Serbian Biochemical Society

President: Marija Gavrović-Jankulović Vice-president: Suzana Jovanović-Šanta General Secretary: Milan Nikolić Treasurer: Milica Popović

Organizing committee Scientific committee

Natalija Polović Milan Nikolić Milica Popović Karla Ilić Đurđić Dragana Robajac Romana Masnikosa Nataša Simin Aleksandra Stefanović Jelena Brkljačić Isidora Protić-Rosić Ana Simović Snežana Spasić Vladimir Mihailović Ana Miltojević Srđan Miletić

Marija Gavrović-Jankulović Mihajlo B. Spasić Vesna Niketić Ivan Spasojević Dejana Mokranjac Neda Mimica-Dukić Snežana Đorđević Suzana Jovanović-Šanta Melita Vidaković Snežana Marković Olgica Nedić Ivanka Karadžić Vesna Spasojević-Kalimanovska Tanja Ćirković Veličković Ivan Gržetić Goran Brajušković Vesna Vučić Niko Radulović

Proceedings

Editor: Ivan Spasojević Cover design: Zoran Beloševac Publisher: Faculty of Chemistry, Serbian Biochemical Society Printed by: Colorgrafx, Belgrade

Serbian Biochemical Society Ninth Conference

with international participation

University of Belgrade – Kolarac Endowment 14-16.11.2019. Belgrade, Serbia

“Diversity in Biochemistry”

106

Bacillus sp. isolated from Japanese food Nattō Kristina Joksimović1*, Aleksandra Žerađanin2, Jelena Avdalović2, Srđan Miletić2, Gordana Gojgić-Cvijović2, Vladimir Beškoski3

1Innovation center of the Faculty of Chemistry, University of Belgrade, Belgrade, Serbia 2Institute of Chemistry, Technology and Metallurgy, University of Belgrade 3Faculty of Chemistry, University of Belgrade

*e-mail:kjoksimovic@chem.bg.ac.rs

Nattō is a traditional Japanese dish made from fermented soybeans and is usually combined with soy sauce. It is very rich in vitamins, amino acids, proteins, sugars, fats, minerals and dietary fibres, and polypeptides consisting of 275 amino acid residues with anticoagulant, fibrinolytic, blood pressure lowering effects and antioxidant activity 1. Bacillus subtilisnatto belongs to the Bacillus subtilis species, and it is the basis for the production of traditional Japanese food. Enzymes and proteins of this strain also show antithrombin effects similar to heparin,as well as antitumor activity. It has also been shown that Bacillus subtilisnatto contains a nattokinase, which exhibits a strong fibrinolytic activity and activates other fibrinolytic enzymes 2. The microorganism was isolated from the Japanese speciality: 1 g of Nattō was added to 9 mL of saline, resuspended and incubated in an aqueous bath at 80°C. A dilution series (10-1-10-9) was made from which 1 mL of culture wastaken and seeded on Petri dish with nutrient agar (peptone 1, 15 g, meat extract, 3 g, sodium chloride, 5 g, dipotassium hydrogen phosphate, 0.3 g, agar, 18 g, distilled water, 1 L) and incubated at 28 °C. Pure, individual colonies were isolated by the method of exhaustion. The isolated microorganism was characterized by API 50 CHB/E tests and 16S rRNAgene sequencing. The results of the API 50 CHB/E test showed that the resulting microorganism belongs to the species Bacillus subtilis with a percentage of agreement of 99.9%, with literature. This was confirmed with 16S rRNA gene sequencing.

Acknowledgements This study was supported by the Ministry of Education, Science and Technological Development, Republic of Serbia, Project No. III 43004.

References 1. Ho H, Nguyen H. Optimization of Bacillus subtilis Natto immobilization process on alginate –

chitosan complex and its application for Natto kinase fermentation. Int J Pharm 2016;5:25-30. 2. Mani V, Ming LC. Tempeh and Other Fermented Soybean Products Rich in Isoflavones. In:

Frias J, Martinez-Villaluenga C, Peñas E. (eds), Fermented Foods in Health and Disease Prevention. Academic Press, Colorado, 2017, pp 453-474.

Isolation and biochemical characterization of Bacillus subtillis Nattō

*kjoksimovic@chem.bg.ac.rs

Introduction

Bacillus subtilis natto belongs to the Bacillus subtilis species and it is the basis for the production of traditional

Japanese food. Nattō is a traditional Japanese dish made from fermented soybeans and is usually combined

with soy sauce. It's very rich in vitamins. Nattō part of 100 g provides 29% of the recommended daily intake of

vitamin K, 22% of vitamin C, 76% of manganese and 48% of daily requirements for iron and dietary fibre. It

contains vitamins, amino acids, proteins, sugars, fats, minerals and dietary fibres, and polypeptides consisting

of 275 amino acid residues with anticoagulant, fibrinolytic, blood pressure lowering effects and antioxidant

activity1. Enzymes and proteins of this strain also show antithrombin effects similar to heparin, as well as

antitumor activity. It has also been shown that Bacillus subtilis natto contains a nattokinase, which exhibits a

strong fibrinolytic activity and activates other fibrinolytic enzymes2.

The results The results of the API 50 CHB/E test showed that the resulting

microorganism belongs to the genus Bacillus subtilis with a

percentage of agreement of 99.9%, with the literature data.

This study was supported by This work was supported by the Ministry of Education, Science and Technological Development, Republic of Serbia, Project No. III 43004.

.

Kristina Joksimović1*, Aleksandra Žerađanin2, Jelena Avdalović2, Srđan Miletić2, Gordana Gojgić-Cvijović2, Vladimir Beškoski3

1 Innovation center of the Faculty of Chemistry, University of Belgrade, Serbia 2 Institute of Chemistry, Technology and Metallurgy, University of Belgrade, Serbia 3Faculty of Chemistry, University of Belgrade,Serbia

Methods The microorganism is isolated from

the Japanese speciality: 1 g of

Nattō is added to 9 mL of saline,

resuspended and incubated in an

aqueous bath at 80 °C. A dilution

series (10-1-10-9) is made from

which 1 mL of culture is taken and

seeded on Petri mug with nutrient

agar (peptone 1, 15 g, meat

extract, 3 g, sodium chloride, 5 g,

dicalium hydrogen phosphate, 0.3

g, agar, 18 g, distilled water, 1 L)

and incubated at 28 °C. Pure,

individual colonies (K1 and K2)

were isolated by the serial dilution

method.

The isolated microorganism was

characterized by API 50 CHB/E

tests.

REFERENCES

1. Ho, H., Nguyen, H. Optimization of Bacillus subtilis Natto Imobilization

Process on Alginate – Chitosan Complex and Its Application for

Nattokinase Fermentation. Int. J. Pharm 2016;5:25-30.

2. V. Mani, L. C. Ming, Tempeh and Other Fermented Soybean Products

Rich in Isoflavones. In: J. Frias, C. Martinez-Villaluenga, E. Peñas,

Fermented Foods in Health and Disease Prevention. Academic Press

2017: 453-474.

Figure 1. The results of the API 50CHB / E test for two

colonies K1 (left) and K2 (right).

Figure 2. Japanese food- Nattō