Clot Lysis Activity of Bacillus subtilis G8 Isolated from Japanese Fermented Natto Soybeans
Applied Food Biotechnology,
Vol. 6 No. 2 (2019),
18 March 2019
,
Page 101-109
https://doi.org/10.22037/afb.v6i2.22479
Abstract
Background and objective: Bacillus spp. with potent fibrin digesting enzymes, especially Bacillus subtilis (natto), has gained favorable insights into the world of cardiovascular health and functional foods. In this study, Bacillus subtilis G8 was isolated from Japanese fermented Natto soybean commercialized in Indonesia. The overall activity of Bacillus subtilis G8 in resolving blood clot was evaluated under variable conditions quantitatively using clot lysis assay.
Material and methods: Cell-free crude extracts of Bacillus subtilis G8 culture were partially purified with acetone precipitation method. Fibrinolytic activity of the enzymes was verified using fibrin plate assay and zymography. The measurement of clot liquefying activity under physiological temperature and pH was performed using chicken blood. The measurement of clot liquefying activity under physiological temperature and pH was performed using chicken blood. The measurement of clot liquefying activity under physiological temperature and pH was performed using chicken blood.
Results and conclusion: Through zymography, Bacillus subtilis G8 expressed four bands of fibrinolytic proteins sized 19 to 30 kDa. Under controlled conditions, a significantly higher clot lysis was seen at 37 and 40°C, compared to lower temperatures (P≤0.05). In contrast, no significant decrease was seen in blood clot at all tested pH (P>0.05). These findings support the use of functional foods for the prevention of blood clot or as a part of blood health restoration.
Conflict of interest: The authors declare no conflict of interest.
- ▪ Bacillus subtilis ▪ Clot lysis assay ▪ Fibrinolytic ▪ Natto ▪ Zymography
How to Cite
References
Kotb E. Activity assessment of microbial fibrinolytic enzymes. Appl Microbiol Biotechnol. 2013;97(15):6647-6665. doi:10.1007/s00253-013-5052-1
Chen C, Yang FQ, Zhang Q, Wang FQ, Hu YJ, Xia ZN. Natural products for antithrombosis. Evid Based Complement Alternat Med. 2015;2015:876426. doi:10.1155/2015/876426
Butcher K, Shuaib A, Saver J, Donnan G, Davis SM, Norrving B, et al. Thrombolysis in the developing world: is there a role for streptokinase? Int J Stroke. 2013;8(7):560-565. doi:10.1111/j.1747-4949.2012.00923.x
Gomaraschi M, Ossoli A, Vitali C, Pozzi S, Vitali Serdoz L, Pitzorno C, et al. Off-target effects of thrombolytic drugs: apolipoprotein A-I proteolysis by alteplase and tenecteplase. Biochem Pharmacol. 2013;85(4):525-530. doi:10.1016/j.bcp.2012.11.023
Johnston SC. The economic case for new stroke thrombolytics. Stroke. 2010;41(10 Suppl):S59-62. doi:10.1161/strokeaha.110.597351
Sikri N, Bardia A. A history of streptokinase use in acute myocardial infarction. Texas Heart Institute Journal. 2007;34(3):318-327.
Sumi H, Yanagisawa Y, Yatagai C, Saito J. Natto Bacillus as an oral fibrinolytic agent: nattokinase activity and the ingestion effect of Bacillus subtilis natto. Food Science and Technology Research. 2004;10(1):17-20. doi:10.3136/fstr.10.17
Weng Y, Yao J, Sparks S, Wang KY. Nattokinase: An oral antithrombotic agent for the prevention of cardiovascular disease. Int J Mol Sci. 2017;18(3):523. doi:10.3390/ijms18030523
Gurewich V. Why so little progress in therapeutic thrombolysis? The current state of the art and prospects for improvement. J Thromb Thrombolysis. 2015;40(4):480-487. doi:10.1007/s11239-015-1217-3
Sumi H, Hamada H, Tsushima H, Mihara H, Muraki H. A novel fibrinolytic enzyme (nattokinase) in the vegetable cheese Natto; a typical and popular soybean food in the Japanese diet. Experientia. 1987;43(10):1110-1111.
Wang CT, Ji BP, Li B, Nout R, Li PL, Ji H, et al. Purification and characterization of a fibrinolytic enzyme of Bacillus subtilis DC33, isolated from Chinese traditional Douchi. J Ind Microbiol Biotechnol. 2006;33(9):750-758. doi:10.1007/s10295-006-0111-6
Eom JS, Lee SY, Choi HS. Bacillus subtilis HJ18-4 from traditional fermented soybean food inhibits Bacillus cereus growth and toxin-related genes. J Food Sci. 2014;79(11):M2279-M2287. doi:10.1111/1750-3841.12569
Park S, Kim MJ, Hong J, Kim HJ, Yi SH, Lee MK. Selection and characterization of Cheonggukjang (fast fermented soybean paste)-originated bacterial strains with a high level of S-adenosyl-L-methionine production and probiotics efficacy. J Med Food. 2014;17(11):1170-1176. doi:10.1089/jmf.2013.3052
Kho CW, Park SG, Cho S, Lee DH, Myung PK, Park BC. Confirmation of Vpr as a fibrinolytic enzyme present in extracellular proteins of Bacillus subtilis. Protein Expr Purif. 2005;39(1):1-7. doi:10.1016/j.pep.2004.08.008
Prasad S, Kashyap RS, Deopujari JY, Purohit HJ, Taori GM, Daginawala HF. Development of an In vitro model to study clot lysis activity of thrombolytic drugs. Thromb J. 2006;4:14. doi:10.1186/1477-9560-4-14
Kamada M, Hase S, Fujii K, Miyake M, Sato K, Kimura K, et al. Whole-genome sequencing and comparative genome analysis of Bacillus subtilis strains isolated from non-salted fermented soybean foods. PLoS One. 2015;10(10):e0141369. doi:10.1371/journal.pone.0141369
Kamada M, Hase S, Sato K, Toyoda A, Fujiyama A, Sakakibara Y. Whole genome complete resequencing of Bacillus subtilis natto by combining long reads with high-quality short reads. PLoS One. 2014;9(10):e109999. doi:10.1371/journal.pone.0109999
Nakamura T, Yamagata Y, Ichishima E. Nucleotide sequence of the subtilisin NAT gene, aprN, of Bacillus subtilis (natto). Biosci Biotechnol Biochem. 1992;56(11):1869-1871.
Inatsu Y, Nakamura N, Yuriko Y, Fushimi T, Watanasiritum L, Kawamoto S. Characterization of Bacillus subtilis strains in Thua nao, a traditional fermented soybean food in northern Thailand. Lett Appl Microbiol. 2006;43(3):237-242. doi:10.1111/j.1472-765X.2006.01966.x
Jeong SJ, Heo K, Park JY, Lee KW, Park JY, Joo SH, et al. Characterization of AprE176, a fibrinolytic enzyme from Bacillus subtilis HK176. J Microbiol Biotechnol. 2015;25(1):89-97.
Yoshimoto T, Oyama H, Honda T, Tone H, Takeshita T, Kamiyama T, et al. Cloning and expression of subtilisin amylosacchariticus gene. J Biochem. 1988;103(6):1060-1065.
Yoshimoto T, Oyama H, Takeshita T, Higashi H, Xu Sl, Tsuru D. Nucleotide sequence of the neutral protease gene from Bacillus subtilis var. amylosacchariticus. Journal of Fermentation and Bioengineering. 1990;70(6):370-375. doi:10.1016/0922-338x(90)90080-g
Jacobs MF. Expression of the subtilisin Carlsberg-encoding gene in Bacillus licheniformis and Bacillus subtilis. Gene. 1995;152(1):69-74.
Peng Y, Huang Q, Zhang RH, Zhang YZ. Purification and characterization of a fibrinolytic enzyme produced by Bacillus amyloliquefaciens DC-4 screened from douchi, a traditional Chinese soybean food. Comp Biochem Physiol B Biochem Mol Biol. 2003;134(1):45-52.
Yao Z, Liu X, Shim JM, Lee KW, Kim HJ, Kim JH. Properties of a Fibrinolytic enzyme secreted by Bacillus amyloliquefaciens RSB34, isolated from doenjang. J Microbiol Biotechnol. 2017;27(1): 9–18. doi:10.4014/jmb.1608.08034
Vijayaraghavan P, Vincent SG. Statistical optimization of fibrinolytic enzyme production by Pseudoalteromonas sp. IND11 using cow dung substrate by response surface methodology. Springerplus. 2014;3:60. doi:10.1186/2193-1801-3-60
Siller-Matula JM, Plasenzotti R, Spiel A, Quehenberger P, Jilma B. Interspecies differences in coagulation profile. Thromb Haemost. 2008;100(3):397-404.
Haggroth L, Mattsson C, Friberg J. Inhibition of the human tissue plasminogen activator in plasma from different species. Thromb Res. 1984;33(6):583-594.
Afifah DN, Sulchan M, Syah D, Yanti, Suhartono MT, Kim JH. Purification and characterization of a fibrinolytic enzyme from Bacillus pumilus 2.g Isolated from Gembus, an Indonesian Fermented Food. Prev Nutr Food Sci. 2014;19(3):213-219. doi:10.3746/pnf.2014.19.3.213
Chen PT, Chiang CJ, Chao YP. Medium optimization for the production of recombinant nattokinase by Bacillus subtilis using response surface methodology. Biotechnol Prog. 2007;23(6):1327-1332. doi:10.1021/bp070109b
Seo JH, Lee SP. Production of fibrinolytic enzyme from soybean grits fermented by Bacillus firmus NA-1. J Med Food. 2004;7(4):442-449. doi:10.1089/jmf.2004.7.442
Kellum JA. Determinants of blood pH in health and disease. Crit Care. 2000;4(1):6-14. doi:10.1186/cc644
Hamm LL, Nakhoul N, Hering-Smith KS. Acid-base homeostasis. Clin J Am Soc Nephrol. 2015;10(12):2232-2242. doi:10.2215/CJN.07400715
Tripathy S. Extreme metabolic alkalosis in intensive care. Indian J Crit Care Med. 2009;13(4):217-220. doi:10.4103/0972-5229.60175
Di Rollo N, Caesar D, Ferenbach DA, Dunn MJ. Survival from profound metabolic acidosis due to hypovolaemic shock. A world record? BMJ Case Rep. 2013;2013. doi:10.1136/bcr-2012-008315
Wang C, Du M, Zheng D, Kong F, Zu G, Feng Y. Purification and characterization of nattokinase from Bacillus subtilis natto B-12. J Agric Food Chem. 2009;57(20):9722-9729. doi:10.1021/jf901861v
- Abstract Viewed: 2089 times
- PDF Downloaded: 970 times