| Nattokinase acts rapidly and has a lasting effect in the body.It is an ideal natural active agent to prevent and treat cardiovascular disease.Nowadays,there are health care products and regular foods containing nattokinase available on the market.Health care products include nattokinase capsules and tablets,which are similar in form to drugs and can easily cause swallowing difficulties and psychological burden for users.Regular foods such as natto and natto powder often have problems with unclear nattokinase activity and unpleasant odor.In this paper,a high-yield nattokinase-producing Bacillus natto was screened in the laboratory and a fermented soy milk with refrigerated shelf life,good flavor,controllable enzyme activity,and convenient consumption was developed using soybeans as raw materials,and animal experiments were used to verify the thrombolytic effect of the fermented soy milk and its impact on the intestinal flora of thrombotic mice,in order to provide a reference for the development of natto products that are rich in nattokinase and convenient to consume.The specific research content and results are as follows:Firstly,a strain of Bacillus natto with high nattokinase yield was screened and analyzed from genetic perspective.Bacillus natto JNFE 0127 and Bacillus natto JNFE 0126 were screened to study their enzyme-producing ability.The results showed that JNFE 0127 had stronger nattokinase production capacity and reached 23361.18 IU/m L.The results of gene analysis showed that JNFE 0127 had 26569 basepairs and 24 coding sequences more than JNFE0126.There were functional gene differences in transmembrane transport,amino acid transport,metabolism and carbohydrate active enzymes between the two strains.The differences of the above functional genes may cause the physiological function differences between JNFE 0127 and JNFE 0126.Secondly,soybean was used as raw material to prepare fermented soybean milk with JNFE0127 and to explore the quality changes during storage.The optimal process parameters were determined through single factor experiments.The optimal process parameters were obtained by single factor experiment: static fermentation method,fermentation time 6 h,homogeneous pressure 20 MPa,the addition of xylitol and erythritol as sweeteners were 1.5% and 3.5%,gellan gum was added as stabilizer at a dosage of 0.20 g/100 m L,the ratio of monoglycerol and sucrose ester as emulsifier was 8:2,p H 6.8.The nattokinase activity and volatile base nitrogen content of fermented soybean milk were 45.59 FU/m L and 0.21 mg/100 m L,respectively,the microbial indicators of the soy milk prepared under optimized conditions complied with GB29921 standards,the soy milk was slightly yellow in color,had no odor,had a moist texture,and was uniform in shape.The study on the storage stability of fermented soy milk showed that the nattokinase activity was 28.04 FU/m L at 4℃ for 21 days,which could meet the requirements of nattokinase intake.Finally,the safety,thrombolytic effect and effect of fermented soybean milk on intestinal tract of mice with thrombus were verified by animal experiments.The results of acute toxicity test showed that the median lethal dose of fermented soy milk was greater than 20 m L/kg.The results of black-tail experiment showed that 24 h after the establishment of thrombus model,the relative length of black-tail of mice in fermented soybean milk groups with 2000 FU or5000 FU nattokinase decreased significantly compared with model groups.After 96 h,the relative length of black tail of mice fermented soybean milk with nattokinase containing 2000 FU or 5000 FU was less than 48 h.The effect of fermented soybean milk group containing 5000 FU nattokinase was better than that of the equal nattokinase content group and fermented soybean milk group containing 2000 FU nattokinase.The results of tail and colon sections showed that fermented soybean milk containing nattokinase could inhibit thrombus in the tail of mice and repair colon tissue.The inflammatory factors and oxidative stress indexes showed that the levels of tumor necrosis factor-α and interleukin-1β were significantly decreased and the levels of interleukin-10 were significantly increased in mice given fermented soy milk containing 2000 FU or 5000 FU nattokinase compared with model mice.Superoxide dismutase level increased significantly,malondialdehyde level decreased significantly,glutathione peroxidase level increased.In conclusion,fermented soy milk has a dose-dependent effect on thrombolysis.Under the premise of the same nattokinase content,the thrombolytic effect of soybean milk system is better than that of pure nattokinase system.The results of 16 S r RNA gene sequencing showed that fermented soy milk could reduce the proportion of firmicutes and bacteroides in the intestinal tract of mice,increase the number of lactic acid bacteria and decrease the number of clostridium,which had a positive effect on the intestinal flora of mice. |
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