Sunteți pe pagina 1din 4

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1976, p.

284-287
Copyright C) 1976 American Society for Microbiology

Vol. 32, No. 2


Printed in U.S.A.

Penetration of Rhizopus oligosporus into Soybeans in Tempeh


ALAN M. JURUS AND WALTER J. SUNDBERG*
Department of Botany, Southern Illinois University, Carbondale, Illinois 62901

Received for publication 19 March 1976

Histological observations were made on the penetration of hyphae of Rhizopus


oligosporus 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 width
of a soybean cotyledon. This previously unreported degree of penetration offers
partial explanation for the rapid physical and chemical changes in soybeans
during tempeh fermentation.
Tempeh is an Indonesian fermented food consisting of soybeans partially digested and
bound together by mycelium of Rhizopus. Although other species are sometimes encountered, Rhizopus oligosporus Saito is most frequently isolated from natural tempeh samples
(5).
Investigations designed to determine the nature of tempeh and reasons for its improved
organoleptic qualities over unfermented soybeans have primarily centered on manufacture
of the food (2, 4, 9, 12), isolation of the most
productive species of Rhizopus (5), and biochemical analysis (4, 13-15). Only Boorsma
(cited in reference 14), van Veen and Schaefer
(14), and Steinkraus et al. (13) have made any
histological observations.
Boorsma (see reference 14) reportedly found
that the hyphae penetrated the bean cells and
liberated their contents. However, he noted little or no dissolution of the soybean cell walls
and concluded that their mechanical resistance
was not affected by the fungus. van Veen and
Schaefer (14) and Steinkraus et al. (13) observed that the fungus seemingly penetrated
only a few superficial layers of cells, surrounding these cells with a network of mycelium. The
curved outer side of the cotyledon was apparently more easily perforated by the mycelium
than was the flat inner side (14). Based on these
and available biochemical data, it was concluded that changes occurring during fermentation resulted from deep penetration of enzymes
via diffusion from the more or less superficial
hyphae (13, 14).
Contrary to observations noted above, our
preliminary investigations showed deep penetration of fungal hyphae into the soybean cotyledons. Further, apparently no information exists regarding the frequency of penetration by

the fungus. Therefore, this anatomical investigation was undertaken to determine both the
approximate frequency of surface hyphal penetration and the depth of penetration by R. oligosporus into the soybean cotyledon in tempeh.
MATERIALS AND METHODS
Tempeh manufacture. The laboratory method of
tempeh production (modified from reference 9) was
used. Williams variety soybeans (50 g) were soaked
in 150 ml of tap water for 24 h. After dehulling,
boiling, and cooling them to room temperature, the
beans were inoculated with R. oligosporus (Southern Illinois University Culture Collection no. Z-3,
originally obtained from C. W. Hesseltine, Northern
Regional Research Laboratory, Peoria, Ill.) by dropping 4 to 7 ml of a spore suspension prepared by
adding 10 ml of distilled water to a sporulating malt
agar slant culture. The soybeans were hand-mixed
to further disperse the spores and were placed in
thin plastic bags perforated once every square centimeter with a sterile sewing needle for proper aeration. Bags of inoculated beans were incubated in a
glass bowl, also containing a small beaker of water
to maintain high humidity, and covered with thin
plastic film (food wrap). Fermentation was allowed
to progress for 80 h at 25C (9), after which the
mycelium appeared to be more or less uniformly
distributed throughout the resulting approximately
5-cm-thick cake.
Microtechnique. Since information was lacking
on the best fixatives for cytological preservation of
tempeh and ultimate differentiation of the fungus,
the following fixatives were initially used: formalinacetic acid-alcohol (6), chromic sulfate-formaldehyde-copper hydroxide and chromic sulfate-formaldehyde-picric acid (1), CRAF III (10), chromic-acetic
acid (6), and 3% glutaraldehyde in phosphate buffer
at pH 7.2. In all cases, control (cooked and dehulled,
but unfermented) soybeans and slices of tempeh
were fixed for 48 h.
Although chromic sulfate-formaldehyde-copper
hydroxide and formalin-acetic acid-alcohol produced
284

VOL. 32, 1976

well-preserved tissue, 3% glutaraldehyde fixation


produced the best cytoplastic detail, particularly
demonstrating the presence and distribution of vacuoles. However, best fungal differentiation after
staining was obtained with chromic sulfate-formaldehyde-copper hydroxide- and formalin-acetic acidalcohol-fixed materials, so these fixatives were used
throughout the rest of the study.
All fixed materials were dehydrated in tertiary
butyl alcohol, embedded in paraffin, sectioned, and
mounted on slides using standard procedures (6).
Preliminary staining procedures tested included
safranin and fast green (6), Johansen's quadruple
stain (7), Stoughton's thionin and erythrosin-orange G (8), Stoughton's thionin and orange G (3),
methyl violet and eosin (Ikata, 1932, cited in reference 7), modified pianese IIIb (11), chlorazol black
E and pianese IIIb (17), and progressive iron hematoxylin (modified from reference 10) counterstained
with orange G. Clearest hyphal differentiation was
obtained with Stoughton's thionin and orange G
and with progressive iron hematoxylin and orange
G, which were subsequently used on all other fixed
materials.
Data collection. Anatomical observations were
made on several beans randomly selected from different slices of each of two final tempeh batches.
The number of penetrations over the cotyledon
surface area was determined as follows. The length
of the surface area examined was delineated with an
eyepiece micrometer. Since each section observed
was approximately 10 um thick (plus or minus tolerance limits of the microtome), an approximation of
surface area was made from these measurements.
Because the maximum observed hyphal diameter
was 26 ,um, the same hypha theoretically could be
observed in four consecutive 10-,um-thick sections at
most. Therefore, to assure that a penetrating hyphal
segment found in one section would not be counted
as a penetration in another, every fifth serial section
was used to obtain surface penetration frequency
data. Means of penetration frequency on the curved
and flat cotyledon surfaces were compared using
Student's t test (16).
The depth of maximum hyphal penetration was
determined by direct measurement with an eyepiece
micrometer. Subsequently, the percentage of maximum mycelial penetration into the cotyledons was
calculated by using these data and the average
width of control soybeans.

RESULTS
The walls and cytoplasm of the soybean cells
and hyphae were all distinct. In sections
stained with Stoughton's thionin and orange
G, the soybean cell walls and cytoplasm were
light brown and greenish blue, respectively.
Although the fungal cell walls were similar in
color to those of the soybean cells, the fungal
cytoplasm consistently contained numerous,
variously sized, violet-stained granules, thus
making the fungal hyphal segments easily distinguishable (Fig. 1, 2, and 4-6). Progressive

RHIZOPUS IN TEMPEH

285

iron hematoxylin and orange G resulted in


similar structural differentiation, although the
color differences obtained were not as striking.
The degree of distortion caused by the fungus, lacking in control preparations (Fig. 3),
was most severe at or near the cotyledon surface (Fig. 6). Portions of the outermost cell
layers of the cotyledon were often completely
permeated with mycelium, creating an indistinct mass. Walls of these cells were shriveled,
the cytoplasm was very distorted, and frequently entire cells were so grossly disrupted
that they were no longer recognizable. Fewer
hyphae and little or no distortion were observed
among the inner cellular layers of the cotyledon.
Penetration and growth of the hyphae, which
grew only between the cylindrical cotyledon
cells, were generally inwardly directed and perpendicular to the cotyledon surface (Fig. 1, 2,
and 5). Hyphal width decreased as the distance
of penetration increased. Little or no lateral
growth was observed, with the exception of occasional lateral branches of acute-angled origin
(Fig. 4). Whether the fungus penetrated the cell
wall and grew along the region of the middle
lamella or grew only within intercellular
spaces already present could not be determined
with certainty. No haustoria were observed.
The sample size and the number and frequency of hyphal surface penetrations encountered, as illustrated in Fig. 1, 2, and 4 through
6, are listed in Table 1. To summarize, there
was one hyphal invasion per 1,400 am' (+390
,um2) on the outer curved surface of the soybean
cotyledon. The inner flat side was penetrated
once every 1,010 ,Um2 (+340 /Im2).
A large number of hyphae infiltrated 300 to
500 ,um into the cotyledons (Fig. 1 and 2), a
distance equivalent to 10 to 17% of their average width. Fewer hyphae penetrated to a depth
of over 600 ,um. The maximum observed hyphal
penetration was 742 ,tm, or approximately 25%
of the average cotyledon width.

DISCUSSION
The deeper penetration demonstrated herein
clearly illustrates a closer physical relationship
between the hyphae and the inner cells of the
soybean cotyledon than previously reported.
Reasons for this discrepancy are unclear, but
the use of a higher fermentation temperature
and shorter incubation time by previous workers (14) might partially explain the observed
difference. Unfortunately, lack of explicit temperature data on fermentation used for histological studies by Steinkraus et al. (13) makes
correlation difficult. Additionally, utilization of

FIG. 1-6. Sections of tempeh and control soybeans at right angles to the bean surface. The soybean surface,
and superficial hyphae where present, are oriented toward the top of each 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 the
soybean tissue (black arrows), the intercellular nature of the hyphae in the soybean (black arrows), and the
distortion of bean cell walls (black arrows) in Fig. 4, 5, and 6, respectively.
286

VOL. 32, 1976

RHIZOPUS IN TEMPEH

TABLE 1. Sample size and number and frequency of


hyphal penetrations
Data

Number of samples
Total surface area examined (Am2)
Number of surface penetrations observed
Mean area per penetration (/mM2)
Standard deviation (Am2)

Curved
outer cotyledon surface

39
2,282,700

Flat inner
cotyledon
surface
20

232,760

1,634

230

1,400

1,010

390

340

several different fixatives and staining techniques during this study may have produced
clearer differentiation, which, in turn, resulted
in recognition of the fungus in the deeper layers
of the cotyledon where the hyphae become quite
thin. Finally, the attention given to orientation
of the tempeh samples and resultant selection
of slides with optimal orientation quality may
also have more clearly demonstrated the presence of hyphae.
The extreme depth of observed mycelial infiltrations at least partially explains the rapid
physical and chemical changes previously
noted during tempeh manufacture. First, the
hyphae may soften the soybeans by mechanically pushing the cells apart prior to, or in
conjunction with, enzymatic degradation. Second, the deep penetration of enzymatic activity
(13, 14) is probably enhanced since the distance
over which diffusion of exoenzymes must occur
is greatly reduced.
These results demonstrate a higher, statistically significant (P 2 0.01) frequency of penetration on the flat cotyledon surface. This is in
contrast to the data of van Veen and Schaefer
(14), whose qualitative results suggested that
the curved cotyledon surface was more easily
perforated. However, these workers may not
have separated the two cotyledons of each seed,
thus potentially resulting both in less exposure
of the flat inner surface to the inoculum and
lower aeration of that surface. Due to the lack
of numerical data on the frequency of penetration by the hyphae in van Veen and Schaefer's
(14) work, quantitative comparison is not possi-

ble.

287

Previous studies on optimal fermentation


time and temperature requirements for tempeh
manufacture (5, 12) were concerned with physiological and organoleptic changes occurring
during the incubation period. The results
herein suggest that histological data on frequency and depth of penetration might be additional parameters for determination of time
and temperature optima.
LITERATURE CITED
1. Cohen, I., and K. D. Doak. 1935. The fixing and staining of Liriodendron tulipifera root tips and their
mycorrhizal fungus. Stain Technol. 10:25-32.
2. Djien, K. S., and C. W. Hesseltine. 1961. Indonesian
fermented foods. Soybean Dig. 22:14-15.
3. Gurr, E. 1956. A practical manual of medical and biological staining techniques, 2nd ed. Interscience Publishers, Inc., New York.
4. Hesseltine, C. W. 1965. A millenium of fungi, food and
fermentation. Mycologia 57:149-197.
5. Hesseltine, C. W., M. Smith, B. Bradle, and K. S.
Djien. 1963. Investigations of tempeh, an Indonesian
food. 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. McGrawHill Book Co., Inc., New York.
8. Margolena, L. 1932. Erythrosin for Stoughton's Thionin
Orange G. Stain Technol. 5:25-26.
9. Martinelli, A. F., and C. W. Hesseltine. 1964. Tempeh
fermentation: package and tray fermentations. Food
Technol. 18:167-171.
10. Sass, J. D. 1958. Botanical microtechnique, 3rd ed. Iowa
State University Press, Ames.
11. Simmons, S. A., and R. A. Shoemaker. 1952. Differential staining of fungus and host cells using a modification 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 products for protein in human foods. U.S. Department of
Agriculture, 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 tempeh-an Indonesian fermented soybean food. Food
Res. 25:777-788.
14. van Veen, A. G., and G. Schaefer. 1950. The influence
of 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 soybean 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 Publishing Co., Belmont, Calif.
17. Wilcox, H. E. 1964. Staining plant tissues with Chlorazol Black and Pianese III-B. Stain Technol. 39:81-86.

S-ar putea să vă placă și