Bv~tecn.Ad~. ~,ol I1, pp 481-4'~3,1¢~93 0,"14-¢',"5(J.

g] $24q~l
Pnnted mGreatBntatrt All PdghtsR~-er'.ed .,- IqtJ3Pergam,,nPre.~.Ltd

TEMPE FERMENTATION: SOME ASPECTS OF

FORMATION OF y-LINOLENIC ACID, PROTEASES AND
VITAMINS

B. BISP[NG, L. HERING, Li. BALrMANN, I. DENTER, S. KEUTH and

H.J. REHM

Inshtut.t~tr Mikrobtolo,~e. Untut'rsttlU Mienster. Cortensstr 3. D-4400 Munster

Germany

During a tempe fermentation the concentrations of linoleic, and ~-linolenic

acids (ALA) d e c r e a s e d while the concentration of o l e i c a c i d increased. During
fatty acid synthesis Rhizopus sp. p r o d u c e d only -r-linolenic acid (GLA) i n s t e a d
of ALA. The amount of GLA in tempe were influenced by varying external
parameters.
The p r o t e o l y t i c capacity of 36 s t r a i n s of the genus Rhizopus isolated from
Indonesian tempe or tempe inocu[a was examined. There was a distinct
increase in the amount of free amino acids during tempe fermentation.
Fermentations with mixed populations of bacteria and Rhizopus y i e l d e d a
lower level of free amino acids, but an increase in total amount of amino
acids. In comparison to intracellular, and e x t r a c e ] l u l a r proteases the p r o t e -
ases of the cell wall fraction are most responsible for proteolytic capacity of
the different Rhizopus strains.
Two isolated strains of Citrobacter freundii were found to be the best
vitamin B12 producers during the soaking of soybeans. In the solid substrate
fermentation the Rhizopus molds formed vitamin B6. riboflavin, and nicotinic
acid. The addition of bacteria to the solid substrate fermentation resulted in
a strong increase of active vitamin B12 in tempe. In the presence of the
Rhizopus mold, the vitamin BI 2 formation by C. [reundf[ was three times
higher than that of a fermentation without the mold,

Ke4twords: soybeans, tempe fermentation, Rhizopus sp., 1,-linolenic acid. amino

acids, proteases, Ci~robacter freund[i, v i t a m i n B1 z formation

481
 B. BISPING ct al

Introduction

Tempe (tempe kedele) is a traditional fermented soybean food in Indonesia

(Fig. 1). It is produced by different stratus of Rhizopus sp., (R. ohgosporus,
R. arrh~zus, a n d R. stolonifer), w h i c h f e r m e n t b o i l e d a n d d e h u l l e d s o y b e a n s .
A well made Indonesian tempe kedele is a
~ ! w h l t ecompact cake. completely covered by
the fungus. The fungus grows right
" through the beans and forms ]t into a
compact mass whmh can be cut into s l m e s
that are frled, cooked, or used in larger
p]eces in soups as a protein rich meat
substitute (STEINKRAUS et al., 1983). Tem-
pe is consumed daily by mill]ons of people
of all classes m Indonesia and forms the
mare source of protein, calor les. and
vitamins in t h e i r d i e t IW[NARNO and REDDY.
1986). Tempe is also consumed m the
Netherlands. and the nut-like taste of
fried tempe is pleasing to the palates of
Europeans and Americans allke (HESSELTI-
NE, t9651.

Fiq. I: Tempefermented ]n banana leaves.

Annual p r o d u c t i o n of tempe in I n d o n e s i a is 500.000 tons (EBINE. 1979) mainly

m private households and smaller companies. There a r e a b o u t 41.000 of these
small companies producing a daily supply of fresh tempe, employing on
a v e r a g e t h r e e w o r k e r s (SHURTLEFF and AOYAGI. 19-/9" WINARNO, 19-/6).

In 1895 PRINSEN GEERLIGS was the first to descrlbe a method of producing

tempe in Java. Later varlous scientists reported in detail every aspect of
this foodstuff (BOORSMA, 1900; HEYENE. 1913; JANSEN, 1923).

In the USA the STEINIKRAUS and HESSELTINE teams made a thorough study of
the preparatlon and chemical composition of tempe, and they isolated and
identified the molds involved in tempe fermentation (STEINKRAUS et al.. 1983).

There a r e two methods of tempe p r o d u c t i o n : the t r a d i t i o n a l I n d o n e s i a n method

and the modern method of tempe p r o d u c t i o n . During both p r o d u c t i o n methods
the soybeans a r e h y d r a t e d , dehut]ed, a c i d i f i e d , boiled, d r a i n e d , surface-dried,
 TEMPE FERMENTATION 493
i n o c u l a t e d with the e s s e n t i a l m~croorganism(s), put into s u i t a b l e f e r m e n t a t i o n
containers, and incubated at appropriate temperature until the cotyledons
h a v e m a s s e d t o g e t h e r to form a compact c a k e {STEINKRAUS, 1985).
In t h e t r a d i t i o n a l fermentation, the soybeans are soaked in w a t e r a n d then
boiled for about 30 m i n u t e s so t h a t the hulls can be removed more e a s i l y .
O t h e r s p r e f e r t h e b e a n s to be p a r b o i l e d p r i o r to s o a k m g . The b e a n s a r e t h e n
d e h u l l e d b y r u b b i n g them with o n e ' s h a n d s or t r a m p l i n g them u n d e r f o o t . T h e y
are then washed in w a t e r and soaked again overnight. During the soaking
process a bacterial acidification of the soaking water takes place, which
leads to a drop in the pH value down to a range of d.5 to 5.3. This
acidification is low enough to prevent the growth of potential bacterial
s p o i l a g e o r g a n i s m s a n d h a s no d e l e t e r i o u s e f f e c t u p o n t h e g r o w t h of t h e mold
(STEINKRAUS et al., 1960). The soaked beans are then boiled again, drained,
cooled, and surface-dried, then inoculated with a culture grown on leaves
(usar leaves of Hibiscus elatus) or with an inocu]um from a previously
produced tempe. The inoculated cotyledons are then wrapped in banana leaves
where fermentation takes place and incubated for up to 48 hours (WINARNO
and REDDY, 1986). Nowadays the leaves are often substituted by perforated
plastic bags so that a poor exchange of gas can take place.

In the modern method of tempe p r o d u c t i o n , the beans are mainly dehulled b y

machines, the cotyledons are inoculated with pure fungal cultures, and
fermented on t r a y s o r in p e r f o r a t e d p l a s t i c bags (STEINKRAUS, 1985).

Tempe is an excellent source of proteins, vitamins, and minerals (SHURTLEFF

and AOYAGI, 1979). D u r i n g the fermentation of soybeans some f e a t u r e s as
fatty acid composition, the amount of oligosaccharides, and the amount of
several v i t a m i n s are improved (NOUT and ROMBOUTS. 1990).
One of the most important q u a l i t i e s of tempe is the h i g h amount of p r o t e i n ,
which claims a level of d0% of the d r y mass. It e x p l a i n s the g r e a t i n t e r e s t in
t h i s food in many d e v e l o p i n g countmes, which f i g h t a g a i n s t p r o t e i n d e f i c i e n c y
of the population. MURATA et al. (1971) reported that the protein content
remains constant d u r i n g fermentation, but the content of free amino acids in
tempe increases up to 85% above that of unfermented soybeans. M e t h i o n m is
the f i r s t l i m i t i n g amino acid whereas the h i g h l y s i n e content supplements the
l y s m e d e f i c i e n c y in cereals.

In 1978 SUDARMADJI and MARKAKIS found 10 g free fatty acids in 100 g tem-
pe. WAGENKNECHT et aL (196]) identified the free fatty acids: palmitic, stea-
ric, oleic, linolelc, and ]inolenic acLds. During the most active phase of funga[
growth, greater amounts of free palmitic acid were found and the amount of
]ino]eic acid was somewhat lower. With the exceptlon of a loss of 40% ]inolenic
4,~1 B. BISPING t't al
acid in the late phase of fermentation, there was obviously no preferred
consumption of any of the acids (SUDARMADJI and MARKAKIS, 1978; BEUCHAT,
1983).

One f a t t y a c i d t h a t h a s a c h i e v e d a g r e a t i n t e r e s t in r e c e n t y e a r s is T-lmo-
]enic a c i d (GLA), t h e a l l - c i s 6 . 9 . 1 2 - o c t a d e c a t r i e n i c a c i d is a p r o s t a g l a n d i n a n d
leucotriene precursor. Further on. GLA [s u s e d therapeutically to d e c r e a s e
t h e c h o l e s t e r o l a n d t r J g l y c e r i d e s c o n t e n t s in blood. GLA d o e s n o t e x i s t in u n -
f e r m e n t e d s o y b e a n s at all.

Special reports about the high vitamin B1 2 content in tempe led to a

popularity among v e g e t a r i a n s in t h e w e s t e r n world, because plants normally
do not contain vitamin B12. Tempe made from a pure Rhizopus oligosporus
c u l t u r e only c o n t a i n s v e r y l i t t l e v i t a m i n B12 c o m p a r e d to t h e p r o d u c t made
by traditional methods, which also include the action of n a t u r a l l y o c c u r i n g
bacteria. One hundred grams of tempe made from a pure culture contains
a b o u t 0.047 [~g of v i t a m i n B~2 a n d p r o v i d e s l e s s t h a n 2% of t h e r e c o m m e n d e d
d a l l y allowance. Commercml s a m p l e s of tempe p r e p a r e d by traditional methods
in Toronto, Canada were found to contain a specific bacterium which
synthesizes wtamin B12 during fermentation (LIEM et al., 19771. CURTIS et
al. (1977) identified this bacterium as Klebsiella pneumomae (nonpathogenic
strain). By a d d i n g it to t h e s t a r t e r culture, t h e v i t a m i n B12 c o n t e n t in t h e
final p r o d u c t could be i n c r e a s e d to more t h a n 14.8 ~g p e r 100 g r a m s .
For f u r t h e r i n f o r m a t i o n a b o u t tempe l i t e r a t u r e s of: KO a n d HESSELTINE (1979),
SHURTLEFF and AOYAGI (1979), STEINKRAUS et al. (1983), BEUCHAT (1983), WI-
NARNO and REDDY 11986), and NOUT and ROMBOUTS (1990) may be consulted.

The p r e s e n t aim of t h e work was to i n v e s t i g a t e t h e r o l e of d i f f e r e n t s t r a i n s

of R h i z o t ~ s s p e c i e s (R. oligosporus, R. arrhizus, and R. stolonifer) relative to
t h e i r p o t e n c y of c h a n g i n g t h e f a t t y a c i d c o m p o s i t i o n of tempe, r e s p e c t i v e l y to
produce GLA. F u r t h e r on we looked for Rhizopus s t r a t u s and methods, which
could improve the protein s~tuation in tempe, and we characterized some
a s p e c t s of t h e i r p r o t e a s e s y s t e m .
Additionally we characterized other microorgamsms, which are involved in
tempe f e r m e n t a t i o n , a n a l y z e d t h e i r s p e c i f i c r o l e in t h i s p r o c e s s a n d t r i e d to
d e f i n e t h e optimal c o n d i t i o n s for a good v i t a m i n p r o d u c t l o n .
 TEMPE FERMENTATION 485
N a t e r i a l s and Nethoda

Bacteria and f u n g i used in these i n v e s t i g a t i o n s were isolated from Indonesian

tempe and s o a k i n g water samples. All isolations were done d i r e c t l y and the
colony f o r m i n g u n i t s were separated b y s t r i k i n g out on a g a r plates.
For s o a k i n g e x p e r i m e n t s cooked soybeans were inoculated with p u r e s t r a i n s
of isolated b a c t e r i a (inoculum level l x l 0 6 / m l ) and soaked f o r 15 h at 30 °C.
The tempe f e r m e n t a t i o n was c a r r i e d out under standardized conditions (HE-
RING et al., 1991). The e x t r a c t i o n of the lJpids was done b y the method of
BLIGH and DYER (1959). The fatty acids were methylated with boron trifluo-
ride (in methanol) by the method of MORRISON and SMITH (1964) and
determined by GC analysis. The amino acid analysis was possible after a
pre-column-derivatization with the Edman-reagent phenylisothiocyanat (PITC)
and carried out by HPLC. The vitamin B~2 determination was done with
microbiological assays following the method of OKADA et al. (1985). This
method distinguishes between physiological active vitamin B12 and analogous
forms, which cannot be utilized by human beings. Other analytical methods
often do not distinguish between these forms of corrinoids.
Also nicotinic acid and nicotinamide were examined by microbiological assays.
The determinations of riboflavin, vitamin B6, and thiamine were carried out
using HPLC with fluorescence detection. For further details see: HIRING eta/.
(1991), B A U M A N N eta/. (1990). DENTER eta/. (1993). and KEUTH eta/. (1993).

Results

The change of the fatty acid composition during a standard type fermentation
is shown in Fig, 2. The tendency of the decrease of palmitic acid, linoleic
acid, and ALA but the increase in oleic acid and GLA could be observed over
a period of 70 hours of fermentation.

~O

50 ~

40

30
" O
20
£3---[] £13 []
10

0
& 2'O .'O 6'o eo
tlrne (h)

Fig. 2: Fatty acid composition during fer=entati0n 0f soybeans us=ng the strain RhJzopus
arrhJzus EN.
4,~h B. BISPING ~'t ~l.
A c o m p a r i s o n of t h e GLA c o n t e n t of tempe p r o d u c e d at 24°C, 32°C. and 30°C
shows the .ncrease of GLA at lower t e m p e r a t u r e s {Fig. 3). The h i g h e s t yield
of GLA c o u l d be r e a c h e d at 2,t°C w~th t h e strain Rhizopus stolonifer c o d e d IIK
(0.8~). At e t e m p e r a t u r e of 36°C t h e a m o u n t of GLA was lower t h a n 0.15% in
all c a s e s .

0.8

O 6 -

O 4- -

O 0
2 -

Hi, Ft I1~ J16 EN HIB SIO IN GT

~.tr-alns

Fiq, 3: ~-Lm0lenlc acid IGLAI content 0[ tempe produced at dLfferent temperatures. The
abbreviations name strams ts01ated from different sources that were determmed as
follows: Rhzzopus ol~gosporus" SID and IN R arrhzzus rI and EN R s(olon~fer: IK
J16 HIB and GT.

The intraceIlular fatty acid c o m p o s i t i o n of Rhizopus oligosporus MS1 g r o w n on

different l i q u i d media in s u r f a c e a n d s u b m e r g e d c u l t u r e s is s h o w n in Fig. 4.

60
i ~60
180
181
50 182
18 3 y
~- 40

=u 30

20

10

USA-soy S~yam re~p~ CTapek Mp-Ob Mp-lub

culture

FlA. 4: Intracellular fatty acid c0mp0sltl0n of Rh~zopus olLgosporus MS1 grown on

different carbon sources ISoyam: homogenized soybean medlum Tempe: mycelium from tempe
Czapek: czapek-dox medium, MP-Ob : malt-peptone medium surface HP-sub." malt-peptone
medlum submerged).

The intracellular fatty acid composition differed widely dependent on the

culture conditions. Rhizopus mycelium grown on soybeans or on liquid
soybean medium showed a fatty acid composition close to tempe. The
differences did not exceed 2%-4% w i t h the exception of olelc acid (+ 10%).
Rhtzopus g r o w n on l i p i d f r e e media h a d a v e r y d i f f e r e n t c o m p o s i t i o n of f a t t y
 TEMPE FERMENTATION 4~7
acids. The l a r g e s t difference between the surface cultures and mycelium from
t e m p e o r s o y m e d i u m is t h e i n v e r s i o n of t h e a m o u n t s of ALA a n d GLA. ALA was
built up only in amounts of 0.4%-1%. while GLA on the opposite reaches
6~-8.5%. The s u b m e r g e d cultures s h o w e d a GLA c o n t e n t within the fatty acid
f r a c t i o n of 20.8%. H o w e v e r no ALA c o u l d be d e t e c t e d .

The a m o u n t of f r e e a m i n o a c i d s after 30 h o u r s of s t a n d a r d fermentation (HE-

RING et al., 1991) was increased up to five£old. On a n average, it c o u l d be
seen that higher p r o t e o l y t i c a c t i v i t y of t h e f u n g i r e s u l t s in a g r e a t e r a m o u n t
of all amino acids, but did not prefer special ones. In Fig. 5 the effect o£
reduced fermentation temperature .s shown. In this case the duration of
fermentations was longer and a good tempe cake was ready after ca 40
h o u r s . But t h e a m o u n t of f r e e a m i n o a c . d s was ,mproved u p to 120%.

24 11
20 :24°C

o , ,
Hib IK EN ~1 C~ MS1

strains

Fig. 5: Comparison of the amount o£ free amlno aclds after fermentatlons at temperatures
of 24°C and 32°C. RhJzopus mlJgosporus: MS1 and CN R. arrhlzus: EN and Fl and R. sto-
lonlfer: Hlb and IK. There was an mcrease of the amount of free amino ac,ds of ca 20~ at
a lower temperature.

When the fermentation was carried out with mlxed cultures of R. oligosporus
and bacteria there was a shght increase of the total a m o u n t of a m i n o acids
compared with the u n f e r m e n t e d beans. In contrast to this. the a m o u n t of free
amino acids decreased drastically. The amount decreased down to a level of
,t32; w h e n Citrobacter freundii took part m t h e mixed c u l t u r e .
It was s h o w n t h a t t h e cell wall b o u n d proteases w e r e most i m p o r t a n t f o r t h e
proteolytic capacity of fermenting Rhlzopus. In Fig. 6 the turnover rate of
the three fractions in r e l a t i o n to t h e d r y weight is g i v e n for 7 strains. On
the average of all strains 76% o£ t h e total proteolytic capacity belonged to
this fraction, 1,1:~ to the extracellular system, and 10% belonged to the
intracellular proteases.
4~ B. BISPING, 1 al
:240 " debr,,
•1• a x,tr o©ollulor
,n t r a¢ w l l ~ l a r
~'00

160

120 -

80

4 0 -

0
J~p M~I~ J'~j~ M 51 ~ s~ legal ~S5

Fig. 6: Turnover rate for the seven most active strams of our cc.llectlon. On an average
76~ of the total proteolytl,: capacity of Rhlzopus belonged t,:, the cell wall bound
[raetlon. ~lth the exception o[ the straLn coded Hala whLch was a member .:f R. arrhJ:~s
all the other strains given in this [Lgure belonged t,:, R oltgosporu~

The bacteria, which are involved in t h e tempe fermentation process, belong

m a i n l y to t h e lactic acid bacteria and to t h e Enterobacteriaceae. Additionally
a few members of the Pseudomonaclaceae a r e present, too. Only lactic acid
bacteria were able to a c i d i f y the soaking water to pH v a l u e s less than 5.0.
]he acidification of t h e beans is necessary to a v o i d g r o w t h of c o n t a m i n a n t
m i c r o o r g a n i s m s , t h a t c a u s e s p o t l a g e a n d t h e low pH g i v e s an a d v a n t a g e to t h e
g r o w t h of t h e Rhizopus mold ,n t h e s o l i d s u b s t r a t e fermentation. Some of t h e
isolates were examined for their ability to form v i t a m i n B~ 2. n , c o t i n l c acid.
nicotinamide, thiamine, and vitamin B6. Two CitroOacter freunda ,solates
showed the best formation of physiologically active vitamin BI~ of all the
tested bacteria. In Fig. V t h e r e s u l t s of t h e v i t a m i n BI~ production of t h e s e
two strains and of two reference strains (PropiontOacterlum freudenrexchll
s s p . ) , w h i c h a r e known a s w t a m m B~ ~ p r o d u c e r s , are presented.

1 1 i,:,r,~l ij ~ , I ~ i-,~

..........................
Ira,-, If ,- 2: ....

Fig. 7: Formation of cmrrLnolds Ic.fanocobalamln and analogues: In the so,,bean ractlon bf

several bacterlal isolates of tempe and b,~ two reference strams after the smakmg
process.
 TEMPE FERMENTATION 489
The c o n c e n t r a t i o n in the soybean f r a c t i o n ranges from 2.3 - 4.9 n g / g (dw)
for t h e two C. [reundii isolates. The small amounts of v i t a m i n B~2 p r o d u c e d
by P. [reudenreichii ssp. indicates that these strains are less adapted to
soybeans, in c o n t r a s t to the o t h e r tempe b a c t e r i a w h i c h is also c o n f i r m e d by
a minor cell number of P. freudenreichii ssp.
Other physiologically active vitamin B~2 forming bacteria, which were
discovered, are Klebsiella pneumoniae, Streptococcus sp., and Pseudomonas
fluorescens (Fig. 8). Aeromonas hydrophila formed analogues only. Besides
v i t a m i n B~= some isolates formed m c o t i n i c acid, n i c o t i n a m i d e , and thiamine.

~1 trjt aJ Qrnc, u n f

li
Fi.q. 8: F0rmatl0n of c0rrin01ds Icyanoc0balam]n and anal0guesl by several bacterial
isolates of tempe after the soaking of soybeans

During solid substrate fermentations with 14 d i f f e r e n t stratus of Rhizopus

oligosDorus, R. arrhizus, and R. stolonifer it could be shown that all molds
formed riboflavin, vitamin B6, nicotinic acid, and nicotinamide. The final
concentration d i f f e r e d from s t r a i n to s t r a i n , whereas the isolates of R. o/igo-
sporus were the best v i t a m i n formers. No mold Formed p h y s i o l o g i c a l l y a c t i v e
vitamin BI=. This is a proof that only bacteria synthesize physiologically
a c t i v e v i t a m i n B12. The a d d i t i o n of b a c t e r i a of the s o a k i n g e x p e r i m e n t s to the
solid substrate fermentation resu[ted in a strong increase of active
v i t a m i n B12 in tempe, and a g a i n C. [reundii was t h e best v i t a m i n B12 former
(Fig. 9). Additionally Klebsiella pneumoniae, which was d e s c r i b e d by LIEN et
al. (19-/V) to form t h i s v i t a m i n , is also a good p r o d u c e r . The f e r m e n t a t i o n was
extended to 96 h for analyzing the influence of time on the vitamin
production. Furthermore 13 o t h e r bacterial isolates including the reference
strain Propionibacterium [reudenceichii ssp. were tested (Fig. 10). P. f r e u -
denreichii formed only traces of wtamm B12 in comparison to the tempe
bacteria, In a c c o r d a n c e to t h i s fact it was found t h a t the number of cells had
4')1) B. B I S P I N G :'t ,d.

reached only a value o[ 10~/g beans in c o m p a r i s o n to tempe bacteria whose

numbers o f ceils had reached values of 10-~/g beans. The addition of b a c t e r i a
led to a further increase of vitamin B~, r~boflav]n and [n some cases of
thiamine.

il~ ,3n ~,:.,~ u e ~ - - I

,: ~.~r,,:.,: o b ,~d,~n,, r,~

Fiq. 9 Formation of corrlnoldS Ician,:,cobalamin and analogues~ by several bacterial

isolates of tempe during the tempe s,:,lld substrate fermentation Icontrol : unfermented
soybeans' Strep, sp. = S[repLo,:occus sp • ~ pneum. = Klebszella pneumoniae E. cloac. =
Enlerobac~er cloacae C. [reun. : CJ~robac[er frecnd~l

:c K.,,~.:i.J~

Fiq. |0: Eormatlon o{ cc,rrlnolds Icyanc,cobalamin, and analoguesl by several bacteria[

isolates of tempe during the tempe solid substrate [ermentat]on qP. [reuden. = Propionl-
bacLer~um freudenreichil E cloacae : Emerobaczer cloacae" P. fluor. : Pseudomonas
fluorescens H. a]vei = Hafnla alve1 " K pneum. = KlebsleIla pneumonlael.
 TEMPE FERMENTATION 491
The i n f l u e n c e of t h e Rhizopus mold on t h e v i t a m i n B l z f o r m a t i o n of C. f r e u n -
dii was i n v e s t i g a t e d also. As shown in Fig. 11 t h e v i t a m i n c o n t e n t of tempe,
t h a t was f e r m e n t e d with t h e mold a n d t h e b a c t e r i u m , was t h r e e t i m e s a s h i g h
in c o m p a r i s o n to a c o n t r o l f e r m e n t a t i o n with C. f r e u n d i i only. The f e r m e n t a -
t i o n time was 34 h o u r s .

c cr~,~,~las

.z~ ~

.:: I--'°°°'°g°" I
L~cvanoco~alorn,n~

.............................. L'.L'. ;

Fig. !1: Formation of c0rrm0ids Icyan0c0balamin, and analogues) during the solid sub-
strate fermentation which was performed in three different ways Isee textl.

Discussion

The f a t t y acid composition of tempe r e p r e s e n t s a composition close to that of

soybeans. In contrast to soybeans the concentration of o]eic acid is
increased, GLA occurs, and the c o n c e n t r a t i o n s of the o t h e r f a t t y acids a r e
reduced a little. The fungi metabolize the soybean lipids and synthesize
r e s p e c t i v e l y change the f a t t y acid composition o n l y a l i t t l e b i t . This depends
on t h e s t r a i n and the e n v i r o n m e n t a l c o n d i t i o n s . If the fungi a r e forced to
synthesize t h e f a t t y acids d e n o v o t h e f a t t y a c i d composition d i f f e r s widely
in dependence on the c u l t u r e c o n d i t i o n s chosen. The a d v a n t a g e of submerged
produced Rhizopus biomass is the h i g h amount of GLA, [JnoleJc, and oleic acid
in c o n t r a s t to s u r f a c e c u l t u r e mycelium. T h e r e a r e possibilities to influence
the fatty acid c o m p o s i t i o n of t h e fermentation product by v a r y i n g external
parameters and also b y u s i n g the p o t e n t i a l of d i f f e r e n t Rhizopus species (HE-
RING et al., 1991). We were able to f i n d several Rhizopus s t r a i n s with high
p r o t e o l y t i c capacities, t h a t could even be improved b y r e d u c i n g f e r m e n t a t i o n
temperature. Enzymatic tests showed t h a t cell wall bound proteases a r e most
of all responsible for proteolytic capacity of fermenting Rhizopus sp.
492 B. BISPINGet ,I
Determination of t h e i r s p e c i f i c a c t i v i t i e s with simple test k i t s should g i v e an
easy method to f i n d more s t r a i n s wlth a high p r o t e o l y t i c a c t i v i t y and for fast
c o n t r o l l i n g fermentation q u a l i t i e s of inocula used. The release of amino acids
could improve the nutritional value of tempe in comparison to unfermented
soybeans. In the presence of c. freundit the amount of free amino acids
decreased (BAUMANN et al.. 1992). It is supposed that the bacteria consumed
the amino acids, which resulted in an increase of vitamin B12 production.
Probably the release of other metabolites l i k e sugars and free f a t t y acids by
the h y d r o l y t i c capacities of the fungus will also c o n t r i b u t e to the increase of
t h i s vitamin (KEUTH et al.. 1993).

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