scribed in the next paragraph, is an example of a highly pea and ricebean, all have a typical starchy cell structure developed processing method . The seeds of yambean (Figure 2). Although their starch grains are all single, are seldom utilized , although the fresh turnip-like roots instead of compound or aggregated , the shape, size and are used as food ; some legume seeds, pods, and leaves, thei r numbers in a cell vary somewhat depending on the are usually eaten as vegetables in their immature form. species. Black gram, cowpea , and green gram , which Clusterbeans are a source of galactomannan (guar gum).

336 Legume seeds for food use

Figure 5 . Transmission electron micrographs of cytoplasm in cotyledon of tropical legume seeds. Bars = I .urn. A: Hyacinth bean, B: Cowpea. S: starch granule, PB: protein body, V: vacuole, LB: lipid body, CW: cell wall.

337 K. Saio and M. Monma

8 B

·~&f· ~ -1-· . , "-<~ ,,. _ - , I ,, ) Figure 6. Transmission el ectron mi crograph of cell wall in cotyledonary cell of benas. Bar = 1 J.tffi . Arrow: plasmodesmata. Figure 7. Flow sheet of ! RESIDUE QlJ. making. CD ~ SO .\KING I~ / GRINDIN G_ _f __s_ c_REENING lJ Figure 8 . Light micro graphs (at identical LJLEACHING I ~ I GRINDING/---{ DRJF.D AN 1 magnifi cati ons) of Qll particles: A: PAS , 8 : CBB. Bar in B = 10 J.l.ffi. Arrow: artifact shown only in intact starch granule.

100 20

Ul c 0 -o a.l ~ 0 .....>- 0 !!' 50 10 (.) Ol N E ---Ol E

0 40 50 60 days after flow ering

F igure 9. Starch content in developing soybean seeds (cv. Enrei).

338 Legume seeds for food use

Adzuki beans in ruJ. making The traditional use of Phaseolea, such as adzuki bean, for Q11 is popular in Japan. A brief procedure of @ making is shown in Figu re 7. Here, the cooking step is th e most important and distinctly affects the quality of the final product. In order to obtain the characteristic texture of an., it is necessary to convert starch grains in th e cell into the a-form without disruption of cell walls, that is, retaining th e so call ed an. particles. Figure 8 shows Q1L particles, in whi ch swollen starch grains are obse rved, being surrounded by intact cell walls. In so lu bilized and heat-coagul ated protein appears as linear ag gregates th at span several starch grains. According to Watanabe and Kozuma ( 10), such protein inhibits gela tinization of starch , in spite of being easily digestible by gl ucoamylase. Japanese are very familiar with tradition al sweets which use Qll. Local varieties of soybeans More than four hundred varieties and strains of soybeans are said to be grown in Japan , and breeding has been systematically carried out since 1910. The main objectives of breeding for the leading varieties are high yield ab il ity, resistance to diseases and insect pests , and chemi cal composition for food use. On th e oth er hand , local vari eti es remain in culti vation on a sma ll scale in each region, deeply rooted in Japanese regional and traditional dietary life, especiall y confectionari es and cooked beans served at festivals. Such local varie ties often have large sized seeds, beautiful colors (green hull s and someti mes even cotyledons, brown, black and speckled) and are easy to cook. Starch g rains are widely distributed in soybean cotyledonary tissue during ripening, but rapidly decrease in number with maturation , as reported previously (4, 6, 9). A typical pattern of change in starch content is shown in Figure 9. EM changes of starch contents in developing seeds reponed previously (9) are shown in Figure I 0 . Japanese eat immature soybean seeds as a vegetable in the periods when size and numbers of starch ' grain s are almost maximum. In the present experiment, _ 1...,m• ~· microstru ctures of cotyledonary tissue in the three varieties, Tanbaguro, Aomame, and Enrei , were ob served with a T EM. The number of starch grains fou nd in the cotyledonary cell s (Figure II) were in th e order Aomame > Tanb aguro > Enrei. The data in Table I were coll ected in the Ex periment Station where these vari eties were produced (Minamide T, Fujimura K, Hata A, Hikino 1: Stability of green color and processing qualities of green soybeans. Personal communication, 1992). lt appears th at the higher the amounts of total sugar, the greater the number of starch grains that were found in the cells. A few papers have reported that local varieties in Japan conta ined higher amounts of total sug Figure 10. Microstructural changes of plastids (starch ars and/or starc h (5, 13). Generally speakin g, the local granule) in developing soybean (cv. Enrei). Bars in A, varieties have high water absorption capacities, are easy 8 , D, E, and F = 1 p.m . Arrow: dividing point of plas to soften by cooking, and have lower beany flavor with tid. A: 15-20 days after nowering (OAF); 8 : 20-25 a sweet taste. As a result, they are used in many tradi OAF; C : 25-30 OAF; D and E: 35-45 OAF; and F: tional Japanese dishes. 50-55 OAF.

339 K. Saio and M. Monma

Figure II . Transmission electron micrographs of cotyledonary cell of soybeans. A: Aomame, B: Tanbaguro, C: Enrei. Bars = 5 J.Lffi. The magnification in A is smaller th an 8 and C to show th e frequent distribution of plastids. Lipid bodies a re scattered in cell matri x as small and electron dense dots. PB: protein body , S: starch granule.

Table 2 . Classification of individual starch grain s smaller in size and locali zed one to a few in plastids. According to Win ton and Winton ( 12). The starch grains in soybean, as in winged bean, can easily be detected with PAS staining with a short red uc Globular (peanut, floury part of mai ze) tion-time with periodic acid. Finally, legume seeds with higher nutritional Lenticular (wheat, rye, barley) quality than cereal grains, are grown on poorer land and Ellipsoidal (legumes) given less attention, and they have been utilized domes 4 Pear-shaped (potato, cann a, banana) tically and industrially in a great variety of foods. 5 Truncated (cassava, sago) However, most legumes, with th e possible exception of 6 Polygonal (maize, rice, sorghum) soybeans, are still under in vestigation because of a wide 7 Bone-shaped (latex of euphorbia) genetic variation and confusion of the names by coun tries and regions. Comparison or sta rch grains between soybeans a nd Microstructural study of legume seeds by observa the other legumes tion un der light and electron microscopes can clari fy Tabl e 2 shows the classification of starch grain s relationships between approximate composi tions an d in seeds by Winton and Winton ( 12) ; they mentioned that structures (e.g., high sta rch content and prevalence of the shape of leg um e starch grains was ellipsoidal , that starch grains) and reveal physical characte ri stics of im those in Canna were th e largest (170 J.Lm) and those in portance in food appli cation s (e.g., thi ckness of cell ri ce were th e small est (2 - 10 J.Lffi) among th e seeds wh ich wal ls). Such studies are useful in considering how leg they examin ed. In relation to legumes, th ey also stated umes are utilized in traditional foods and also how they that starch grains in co mmon beans reached up to 60 J.Lffi may be applied to no vel foods in th e future. and those in adzuki bean up to 80 J.Lill , but they men Acknowledgements tioned th at soybeans contained only traces of starch. In our experimen ts, starch grains in ch ickpea, green gram , The Tropical Agricultural Research Center , black gram , cowpea, and benas ranged from 5-40 J.l-ffi , Tsukuba, and the Agricultural Experimental Station of relatively smal ler; on the other hand, those in hyacinth Hyogo Prefecture, Wadayama, Hyogo, and Dr. T. bean, pigeon pea, ri cebean and swordbean were relatively Watanabe, Tokyo Metropolitan Food Technological larger, ranging from 15 -80 I'm. Research Center, kindly provided the experimental sam In contrast, the starch grains in soybeans, even ples. We thank Dr. W .J. Wolf, National Center for the ones remaining after harvest, are consid e red to have Agricultural Ut il ization Re search, Peoria, lL, USA, and a temporary storage fu nction , being different fro m the Ms. J-K. Kim , Korea University, Seoul , Korea for assis ones in starch· ri ch leg umes. Soybean starch grains are tance and advice.

340 Legume seeds for food use

References 9. Saio K, Kondo K, Sugimoto T (1985). Changes in typical organelles in developing cotyledons of I . Central Food Technological Research Institute soybeans. Food Microstruct. 4, 191 - 198. (1986). Traditional Foods, Some Products and Techno 10. Watanabe T, Kozuma Y (1982). Studies on logies. Sharada Press , Bangalore, India. red bean Q.!1 (I). Some properties of protein and starch 2. In ternational Crops Research Institute for th e in Qll. Kiyou of Kyoritsu Women 's University, Tokyo, Semi-Arid Tropics ( 199 1). Uses of Tropical Grain Leg Japan 28, 29-39. umes. Proceedin g of a Consultants Meeting (1989). II. WatanabeT, Kozuma Y, Watanabe K (1982). ISBN 92-9066- 180-1, ICR-0005. India. Studies on red bean Q!1 (2). Process of {J.1L particle forma 3. Kim J-K, Saio K (1988). Microstructure prop tion. Kiyou of Kyoritsu Women' s Unive rsity 28, 4 1-50. erties of tropical legume seeds. Korean J. Food Sci. 12. Winton AL, Winton KB (1945). Structure and Techno!. 20, 72-78. Composition of Foods. John Wiley and Sons. New York. 4 . Kondo K, Sugimoto T, Saio K (1986). Changes 13. Yasui T (1985). Dissimilarity in low molecu in storage protein components of developing soybean lar weight carbohydrate composition of the seeds of cul seeds (Glycine max var. Enrci). Agric. Bioi. Chern. 50, ti vated soybean and wild soybean. Ag ric. Bioi. Chern. 581 -587. 49, 933-937. 5. Miyazaki S , Yagasaki K, Yasui T (1985). The rapid determination of starch in soybean seeds with io Discussion with Reviewers dine-starc h staining. Jpn . J. Crop Sci. 54, 177-180. 6. Mo nnma M, Sugimoto T, Monnma M, Kawa Editor: Please explai n the use of rather thick sections mura Y , Saio K (1990). Starch breakdown in developing "about 0 .2-0.3 Jlm thick, to retain starch grains in the soybean seeds (Glycine ma.t cv. Enrei). Agric. Bioi. ti ssues" for TEM examination. Chern . 55 , 67-71. Authors: If we prepare ultrathin slices, the big starch 7. Saio K ( 198 1) . Mi crostructure of traditional grains in the cytoplasmic matrix, whi ch are quite diffi Japanese soybean foods. Scanning Electron Microsc. c ult to fix with any regent, drop out during cutting with 1981 ; Ill : 553-559. Ultratome resulting in a section with many holes. T his 8. Saio K, Suzuki H, Kobayashi T, Namikawa M is why we prepared thicker sections, although they do (1984) . Mi c rostruc tural changes in winged bean and not give beautiful micrographs under TEM. soybean during fermentation into miso. Food Mi c ro struct. 3 , 65-71.

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