Agricultural and Biological Chemistry

ISSN: 0002-1369 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/tbbb19

Antioxidative Stability of Tempeh and Liberation of

Isoflavones by Fermentation

Hiroshi Murakami, Tomomi Asakawa, Junji Terao & Setsuro Matsushita

To cite this article: Hiroshi Murakami, Tomomi Asakawa, Junji Terao & Setsuro Matsushita
(1984) Antioxidative Stability of Tempeh and Liberation of Isoflavones by Fermentation,
Agricultural and Biological Chemistry, 48:12, 2971-2975

To link to this article: http://dx.doi.org/10.1080/00021369.1984.10866635

Published online: 09 Sep 2014.

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 Agric. Bioi. Chem., 48 (12), 2971 ~2975, 1984 2971

Antioxidative Stability of Tempeh and Liberation

of Isofiavones by Fermentation

Hiroshi MURAKAMI, Tomomi ASAKAWA,* Junji TERAO

and Setsuro MATSUSHITA
Research Institute for Food Science, Kyoto University, Uji, Kyoto 611, Japan
*Doshisha Women's College, Imadegawa, Kamigyo-ku, Kyoto 602, Japan
Received April 13, 1984

Dried tempeh is known to be remarkably stable to lipid oxidation. An isofiavone has been
isolated and is considered to be one of the antioxidants in tempeh. However, the true origin of the
antioxidative activity of tempeh is still obscure. In this paper, isofiavones and their glucosides in
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tempeh were analyzed by HPLC, and the liberation of isofiavones from glucosides occurred during
fermentation was made clear. The main isofiavones responsible for the antioxidative activity in
tempeh were deduced to be daidzein and genistein.

Tempeh is a fermented soybean food, which not clear that sapogenins are liberated during
is indigenous to Indonesia. Dried tempeh is fermentation. In this paper, our attention is
known to be remarkably stable to lipid oxida- focused only on isoflavones.
tion compared with unfermented soybeans. A Soybeans are known to contain several
powerful antioxidant has been isolated from isoflavones and their glucosides. 7 ,8) These
tempeh and has been recognized as 6,7,4'- compounds were proved to have anti oxidative
trihydroxyisoflavone. 1,2) This compound has activity.9) The quantitative determination of
been proven to be one of the most active soybean isoflavones has been performed using
antioxidants among natural flavonoids in gas liquid chromatography (GLC)10) and high
aqueous suspension of linoleic acid. However, performance liquid chromatography
this new trihydroxyisoflavone did not prevent (HPLC)Y -16) Determination ofisoflavones in
autoxidation of soybean oil and soybean meal soybean meal, protein concentrates and pro-
when the synthesized compound was added. 3 ) tein isolates has also been reported. In this
It showed little hemolysis-preventing activity study, isoflavones in tempeh were analyzed by
in rats fed a vitamin E-deficient diet in vivo. On HPLC and identified with the aid of gas
the other hand, extracted tempeh oil showed chromatograph-mass spectrometry (GCjMS).
effective antioxidative activity when added to The anti oxidative stability of tempeh oil is
refined oils.4) The true cause of the anti- discussed in relation to the appearance of
oxidative stability of tempeh is still obscure. isoflavones.
The stability of tempeh to autoxidation in-
creased during fermentation. 3 ) Generally, anti- MATERIALS AND METHODS
oxi<iative activity is shown by phenols or
amines. Tocopherols and isoflavonoids are the Materials. Tempeh (fermented for 40hr with Rhizopus
main phenols found in soybeans. Among these o/igosporus) and soybeans (dehusked and steamed) were
compounds, tocopherols might not be mod- obtained from Marusanai Co. (Okazaki). A part of these
samples was freeze-dried for stability tests and another
ified by the fermentation process, but lipo- 'part was dried by blowing warmed air at 50°C through it
philic aglycone of isoflavonoids is liberated for extraction of isofiavones. Both dried samples were
by p-glucosidase during fermentation. 5 ) powdered by a coffee mill. Heated and dried meal was
Saponins show antioxidative activity,6) but it is defatted with hexane. Well-fermented and spray-dried
 2972 H. MURAKAMI et al.
tempeh meal was provided by Marusanai Co. This prepa-
ration was also defatted with hexane. n-Butyrophenone
and TMS-HT (hexamethyldisilazane and trimethyichlo-
rosilane) were purchased from Tokyo Kasei Co.
Incubation of tempeh and soybean meals. Freeze-dried
tempeh and soybean meals (100 g) were placed separately
in 18cm diameter culture dishes and were incubated at
370C in the dark. Lipids were extracted from 109 of the
samples with ethyl ether every two weeks. The yield of
lipids was about 2.1 g. The PV of the extracted lipids were
determined.
HPLC of isofiavones. Isoflavones and their glucosides
were extracted twice from soybean and tempeh meal (3 g)
with 80% methanol (30 ml). The extracts were dried by
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vacuum distillation. The residue was dissolved in 5 ml of
80% methanol and was placed in a refrigerator. The white
precipitate was filtered off, and the clear filtrate was made
up to 10ml with 80% methanol.
HPLC was carried out with a Shimadzu LC-4A, equip-
ped with a stainless column (6 x 150 mm) ofYMC-Pack A-
312 containing a precolumn (4 x IOmm). A UV Shimadzu
SPD-2A variable wave length UV detector was used to
monitor the eluent at 262 nm, The solvent flow rate was
1 ml/min. Separations were carried out with a linear
gradient of methanol in water from 20 to 60% as shown in
Fig. 2. n-Butyrophenone was added as the internal
standard.
GC/ MS analysis. The isoflavone fraction of HPLC elu-
ent was collected and the solvent was evaporated in vacuo.
The dried residue was silylated with TMS-HT in pyridine.
GCjMS was carried out with a Shimadzu LKB 9000 gas
chromatograph-mass spectrometer and a PAC 300DG
mini-computer. The column used was a glass tube (3 mm x
2m), packed with 2% silicone OV-17 on Neopak lA, AW,
DMCS, 60-80 mesh. tJelium gas was used at 30ml/min.
The column-temperature was programmed from 150 to
250°C (5°Cjmin). Operation conditions were as follows:
ion source temperature of 290°C, separator temperature of
280°C, ionizing electron energy of 22eV, trap current of
60 p.A and accelerator voltage of 3.5 kV.
RESULTS
Stability of tempeh oil
Tempeh and soybean meals were incubated
to test the oxidative stability of their oils. As
shown in Fig. 1, oils in tempeh were very
stable to oxidation.
Determination of isoflavones
Eighty % methanol extracts of soybean
and tempeh were analyzed with HPLC. The
150
Soybeans
>
II..
Tempeh
0468101214
Weeks
FIG. 1. Changes of Peroxide Value of the Oils Extracted
from Tempeh and Soybean Meals.
isoflavone glucosides having fewer hydroxyl
groups are eluted faster than the glucosides
having more hydroxyl groups. And isoflavone
glucosides are eluted faster than free iso-
flavones. 12 ) The results are shown in Figs. 2, 3
and 4. Soybean extract (Fig. 2) contained
large amounts of isoflavone glucosides, but
tempeh extract (Fig. 3) showed a decrease of
glucosides and an increase of isoflavones.
Glucosides a, band c and aglycones aI, b l and
c were able to be assigned to daidzin,
l
glycitein-7-0-gucoside and genistin and daid-
zein, glycitein and genistein on the basis of
several references ll - 16 ) and of the following
GCfMS experiments. Well fermented tempeh
(Fig. 4) contained no isoflavone glucosides but
large amounts of isoflavones.
GCjMS of isoflavones
Isoflavone fractions were analyzed by
GCfMS. From derivatization procedures, two
peaks, A and B, were found to correspond to
trimethylsilylated derivatives of isoflavones.
The mass spectrum A (Fig. 5) showed frag-
ment ions at m/z 398 (M+) and 383
(M+ -CH3). From the spectrum, this com-
pound was identified as daidzein which has
two OH groups. The mass spectrum of B(Fig.
6) showed fragment ions at m/z 471
(M+ -CH3), 414 (M+), 399 (Mt+ -CH3)'
The structure of MI is a TMS-derivative of
isoflavone which has one OH group and two
OTMS groups. Trimethylsilylation of neigh-
 Isoflavones in Tempeh 2973

a c

E
= .--- - - - - - - -5- _... - - -; 60
'"
CD

'" b !
50 :r
o
c'
40
'"
::E

30

20
, ,
b 10 20 30 40 50 60
Time(min)

FIG. 2. HPLC Elution Diagram of an Aqueous Methanolic Extract (80%) of Defatted Soybean Meal.
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The solution, 200 jll, was injected onto the column. Internal standard, 20 jlg, was added to the sample. The
amount of isoflavones and their glucosides are estimated from their UV absorptions to be roughly 30 and
320 mg in 100 g defatted soybean meal. a, daidzine; b, glycitein-7-0-glucoside; c, genistin; a', daidzein; b',
glycitein; c', genistein; s, internal standard.

a c
c'

a'

, , , ,
10 20 30 40 50 60 ';0
Time(min)

FIG. 3. HPLC Elution Diagram of an Aqueous Methanolic Extract (80%) of Defatted Tempeh Meal.
The amount of isoflavones and their glucosides are estimated to be roughly 130 and 220 mg in 100 g defatted
tempeh meal.
c'
a'

, ,
10 20 30 40 50 60
Time(min)

FIG. 4. HPLC Elution Diagram of an Aqueous Methanolic Extract of Defatted Well-fermented Tempeh
Meal.
 2974 H. MURAKAMI et al.

~
oxidative stability of tempeh oil depends on
the liberation of lipophilic aglycons from iso-
flavone glucosides which exist originally in
soybeans and proved the appearance of p-
300 400
glucosidase in tempeh during fermentation.
100 To clarify this phenomenon, isoflavones and
TMS°-O:)..o.OTMS M = 398 their glucosides in tempeh and non treated
50 ° soybeans were analyzed by HPLC in this
paper. There are several studies ll -16) on HPLC
analysis of isoflavones and their glucosides.
Use of 80% methanol seems to be recom-
FIG. 5. Mass Spectrum of Isoflavone A.
mended for the extraction. 16) As shown in
Figs. 2 and 3, soybean contained large
414
amounts of isoflavone glucosides but tem-
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399
471
300 400
100
TMSO'("y0) ~
~~OTMS M = 486
50
°~ °
o 100 200
FIG. 6. Mass Spectrum of Isoflavone B.
boring hydroxyl groups may be difficult due to
steric hindrance except a trace amount is com-
pletely silylated. Therefore, .this compound is
considered to contain three TMS groups, but
one of them is not appreciably trimethylsilyl-
ated by TMS-HT. Trimethylsilylation with
N,O-bis-(TMS)-acetamide and TMS-imida-
zole also showed the difficulty of complete
silylation of three OH groups. From these
considerations, this compound can be attrib-
uted to genistein.
DISCUSSION
Tempeh oil has been reported to be stable
for oxidation. The sample used in this experi-
ment was also quite stable for oxidation as
shown in Fig. 1. This phenomenon was con-
sidered to depend on the existence of anti-
oxidants. One of them has been isolated and
identified as 6,7,4' -trihydroxy isoflavone. 1 ,2)
Murata, et al. 3 ,5) considered that the anti-
peh lost them and isoflavones increased sig-
nificantly. In the case of well fermented tem-
peh (Fig. 4), almost all glucosides disappeared
and changed to aglycones. Three compo-
nents involving daidzein and genistein were
found to be major aglycones resulting from
the fermentation process of tempeh.
Our HPLC data of tempeh extract support
300 the explanation by Murata 3 ) that the stability
of tempeh to oxidation is generated by the
liberation of lipophilic isoflavones from gluco-
sides by p-glucosidase. However, the main
components responsible for the stability may
be genistein and daidzein. These isoflavones
are known to possess anti oxidative activity.4)
Acknowledgm~nts. The authors thank Dr. Kiku
Murata, a Professor Emeritus of Osaka City University,
for her valuable suggestions. They also thank the
Marusanai Co., Okazaki, for their kind gift of tempeh.
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 Isoflavones in Ternpeh 2975

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