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Production of Tempeh from various indigenous
Ethiopian legumes
Article in World Journal of Microbiology and Biotechnology · January 1991
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2 authors:
Mogessie Ashenafi
Martin Busse
College of Development Studies, Addis Abab…
Coburg University of Applied Sciences and Arts
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World Journal of Microbiology and Biotechnology 7,72-79
Production of tempeh from various
indigenous Ethiopian beans
M. Ashenafi and M. Busse
Tflmpeh was prepared from unacidifled and
acidified horse-bean, pea, chickpea and
soybean. At the completion of fermentation,
the various beans were compactly bound
together by white mycelia, had a bland or
yeast-like odour and a firm texture that
sliced readily. Weight Increase was noted
during soaking (75 to 80%) and fermentation
resulted In a slight decrease In weight (1.4
to 5.4%). A sharp increase in pH and
temperature was noted following active
mycelial growth on the bean mass. Fermen­
tation resulted in loss of dry maHer. Only
slight changes were observed In total crude
protein. Water-soluble solids and water­
soluble proteins Increased by more than 60
and 40%, respectively. In sensory evalu­
ation tests, sauces made of the various
products compared favourably with sauces
made of meat or egg. Acceptable and
nutritious tempeh can, thus, be produced
from Indigenous Ethiopian beans.
M, Ashenafi is with the Department of Basic
SCiences. Awassa College of Agriculture,
Addis Ababa University. P,O, Box 5,
Awassa. Ethiopia; M, Busse is with. the
Bakteriologisches Institut, S,V.F,A,. Weihen­
stephan. Technische Universital Munchen,
0-8050 Freising, Germany. M. Ashenafi is
the corresponding author,
•
©
1991 Rapid Communications of Oxford Ltd,
The use of microorganisms to process foods goes back to ancient times, Ferment~d
foods are essential parts of diets in all regions of the world, A number of food
fermentation processes, including those that yield dairy products, sausages, pickles,
sauerkraut and bread have been extensively investigated and documented. But
many other foods prepared by the action of diverse species of fungi, bacteria and
yeasts on plant materials are little known outside their native countries (Hesseltine
& Wang 1980).
Food fermentation processes have several advantages, such as improving
keeping quality and flavour (Hesseltine 1983), and addition of desirable nutrients
not present in the original product (Steinkraus 1983a). Typically, fermentation
does not greatly alter the amino acids in cereals and soybeans, but often it makes
proteins more available (Au & Fields 1981). In many fermentations using
soybeans, undesirable beany flavours are destroyed (Van Veen & Steinkraus 1970),
trypsin inhibitor is inactivated (Hesseltine 1983) and flatulence factors are elimi­
nated (David & Verma 1981). Finally, in many instances, the use of microorgan­
isms to produce tasty, nutritious food requires less energy than conventional
processing (Steinkraus 1983b).
One of the several methods of legume fermentation that generate meat-like
flavours and improve nutritional value is the tempeh process where partially cooked
soybean cotyledons are knitted into a compact cake that can be used as protein-rich
meat substitutes in soups. Indonesian timpeh offers the world a process of making
low-cost protein-rich meat analogues of excellent nutritional quality (Steinkraus
1983b). Tempeh has been suggested as a possible source of low-cost protein for
child-feeding programs in developing countries (Autret & Van Veen 1955).
Although the most important and popular typ~ of tunpeh is made from soybeans,
it may also be made from a variety of other substrates (Djien & Hesseltine 1979).
The diet of the average Ethiopian consists of only a limited number of foods.
,For the most part, these are based on cereals supplemented, in certain cases, by
a stew made of mainly pulses. Although the protein content of these legumes is
about 20 to 25%, the traditional processing involved in preparing the stew
decreases the protein content down to 14 to 17% (Agren et al. 1987).
Thus it was attempted, in this study, to prepare tetnpeh from some of the legumes
commonly consumed in Ethiopia, namely horse-bean, pea and chickpea, evaluate
72
'-~--------------~....- -.. - -...- -.. - -...
Tempeh from Ethiopian beans
its physical properties and nutritional quality and assess its acceptability if
introduced into the country.
Materials and Methods
Preparation of Tempeh from the ~Various Legumes
Tempeh was prepared following the methods of Steinkraus et al. (1965) with some
modifications as described by Ashenafi & Busse (1989). For every experiment,
200 g hull-free, cracked horse-bean, pea or chickpea seeds were thoroughly washed
with hot water and soaked in 600 ml tap water at 30°C for 24 h in 1000 ml beakers.
Equal amounts of the various beans were also processed in a similar way, except
that 1.5 ml of glacial acetic acid was added to the soak water. Soaking was at
30°C for 24 h. Soybeans were processed similarly for comparative purposes. The
soaked beans were then cooked in the soak water at 100 c C for varying periods
(see Table 1).
After cooking, the beans were drained while still hot and immediately spread
on a layer of sterile cloth and covered with another layer of sterile cloth, thus
giving them a fairly dry surface. The beans were allowed to cool to about 37°C
and mixed with spores of Rhizopus oligosporus (CBS 338.62) to give 1.2 x 106
spores/100 g cooked beans. R. oligosporus cultures were obtained from Centraalbur­
eau voor Schimmelcultures, The Netherlands.
Spores of R. oligosporus were harvested with 5 ml sterile water from a growth
on slants of 4% malt extract/0.5% peptone/1.5% agar, at pH 5.4 after incubation
at 30°C for 5 days.
Inoculated beans, 200 g, were packed into thin polyethylene bags which had
perforations, made with a sewing needle, at 1 cm intervals on both sides (Djien
& Hesseltine 1979). The bags were sealed and incubated at 30°C for about 35 to
40 h until the beans were tightly bound together into a compact cake by white
mycelia. On completion of fermentation, the extent of Rhizopus growth on
the beans and the odour and texture of the fermented products were evaluated.
Determination of Temperature and pH
The temperature of the fermenting beans was measured by placing the probe of
a digital temperature measuring device (THERM 2283-2, West Germany) inside
Table 1. Changes in weight during the different steps of tempeh processing from various
beans.
Weight (g)
Substrate
----Dry beans
Soaked and
cooked beans
Tempeh
Horse-bean
Unacidified
Acidified
200
200
372 (86%)
352 (79%)
352 (-5%)
344 (-4%)
Pea
Unacidified
Acidified
200
200
371 (86%)
365 (83%)
364 (-2%)
360 (-1%)
Chickpea
Unacidified
Acidified
200
200
359 (80%)
350 (75%)
344 (-4%)
340 (-3%)
Soybean
Unacidified
Acidified
200
200
407 (104%)
408 (104%)
400 (-2%)
396 (-3%)
Figures in parentheses indicate percentage increase or decrease in weight.
73
M. A shenaji and M. Busse
the fermenting bean mass. The pH of the samples was measured by placing the
electrode of the pH meter into a slurry containing 1 in 5 dilution of the sample
in sterile water.
Proximate Ana!ysis
Raw dehulled horse-bean, pea, chickpea and soybean and tempeh made from the
various beans were analysed for dry matter, crude protein, crude fat, crude fibre,
ash, nitrogen-free extracts, soluble proteins and soluble solids, as described by
Krishina & Ranjhan (1980).
Sensory Evaluation
The various fermented products were cut into small pieces and separately cooked
into wOf, a traditional Ethiopian hot spiced stew. A total of 100 students of A wassa
College of Agriculture were requested to evaluate the odour, flavour and overall
acceptability of the stew by sampling it with enjerra, a traditional Ethiopian pancake
made of flours of Eragrostis tef. A nine-point hedonic scale was used, ranging
from 'like extremely' (9 points) to 'dislike extremely' (1 point). The results were
analysed using analysis of variance, and where two preparations were evaluated,
the mean scores were compared using the t-test (Ihekoronye & Ngoddy 1985).
Results
Physical Properties of the Various Products
On completion of fermentation, the cake was composed of the cooked beans
compactly bound together by white mycelia. Sporulation did not occur unless
incubation was continued for more than 45 h. During the fermentation process,
mycelial growth was relatively slower in acidified beans than in unacidified ones.
In general, excellent mycelial growth was seen, although, in a few cases, a slimy
product with a foul odour was produced in unacidified horse-bean and pea tempeh.
The odour of the fermented products was bland or yeast-like. Tempeh from
horse-bean, pea and soybean, in general, had a bland odour. Tempeh from chickpea
tended to have yeast-like odour. Tempeh made from the various beans, generally,
had a firm texture and could be readily sliced. Chickpea tempeh tended to be much
firmer than the other products.
Yield of Tempeh
All beans increased in weight (75 to 86%) during soaking (Table 1). Weight
increase in horse-bean, pea and chickpea soaked in unacidified water was sig­
nificantly higher than in beans soaked in acidified water (P < 0.01). Fermentation
resulted in loss of weight (1.4 to 5.4%) in all beans.
pH and Temperature Changes
The initial average pH of the various unacidified beans ranged between 5.0 and
5.5 and started to rise after about 18 h of fermentation until it reached the final
value of 6.4 to 6.7 for chickpea and soybean tempeh and 7.2 to 7.4 for horse-bean
and pea tempeh. Rise in pH always corresponded to active mycelial growth on the
substrates.
The a verage pH of the various acidified cooked beans ranged between 4.8 and
5.2. Similar increase in pH was noted during the fermentation process. Acidifica­
tion of beans usually delayed the appearance of mycelia by about 1 h, thereby
delaying the corresponding rise in pH. The final pH of the acidified fermented
products was 6.8 to 7.4 for horse-bean and pea tempeh, and 6.4 to 6.7 for chickpea
and soybean tempeh.
74
Tempeh from Ethiopian beans
Temperature of the fermenting beans did not increase until active mycelial
growth started. It then rose steadily until the end of fermentation, where it reached
lOoe to 12°C above the incubation temperature (Figure 1).
Proximate Analysis of the Various Tempeh Products
The fermentation of the various beans into tempeh resulted in loss of dry matter
and decrease in crude fat and nitrogen-free extracts (Table 2). Although changes
in total crude protein were slight, a marked increase in water-soluble proteins and
water-soluble solids was noted.
45
45
PT
HBT
40
40
~ 35
35
-o
o
.........
0..
E
30~~-+--~~-+~--+-~
o
30~~-+--~~-+~--+-~
o
5 10 15 20 25 30 35 40
Time (h)
45
45
CPT
00
5 10 15 20 25 30 35 40
Time (h)
SBT
h
40
40
35
35
30~~-+--~+--+-4--+-~
30 i!?---I--+--+I--1---11---4---+1--11
o 5 10 15 20 25 30 35 40
Time (h)
........­
0..
E
(])
I­
o
5 10 15 20 25 30 35 40
Time (h)
Figure 1. Temperature changes during the fermentation of unacidified (0) and acidified (6) horse-bean tempeh (HBT), pea tempeh (PT),
chickpea tempeh (CPT) and soybean tempeh (SBT).
75
M. A shenaji and M. Busse
Table 2. Proximate analysis of Tempeh made from the various beans.
Dry
maHer (%)
Crude
protein (%)*
Crude
fat (%)*
Crude
fiber (%)*
Ash (%)*
NFE (%)*
Soluble
protein (%)*
Soluble
solids (%)*
Horse-bean (raw)
Tempeh
% increase
95.5
42.2
-55.8
26.8
28.1
+4.6
2.3
0.9
-60.8
6.8
10.9
+37.6
3.9
4.6
+15.2
69.8
55.5
-20.5
4.4
11.2
+60.7
7.3
12.4
+41.1
Pea (raw)
Tempeh
% increase
89.8
39.7
-55.8
22.6
25.7
+12.1
1.6
1.0
-37.5
1.6
2.6
+38.5
3.8
4.6
+17.4
70.4
66.1
-6.1
3.7
9.8
+62.3
4.1
7.4
+44.6
Chickpea (raw)
Tempeh
% increase
94.3
39.5
-58.1
22.6
24.1
+6.2
6.5
4.3
-38.9
3.0
4.8
+37.8
3.1
4.2
+26.2
64.8
62.6
-3.4
3.8
10.2
+62.7
4.3
7.6
+43.4
Soybean (raw)
Tempeh
% increase
93.7
34.2
-63.5
34.7
38.4
+9.6
16.7
12.1
-27.6
4.2
6.7
+37.3
4.0
5.1
+21.6
40.4
37.7
-6.7
3.8
11.3
+66.4
12.9
25.1
+48.6
* Dry matter basis.
Sensory Evaluation of the Products
Frequency distribution of scores for the various tempeh stews is presented in Figure
2. Stew made of horse-bean, pea or chickpea tempeh was liked to varying degrees
by a large proportion of the judges (Figure 3). Only a small proportion detected
a beany flavour in the various stews (Table 3). The various stews compared
favourably with stew made of meat or egg. There was a significant difference
between samples and judges (P < 0.05). Stew made of horse-bean tempeh scored
40
Horsebean tempeh
Pea tempeh
--
30
D
I/)
Q)
en
"0
.20
Chickpea tempeh
20
'#.
lliillillillill
9
8
7
6
5
4
~ 3
10
2
c=J
o
Hedonic scale
Figure 2. Frequency distribution of hedonic scores given to stews made of various tempeh products: 9---like extremely; 8---like very
much; 7-like moderately; 6--like slightly; 5-neither like nor dislike; 4--dislike slightly; 3--dislike moderately; 2-dislike very much;
1-dislike extremely.
76
TempehJrom Ethiopian beans
Table 3. Hedonic scores and tastes of various stews made from horse-bean, pea, and
chickpea tempehs.
Basis of stew
Hedonic score*
X
Horse-bean Tempeh 7.14
Pea tempeh
6.52
6.72
Chickpea tempeh
* 9--like
% ludges
SO
Max
Min
Stew from
meat
1.53
1.81
1.69
9
9
9
2
1
38
Stew from
egg yolk
Stew from
beans
88
42
12
7
20
17
5
71
extremely; 5-neither like nor dislike; 1-dislike extremely.
significantly higher than the other two stew samples (P < 0.05). No significant
difference was observed between scores for stew samples made of pea or chickpea
tempeh.
Discussion
Fresh tempeh made from the various beans was bound into a white compact cake
as reported for soybean tempeh (Hesseltine & Djien 1979; Kronenberg 1984;
Sutardi & Buckle 1985). Good mycelial growth on horse-bean was previously
reported by David & Verma (1981) and Berghoffer & Werzer (1987). However,
Robinson & Kao (1977) reported that chickpea and soybean tempeh had a yellowish
colour, whereas horse-bean tempeh was greyish. According to our observations,
the final colour of the finished product seems to depend on the extent of mycelial
growth on the fermenting beans.
Incubation for more than 45 h was usually followed by sporulation, although
there is a report where sporulation started after 26 h of horse-bean tempeh
fermentation (David & Verma 1981). Acidification of soak water resulted in a
better mycelial growth and delayed sporulation. An earlier sporulation is not
desired because it is usually accompanied by signs of deterioration, such as loss
of pleasant taste, smell of ammonia and darkening of colour (Sudarmadji &
Markakis 1978).
The odour of fresh tempeh may vary depending on the type of substrate or even
the variety of the same substrate. The odour of soybean tempeh is, for example,
expressed as bland or slightly nutty (Hesseltine & Djien 1979), mushroom-like
(Kronenberg 1974) or clean and pleasant (Steinkraus et al. 1960). Chickpea and
horse-bean tempeh were reported to have alcoholic and ester-like aromas (Robinson
& Kao 1977).
The various types of tempeh in this study were firm and could be readily sliced,
although Robinson and Kao (1977) reported a soft texture for horse-bean tempeh.
The physical appearance of tempeh depends on the degree of microbial spoilage
during fermentation (unpublished data). Unless the count of aerobic spore formers
is not high enough to cause visible spoilage resulting in a slimy and putrid product,
a relatively firm tempeh can be produced from all beans.
Although unacidified beans have undergone some acidification during soaking,
spoilage was often seen during the fermentation of unacidified horse-bean and
pea tempeh. A proper acidification of the beans during soaking or use of a suitable
lactic acid bacteria during fermentation (Ashenafi & Busse 1989) may inhibit an
early proliferation of aerobic spore fonners, favour a good growth of mycelia and
guarantee the production of an acceptable tempeh.
Soaking and cooking are reported to result in loss of material by leaching
(Steinkraus et al. 1960). Material loss in the form of heat was also observed during
fermentation (Steinkraus et al. 1960). The sharp rise in temperature in our
77
Ai. A shenafi and M. Busse
fermenting beans is in agreement with the observations of other workers (Stein­
kraus et al. 1960; Berghoffer & Werzer 1986).
Deep penetration of Rhizopus hyphae in the bean cotyledon (Jurus and Sundberg
1976) and the production of extracellular enzymes by the mould (Wang &
Hesseltine 1965) result in an extensive breakdown of the bean constituents
(Steinkraus 1983a,b). Increase in soluble protein in the various beans in this study
is in agreement with other reports (Steinkraus et af. 1960; Van Buren et af. 1972;
Robinson & Kao 1977; Berghoffer & Werzer 1986; Paredes Lopez et af. 1987).
Utilization of nitrogen-free extracts by the growing fungal mycelia may result
in an increase of reducing sugars (Berghoffer & Werzer 1986). Decrease in crude
fat is due to the lipolytic activity of R. oligosporus, which results in an increase of
free fatty acids (Wagenknecht ef al. 1961).
Since plant proteins are generally considered to be less available to humans, the
high level of water-soluble proteins in the fermented beans is of significant
importance in improving the diets of many people in developing countries.
The results of the sensory evaluation tests imply that the product, although not
yet introduced into Ethiopia, has a chance of being accepted by the population.
The high comparability of the tempeh stews to meat and egg stews indicates the
possibility of using tempeh as an analogue to the otherwise expensive animal
proteins. Acceptability of fermented fempeh made from various beans is also
reported by other workers (Berghoffer & Werzer, 1986; Paredes Lopez ef af. 1987).
Acknowledgements
Due thanks go to the Nutrition Laboratory of the Department of Animal Sciences
and Technology, Awassa College of Agriculture, for performing the proximate
analysis, and the Nutrition Laboratory of the Department of Home Science and
Technology, Awassa College of Agriculture, for conducting the sensory evaluation
tests. The technical assistance of Haile Alemayehu and Etenesh Tesfaye IS
acknowledged.
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Tempehfrom Ethiopian beans
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79
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