Food Reviews International

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Tempehtechnology and food value

B. K. Mital & S. K. Garg

To cite this article: B. K. Mital & S. K. Garg (1990) Tempehtechnology and food value, Food
Reviews International, 6:2, 213-224, DOI: 10.1080/87559129009540867

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 Food Reviews International, 6(2), 213-224 (1990)

TEMPEHTECHNOLOGY AND
FOOD VALUE

B. K. MITAL
Department of Food Science and Technology
College of Agriculture

S. K. GARG
Department of Microbiology
College of Basic Sciences and Humanities

G. B. Pant University of Agriculture and Technology

Pantnagar 263 145, India

ABSTRACT
Tempeh is a greyish-white product obtained by fungal fermentation
of dehulled, hydrated, and partially cooked soybean cotyledons. It
is free of cholesterol, a good source of vitamins and minerals, and
is highly digestible. Tempeh can serve as an excellent substitute for
animal protein products and it therefore holds promise to combat
malnutrition in countries where proteins and calories are in short
supply.

Research Paper No. 5837 through the Experiment Station, G. B. Pant University of Agriculture and
Technology, Pantnagar, U.P. India.

213

Copyright 1990 by Marcel Dekker, Inc.

214 MITALANDGARG

INTRODUCTION

Tempeh (pronounced TEM-pay) is a mold-fermented soybean food. Occasionally

other legumes, seeds, and cereal grains are also used along with soybeans. The
mold fermentation results in a matrix of dense, cottony, mycelia in which cooked
soybeans are embedded, forming a compact greyish-white cake. Tempeh possesses
a pleasant aroma which can be described as nutty, cheesy, mushroomy, etc.
Tempeh is a popular fermented food of Indonesia. However, in the last two
decades it has attracted the attention of the Western world, particularly North
America, because of its unique flavor, sliceable meat-like texture, and nutri-
tional attributes. In 1983 the U.S. industry produced approximately 2 million
pounds of tempeh for a retail turnover of $5 million (1). The nut-like flavor of
fried tempeh appeals to Europeans and Americans. It is free of cholesterol, a
good source of vitamins and minerals, and highly digestible, and is also consid-
ered to impart growth-stimulating effects. It can serve as an excellent substitute
for animal protein products such as meat, chicken, fish, etc.
The present review aims to summarize methods of preparation, physicochem-
ical changes during the process of fermentation, and its nutritional attributes.

PREPARATION METHODS

Tempeh manufacture, whether traditional or modern, requires that soybeans be

hydrated, dehulled, cooked, inoculated with mold, and incubated at appropriate
temperature until cotyledons are knitted together into a cake. The cake is then
sliced and deep-fat fried until the surface is crisp and golden brown or cut into
chunks and used as a protein-rich meat substitute in soups.
The traditional fermentation is very simple. It is a household art in Indonesia
and therefore varies from place to place. However, the essential steps of tempeh
fermentation are the same. The soybeans are washed and soaked in water over-
night, during which time the beans double their dry weight and undergo bac-
terial acid fermentation reducing the pH to 5.0 or lower. An alternate process
is to boil the beans in water and then allow the beans to soak overnight. The
general purpose of boiling is to facilitate hull removal. The hulls are then re-
moved manually and the loosened hulls are floated away with water. It is essen-
tial that all hulls be loosened from the cotyledons as the mold cannot grow on
intact beans. The dehulled beans are then boiled with excess water for varying
lengths of time (usually about 30 min), cooled, surface dried, and inoculated
with tempeh from previous fermentation or with mold grown on dried leaves.
Traditionally, the inoculated cotyledons are then wrapped in small packets using
wilted banana leaves and allowed to ferment at room temperature for a day. By
this time the cotyledons are bound together by the growth of white mycelium to
form a cake which has a pleasant flavor.
TEMPEH 215

About 20-30 years ago most of tempeh production in Indonesia was based
on this traditional process with minor modifications. Steinkraus (2, 3) devel-
oped a pilot-plant process which resulted in modernization of the process in that
country. In this process soybeans are shrivelled at 104 C for 10 min to facilitate
hull removal. The hulls are then cracked using a burr mill and separated from
cotyledons by an aspirator or gravity separator. Alternately, the beans are soaked
and dehulled wet by pressing them through an abrasive vegetable peeler. Acidi-
fication of beans is accomplished by adding 197o (v/v) lactic acid to the soak or
cook water or by inoculating the soak water with Lactobacillus plantarum to
promote natural acidification. Fermentation with lactobacilli has also been re-
ported to reduce the content of oligosaccharides responsible for flatulence (4,
5). Steinkraus et al. (6) found that addition of lactic acid to soak and cook water
lowered the pH of beans to 4.0-5.0, which favored the growth of the mold and
inhibited the growth of bacterial contaminants. Acidification is an essential step
in the manufacture of tempeh. The pH of unacidified soybeans is in the range
of 6.5-6.8, which is suitable for the growth of variety of organisms including
spoilage and food-poisoning types. Tanaka et al. (7) found that Clostridium
botulinum (types A and B) produced toxin and that Staphylococcus aureus, Sal-
monella typhimurium, and Yersinia enterocolitica grew well when they got entry
during tempeh preparation through unacidified soybeans.
In another process cotyledons are boiled or steamed for 15 to 20 min, drained,
cooled, surface dried, and inoculated with powdered culture of tempeh mold.
The inoculated beans are spread in trays, covered, and incubated at 37 C for
about 20 h at 75-85% relative humidity. Martinelli and Hesseltine (8) developed
a process in which inoculated cotyledons were packaged in perforated plastic
bags and then incubated. The process has been found very useful in commercial
manufacture of tempeh (9).
After completion of fermentation, tempeh cake has an attractive, bland, slightly
nutty flavor. It is then sliced, dipped in salt solution, and deep-fat fried. At times
the slices are dipped in a water suspension of rice flour, salt, and spices before
frying. The fried tempeh has a dark-brown, appealing color.
The keeping quality of tempeh is short at ambient temperature. Therefore, it
is consumed the same day. If kept at room temperature for longer periods, the
color turns dull brown, the surface becomes wet and slimy due to bacterial growth,
and the product develops bitterness. Prolongation of storage under such condi-
tions results in disagreeable putrid flavor. However, deep-fat fried tempeh has a
longer shelf-life. If kept in closed containers or sealed plastic bags, the crisp slices
remain tasty for weeks. Sometimes the cake is sliced into thin pieces and dried
under the sun. The dried pieces remain edible for several days.

FOOD VALUE
Raw mature soybeans are indigestible and bitter in taste. However, fermentation
of soybeans with Rhizopus oligosporus transforms beans into a highly digestible
216 MITALANDGARG

and delicious product with unique texture and appearance. One of the most im-
portant functions of the mold in such food fermentations is the synthesis of en-
zymes (10) which decompose complex compounds such as starch and protein into
smaller molecules. Such enzymatic activity may also decrease or eliminate anti-
nutritional components such as phytic acid and, consequently, improve the food
value of fermented product. Fresh tempeh contains approximately 19-20% pro-
tein, which compares well with the protein content in chicken (21 %), beef (20%),
hamburger (13%), whole eggs (13%), and whole milk (3%). The nutrient com-
position of tempeh is shown in Table 1.
Tempeh resembles closely food products from meat and milk in nutritional
attributes (Table 2). Soybean protein is rich in lysine. Therefore, soybean foods
such as tempeh can be effectively used to supplement typically lysine-deficient
cereal diets. The essential amino acid pattern of tempeh is shown in Table 3.
Tempeh is free from cholesterol and approximately 80% of its fat consists of
unsaturated fatty acids (16). It is a good source of minerals and B vitamins, par-
ticularly of B,2, which is generally absent in foods of plant origin (Table 4).

Table 1. Nutrient Composition of Tempeh (per 100 g)

Nutrient Fresh (11) Freeze-dried (12) Deep-fat fried
Food energy (cal) 157
Moisture (%) 60.4 1.9 50.0
Protein (7o) 19.5 46.2 23.0
Fat(7o) 7.5 23.4 18.0
Carbohydrates (%) 9.9 25.8 8.0
Fiber (%) 1.4 2.7 2.0
Ash (%) L3 2^7 1.0
Source. Shurtleff and Aoyagi (1).

Table 2. Nutritonal Attributes of Tempeh

Characteristics Value Ref.
PER 2.42 Wang et al. (13)
NPU 58.70 Bai et al. (14)
Digestibility 86.10 Djurtoft and Jensen (15)
Chemical score 63.90 Djurtoft and Jensen (15)
Biological value 58.70 Djurtoft and Jensen (15)
TEMPEH 217

Table 3. Comparison of Amino Acid Pattern of Tempeh with FAO/WHO Reference Pattern
(mg/g nitrogen)
FAO/WHO Tempeh 6 Percent of FAO/WHO
Amino acid pattern" (soybean) pattern in tempeh c
Methionine/cystine 220 171 78
Threonine 250 267 107
Valine 310 349 113
Lysine 340 404 119
Leucine 440 538 112
Phenylalanine and tyrosine 380 475 125
Isoleucine 250 340 136
Tryptophan 60 84 140
"Provisional amino acid scoring pattern. Technical Report Series 522, WHO (1973).
*Murata et al. (12).
c
Shurtleff and Aoyagi (1).

Table 4. Vitamin and Mineral Contents in Fresh Tempeh

Amount per U.S. Percent of RDA
Nutrient 100 g RDA in 100 g tempeh
Vitamin A (IU) 42 5000 1
Thiamine (mg) 0.28 1.5 19
Riboflavin (mg) 0.65 1.7 28
Niacin(mg) 2.52 20 13
Pantothenic acid (mg) 0.52 10 7
Pyridoxine G*g) 830 2000 42
Folic acid (jig) 100 400 25
Cyanocobalamine Qig) 3.9 3.0 130
Biotin Qig) 53 300 18
Calcium (mg) 142 1000 14
Phosphorus (mg) 240 1000 24
Iron (mg) 5 18 28
Sources. Steinkraus et al. (2), Roelofson and Thalens (17), Murata et al. (18), Napavarn et al. (19), Liem et
al. (20).

Liem et al. (20) found that tempeh contained 1.5-6.3 fig B12 per 100 g. They
also observed that its content could be increased to as high as 14.8 /*g/100 g if
Klebsiella pneumoniae is incorporated in the starter. In addition, soybeans also
contain (per 100 g dry weight) magnesium (236 mg), potassium (15.04 mg), zinc
(3.8 mg), and manganese (1.2 mg).
218 MITAL AND GARG

The high digestibility of tempeh may be ascribed to predigestion of soybean

nutrients and their breakdown into soluble solids and nitrogen by the mold dur-
ing fermentation. The beans, therefore, become soft and tender. The proteases,
produced by the mold, break down the proteins into amino acids and other water-
soluble products which are readily assimilated by the body. Similarly, the Upases
produced by the mold break down neutral fat into easily assimilable fatty acids.
The operations of soaking, cooking (21), and fermentation during tempeh pro-
duction greatly reduce the oligosaccharides such as melibiose, raffinose, stach-
yose, and verbascose that are believed to cause flatulence. The cooking process
also inactivates trypsin inhibitors. The tempeh-making process masks beany
flavor, and imparts pleasing flavor and aroma which make the product highly
acceptable even to people not traditionally accustomed to soybean foods.

MICROBIOLOGY OF FERMENTATION

Traditional tempeh manufacture involves two distinct fermentations. During

hydration of beans under conditions prevailing in the tropics, bacterial growth
occurs which results in acidification of beans. During this process the pH of the
beans is lowered to 4.5-5.3. This inhibits the growth of bacterial contaminants.
It has been postulated that bacterial fermentation might be eliminating part of
the galacto-oligosaccharides of the raffinose family considered responsible for
flatulence when soybeans are consumed (22). Besides this, the initial lower acidic
conditions favor the growth of mold by inhibiting pH rise due to liberation of
ammonia as the mold is strongly proteolytic. Above pH 7.0, sufficient ammonia
is liberated to kill the mold.
Hesseltine et al. (23) found that soybeans contain a heat-stable, water-soluble
compound which inhibits the growth of the mold. The same compound also in-
hibits the proteolytic enzymes of the mold. These findings suggest that soak
water should be discarded to accomplish desirable tempeh fermentation (24).
The mold involved in tempeh fermentation belongs to the genus Rhizopus.
Of this genus, R. oligosporus, R. stolonifer, R. oryzae, and R. arrhizus can
bring about desired mycelial growth to knit soybean cotyledons into a compact
mass (25, 26). However, the most desirable strain for tempeh manufacture is
R. oligosporus NRRL 2710. This strain was isolated in the laboratory of Dr.
K. H. Steinkraus of Cornell University and identified in the laboratory of Dr.
C. W. Hesseltine, Northern Regional Research Laboratory, Peoria, Illinois (22).
The characteristics which make R. oligosporus NRRL 2710 most suitable for
tempeh fermentation are its ability to grow rapidly at temperatures between
30 and 42 C, ability to ferment sucrose, high proteolytic and lipolytic activities,
ability to produce strong antioxidants, and ability to impart pleasing flavor and
aroma.
TEMPEH 219

Tempeh produced under commercial conditions contains vitamin B12 (20, 27).
This vitamin is altogether absent when pure culture of R. oligosporus is used
for tempeh manufacture. Curtis et al. (27) identified the culture responsible for
B12 production in tempeh as Klebsiella pneumoniae. Later on they observed that
this organism must also be used if tempeh is to serve as an essential source of
vitamin B,j in the diet of vegetarians. It can produce as much as 150 mg of B n /g
of soybean substrate. Liem (28) found that increasing the cobalt content of soy-
bean substrate above 100 ppb exhibited a directly proportional increase in B12
content of tempeh.
Variety of molds and bacteria may be found in tempeh depending upon the
microflora of the raw soybeans; methods of soaking, cooking, and incubation;
and sanitary and hygienic conditions during manufacture. However, only one
mold species is essential for the production of quality tempeh (2, 3, 6, 29). This
mold does not require much aeration, as do many other molds. In fact, too much
aeration may cause sporulation.

PHYSICOCHEMICAL CHANGES DURING FERMENTATION

A number of interesting changes occur during fermentation. Steinkraus et al.

(6) found that the temperature of the beans rises by 5-7 C above that of incuba-
tion temperature as the mold begins to grow rapidly during tempeh fermenta-
tion, and then falls as the mold growth subsides. The soluble solids increase from
approximately 13% to 28% during 72-h fermentation. Soluble nitrogen increases
from 0.5% to 2.5% while the total nitrogen remains relatively constant. The
initial pH of soybeans is 5.0, which rises progressively to above 7.0, and free
ammonia may be noted in the later stages of fermentation.
The neutral fat of soybeans is composed of palmitic, stearic, oleic, linoleic,
and linolenic acids, with linoleic acid predominating. R. oligosporus is strongly
lipolytic and hydrolyzes one third of the fat during fermentation at 37 C. The
proportion of fatty acids liberated during fermentation remains more or less
the same as originally present in soybeans with the exception of linolenic acid,
which is reduced by about 40% (16). The acid number of soybeans increases
approximately 30- to 50-fold during tempeh fermentation (12, 16).
Wang and Hesseltine (30) reported that R. oligosporus produces two proteo-
lytic enzymesone with optimum pH at 3.0 and the other at 5.5. However, both
have temperature optima at 50-55 C. The maximum activity of acid protease
was observed at 25 C after 96 h on wheat bran with 50% moisture (31). The pH
of soybeans increased to 7.1 as a result of proteolysis and deamination of amino
acids by mold.
The principal fermentable sugars in soybeans are water-soluble, low molecular
weight oligosaccharides such as sucrose (5.0%), raffinose (1.1%), and stachyose
220 MITAL AND GARG

(3.8%) (32). Shallenberger (33) observed substantial hydrolysis of stachyose dur-

ing tempeh fermentation by R. oligosporus. Rhizopus oryzae produces high
amounts of amylase compared to R. oligosporus. However, amylase activity is
not of significance since mature soybeans do not contain starch. Of the many
Rhizopus spp. used for tempeh production, Rhizopus arrhizus NRRL 1526 pro-
duces pectinase. In contrast, strains of R. oligosporus tested exhibit little or no
pectinase activity.
Wagenknecht et al. (16) found that R. oligosporus exhibits strong lipase ac-
tivity but does not utilize the fatty acids liberated as a result of hydrolysis of lip-
ids. They concluded that either this organism does not possess the enzyme systems
to metabolize these fatty acids or these fatty acids are not permeable to the cyto-
plasmic membrane.
Proteases are perhaps the more important enzymes in tempeh fermentation.
The ability of Rhizopus spp. to produce proteolytic enzymes varies greatly among
strains. During tempeh fermentation, lysine and methionine contents have been
reported to be decreased by 25% and 10%, respectively (2). Murata et al. (12)
noted that some amino acids increased while others decreased as a result of mold
activity. However, they observed an overall increase in the content of free amino
acids as the fermentation progressed. Stillings and Nackler (35) did not find any
change in the essential amino acid index during fermentation. Sorensen and
Hesseltine (34) observed that ammonium salts and amino acids such as proline,
glycine, aspartic acid, leucine, etc., serve as an excellent source of nitrogen for
R. oligosporus, but this organism does not specifically depend on any of them
for growth.
Phytic acid present in soybeans hinders mineral absorption in intestinal tract.
Phytase, which can hydrolyze phytic acid into myoinositol and orthophosphate,
has been isolated from molds (36, 37) and also from some legume seeds (38).
Sudarmadji and Markakis (39) observed 22% reduction in phytic acid during
tempeh fermentation. They attributed it to phytase activity of R. oligosporus.
The vitamin content of soybeans increases substantially as a result of tempeh
fermentation. Various researchers have reported 2-fold increase in riboflavin,
7-fold increase in niacin, and 33-fold increase in B,2 (2, 17, 19). Steinkraus et al.
(2) found that thiamine decreased whereas pantothenate remained the same dur-
ing tempeh fermentation. However, Murata et al. (12, 18, 40) recorded 2- to
4-fold increase in pantothenate, biotin, and folate compounds. The peroxide
value of soybeans decreased from the range 18.3-201.9 to 0.0-1.1 as a result of
fermentation with R. oligosporus (2).

Antioxidants

An antioxidant was isolated from tempeh and characterized as an isoflavone

different from genistein and diadzein (41, 42). Ikehata et al. (43) identified this
TEMPEH 221

flavone as 6,7,4-trihydroxyisoflavone (Factor 2). The antioxidative effect of this

compound was found to be the same as that of DL-tocopherol but 3 times that
of genistein. Murata (44) observed that flavonoid compounds are not the only
antioxidants in tempeh; other antioxidant factors soluble in oil may also be pres-
ent. Zilliken and Jha (45) also identified and synthesized a novel class of iso-
flavones from tempeh with high antioxidant activity.

Flavor Profile

The flavor profiles of fermented foods are generally complicated. Variations in

manufacturing practices significantly alter the flavor profile. The typical flavor
of a fermented product depends upon metabolites such as pyridine derivatives,
diacetyl, acetoin, 2,3 butylene glycol, valeric acid, etc. (9). Moroe et al. (46) ob-
served that major flavor compounds of tempeh include maltol and higher fatty
acids (palmitic, stearic, oleic, linoleic, and linolenic acid) and that their con-
centration increased 2.6 and 1.4 times, respectively, as a result of mold activity.

USE OF ALTERNATE LEGUMES AND GRAINS

The principles of tempeh fermentation have been exploited to make tempeh-like

foods by substituting soybeans with a number of other beans (winged beans,
Jack beans, velvet beans, pigeon pea, sweet lupine, yellow pea, broad peas, cow
peas, horse beans, and chick pea), bean fractions (mung bean starch), residues
obtained during soy milk/tofu manufacture, cereals (wheat, barley), and mixtures
of cereals and beans (14, 47-50). Table 5 shows some tempeh-like products pre-
pared from substrates other than soybeans.
Tanuwidjaja (55) developed a process for making tempeh using defatted soy
flour (3.5% fat) as the substrate. He observed that the product was a solid cake

Table 5. Tempeh-Like Products from Substrates Other Than Soybeans

Product Substrate Ref.
Tempeh kecipir Winged bean seeds Gandja (49)
Tempeh gembus Residuesoybean curd Bates et al. (51)
Wheat tempeh Wheat Wang and Hesseltine (52)
Tempeh bongkrek Coconut press cake Gandja and Hermana (53)
Tempeh benguk Mucuna pruiens Gandja (54)
222 MITAL AND GARG

covered with white mold mycelium with typical flavor and texture. Shurtleff
and Aoyagi (1) have also described methods for making tempeh-like foods using
substrates other than soybean.

PRODUCTION OF ANTIMICROBIAL SUBSTANCES

Wang et al. (56, 57) reported that R. oligosporus NRRL 2710 produced a heat-
stable antimicrobial substance active against Gram-positive bacteria including
both microaerophilic and anaerobic bacteria such as Streptococcus cremoris,
Staphylococcus aureus, Bacillus subtilis, Clostridium perfringens, and Cl. sporo-
genes. The only Gram-negative bacterium sensitive to this substance was Kleb-
siella pneumoniae. The compound contains polypeptides. Its activity is not in-
fluenced by pepsin or R. oligosporus protease but is slightly decreased by trypsin
and peptidase. However, it is readily inactivated by pronase.

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