Fermentation of Sweet Corn Produced A Very High
Concentration of Vitamin K₂

PB0557

H. SUMI1, Y. YANAGISAWA2, S. NAITO1, T. MITSUO3 and C. YATAGAI1
1 Kurashiki University of Science and the Arts, Kurashiki, Japan, 2 Chiba Institute of Science, Choshi, Japan, 3 Mitsuo Clinic, Tokyo, JapanINTRODUCTIONRESULTSCorn is one of the world's major cereal crops, with a production volume
comparable to that of wheat and rice.

Fermentation of sweet corn was performed with M bacteria at 37oC for 48 hours. The results showed a very high concentration of vitamin K2(1,890µg/100g: dry weight).
The thrombolytic activity of the ferment was quite resistant to heat at 100-150oC for 30 min, but the substrate I/substrate II was about 2.0 in resolution
against the synthetic amide substrate, indicating that it was nattokinase.
N bacteria were also used for comparison under various conditions. The morphology was better puree, pH increased, and fermentation at 45oC for 4-8
days showed a maximum vitamin K2 value of 406.0µg/g (dry weight).

In this study, we report on a fermentation test using Bacillus subtilis thatshowed high production of vitamin K2, which is used as a preventive drugfor osteoporosis, as well as thrombolytic activity.

a)

b)
450.0

Whole kernel corn from Meidi-ya (Tokyo, Japan) was used as the corn,and two types of Bacillus subtilis, M and N, cultured in the laboratory, wereused.

Vitamin K2
For vitamin K2, the following procedures were carried out according to themethod previously reported. HPLC and an ODS column (4.6×250mm)
were used to operate the system at an excitation wavelength of 320nm andfluorescent wavelength of 430nm through the reduction fluorescent coloring
method with a platinum-alumina catalyst column.
Amidase activity
An amount of 0.8ml of BSB (0.17M of borate-saline buffer: pH7.8) wasadded to 0.1ml of nattokinase and underwent pre-incubation at 37oC for
2min. An amount of 0.1ml of the synthetic substrate, conditioned for a final
concentration of 5×10-4M, was added to this mixture and incubation wasperformed at 37oC for 250sec. The absorbance of the pNA generated wasmeasured at 405nm with spectrophotometer.

Sweet corn fermented product showed a strong thrombolytic activity onfibrin plate, upper: fermented corn, lower: control corn.

400.0

400.0

350.0

350.0

300.0

300.0

250.0
200.0
150.0

200.0
150.0
100.0

50.0

50.0

0.0

0.0
①-2

②-1

②-2

③-1

③-2

④-1

Supernatant

250.0

100.0

①-1

0

④-2

4

8

1216

1216

day

450.0

Non heated

100oC

120oC

150oC

Fig.2 Thermal stability of nattokinase
Table Substrate specificity of nattokinase
Substrate

μmol/min/g

Bz-Ile-Glu-(OR)-Gly-Arg-pNA (I)
Suc-Ala-Ala-Pro-Phe-pNA (II)MeO-Suc-Arg-Pro-Tyr-pNA
H-D-Ile-Pro-Arg-pNA
H-D-Val-Leu-Lys-pNA

3440.46
1775.72549.360.00
0.00

Substrate concentration:5×10-4M; 0.17M BSB (pH 7.8)

CONCLUSIONS
The main feature of sweet corn fermented product is that it has less odor or stickiness compared to ordinary stringy natto. In addition, thethrombolytic activity of this ferment was extremely stable under heat.
The vitamin K2 content of natto (wet weight) in 600µg/100g, the maximum value of vitamin K2 was 40,600µg/100g (dry weight) of the corn
fermented product is calculated to be several tens of times higher than that. This is equivalent to approximately 0.37g of our fermented product,
based on the daily requirement of 150µg for adults, and is considered to be a very good functional food.

vitamin K2 (µg/g)

Fibrin plate
Thrombolytic activity was measured by using the fibrin plate method.
Amounts of 10ml of 0.5% fibrinogen solution (0.17M of borate-saline buffer:pH7.8) and 500μl of 50U/ml thrombin were used to prepare fibrin plates,
where 30μl of each specimen was placed. The solution was pre-incubatedat 37oC for 24hr and then the lysis area formed was measured (mm2).

Fig.1 Thrombolytic activity

Supernatantvitamin K2 (µg/g)

vitamin K2 (µg/g)

METHOD

450.0

450.0

Precipitate

400.0

400.0

350.0

350.0

vitamin K2 (µg/g)

AIM

300.0
250.0
200.0
150.0

Precipitate

300.0

250.0
200.0
150.0

100.0

100.0

50.0

50.0

0.0
①-1

①-2

②-1

②-2

③-1

③-2

④-1

④-2

0.0
0

48day

Fig.3 Vitamin K2 content in fermented sweet corn
Bacillus subtilis (N bacteria) were used for comparison under various conditions.
a) After fermentation for 4 days at 45oC, more than 99% of vitamin K2 were observed in precipitate fraction.
-1, Whole mixture or -2, Puree was used for applied sample.
b) Maximum arise of vitamin K2 were detected at 4-8 days, in contract 8-16 days gradually decreased.

REFERENCES• SUMI H et al. A novel fibrinolytic enzyme (nattokinase) in the vegetable cheese Natto; a typical and popular soybean in food of theJapanese diet. Experientia 1987;43: 1110-1111• ASTRUP T et al. The fibrin plate method for estimating fibrinolytic activity. Arch Biochem Biophys 1952; 40: 346-351• YANAGISAWA Y et al. Natto Bacillus contains a large amount of water-soluble vitamin K (menaquinone-7). J Food Biochem 2005; 29: 267277• SUMI H et al. Accumqulation of vitamin K (menaquinone-7) in plasma after ingestion of natto and natto bacilli (B. subtilis natto). Food Sci
Technol Res 1999; 5: 48-50• YATAGAI C et al. Nattokinase-promoted tissue plasminogen activator release from human cells. Pathophysiol Haemost Thromb 2009; 36:227-232• Standard tables of food composition in Japan -2020- (Eighth Revised Edition), Ministry of Education, Culture, Sports, Science andTechnology, 2020