Screening Of Efficient Nattokinase Strains And Natto Production

Natto is a Japan traditional fermented food that has been 2000 years of history. Hunders of physiologically active substances in nattohave been found,including small molecules protein, peptides, short peptide and amino acids, kinds of biological compounds which have special physiological functions, many special physical and chemical properties and physiological activity.Its fermentation product nattokinase a serine protease, has strong ability of dissolving thrombosis.In recent years,domestic and overseas scholars have studied the genetic sequence and physiological and biochemical characteristicsof nattokinase,and a series of researches showed that nattokinase can be developed to become a new kind of dissolve clots medicine.Natto efficacy is mainly from Bacillus subitis, which was recognized as the "first probiotics" for human.At present,most of the studies have been done on strain improvement and fermentation condition optimization as well as the purification of nattokinase productivity.However,high-yielded strains of nattokinase Bacillus subtilis still need to be screened from nattos produced in japan.There is an urgent need to develop the technology of genetic engineering for industrial production on nattokinase.This study screened and solated the strains that produce fibrinolytic enzyme from 14 fermented foods.From the first step of casein flat screen,flat line drawings,we got 52 strainsthat had fibrinolytic activity. After fibrin flat screening and fermentation enzyme measured,we got six strainsthat had high enzyme activity.The enzyme activity produce from Japan natto strans,Sichuan kimch, Soy sauce, NeiMenggu auspicion, Qing Dao beer, ZhongXian ferment bean curd are 2261.3 U/mL,2105.9 U/mL,1965.9 U/mL,1725.1 U/mL,1463.0 U/mL,898.3 U/mL, respectively.Further through the experiment of the fibrin plate, the strain FNK-3 isolated from natto, was found to have athe highest efficiency in dissolving thrombosis. The fibrinolytic enzyme was obtained from fermented liquid produced through liquid fermentation of the strain. The high purity of fibrinolytic enzyme was obtained with the activity equal to 2261 U/mL of urokinase activity, through purification of Subsection salting-out of ammonium and Gel chromatography of sephadexG-50 andSepharose CM Fast Flow. A 1268 bp of 16S ribosomal DNA sequence was obtained from the strain FNK-3 with the universal primer of bacterial 16S ribosome DNA. Sequence analysis carried out by Blast on NCBI website shows that FNK-3 shared 98.74% homology with Bacillus subtilis. The phylogenetic analysis with PAUP4.0 and MAGE4 software shows that the strain FNK-3 and Bacillus subtilis are on the same branch of the constructed phylogenetic tree. The strain FNK-3 was deduced to belong to Bacillus subtilis with the combination of morphological and physiological characters.The strains on the natto production process test was studied for the factors such as immersion ime, cooking time, fermentation temperature, fermentation time, inoculation quantity, growth factors for single factor experiment analysis.The best process for production of beans through the sensory evaluation and measuring thrombolysis circle diameter curve were immersion time 18 hour, Cooking time121℃,45min, fermentation temperature 43℃, fermentation time24 hour, inoculation quantity 4%, growth factors 0.015g/100g.The study of how to screen nattokinase high-yielding strains and optimized production process of natto,for industrial ferment produce nattokinase and natto production laid solid foundation.