Decomposition of soybean oligosaccharides by intestinal bacteria. II. Comparison of twenty strains of Escherichia coli for consuming each sugar in the sugar mixture extracted from defatted soybean meal-香川大学学術情報リポジトリ

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Vol. 20, No. 1 (1968)

DECOMPOSITION OF S O Y B E A N

OLIGOSACCHARIDES

BY INTESTINAL B A C T E R I A *

I1 C o m p a r i s o n o f T w e n t y Strains o f Escherichia coli

for

C o n s u m i n g Each . Sugar i n the Sugar M i x t u r e

Extracted

from Defatted

S o y b e a n M e a l

Sin'itir6

KAWAMURA

and Tadasi

KASAI

As the screening of 17 strains of E. coli reported in Part 1 (1) of this series was preliminary, 20 strains (3

new strains added t o the former 17) were once again compared for the decomposition of sugars extracted from soybean meal.

1. Experimantal Procedures

Twenty strains of E. coli were used. They are shown in Table 1. I A M stands for Institute of Applied Table 1. Strains of Esclzerichia coli examined

No. in this No. of

paper IAM Other descriptions

B-151-1; coli-2; from IID B-151-2; coli-5; from Chisso Ken.

B-151-3; coli-6; from IID, strain Taki No. 2. B-151-4; coli-7; from IID, strain Shoku No. 2 . B-151-5; coli-8; from IID, intermediate type. B-151-6; 2-7; frmo antibiotic test strain.

B-151-7; ML-3; from Pasteur Institute, colicinogenic strains.

B-151-8; Bordet; from Pasteur Institute, colicinogenic (sensitive) strain.

B-151-9; from ATCC, 11246; from V.A. Najjar, produces lysine, glutamine, histidine, and arginine decarboxylasses.

B-151-10; from ATCC, 3655, antifungal test strain.

B-152-1; from IAM, K-12; from ATCC, 10798; from C. E. Clifton, K-12. B-152-2; from IAM, B; from ATCC, 11303; from S. E. Luria, B.

B-152-3; from ATCC, 7009 (E. communior); from F. S. Orcutt, strain F, Virginia Polytech. Inst.

B-152-4; from ATCC, 745 (E. communior).

B-152-5; from ATCC, 206 (Bacterium coli-communior)

.

B-152-6; from IAM, nitric acid determinative strain. B-152-a; ML 308

B-152-b; Hfr 2252 B-152-C; W 677 B-152-d; I F 0 3366

The scientific name for Nos. 13, 14, and 15 is E. coli subsp. communior (TOPLEY et WILSON) Yale, and that for the rest is E. coli (MIGULA) CASTELLANI et CHALMERS.

ATCC: American Type Culture Collection, 12301 Park Lawn Drive, Rockville, Maryland 20852, U. S. A.

IAM: Institute of Applied Microbiology, University of Tokyo. IID: Institute for Infectious Diesases, University of Tokyo. IFO: Institute for Fermentation, Osaka.

*This research has been financed in part by the grant (FG-Ja-123) made by the United States Department of Agriculture under Public Law 480.

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34

Tech.

Bull. Fac.

Agr.'I(agawa

h i v .

Microbiology, The University of Tokyo. Preliminarily one strain (No. 20) of E. coliwascultivated on some

soybean media, and then 20 strains were compared on the medium,(see below, (1) of 1.1.) containing soy-

bean sugar mixture, peptone, and salts.

1.1.

Cultivation of E.

coli strain No. 20 on soybean media

This strain was cultured on 5 different media at 30" for 96 hours by shaking.

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Semisynthetic medium containing the sugar mixture extracted from defatted meal of Hampton

soybeans. The components are shown in Table 2, The sugar mixture was extracted from defatted soybean

Table

2

Culture

medium

for

1.1. (1)

(NH‘IIzSO~

0.2 g

KzHP04 0.7

KHzP04 0.3

Peptone

0.5

MgS04.7HzO

0.02

Sugar mixture

' 20 ml

(ca.

210 g sugars) Fill

up

to 100 ml with

well water.

meal with hot ethanol and cold water and purified by deproteinizatioil and desalting. The extract from

100

g

defatted soybean meal was concentrated to 100 ml., This contains about 10 g sugars (sucrose, ~ a f f i -

nose, and stachyose in 6.4:1.1:3.7 approximately).(2)

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Defatted soybean meal. Add 25 parts water (well water) to

'I part ae'fatted soybean' meal. ~ d d

soy-

bean oil corresponding to 0.1

%

in order to prevent foaming. sterilize with anautqclave at.120" for 10 rnin.

(3) Hot water extract of soybeans. Add 10 parts water to Ilpart full-fat soybeans. Grush after soaking

ove~night. Extract by boiling for 30 min. Sterilize the jiltrate with cloth,

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Concentrated soybean extract. Concentrate the extract ,of,(3) two-fold.

(5) Defatted soybean meal plus~potassiium phoSphate. Add 6 g'defatted 'soybean' meal and 1.914 g

K 2 H P 0 4

to 150 in1 water.

Each of ,these media (100

ml

x

2)in cultivatioq flasks (500

ml)

was sterilized by autoclaving at 120" for

10 min. After cooling a loop of the strain No. 20 was inoculated to one of pairs o'f flasks. Cultivation was

made by shaking at 30". Five tnl was taken out after 0.

6,

12,

'24,48,

72, and 96 hours.

The growth of bacteria was rneas~red~bydreading

the absorbance at 620

rnp

by Hitachi spectrophotom-

eter Model-101 against each medium without inoculation as the blank.

1.2.

Culture medium

This is the same with that given in Table 2, except that the sugar; solution was half, i.e. 10 ml of the con-

centrated mixed, sugal-.solutionfextr~acted.

from defatted soybean as, described in,l.l.+(l) and ca. 90 rqlgof

well water was added.

1.3.

Seed cuItivation

'

The medium without sugar mixture (5 ml) wasetaken in test tubes and,was.sterilized by autoclaving,at

120" for 10 min. Inoculate with a loop of each strain. CuItivate,at '37" for'~8~ho1irs'by

sliaking.

1.4.. Main cultivation

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Cultivate at 37' by shaking. Measure the sugars left after 20,40, and 60 ho,urs for each strain.

1.5. Determination of sugars left in the culture liquid .

Add 15 ml 99% ethanol to each test tube to stop the enzymatic reaction. Stir and centrifuge. Cor- centrate 10 ml of the supernatant to 0.5 ml with a vacuum freeze drier.

Spot 20 ,u1 of theconcentrate to a sheet of filter paper. Develop with butanol-acetic acid-water (4:1:2) 3 times. Spray with p-anisidine hydrochloride reagent. Determine with a densitometer.

2. Results and Discussion

2.1. Growth of the strain No. 20 on soybean media

See Table 3. In case of media other than (I), we obtained suspensions where bacteria and other particles

'

Table 3 Growth of bacteria on soybean media

Medium (1) (2) (3) (4) ( 5 ) Cultivated for 6 hrs. 0.03 0.80 0.58 0.90 0 5 0 12 0.02 0.77 0.70 0.94 0.95 24 0.75 6.64 0.66 0.63 1 .OO 48 1.45 0.64 0.78 0.95 1.03 72 1.60 0.83 0.62 0.97 1.25 96 1.55 0.70 1.12 0.90 1.18

moved vig~rously up and down. Thus it ~ 3 s very difficult to measure the absorbance. The value in the table is the initial largest value. In case of the medium (1) turbidity seemed to increase in parallel with the multiplication of bacteria. In the other media this correlation is not clear.

. From Table 3, we see that this strain grew best on the medium (1). The nutrients for this strain are con- tained in aqueous extract of soybeans, as the growth on the medium (3) was a little better than on the medium (2). One of the shortage of soybean media is decifiency in potassium and phosphate as shown by better growth on the medium (5) than on (2). In any case we planned to study further on the medium (1).

2.2. Comparison of 20 strains on sugar mixture from soybeans

The results are shown in Table 4 and Figs. 1-20. In the figures the abscissa, shows the time of cultivation. In general the sugars were determ~ned after 20,40, and 60 hours. In cases of the strains (Nos. 4,7, 11, 12, 13, 14, 15, 16, and 17) which consumed stachyose completely in 10 hours, the 3 sugars were determined again after 2.5, 5, and 10 hours.

The ordinate in figures shows relative amounts of remaining sugars. The numerical values in Table 4 represent the densitometer readings which are relative values for each sugar. However, the densitometer readings cannot be compared among different sugars. For example 100

r

sugar gives the desitomet'er reading of 1 : 0.531 : 0.266 for sucrose, raffinose, and stachyose. This means that the amount of sugar should be multiplied by 1, 1.88, and 3.76 for sucrose, raffinose, and stachyose. It is more complicated, however, when the sugar mixtures contain different sugars in unequal amounts as sqybean sugar extract. Now the ordinate in figures is expressed as follows. We know that Hampton soybeans contain sucrose, raffinose, and stachyose in approximate ratios of 6.4: 1.1 : 3.7. ( 2 ) This proportion is taken on the ordinate

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Tech. Bull. Fac. Agr. Kagawa Univ.

Fig. 1. Strain No. 1 . Fig. 2. Strain No. 2. Fig. 3. Strain No. 3. Fig. 4. Strain No. 4.

Fig. 5. Strain No. 5. Fig. 6. Strain No. 6. Fig. 7. Strain No. 7. Fig. 8. Strain No. 8.

Fig. 9. Strain No. 9. Fig. 10. Strain No. 10.

-

Sucrose

Stachyose

-

Raffinose

Figs. 1-10. Comparison of strains Nos. 1-10 of on sugar mixture from soybeans. See the text 2.2. for explanation.

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Vol. 20, No. 1 (1968)

Fig. 11. Strain No. 11. Fig. 12. Strain No. 12 Fig. 13. Strain No. 13. Fig. 14. Strain No. 13.

(K-12). (B) (subsp. communior)

.

(subsp. communior)

.

Fig. 15. Strain No. 15. Fig. 16. Strain No. 16. Fig. 17. Strain No. 17. Fig. 18. Strain No. 18. (subsp, communior)

.

Fig. 19. Strain No. 19.

TlhlE l l ~ r )

Fig. 20. Strain No. 20.

0---0 Sucrose

-

Stachyose

-

Raffinose

Figs. 11-20. Comparison of strains Nos. 11-20 on sugar mixture from soybeans. See the text 2.2. for explanation.

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Vol. 20, No. 1 (1968)

3. Summary

A strain of E. coli (No. 20) grew well on the medium containing sugar mixture extracted from defatted soybean meal, when supplemented with peptone and some inorganic salts (shaking culture). All the 20 strains were compared for the ability of consuming this sugar mixture. Some of them consumed sucrose very slowly and yet consumed all the three oligosaccharides rapidly.

The preliminary experiment (1) showed clearly that some strains (No. 13 and 16) consumed raffinose, and the selected strain (No. 13) consumed also galactose, sucrose, glucose, and fructose. However, the a-gal- actosidase activity as measured by decomposition of melibiose was very low, when it was measured in the culture liquid. These cultures were made at 37" by settling for 72 hours.

The cultivation of bacteria was made by shaking in the experiment reported in this paper. Thus we described on extensive experiments on 20 strains of E. coli. The experimental results on the availability of various sugars in mixture as extracted from defatted soybean meal are shown in Table 5 and Figs. 1-20.

Acknowledgments

Thanks are due to the members of Northern Regional Research Laboratory of the United States De- partment of Agriculture, Peoria, Illinois, especially to Dr. R. J. DIMLER, Dr. John C. COWAN, and Dr. Walter J. WOLF, for their encouragements.

The authors are grateful for donation of the strains of E. coli to Prof. Kei ARIMA and Prof. Hiroshi IIZUKA of The University of Tokyo.

Valuable discussions were made with Dr. Teiiti NARASAKI and Dr. Hiroshi SUZUKI, Assistant-Profes- sors of Kagawa University. Sincere thanks are expressed to them.

A brief summary was presented to the 7th International Congress of Biochemistry, Tokyo, August 1967,

(4) concerning a-galactosidase activity of E. coli in relation to soybean oligosaccharides. An outline of this study was presented before the Annual Meeting of Japanese Biochemical Society at Sakai, Osaka, on Nov. 6, 1967. ( 5 )

References

(1) KAWAMURA, S., MIYAKE, T., NARASAKI, T. : Kagawa Daigak~r Nogakubu Gak~rzyutu Hokolcu (Tech. Bull. Fac. Agr. Kagawa Univ.), 20,25, (1968) (in English).

(2) KAWAMURA, S.: Ibid., 18, 117-31 (1967). (3) IIZUKA, H.(ed.) : Catalogue of Cultures, Additional

Edition 1966, 77-8, Tokyo, The Japanese Federa- tion of Culture Collections of Microorganisms (1966).

(4) KAWAMURA, S., SUZUKI, H., KASAI, T. : Possible nutritional availability of soybean oligosaccharides. Intern. Congr. Biochem., 7th Congr., Tokyo, Ab- stracts, I-6(p.914) (1967).

(5) KAWAMURA, S., KASAI, T., TADA, R.: Utilization of soybean oligosaccharides by intestinal bacteria (abstract), Seikagaku (J. Japan. Biochem. Soc.),

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Tech. Bull. Fac. Agr. Kagawa Univ.

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