PB1224

Cell-Lysis (Lysozyme) Activity in Japanese Fermented Food-Natto
H. SUMI1, M. IMAI1, T. MITSUO2 and C. YATAGAI1
1 Kurashiki University of Science and the Arts, Kurashiki, Japan, 2 Mitsuo Clinic, Tokyo, JapanPURPOSE / OBJECTIVES

Table1 The cell lysis activity in species of Bacillus natto
Natto bacteria
Natto bacteria* (for starter)
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TempehE.coli

MATERIALS & METHODS
Bacillus natto (12 species) were isolated in our laboratory. It
was cultured on 3% dry broth medium (Nippon Seiyaku) as thebasic medium, and incubated for 48hr in a static culture or 24168hr with shaking (100rpm).
Thrombolytic activity was measured by using the fibrin plate
method. Amounts of 10ml of 0.5% fibrinogen solution (0.17Mof borate-saline buffer:pH7.8) and 500μl of 50U/ml thrombin
were used to prepare fibrin plates, where 30μl of each sample
was placed. The solution was incubated at 37oC and then thelysis area formed was measured (mm2).

No.1
No.2
No.3
No.4
No.5
No.6
No.7
No.8
No.9
No.10
No.11
No.12

0.6

The cell lysis (lysozyme) activity (CL50) was 0.21μg/ml.
Micrococcus luteus 1.10mg/3ml phosphate buffer,
pH6.2 and incubated for 2hr.

0.4

000.5

11.5

22.5Lysozyme (µg/ml)

1.0
0.8
0.6
0.4
0.2

Table 2 The cell lysis activity in Bacillus natto (No.1)

3% dry broth, 1% ammonium sulfate
3% dry broth

0Incubation time (hr)

pH
6.3
6.4

024

48

168―

603

1,523―

―

243

162
7

384
95
10

0.0479510152025Dilution

Fig.2 The cell lysis activity in 10 commercial natto

74

Each value represent lysozyme activity (CL50 μg/ml).

RESULTSSUMMARY/CONCLUSION

10864250.25

200.20150.15100.1050.05

20102030405060Fraction No.

Fig.1 Isoelectric focusing pattern (LKB) of Bacillus natto (No.1)
Column method (110ml) at 900V for 19hr, 5oC.
Lysozyme ( ― ), protein ( ― ), pH (---).

Although the cell-lysis activity in natto probably extends to all microorganisms or evencancer, the relationship between this bacteriolysis activity and antibacterial activity andnattokinase activity is still unknown and remains a future challenge.
Abs 280nm

12Lysozyme (1/Abs 620nm)

14pH

The cell-lysis activity after static incubation showed that nolysis was observed in general bacteria, but No.1-3 bacteria
showed particularly strong activity among the 12 species ofBacillus natto (Table 1).
From the shaking culture of No.1, the yield of cell-lysis activity
was equivalent to 1.5g of lysozyme per liter. It was found thatthe bacteria (Bacillus) could utilize ammonium sulfate as anitrogen source, and that the activity was transient and verystrong at 37oC for 24 to 48hr of incubation, after which it
gradually decreased. The optimal pH of this enzyme was about
6.0, and pI was about 9.5 by isoelectric point electrophoresis(LKB column method) (Fig.1).
There was a fairly large difference in cell-lysis activity amongcommercial natto (10 varieties), but three products (No.1-3)
had cell-lysis activities of 1.70, 0.83, and 0.53µg/ml, in orderfrom strongest to weakest (Fig.2).

0.80.2

It was cultured on 3% dry broth medium incubated for 48hr. The cell lysis (lysozyme) activity (CL50) was 1.10mg/ml.
Micrococcus luteus 1.15mg/3ml phosphate buffer, pH6.2 and incubated for 24hr.

Shaking culture
The cell-lysis activity (CL₅₀, µg/ml) was calculated from theenzyme concentration required to lyse 50% of Micrococcus
luteus by the method of Shugar et al. Egg lysozyme waspurchased from Sigma Co. Ltd..

Lysozyme activity (µg/ml)
307.7
240.0
10.9
7.2
―
――
―
―
―
―
―
―
―

Abs 620nm

SpeciesAbs 620nm

Commercially available natto has strong nattokinase, whichworks for thrombolysis when eaten, while it has been reported
to have antibacterial activity against Escherichia coli or O-157
bacterial.
In this work, we focused on the cell-lysis (lysozyme) activity innatto, which is usually consumed by Japanese people.

1REFERENCES• SUMI H. Advanced food ingredients and functions CMC, Tokyo1997; p.190-196• SUMI H et al. Anti-bacterial component dipicolinic acid measured in Natto and Natto Bacilli. Nippon Nogeikagaku Kaishi
1999; 73: 1289-1291• SUMI H et al. Anti-bacterial and cell activation ability of Bacillus natto. New Food Industry 2019; 61: 95-98• SUMI H. Discovery of new ingredient “lysozyme” expected to revitalize the market. The health industry news 2001; 976: 26• SHUGAR D. The measurement of lysozyme activity and the ultra-violet inactivation of lysozyme. Biochim Biophys Acta 1952;
8:302-309