“TEMPEH”

for
Industrial Food Fermentations
FOS 6455
July 1, 2002

Instructors
Dr. RP Bates
And
Dr. RH Schmidt
By
Sharyn Passeretti
 1

Soy History:
The soybean originated thousands of years ago in the north and central China regions
of Eastern Asia. The cultivated varieties of this legume were introduced to surrounding
countries via trade routes and [more commonly] through warring nations. Diet and
agriculture would have been one of many changes the conquered people would had
altered to appease the current rulers life style [sometimes the rulers would adopt
practices from the subjects as well]. As a result soy products were developed and
integrated into the Asian diet, with many items still actively used to this day. An
interesting contrast is the different way soy is consumed across the world [7].

I
ntheAsi
ancount
ri
es,soyi
sconsumedasa“
soyf
ood”(
Tempeh,t
ofu,Mi
so,Nat
to)
,yet
in North America, soy is a protein source incorporated into a variety of foods ranging
from meats, to dairy and pastries [7].

Traditional soy protein foods are divided into two main categories: non fermented and
fermented. The non-fermented include tofu, and soymilk [note there are fermented
forms of this made]. Soysauce, tamari, Miso, and Tempeh fall into the fermented class.
While most of these products originated in China, Tempeh originated in Indonesia [7].

What is tempeh?
Tempeh is a solid-substrate fermentation patty consisting of cooked acidified beans
and/or grains. This product has been a protein staple in Indonesia for centuries [10].
While Soy is the most frequently made variety of tempeh, other combinations have been
known to include, barley, oats, rye, quinoa, and chickpeas to name a few [2].

Nutritional factors:
Soy tempeh is a great source of high fiber, cholesterol free protein, which can be easily
used as a meat alternative. These nutritional benefits are clearly illustrated in Table 1
on the following page. An additional benefit of Soy Tempeh is that it contains a
significant amount of phytoestrogens, which may reduce the risk of certain forms of
cancer.
 2

Table 1:Nutritional Comparison Chart (100g serving)

Product Protein SatFat TotFat Chlst Fiber

Soy Tempeh 13g .6g 3g 0mg 7g
Ground Beef 15g 9.0g 18g 58mg 0g
Lean Ground Beef 18g 4.5g 9g 56mg 0g
Chicken Breast 14g 1.0g 4g 45mg 0g
Chicken Drumstick 10g 2.0g 6g 45mg 0g
From Nutrition Almanac, McGraw-Hill, and Turtle Island Foods Lab Reports. [10] protein comparison to
egg albumin

Another factor regarding tempeh is the high quality and digestibility of its protein.
The high digestibility is due to the Rhizopus oligosporus production of enzymes
protease and lipase, resulting in the breakdown of proteins to amino acids and fats are
hydrolyzed into fatty acids. Because of the proteins semi digestive state, it makes a
nt
good protein source for people with gastro-i est
inalupset
s(i
.e.POW’
s,AI
DS,t
hir
d
world countries) [5]. Other reports noted:
 Increased levels of niacin, riboflavin and vitamin B12 in the fermented product [Van
Veen and Steinkraus, Food Tech., 19, 63 (1965)].
 A possible relation between disease resistance and tempeh consumers due to
antibiotic production by Rhizopus oligosporus [Wang et. Al., Proc. Soc. Exp. Biol.
Med., 131, 579 (1969)
The protein and lipid breakdown resulting from the enzyme diffusion compounded by
the Rhizopus oligosporus mycelial penetration changes the texture of the beans thereby
affecting the final product [5]. The Hyphae penetrate the bean perpendicular to the
cotyledon surface. This seems to cause an increase in turgor pressure, pushing the
soybean cells apart as the enzymes partially degrade them. This causes the bean
surface to soften, forming a mass that is bound by the mycelial growth known as
tempeh [6].

HOW TEMPEH IS MADE:

As mentioned earlier, tempeh is a solid substrate fermentation [SSF] process. This is
basically the use of the water-insoluble materials to be used for microbial growth in a
low water or water free environment. SSF allows a favorable microbial environment of a
diffusible nutrient bed with a gas surface [6].
 3

Phase I

Phase II

Diagram 1: general outline of soy tempeh production developed at Northern

Regional Research Centre, Peoria, IL.
NOTE: while the diagram does use dehulled full fatted soy grits, whole soy is more
commonly used. The only difference will be a longer soaking and cooking time and a
dehulling process before the bean is cooled and inoculated.

Phase I fermentation:
As seen in diagram1, tempeh involves two phases of fermentation. Phase I involves
washing the beans then soaking them overnight [>8hrs] in acidified water (0.60-0.85%
lactic acid or 0.25% acetic acid). The soaking water is discarded, the beans placed in a
cooking container and acidified water is added in a 1:3 ratio of bean to water. The
product is boiled for 90 minutes, drained, and cool water is added. From this point the
beans will be dehulled by machine or manually, then cooled to about 37°C [8].
Phase I of the fermentation process is important because;
 It drops the pH of the bean/grain product. Dropping the pH of the product creates
an environment that simultaneously represses microbial growth, while facilitating
rapid mold growth. This pH drop also affects the protein solubility ratio.
 It changes the solubility of the protein. The increase in the product acidity, results
in lower protein solubility [7]. It is important to note that there are different pH ranges
for the different beans/grains, listed below are some standard pH levels for some
popular selections:
 4

o 3.0-4.0—Barley, Oats, Rye

o 4.0-5.0—Soy
o 5.0-6.5—Quinoa
o 5.7—Wheat
 Eliminates enzyme and antinutritional factors during the boiling step of phase
I fermentation. One of the most important enzymes in soybeans is lipoxygenase.
This is an enzyme that catalyses the oxidation of poly-unsaturated fatty acids, which
causest
he“
beany
”or“
pai
nt”l
i
kef
lav
orsofmanyr
awseeds[
7].Theant
inut
ri
ti
onal
factors consist of lectins and two forms of trypsin inhibitors. While dietary ingestion
of lectins seems to have neither a positive or negative effect upon the human
system, the same cannot be said for trypsin inhibitors. In soybeans there are two
forms of trypsin inhibitors, the Kunitz inhibitor [20000 daltons] and the Bowman-Birk
inhibitor [8000 daltons]. One is heat labile proteinous structure, while the other exist
in an inactive, bound, heat resistant form [6]. They are free fatty acids in nature,
which are liberated from oil in the soybeans by the lipase activity of Rhizopus
oligosporus. Once they are released, boiling or fat frying can easily destroy them.
Since trypsin ingestion has been associated with impaired growth, increased
pancreatic secretion and hypertrophy of the pancreas inactivation is nutritionally
important [6,7]. Yet there is the question as to trypsin toxicity in humans, simply
becauset
her
ehaven’
tbeenexper
iment
sper
for
medwi
thhumansubj
ect
s.Luc
kil
y
these potentially harmful [or unpalatable in the case of lipoxygenase] factors are
inactivated in the boiling step of phase I fermentation, and the final cooking process
before ingestion [6,7].

Phase II fermentation:
Inoculation
After the beans have been dehulled, drained and cooled to about
37°C, phase II fermentation begins by inoculation with Rhizopus spp
of mold. There are two methods of inoculum, mass sporulation, and
tempeh to tempeh. Mass sporulation is a preserved mass with a total
viable count [usually 89 X 107/g] that has been cultivated on a
carbohydrate-based agar (malt extract, Sabouraud dextrose, ect.).
 5

This is suspended in cool sterilized water, and then applied to the acidified cooked
beans. If the spores come freeze dried, the inoculation directions were usually on the
package (usually 1t (of 1X107 spores) to 1lb of dried beans). The tempeh to tempeh
i
nocul
ati
ons
impl
yinvol
vesas
uspensi
onoft
hepr
evi
ousday
’st
empehcak
einwat
er,
then spraying it over the processed beans [4]. The most common strain of Rhizopus
oligosporus used in the production of tempeh is Rhizopus oligosporus NRRL 2710.
This strain provides the best hyphae length and penetration of the cotyledon, resulting
in better cake formation. Another benefit of NRRL 2710 is that it caused the pH to drop
to about 4.5-4.0 at 15 hours, an advantage for limiting unwanted microbial growth [2].
The origin of this tempeh starter originates in Indonesia with the use of Hibiscus leaves
of the waru (hibiscus spp) trees in the making of Usar [the traditional inoculum for
tempeh] [1]. Geographic specificity of the Rhizopus spp was validated through a study
involving several species of waru from different regions of Indonesia, the Netherlands,
Nigeria, and Ghana. Incubation experiments were run on the leaves consisting of
leaves with no soybean, and leaves with acidified and cooked soybeans. The results of
the experiment elicited two significant factors:
 The necessity of the soybean in order for a mold to grow—the leaves without
the soybean had no visible growth with the exception of Nigeria [Cladosporium spp],
and Ghana two of the 4 had Cladosporium spp the other two had no growth [1].
 The significant difference between the mycoflora of Hibiscus leaves from the
different regions. This was seen in the almost exclusive cultivation of the Rhizopus
spp (mainly R oligosporus and R oryzae) with the Indonesian leaves. Only two of
the Netherlands varieties cultivated Rhizopus (R stolonifer and R oryzae), and one
sample from Ghana cultivated R orzyae. Otherwise the remaining samples
produced Aspergillus, Neurospora, or Fusarium spp [1].

Packing
Immediately after inoculating the beans are tightly packed into a 2.5cm thick cake using
one of the following types of containers:
 Perforated banana leaves and tied into place.
 20 X 38cm plastic bag with perforations about 0.5cm apart
 Shallow metal trays, with perforations about 0.6mm in size about 1 cm apart.
 6

 9-15cm ventilated petri dish

 Tetra brix containers perforated about 1cm apart
Proper surface area and thickness of the cake formation is essential in tempeh
production. If the fermenting soybean cake is too thick, the beans in the center will not
get enough oxygen. This results in the absence of mycelial growth toward the center of
the product, and a lack of proper cake formation due to hardness of the beans in the
cakes center. Insufficient oxygen also results in the lack of alkali-generating metabolic
reactions mainly protein metabolism [the oxidation of amino acids] and the oxidation of
anions [i.e. lactic acid] present in acidified cotyledons [3]. Without these reactions the
pH of the product will not raise, and protein solubility cannot occur. The solubility of
soybean protein in water is strongly affected by the pH. If the pH is not brought to about
6.5-7.0, the protein will not become soluble, resulting in a hard bean end product.

Incubation
At this point the packages are placed into a 60-65% humidity fermentation room set at
31°C. A container of water is placed in the chamber to maintain humidity, and the
product is allowed to incubate for 18-22 hours, or until a vigorous white mycelial growth
has occurred. It is important not to over ferment this product. This will cause
production of ammonia causing the pH to increase (7.5-9.25), resulting in off odors, and
increase in microbial susceptibility.
Finally the tempeh cakes are removed from their forms, and blanched in boiling water to
kill the mold and stop enzymatic reactions.

There has been another method of tempeh preparation involving a device patented by
Gunter Pfaff. This method explained below in Figure 1 utilizes stainless steel trays that
are partially submerged in a circulating waterbath. The insulated lid covers the tray,
sealing it from the environment. An air injection system is applied, circulating air over
the incubating tempeh and exiting the opposite end. This process not only provides
excellent fermentation conditions for the production of tempeh, the waterbath incubator
allows pasteurization at the end of the incubation period by raising the temperature to
80°C for 15 minutes. The article also mentioned the possibility of using the stainless
 7

steel trays to pre-marinate the tempeh with different flavors before being packaged or
frozen.

Figure 1: drawing and photo of patented tempeh process

From this point the tempeh can either be used immediately for cooking or packaged for
storage.
What to look for in a tempeh product [courteously of Turtle Island Foods, Inc.]
•
The soy beans/grains should be bound together into tight cakes with no mold other
than the white Rhizopus culture. Small black spots may occasionally appear. These
spots do not necessarily indicate spoilage. They are part of the cultures sporulation
cycle.
•
Look for a non-slimy texture and fragrant, mushroom-like aroma
•
If frozen in the package, Tempeh keeps well for up to one year. Once thawed, it will
keep for about one month in the fridge.
•
Tempeh can be used in any mushroom recipe, or in place of them in certain recipes.

Good for the earth reasons:

In closing, I would like to quote two paragraphs from different articles concerning
environmental issues, one stating a situation, the other an ignored solution. Because I
feel that ultimately our true responsibility as future Research Chefs, Food Chemists, and
technologists lies in the integration of new product ideas with environmentally friendly
GMP’
s.

“Hungerandmanyoft hewor l
ds’envi ronment alpr oblemsar edi rectlylinkedt ot he
animal agricultural system. Millions of forested acres are burnt to clear land to raise
cattle in developing countries each year. This depletes the ozone lay by releasing
carbon dioxide. Since 1970 over 1.4 billion tons of carbon dioxide has been released
from rainforest land cleared and burned for cattle animal protein. Over 20% of the
 8

worl
d’tot almet haneemiss
ionscomedirect
lyf
rom cat
tl
e.Li
vest
ockpr
oduct
ion is also a
ver
yinef fic
ientus eoft
heearth’
spreci
ouslandandwaterr
esour
ces”~Turt
leIs land
Foods, Inc. [10]

“Itcanbecal culatedt hat,thequant i

tyofpr ot ei nint hey ear
lywor l
dpr oduct ionof
soybeans, if it could be totally and directly utilized for human consumption, would be
sufficient for providing roughly one third of the global need for food protein. This makes
the soybean one of the largest potential sources of dietary protein. However, the bulk of
the soybean oil meal is used in animal feed for the production of meat and eggs.
Despite considerable public and commercial interest in soybean products as food, the
proportion of soybean protein consumed directly in human nutrition is still relatively
smal l
”~Zeki ,Ber k,IsraelIns ti
tuteof Technology

References:
Literature
1. Nout, MJ Robert, Tri D Martoyuwono, Peter CJ Bonnie and George T Odamtten.
1992, Hibiscus Leaves for the Manufacture of Usar, a Traditional Inoculum for
Tempe. Journal of Science Food Agriculture 58, 339-346.
2. Peñaloza Walter, Chistopher L Davey, John N Hedger and Douglas B Kell.
1992,Physiological Studies on the Solid-state Quinoa Tempe Fermentation,
Using On-line Measurements of Fungal Biomass Production. Journal of Science
Food Agriculture, 59, 227-235.
3. Sparringa RA, JD Owens. 1999, Causes of Alkalinization in Tempe Solid
Substrate Fermentation. Enzyme and Microbial Technology, 25, 677-681
4. Usmani Naseem F, Radia Noorani. 1986, Studies on Soy Bean Tempeh. Part I:
Optimization of Facotrs Effecting Fermentation in Commercial Production of
Tempeh with Respect to Pilot Plant Studies. Part II: Propagation and
Preservation of Rhizopus oligosporous spores for Commercial Production of
Tempeh from Soybean. Pakistan Journal of Science and Industrial Research,
Vol. 29 no 2 [April].
5. Varzakas T. 1986, Rhizopus oligosporus mycelial penetration and enzyme
diffusion in soya bean tempe. Process Biochemistry Vol. 33, No. 7 pp. 741-747,
1998.
6. Wadud Surruyz, Hussan Ara and Saida Kosar. 1986, Studies on the Preparation
Of Tempeh and Tempeh Kababs. Pakistan Journal of Science and Industrial
Research, Vol. 29 no 3 [June].
Electronic
7. http://www.fao.org/docrep/t0532E/t0532e10.htm. Zeki Berk, Israel Institute of
Technology, Haifa Israel. Technology of Production of edible flours and protein
products from soybeans. Chapters 1 & 9.
8. http://www.micropix.demon.co.uk/tempeh/. Phytotech tempeh information and
product website.
9. http://www.hort.purdue.edu/newcrop/proceedings1996/V3-509.html. Pfaff Gunter
and B. Shipley. New Technology for Making Tempeh: A Cultured Soyfood.
10. http://www.tofurkey.com/discover/what_is_tempeh.htm. Turtle Island Foods, Inc.