APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 1994, p. 1495-1499 Vol. 60, No. 50099-2240/94/$04.00+0Copyright C) 1994, American Society for Microbiology

Vitamin B12 Production by Citrobacter freundii or Klebsiella pneumoniaeduring Tempeh Fermentation and Proof of Enterotoxin

Absence by PCRSYLVIA KEUTH AND BERNWARD BISPING*

Institut ffir Mikrobiologie, Westftlische Wilhelms-Universitat Munster, D-48149 Munster,Federal Republic of Germany

Received 4 January 1994/Accepted 19 February 1994

The influence of some fermentation parameters on vitamin B12 formation by strains of Citrobacterjfreundiiand Klebsiella pneumoniae isolated from Indonesian tempeh samples during tempeh fermentation wasinvestigated. A decrease in fermentation temperature from 32 to 24°C led to a decrease in vitamin B12formation. Inoculation of soybeans with different numbers of cells of C. freundii at the beginning ofsolid-substrate fermentation showed that only the velocity of vitamin formation and not the final amount ofvitamin formed depended on the number of cells. The addition of cobalt and 5,6-dimethylbenzimidazoleincreased the vitamin B12 content of tempeh. Nevertheless, levels of incorporation of the two precursors intothe vitamin B12 molecule were very low. Neither C. freundii nor K. pneumoniae possessed the genes encoding theenterotoxins Shiga-like toxin SLT IA, heat-labile enterotoxin LT Ih, and heat-stable enterotoxin ST Ih, asindicated by PCR. This result supports the suggested use of these two strains to form vitamin B12 duringtempeh fermentation in Indonesia.

Tempeh (tempe kedelai) is a traditional fermented soybeanfood in Indonesia, where it serves as a cheap basic foodstuff inthe nutrition of the Indonesian population. It is produced bycooking and hulling soybeans. After a soaking process, thesoybeans are cooked once more. For solid-substrate fermen-tation (SSF), they are inoculated with molds of Rhizopus spp.In the traditional process, soybeans are soaked overnight, andspontaneous bacterial acidification takes place. In modernindustrial processes in the Western world, bacterial acidifica-tion is replaced by artificial acidification with lactic acid.Tempeh is highly significant because of its high content ofamino acids, fatty acids, and vitamins (3, 6, 17, 18, 20, 33). Themost important vitamin is vitamin B12, which is normally notfound in vegetarian foodstuffs (16) and which is formed bybacteria that accompany the fermentation process (20, 22, 28).

In a screening for vitamin B12-producing bacteria isolatedfrom Indonesian tempeh samples, a strain of Citrobacterfreundii was found to produce the highest vitamin B12 concen-tration during the tempeh SSF. A Klebsiella pneumoniae strainalso formed large amounts of this vitamin (20). Besides vitaminB12, these two strains also formed other water-soluble vita-mins, such as riboflavin and vitamin B6. C. freundii alsoproduced vitamin B12 during the soaking of soybeans (8).Some strains of the family Enterobacteriaceae are known to

be capable of producing enterotoxins (5, 14, 19). Therefore, weinvestigated whether the aforementioned C. freundii and Kpneumoniae strains possess three known genes for enterotoxinproduction: the genes for Shiga-like toxin SLT IIA (30), forheat-labile enterotoxin LT Ih (34), and for heat-stable entero-toxin ST Ih (24).The aim of the present work was to investigate the influence

of fermentation parameters and additions on vitamin B12formation. Furthermore, it was important to know whether thetested C. freundii and K pneumoniae strains possess genes

* Corresponding author. Phone: 49-251-839821. Fax: 49-251-838388.Electronic mail address: bisping@uni-muenster.de.

encoding enterotoxins, because the intention is to use thesestrains for tempeh fermentation.

MATERUILS AND METHODS

Microorganisms. The mold Rhizopus oligosporus (isolateTebo), which was isolated from Indonesian tempeh (17) andwhich provides high yields of water-soluble vitamins (20), wasused for tempeh fermentation. C. freundii (isolate 259) and Kpneumoniae (isolate 274) were also added at the beginning ofthe fermentation process because they had been characterizedas producing high levels of vitamin B12 during SSF (20).

Microorganisms isolated from tempeh samples which hadbeen soaked according to the traditional process in our labo-ratory were identified on the basis of the key to the genera inBergey's Manual of Determinative Bacteriology (7) and checkedagainst the information in Bergey's Manual of Systematic Bac-teriology (21, 32).

For PCR analysis, the following reference strains were used:Escherichia coli C600 W34 (SLT IIa'), E. coli 2348/I (SLTIIA-), E. coli 0:128H- (LT- ST'), E. coli 0:6H- (LT'ST'), and E. coli G 1253 (LT' ST-); they were kindlyprovided by H. Karch, Institut fur Hygiene und Mikrobiologie,University of Wurzburg, Wurzburg, Federal Republic of Ger-many. E. coli C600 EWD 299 (LT' ST-) and E. coli HB101SLM 004 (LT- ST') have been described by Moseley et al.(23) and were kindly provided by them.

Process of tempeh fermentation. The modern tempeh fer-mentation process was carried out in the standardized way asdeveloped by Hering et al. (17). The beans (300 g, wet weight)were inoculated with a spore suspension (1.8 ml) of Roligosponrs Tebo (106 spores ml of 0.9% NaCl-1 correspond-ing to 6 x 103 spores g of beans-1). In fermentations withC. freundii or K pneumoniae 1.8 ml of a cell suspension (107cells ml of 0.9% NaCl 1, corresponding to 6 x 104 cells g ofbeans-') was added. After that, the beans were fermented at32°C for 34 h.The traditional fermentation process was carried out as

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TABLE 1. Nucleotide sequences of primer pairs and PCR conditions used for the amplification of enterotoxin genes

PCR conditions Expected

Primer Nucleotide sequence Denaturing Annealing Extension fragment°C s °C s °c s (bp)

SLT IIA start 5'-CCCGGATCCATGAAGTGTATATTATTTAAATGG-3' 94 45 53 60 72 60 960SLT IIA stop 5'-CCCGAATTCTTATTTACCCGTTGTATATAAAAA-3'

LT Ih start 5'-GCGTTACTATCCTCTCTATG-3' 94 40 49 60 72 45 320LT Ih stop 5'-ATTGGGGGTTTTATTATTCC-3'

ST Ih start 5'-TCCCTCAGGATGCTAAAC-3' 94 30 47 60 72 30 244ST Ih stop 5'-GCAACAGGTACATACGTT-3'

described by Baumann et al. (3) with C. freundii only. Soybeans(one part) were washed and cooked with demineralized water(three parts) for 30 min. After a period of cooling down, thesoaking water was inoculated with C. freundii (3 x 107 cells mlof soaking water- '), and the beans were soaked at 30°C for 15h. After separation of the beans and soaking water, the beanswere hulled, cooked again for 30 min, surface dried, anddivided into two portions. One portion was autoclaved beforeSSF with the Rhizopus sp., and the other portion was notsterilized.The portion of the artificially soaked soybeans and the two

portions of the traditionally soaked soybeans were divided intothree parts each. One part was inoculated with the Rhizopus sp.and C. freundii, the second part was inoculated with theRhizopus sp. only, and the third part was inoculated neitherwith the Rhizopus sp. nor with C. freundii (control).As five fermentations under the same conditions had shown

a maximal relative standard deviation of .9.2%, we did notreplicate each fermentation.

Variation of fermentation parameters. For analyzing theinfluence of incubation temperature, tempeh fermentationswere carried out at 24, 28, and 32°C. The influence of differentnumbers of bacterial cells on vitamin B12 formation wasinvestigated by inoculating soybeans with suspensions of 103 to108 cells ml-' (equivalent to 7 x 10° to 7 x 105 cells g ofbeans- 1).

Cobalt(II)-sulfate-heptahydrate and 5,6-dimethylbenzimida-zole at a concentration range of 0 to 400 mg of bacterialsuspension liter- ' (equivalent to 0.135 to 2.7 pLg g of beans- l)were added to the bacterial suspension, with which the beanswere inoculated before SSF with the Rhizopus sp. The yieldwas determined by dividing the number of vitamin B12 mole-cules formed by the number of precursor molecules added.

Determination of bacterial growth in tempeh. The growth ofbacteria in tempeh was determined as described by Hausler(15). Bacteria were spread out on plate count agar (E. Merck,Darmstadt, Federal Republic of Germany) supplemented withcycloheximide (100 mg liter- ').

Vitamin B12 analysis. Vitamin B12 analysis was done with amicrobiological assay as described by Okada et al. (28); thisassay is considered to distinguish between different forms ofcorrinoids: physiologically active vitamin B12 (cobalamins),which can be used by humans, and analogous forms, whichcannot be so used. All vitamin B12 assays were executed intriplicate at two different dilutions, each analyzed eight times.The maximal relative standard deviation was .7.5%.PCR analysis. For PCR analysis, C. freundii (259), K

pneumoniae (274), and the reference strains were cultivated in25 ml of LB medium, containing (grams liter- 1) tryptone (10),yeast extract (5), and NaCl (10), (pH 7.5), at 27°C on a shaker

(model G 76 Gyrotory water bath shaker; New BrunswickScientific Co., Edison, N.J.) for 15 h at 200 rpm. In the case ofSLT IIA, ampicillin (100 ng ml-1; Sigma Chemical Co., St.Louis, Mo.) was added to one of two parallel cultures becausethe formation of SLT IIA is sometimes enhanced in thepresence of this antibiotic.The oligonucleotides used as primers for the amplification of

a part of the SLT IIA gene were described by Gunzer et al.(13). The primers used for the amplification of a part of the LTIh gene were derived from the B subunit (34). For theamplification of a part of the ST Ih gene, the primers werederived from the ST sequence (24). The nucleotide sequencesof the primer pairs and the PCR conditions used for theamplification of the three genes are listed in Table 1. Allprimers, which were purified by high-performance liquid chro-matography, were purchased from MWG-Biotech (Ebersberg,Federal Republic of Germany). The PCR analysis was done asdescribed by Schmidt et al. (30).

RESULTS

Influence of fermentation temperature. With both bacterialstrains, fermentations performed at 32°C resulted in the high-est vitamin B12 content (Table 2). Lowering the incubationtemperature from 32 to 28°C and from 32 to 24°C resulted ina decrease in vitamin B12 content in tempeh. A further effect ofthe lowered temperature was a prolongation of the fermenta-tion time for the Rhizopus sp. from 34 h (32°C) to 50 h (28°C)and to 67 h (24°C). C. freundii formed higher vitamin B12concentrations than K pneumoniae at all temperatures inves-tigated.

Influence of cobalt and 5,6-dimethylbenzimidazole. Theaddition of cobalt resulted in an increase in vitamin B12formation (Fig. 1). Fermentations with K pneumoniae whichwere supplemented with low concentrations of cobalt(II)-sulfate-heptahydrate (0 to 100 mg liter- ') especially showed asteep increase in vitamin B12 content from 73 to 170 ng g of dryweight- l. The increase in vitamin B12 content in fermentations

TABLE 2. Formation of vitamin B12 by C. freundii (259) andK pneumoniae (274) at different fermentation temperaturesa

Temp Cyanocobalamin (ng g of dry wt- ')(OC) C freundii K pneumoniae

24 114 7428 130 9532 152 135

" The maximal relative standard deviation was -7.5%.

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VITAMIN B12 FORMATION IN TEMPEH 1497

0 250-

p'3200 -

*E 150-(a

-Q 100-0

0co1 50->U

0 100 200 300 400cobalt(I1)-sulfate-heptahydrate (mg/I)

FIG. 1. Formation of vitamin B12 (cyanocobalamin) during tempehfermentations by C. freundii (259) (0) and K pneumoniae (274) (-)after the addition of cobalt(II)-sulfate-heptahydrate. The maximalrelative standard deviation was .7.5%. dwt, dry weight.

with C. freundii developed in a straight line with increasingcobalt concentrations, and vitamin B12 content reached a

maximal level of 290 ng g of dry weight'- Nevertheless, theincorporation of the precursor was not really effective, as theresulting yields were very low and reached a maximal value ofonly 0.014 (data not shown).The addition of 5,6-dimethylbenzimidazole also resulted in

an increase in vitamin B12 formation, especially at low concen-

trations (0 to 100 mg liter- 1) (Fig. 2). In contrast to fermen-tations with K pneumoniae, fermentations with C. freundiishowed a further increase with concentrations of up to 300 mgliter- . The yields for the incorporation of 5,6-dimethylben-zimidazole into vitamin B12 were also very low and reached a

maximal value of only 0.013 (data not shown). The numbers ofcells were not influenced by the addition of the two precursorsand were comparable in all fermentations.

Influence of inoculation of soybeans with different numbersof cells of C. freundii. The effect of inoculum size was investi-gated with the most effective vitamin B12-producing organism,C. freundii. Although the initial numbers of cells differed from101 to 106 (g of wet weight-'), after 27 h all cell numbersapproached i0O to 1010 (g of wet weight -) (Fig. 3A) as a

result of more rapid growth rates for small inocula. After 27 h

300

n 250-

U) 200 -

c

*s 150-

co-Q 100-0

U

0

C0 50-

cou

0 -

0 100 200 3005,6-dimethylbenzimidazole (mg/I)

460

FIG. 2. Formation of vitamin B12 (cyanocobalamin) during tempehfermentations by C. freundii (259) (0) and K pneumoniae (274) (-)after the addition of 5,6-dimethylbenzimidazole. The maximal relativestandard deviation was .7.5%. dwt, dry weight.

0)

DILU

=a)

u0

L

-0

E

time (h)B

200-

-n

_'150

0010 2 0 4

-Q

0

(0

0

0 10 20 30 40

time (h)FIG. 3. (A) Numbers of cells of C. freundii (259) during tempeh

fermentation after inoculation of soybeans with different numbers ofcells. wwt, wet weight. (B) Dependence of the formation of vitamin B12(cyanocobalamin) on the inoculation of soybeans with different num-bers of cells of C. freundii (259). The maximal relative standarddeviation was c7.5%. dwt, dry weight. Symbols (for both panels): K,

loll; A, 107; *, 106; FO, 105; *, 104; O, 103-

the vitamin B12 contents of the different fermentations reachednearly the same range of 150 to 160 ng g of dry weight- 1, inaccordance with the increase in the cell numbers (Fig. 3B).

Influence of the preparatory treatment of soybeans for SSFon vitamin B12 formation. Different techniques were comparedwith regard to vitamin B12 formation during SSF. The fermen-tation carried out with lactate soaking of soybeans and in whichthe soybeans were inoculated with the Rhizopus sp. and C.freundii for SSF resulted in the highest vitamin B12 level (Table3). Tempeh prepared with traditionally soaked (soaking waterinoculated with C. freundii) soybeans which were also inocu-lated with the Rhizopus sp. and C. freundii before SSF showedlow vitamin B12 levels.There was also a difference between tempeh prepared with

autoclaved and nonautoclaved soybeans. In a sample preparedwith autoclaved soybeans, only C. freundii could be detectedafter SSF with C. freundii. In a sample prepared with nonau-

toclaved soybeans inoculated with C. freundii, Bacillus cereus

was also isolated. Tempeh prepared with traditionally soaked(with C. freundii), nonautoclaved soybeans inoculated with theRhizopus sp. and C. freundii for SSF contained higher vitaminB12 concentrations than tempeh prepared with traditionallysoaked (with C. freundii), nonautoclaved soybeans inoculatedwith only the Rhizopus sp.

U

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1498 KEUTH AND BISPING

TABLE 3. Formation of vitamin B12 (cyanocobalamin) andnumbers of cells during tempeh fermentations carried out under

various conditions'

Cyanocobalamin Total plate count ofMethod of fermentation" (ng g of dry C. freundii on plate

wt-) produced count agar (109)

R. oligosporus + C. freundiiLactate soaking 53.9 50.0Autoclaving 48.8 35.0No autoclaving 33.0 50.0

R. oligosporusLactate soaking NDC NDAutoclaving ND NDNo autoclaving 23.2 5.0

ControlLactate soaking ND NDAutoclaving ND NDNo autoclaving 3.5 0.1

aThe maximal relative standard deviation was .7.5%.* For no autoclaving, beans were soaked overnight with C. freundii and cooked

but not autoclaved after soaking. For autoclaving, beans were soaked overnightwith C. freundii, cooked, and autoclaved. For lactate soaking, beans were soakedwith lactate and autoclaved after soaking and cooking.

ND, not detectable.

Control soybeans (without any inoculation before SSF)soaked in the traditional way (with C. freundii) and notautoclaved also contained vitamin B12. In these beans, C.freundii, B. cereus, and Micrococcus luteus could be detected.Fermentations carried out with traditionally soaked and withlactic acid-soaked beans, both of which were autoclaved aftersoaking, resulted in no vitamin B12 when inoculation for SSFwas done with the Rhizopus sp. alone.

Detection of the enterotoxin-encoding genes by PCR. Testsfor the three enterotoxin (SLT IIA, LT Ih, and ST Ih) geneswere negative for C. freundii and for K pneumoniae. Figure 4shows the test for the enterotoxin ST Ih gene.

DISCUSSIONThe fact that vitamin B12 production by C. freundii or K

pneumoniae is increased by raising the fermentation tempera-ture is in contrast to the amounts of free amino acids (2, 6) andthe content of -y-linolenic acid (17) in tempeh, which are

improved by lowering the fermentation temperature from 32to 24°C. As vitamin B12 production is coupled to the growth ofthe bacteria (11), it is obvious why more vitamin B12 is formedby the bacteria at 32 than at 24°C.The increase in vitamin B12 concentrations with the addition

9 8 7 11 11 1 3 12

- 516- 344

220

FIG. 4. PCR products of the gene for heat-stable enterotoxin STIh. 1, C. freundii 259; 3, K pneumoniae 274; 7, E. coli 0:128H- (LT-ST'); 8, E. coli 0:6H- (LT' ST'); 9, E. coli G 1253 (LT' ST-); 11,E. coli HB101 SLM 004 (LT- ST'); 12, ladder (in base pairs).

of cobalt shows that the cobalt content in soybeans is subop-timal for vitamin B12 production by C. freundii or K pneu-moniae. Although the addition of cobalt and 5,6-dimethylben-zimidazole leads to an increase in vitamin B12 production byboth strains, their incorporation is poor, as shown by the lowyields. Moreover, supplementation with these precursors is notnecessary, as the vitamin B12 concentration produced in tem-peh by either of these two strains would be high enough tomeet the daily requirement of an adult person.

Inoculation of soybeans with different numbers of cells of C.freundii showed that vitamin B12 production is correlated withthe growth of the bacterium. This was also confirmed forPropionibacterium freudenreichii (11). The rate of vitamin B12production by C. freundii depended on the number of cells inthe inoculum but not the final amount.The low level of vitamin B12 in the fermentation with

traditionally soaked, nonautoclaved soybeans additionally in-oculated with C. freundii before SSF can be explained by theexistence of a mixed culture in this tempeh sample. In mixedcultures with other bacteria C. freundii does not produce asmuch vitamin B12 as it does in pure cultures, as proved inanother fermentation (data not shown).The appearance of C. freundii and B. cereus in tempeh

prepared with traditionally soaked, nonautoclaved soybeanscan be explained by the nonsterile conditions of the tempehfermentation process, which imitates the process performed inIndonesia. The results show that bacteria which are presentduring the soaking of soybeans can be transferred to SSF. InIndonesia, this fact is important, as the soybeans are notinoculated with vitamin B12-forming bacteria. In conclusion,vitamin B12 formation only takes place if vitamin B12-formingbacteria that occur by chance are transferred from the soakingstage by handling of the soybeans under nonsterile conditionsafter the second cooking.Rhizopus spp. have a positive effect on bacterial growth. The

reason is the hydrolyzing capacity of the mold, which suppliesgrowth substrates, such as amino acids, for the nutrition of thebacteria (2, 20). Furthermore, protease inhibitors, such as theBowman-Birk inhibitor, are found in soybeans and can inhibitbacterial serine proteases (4). In contrast, the fungus formsproteases of the aspartate type; these are not sensitive to theinhibitor (1) and therefore can release amino acids in thepresence of serine protease inhibitors.

In conclusion, the comparison of the different preparatorytreatments of soybeans for SSF shows that the best way ofreaching a high vitamin B12 level is lactate soaking of soybeansand inoculation with a vitamin B12-producing bacterium, suchas C. freundii 259, before SSF.The family Enterobacteriaceae includes many pathogenic

organisms, such as Salmonella typhi, Shigella dysenteriae, Kpneumoniae, and Yersinia pestis. In addition, strains of E. coliand C. freundii, which belong to the intestinal flora of humans,can cause diarrhea via the production of enterotoxins (9, 10,12, 19, 23, 30, 31). Although tempeh contains many membersof the family Enterobactenaceae (25-27, 29), no reports existabout diarrhea occurring after the consumption of tempeh.Nevertheless, it had to be determined whether the two strainsof C. freundii and K pneumoniae used here possess the abilityto produce enterotoxins before they could be used for tempehfermentation. As both strains were negative for three knownenterotoxins and as they were originally isolated from Indone-sian tempeh, it can now be stated that their use in tempehfermentation should have no negative effect on tempeh con-sumers.

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VITAMIN B,. FORMATION IN TEMPEH 1499

ACKNOWLEDGMENTS

We thank the Federal Ministry of Research and Technology, Bonn,Federal Republic of Germany, for supporting these investigations.We thank H. Karch, Institut fur Hygiene und Mikrobiologie,

University of Wurzburg, Wurzburg, Germany, for supporting theinvestigations of enterotoxin production. We extend our sincerestthanks to our academic teacher, H. J. Rehm, for his constant support,his helpful incitations, and the ability to work in his laboratory.

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