Screening Of A Bacillus Subtilis Strain Producing Both Nattokinase And Milk Clotting Enzyme,and Its Application In Fermented Milk

Bacillus subtilis is a food grade microorganism which is Generally Recognized As Safe(GRAS),and it is widely found in traditional fermented foods.Natto is a traditional food fermented with B.subtilis.In recent years,natto is gaining more popularity for it's rich in nattokinase,which possesses anti-thrombosis function.Nattokinase isolated from B.subtilis fermentation broth has been used in anti-thrombolytic drugs and functional foods.Meanwhile,B.subtilis is probiotic which can enter the intestine through oral intake.It could not only adjust the intestinal flora and enhance intestinal immunity,but also showed the functions of anti-inflammation,anti-tumor,and anti-aging.The major sources of intestinal B.subtilis are fermented food and probiotic preparations.Presently,the dosage forms of the B.subtilis probiotic products are mostly lyophilized powders,while the nattokinase products are mostly capsules and pills.The singularity in product categories is limiting the application of nattokinase and B.subtilis probiotic products.Thus,the development of new functional foods containing B.subtilis and/or nattokinase would be attractive.In this study,a B.subtilis strain which produced both nattokinase and milk clotting enzyme(MCE)was screened from naturally fermented natto.Afterward,a solidified fermented milk with anti-thrombosis function was prepared via the milk clotting enzymatic activity of this strain,and the function of anti-thrombosis in vivo was evaluated with model mice.And the B.subtilis fermented milk was evaluated as a probiotic preparation,Besides,because nattokinase could be largely deactivated by the acidic environment of the gastric juice after oral administration,a novel microparticle loaded with nattokinase with the bilayer shell-core structure possessed the characteristics of acid-resistant in the gastric lumen and sustained-release in the intestinal lumen was designed to enhance its oral effectiveness.The microparticle loaded with nattokinase was prepared,and its anti-thrombosis activity in vivo was evaluated with model animals.The major results of this research were as follows:Firstly,a strain which produced both nattokinase and milk clotting enzyme(MCE)was screened and isolated from home-made natto,which was prepared with the traditional fermentation procedure using straws as the bacterial source.The isolated strain was identified to be B.subtilis according to the results of the morphological observation,biochemical characteristic tests,and 16 S r DNA sequencing.This strain was then named as B.subtilis JNFE0126 and preserved at China General Microbiological Culture Collection Center(CGMCC),and the preservation number was CGMCC 18057.With optimal fermentation conditions,the thrombolytic activity of the fermentation broth reached 14512.4 IU/mL,and the milk clotting activity reached 875.5 SU/mL.The genes of nattokinase and MCE of the strain were amplified with the PCR primers which were designed according to the DNA sequence of nattokinase and M4 metallopeptidase from B.subtilis reported in former literature.Then,the nattokinase and MCE DNA sequences from B.subtilis JNFE0126 were compared with those in NCBI database,and both sequences were highly homologous with the reported data.Secondly,nattokinase and MCE were isolated from the fermentation broth of B.subtilis JNFE0126 after 60 h and 96 h of fermentation,respectively.Each enzyme was isolated and purified using ammonium sulfate precipitation,ion exchange chromatography,and then gel filtration chromatography.The active peak was collected and lyophilized as purified enzyme,and the purity was examined with SDS-PAGE.The results showed that the purified nattokinase was 27 KD,and the purified MCE was 42 KD.The thrombolytic activity of the purified nattokinase was 8722 IU/mg.The optimal pH and temperature were pH 7.0 and 40?;The milk clotting activity of the purified MCE was 263.5 SU/mg.The optimal pH and temperature were pH 6.5 and 55?.On the basis of the results above,B.subtilis JNFE0126 was used to develop a solidified fermented milk.The optimal formula and fermentation conditions were as follows: fresh milk?soy protein 2%?sucrose 4%?hydroxypropyl distarch phosphate 1.5%?pectin 0.1%?PGA 0.1%,fermented at 41? for 8 h.With the optimized formula,the B.subtilis fermented milk possessed ideal appearance and texture properties,with white tender appearance and delicate taste.The thrombolytic activity in the fermented milk reached 215.1 IU/mL.When stored at 4 ?,the thrombolytic activity remained over 66% after 14 d of storage,and over43% after 60 d of storage.More importantly,the in vivo oral anti-thrombosis test showed that intake of the fermented milk by the thrombosis-model mice could prevent the development of thrombosis significantly.The intestinal microbiota adjustment and inflammatory bowel disease(IBD)attenuation function of the B.subtilis fermented milk was evaluated using dextran sulfate sodium salt(DSS)model mice,and the mechanism underlying was investigated.The results from the symptom observation on the model animals and the histological observation of the intestinal mucosa showed that oral intake of the B.subtilis fermented milk could significantly attenuate the severity of IBD,reducing pathological injuries of the intestinal mucosa including bleeding,inflammation,and ulcers;The results from the intestinal microbiota analysis suggested that oral intake of the B.subtilis fermented milk could regulate the flora balance composition disturbed by DSS induced IBD by increasing the diversity of the microbiota and inhibit the proliferation of pathogenic species.Meanwhile,the B.subtilis fermented milk could reduce the inflammatory response of the intestinal mucosa,induce proliferation of the intestinal stem cell,and promote reconstruction of the mucosal barrier,which could prevent the invasion of pathogenic microbes and inflammatory cytokines.As a comprehensive effect of these multiple mechanisms,oral administration of the B.subtilis fermented milk showed significant function in prevention and treatment of IBD.Besides,a novel functional oral delivery system of nattokinase was developed,in which chitosan microparticles were used as the carrier core to load nattokinase via genipin(a natural nontoxic crosslinker extracted from gardenia,the traditional Chinese medicinal plant)crosslinking and then covered by a casein-based protective shell via transglutaminase(TG)crosslinking.The microparticle was prepared with a layer-by-layer crosslinking procedure.The microstructure observation results suggested that the bilayer shell-core structure was successfully constructed,and the nattokinase,which was crosslinked with the chitosan cores,was covered well by the casein protective shell.The releasing dynamic results in the simulated gastric and intestinal juice in vitro suggested that the microparticle could protect nattokinase in the acidic gastric juice and release nattokinase gradually in the intestinal juice.The in vivo tracing of the nattokinase microparticles administrated by model animals showed that the microparticle could release active nattokinase in the small intestine continuously for more than 12 h.The anti-thrombosis function in vivo was evaluated with black-tail model mice,and the results suggested that oral administration of the nattokinase microparticle could significantly inhibit thrombus formation in the blood vessels in tail and promote degradation of the thrombus.In conclusion,the fermented milk prepared with B.subtilis JNFE0126 could prevent thrombosis and regulate intestinal function.This fermented milk would be a promising functional food.Besides,the nattokinase microparticle prepared in this research could enhance the oral effectiveness of nattokinase,and can be applied as a functional additive in anti-thrombosis functional foods.