APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1976, p. 284-287Copyright C) 1976 American Society for Microbiology

Vol. 32, No. 2Printed in U.S.A.

Penetration ofRhizopus oligosporus into Soybeans in TempehALAN M. JURUS AND WALTER J. SUNDBERG*

Department ofBotany, Southern Illinois University, Carbondale, Illinois 62901

Received for publication 19 March 1976

Histological observations were made on the penetration ofhyphae ofRhizopusoligosporus into soybean cotyledons in tempeh, an Indonesian soybean food.Hyphal penetrations averaged one per 1,400 ,um2 (±+390 /tm-) on the curved(outer) cotyledon surface and one per 1,010 ,tm2 (+340 ,um2) on the flat (inner)one. Hyphae infiltrated to a depth of 742 ,um, or about 25% of the average widthof a soybean cotyledon. This previously unreported degree of penetration offerspartial explanation for the rapid physical and chemical changes in soybeansduring tempeh fermentation.

Tempeh is an Indonesian fermented food con-sisting of soybeans partially digested andbound together by mycelium of Rhizopus. Al-though other species are sometimes encoun-tered, Rhizopus oligosporus Saito is most fre-quently isolated from natural tempeh samples(5).

Investigations designed to determine the na-ture of tempeh and reasons for its improvedorganoleptic qualities over unfermented soy-beans have primarily centered on manufactureof the food (2, 4, 9, 12), isolation of the mostproductive species of Rhizopus (5), and bio-chemical analysis (4, 13-15). Only Boorsma(cited in reference 14), van Veen and Schaefer(14), and Steinkraus et al. (13) have made anyhistological observations.Boorsma (see reference 14) reportedly found

that the hyphae penetrated the bean cells andliberated their contents. However, he noted lit-tle or no dissolution of the soybean cell wallsand concluded that their mechanical resistancewas not affected by the fungus. van Veen andSchaefer (14) and Steinkraus et al. (13) ob-served that the fungus seemingly penetratedonly a few superficial layers of cells, surround-ing these cells with a network of mycelium. Thecurved outer side of the cotyledon was appar-ently more easily perforated by the myceliumthan was the flat inner side (14). Based on theseand available biochemical data, it was con-cluded that changes occurring during fermenta-tion resulted from deep penetration of enzymesvia diffusion from the more or less superficialhyphae (13, 14).Contrary to observations noted above, our

preliminary investigations showed deep pene-tration of fungal hyphae into the soybean coty-ledons. Further, apparently no information ex-ists regarding the frequency of penetration by

the fungus. Therefore, this anatomical investi-gation was undertaken to determine both theapproximate frequency of surface hyphal pene-tration and the depth of penetration by R. oli-gosporus into the soybean cotyledon in tempeh.

MATERIALS AND METHODS

Tempeh manufacture. The laboratory method oftempeh production (modified from reference 9) wasused. Williams variety soybeans (50 g) were soakedin 150 ml of tap water for 24 h. After dehulling,boiling, and cooling them to room temperature, thebeans were inoculated with R. oligosporus (South-ern Illinois University Culture Collection no. Z-3,originally obtained from C. W. Hesseltine, NorthernRegional Research Laboratory, Peoria, Ill.) by drop-ping 4 to 7 ml of a spore suspension prepared byadding 10 ml of distilled water to a sporulating maltagar slant culture. The soybeans were hand-mixedto further disperse the spores and were placed inthin plastic bags perforated once every square centi-meter with a sterile sewing needle for proper aera-tion. Bags of inoculated beans were incubated in aglass bowl, also containing a small beaker of waterto maintain high humidity, and covered with thinplastic film (food wrap). Fermentation was allowedto progress for 80 h at 25°C (9), after which themycelium appeared to be more or less uniformlydistributed throughout the resulting approximately5-cm-thick cake.

Microtechnique. Since information was lackingon the best fixatives for cytological preservation oftempeh and ultimate differentiation of the fungus,the following fixatives were initially used: formalin-acetic acid-alcohol (6), chromic sulfate-formalde-hyde-copper hydroxide and chromic sulfate-formal-dehyde-picric acid (1), CRAF III (10), chromic-aceticacid (6), and 3% glutaraldehyde in phosphate bufferat pH 7.2. In all cases, control (cooked and dehulled,but unfermented) soybeans and slices of tempehwere fixed for 48 h.

Although chromic sulfate-formaldehyde-copperhydroxide and formalin-acetic acid-alcohol produced

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RHIZOPUS IN TEMPEH 285

well-preserved tissue, 3% glutaraldehyde fixationproduced the best cytoplastic detail, particularlydemonstrating the presence and distribution of vac-uoles. However, best fungal differentiation afterstaining was obtained with chromic sulfate-formal-dehyde-copper hydroxide- and formalin-acetic acid-alcohol-fixed materials, so these fixatives were usedthroughout the rest of the study.

All fixed materials were dehydrated in tertiarybutyl alcohol, embedded in paraffin, sectioned, andmounted on slides using standard procedures (6).

Preliminary staining procedures tested includedsafranin and fast green (6), Johansen's quadruplestain (7), Stoughton's thionin and erythrosin-or-ange G (8), Stoughton's thionin and orange G (3),methyl violet and eosin (Ikata, 1932, cited in refer-ence 7), modified pianese IIIb (11), chlorazol blackE and pianese IIIb (17), and progressive iron hema-toxylin (modified from reference 10) counterstainedwith orange G. Clearest hyphal differentiation wasobtained with Stoughton's thionin and orange Gand with progressive iron hematoxylin and orangeG, which were subsequently used on all other fixedmaterials.Data collection. Anatomical observations were

made on several beans randomly selected from dif-ferent slices of each of two final tempeh batches.The number of penetrations over the cotyledon

surface area was determined as follows. The lengthofthe surface area examined was delineated with aneyepiece micrometer. Since each section observedwas approximately 10 um thick (plus or minus toler-ance limits of the microtome), an approximation ofsurface area was made from these measurements.Because the maximum observed hyphal diameterwas 26 ,um, the same hypha theoretically could beobserved in four consecutive 10-,um-thick sections atmost. Therefore, to assure that a penetrating hyphalsegment found in one section would not be countedas a penetration in another, every fifth serial sectionwas used to obtain surface penetration frequencydata. Means of penetration frequency on the curvedand flat cotyledon surfaces were compared usingStudent's t test (16).

The depth of maximum hyphal penetration wasdetermined by direct measurement with an eyepiecemicrometer. Subsequently, the percentage of maxi-mum mycelial penetration into the cotyledons wascalculated by using these data and the averagewidth of control soybeans.

RESULTSThe walls and cytoplasm of the soybean cells

and hyphae were all distinct. In sectionsstained with Stoughton's thionin and orangeG, the soybean cell walls and cytoplasm werelight brown and greenish blue, respectively.Although the fungal cell walls were similar incolor to those of the soybean cells, the fungalcytoplasm consistently contained numerous,variously sized, violet-stained granules, thusmaking the fungal hyphal segments easily dis-tinguishable (Fig. 1, 2, and 4-6). Progressive

iron hematoxylin and orange G resulted insimilar structural differentiation, although thecolor differences obtained were not as striking.The degree of distortion caused by the fun-

gus, lacking in control preparations (Fig. 3),was most severe at or near the cotyledon sur-face (Fig. 6). Portions of the outermost celllayers of the cotyledon were often completelypermeated with mycelium, creating an indis-tinct mass. Walls of these cells were shriveled,the cytoplasm was very distorted, and fre-quently entire cells were so grossly disruptedthat they were no longer recognizable. Fewerhyphae and little or no distortion were observedamong the inner cellular layers of the cotyle-don.

Penetration and growth of the hyphae, whichgrew only between the cylindrical cotyledoncells, were generally inwardly directed and per-pendicular to the cotyledon surface (Fig. 1, 2,and 5). Hyphal width decreased as the distanceof penetration increased. Little or no lateralgrowth was observed, with the exception of oc-casional lateral branches of acute-angled origin(Fig. 4). Whether the fungus penetrated the cellwall and grew along the region of the middlelamella or grew only within intercellularspaces already present could not be determinedwith certainty. No haustoria were observed.The sample size and the number and fre-

quency of hyphal surface penetrations encoun-tered, as illustrated in Fig. 1, 2, and 4 through6, are listed in Table 1. To summarize, therewas one hyphal invasion per 1,400 am' (+390,um2) on the outer curved surface of the soybeancotyledon. The inner flat side was penetratedonce every 1,010 ,Um2 (+340 /Im2).A large number of hyphae infiltrated 300 to

500 ,um into the cotyledons (Fig. 1 and 2), adistance equivalent to 10 to 17% of their aver-age width. Fewer hyphae penetrated to a depthof over 600 ,um. The maximum observed hyphalpenetration was 742 ,tm, or approximately 25%of the average cotyledon width.

DISCUSSIONThe deeper penetration demonstrated herein

clearly illustrates a closer physical relationshipbetween the hyphae and the inner cells of thesoybean cotyledon than previously reported.Reasons for this discrepancy are unclear, butthe use of a higher fermentation temperatureand shorter incubation time by previous work-ers (14) might partially explain the observeddifference. Unfortunately, lack of explicit tem-perature data on fermentation used for histo-logical studies by Steinkraus et al. (13) makescorrelation difficult. Additionally, utilization of

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FIG. 1-6. Sections oftempeh and control soybeans at right angles to the bean surface. The soybean surface,and superficial hyphae where present, are oriented toward the top ofeach figure. Scale lines equal 100 and 50,um in Fig. 1-3 and 4-6, respectively. (Fig. 1-2) Sections showing surface hyphal penetrations (white arrows)and depth of hyphal intrusion (black arrows) into soybeans. (Fig. 3) Section of control soybean. (Fig. 4-6)Surface penetrations of soybeans by hyphae (white arrows). Note the acute-angled hyphal branch within thesoybean tissue (black arrows), the intercellular nature of the hyphae in the soybean (black arrows), and thedistortion of bean cell walls (black arrows) in Fig. 4, 5, and 6, respectively.

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RHIZOPUS IN TEMPEH 287

TABLE 1. Sample size and number and frequency ofhyphal penetrations

Curved Flat innerData outer cotyle- cotyledon

don surface surface

Number of samples 39 20Total surface area exam- 2,282,700 232,760

ined (Am2)Number of surface pene- 1,634 230

trations observedMean area per penetra- 1,400 1,010

tion (/mM2)Standard deviation (Am2) 390 340

several different fixatives and staining tech-niques during this study may have producedclearer differentiation, which, in turn, resultedin recognition of the fungus in the deeper layersof the cotyledon where the hyphae become quitethin. Finally, the attention given to orientationof the tempeh samples and resultant selectionof slides with optimal orientation quality mayalso have more clearly demonstrated the pres-

ence of hyphae.The extreme depth of observed mycelial infil-

trations at least partially explains the rapidphysical and chemical changes previouslynoted during tempeh manufacture. First, thehyphae may soften the soybeans by mechani-cally pushing the cells apart prior to, or inconjunction with, enzymatic degradation. Sec-ond, the deep penetration of enzymatic activity(13, 14) is probably enhanced since the distanceover which diffusion of exoenzymes must occuris greatly reduced.These results demonstrate a higher, statisti-

cally significant (P 2 0.01) frequency of pene-tration on the flat cotyledon surface. This is incontrast to the data of van Veen and Schaefer(14), whose qualitative results suggested thatthe curved cotyledon surface was more easilyperforated. However, these workers may nothave separated the two cotyledons of each seed,thus potentially resulting both in less exposureof the flat inner surface to the inoculum andlower aeration of that surface. Due to the lackof numerical data on the frequency of penetra-tion by the hyphae in van Veen and Schaefer's(14) work, quantitative comparison is not possi-ble.

Previous studies on optimal fermentationtime and temperature requirements for tempehmanufacture (5, 12) were concerned with physi-ological and organoleptic changes occurringduring the incubation period. The resultsherein suggest that histological data on fre-quency and depth of penetration might be addi-tional parameters for determination of timeand temperature optima.

LITERATURE CITED

1. Cohen, I., and K. D. Doak. 1935. The fixing and stain-ing of Liriodendron tulipifera root tips and theirmycorrhizal fungus. Stain Technol. 10:25-32.

2. Djien, K. S., and C. W. Hesseltine. 1961. Indonesianfermented foods. Soybean Dig. 22:14-15.

3. Gurr, E. 1956. A practical manual of medical and bio-logical staining techniques, 2nd ed. Interscience Pub-lishers, Inc., New York.

4. Hesseltine, C. W. 1965. A millenium of fungi, food andfermentation. Mycologia 57:149-197.

5. Hesseltine, C. W., M. Smith, B. Bradle, and K. S.Djien. 1963. Investigations of tempeh, an Indonesianfood. Dev. Ind. Microbiol. 4:275-287.

6. Jensen, W. A. 1962. Botanical histochemistry. W. H.Freeman and Co., San Francisco.

7. Johansen, D. A. 1940. Plant microtechnique. McGraw-Hill Book Co., Inc., New York.

8. Margolena, L. 1932. Erythrosin for Stoughton's ThioninOrange G. Stain Technol. 5:25-26.

9. Martinelli, A. F., and C. W. Hesseltine. 1964. Tempehfermentation: package and tray fermentations. FoodTechnol. 18:167-171.

10. Sass, J. D. 1958. Botanical microtechnique, 3rd ed. IowaState University Press, Ames.

11. Simmons, S. A., and R. A. Shoemaker. 1952. Differen-tial staining offungus and host cells using a modifica-tion of Pianeze IlIb. Stain Technol. 27:121.

12. Steinkraus, K. H., D. B. Hand, J. P. Van Buren, and L.R. Hackler. 1961. Pilot plant studies on tempeh, p.83-92. In Proceedings of conference on soybean prod-ucts for protein in human foods. U.S. Department ofAgriculture, Agricultural Research Service 71-22,Washington, D.C.

13. Steinkraus, K. H., Y. B. Hwa, J. P. Van Buren, M. I.Provvidenti, and D. B. Hand. 1960. Studies on tem-peh-an Indonesian fermented soybean food. FoodRes. 25:777-788.

14. van Veen, A. G., and G. Schaefer. 1950. The influenceof the tempeh fungus on the soya bean. Doc. Neerl.Indones. Morbis Trop. 2:270-281.

15. Wagenknecht, A. G., L. R. Mattick, L. M. Lewin, D. B.Hand, and K. H. Steinkraus. 1961. Changes in soy-bean lipids during tempeh fermentation. J. Food Sci.26:373-376.

16. Weinberg, G. H., and J. A. Schumaker. 1969. Statistics:an intuitive approach, 2nd ed. Brooks/Cole Publish-ing Co., Belmont, Calif.

17. Wilcox, H. E. 1964. Staining plant tissues with Chlora-zol Black and Pianese III-B. Stain Technol. 39:81-86.

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