Application of bacillus subtilis plasmin in preparation of anti-aging and life-prolonging medicine

Abstract

The invention discloses an application of bacillus subtilis plasmin in preparation of anti-aging and life-prolonging drugs, the bacillus subtilis plasmin is prepared by fermentation of bacillus subtilis QK02 strain, the strain is preserved in the China Center for Type Culture Collection, and the strain number is CCTCC NO: M203078. The bacillus subtilis plasmin is matched with spermidine, taurine and pharmaceutically acceptable auxiliary materials to prepare a liquid preparation, a capsule or a tablet. The bacillus subtilis plasmin disclosed by the invention has good effects of resisting aging and prolonging the life, and the activity is remarkably improved and the life is prolonged after the nematodes are fed with the bacillus subtilis plasmin; the antioxidant capacity, the immunity and the anti-inflammatory capacity of natural aging organisms can be enhanced, and the compound can be widely applied to anti-aging and life-prolonging medicines.

Description

Technical Field

[0001] The present invention relates to the field of anti-aging and life-extending drugs, and more specifically, to the use of Bacillus subtilis fibrinolytic enzyme in the preparation of anti-aging and life-extending drugs. Background Art

[0002] In the most ideal case, the length of life is mainly determined by the rate of aging. Aging is controlled by interrelated processes occurring at the tissue, cell, molecule and genetic levels. As the body's tissues and organs age, their structures and functions gradually undergo irreversible degenerative changes such as protein degradation, tissue atrophy, reduced metabolic rate and abnormal calcium metabolism. With the passage of age, diseases caused by aging, such as Alzheimer's disease, Parkinson's disease, cardiovascular and cerebrovascular diseases and cancer, are growing exponentially around the world. With the continuous increase in the base and proportion of the elderly population, and the increasing demand for anti-aging and life extension, it is of great social significance to conduct research on the mechanism of human aging and the development of anti-aging drugs, slow down the rate of human aging within the limits determined by genetic characteristics, and prolong the life of the body.

[0003] At present, drugs such as rapamycin, aspirin, metformin and resveratrol have been proven to delay the lifespan of many species, but considering the use of drugs to delay aging in humans, there is still a lack of large-scale, long-term clinical experimental data. The above drugs all have certain shortcomings and cannot be used safely and effectively for a long time. For example, rapamycin can cause adverse reactions such as hyperglycemia, hyperlipidemia, renal toxicity, impaired wound healing, decreased platelet count and immunosuppression; aspirin can easily cause gastrointestinal symptoms such as nausea, vomiting, discomfort and pain, and can also cause abnormal allergic reactions such as rash, angioedema and asthma; long-term high-dose use of metformin can induce lactic acidosis; taking large doses of resveratrol can cause gastrointestinal and liver dysfunction in humans, etc.

[0004] Immunity and inflammation are closely related to aging and are important directions for anti-aging research. The immune system gradually ages with age, and immune aging has many manifestations, including the loss of protective functions of the skin and mucous membranes with age, chronic inflammation of the body, reduced immune response to pathogens, decreased new immature lymphocytes, and impaired diversity of lymphocyte subsets. In addition, the liver is the largest solid organ in the human body and the largest gland. The rudiments of the liver develop from the foregut germ layer. The embryonic liver is mainly hematopoietic and is converted into a metabolic organ after birth. The liver participates in the synthesis, decomposition, excretion and other metabolism of various substances in the body. When the liver function is impaired, the whole body will be affected. The liver plays a vital role in regulating the aging process.

[0005] Therefore, there is an urgent need to develop a safe and effective anti-aging and life-extending drug. Summary of the invention

[0006] In order to achieve these purposes and other advantages according to the present invention, a preferred embodiment of the present invention provides a use of Bacillus subtilis fibrinolytic enzyme in the preparation of anti-aging and life-extending drugs.

[0007] The preparation process of the Bacillus subtilis fibrinolytic enzyme is as follows: the Bacillus subtilis strain is activated with a fresh slant for 18 hours to 24 hours, a loop of bacterial cells is taken from the slant and inoculated into a triangular flask filled with LB liquid culture medium, and cultured on a shaking table at 37°C and 200r / min for 18 hours; the cultured strain is inoculated into a fermenter at 1wt% and fermented for 24 hours, and the temperature in the fermenter is controlled at 37°C and the dissolved oxygen is controlled at 20-50%; the fermentation liquid is concentrated by centrifugation, microfiltration, and ultrafiltration, and then ethanol precipitation, ion exchange chromatography purification, and spray drying to obtain the Bacillus subtilis fibrinolytic enzyme.

[0008] According to a preferred embodiment of the present invention, the Bacillus subtilis fibrinolytic enzyme is produced by fermentation of Bacillus subtilis QK02, which has been preserved in the China Center for Type Culture Collection with a strain number of CCTCCNO:

[0009] The Bacillus subtilis with the deposit number of CCTCCNO: M203078 is deposited in the National Center for Type Culture Collection, named Bacillus subtilis QK02, classified as Bacillus subtilis, deposited in Wuhan, China, and deposited on November 17, 2003.

[0010] According to a preferred embodiment of the present invention, the Bacillus subtilis fibrinolytic enzyme can be combined with pharmaceutically acceptable excipients to form a liquid medicine, capsule or tablet.

[0011] According to a preferred embodiment of the present invention, the Bacillus subtilis fibrinolytic enzyme is combined with spermidine and taurine, as well as pharmaceutically acceptable excipients, to form a liquid medicine, capsule or tablet.

[0012] The present invention includes at least the following beneficial effects: the Bacillus subtilis fibrinolytic enzyme of the present invention has a good effect on anti-aging and life extension, and significantly improves vitality and life extension after feeding nematodes; it can enhance the antioxidant capacity, immunity and anti-inflammatory capacity of naturally aging organisms, and can be widely used in anti-aging and life extension drugs.

[0013] Other advantages, objectives and features of the present invention will be embodied in part through the following description, and in part will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is a graph showing the lifespan of Caenorhabditis elegans.

[0015] Figure 2 Mouse liver tissue section (×40).

[0016] (A: control group; B: model group; C: low-dose group; D: medium-dose group; E: high-dose group; F: Vc group) DETAILED DESCRIPTION

[0017] The present invention is further described in detail below in conjunction with the accompanying drawings so that those skilled in the art can implement the invention with reference to the description.

[0018] The following description is used to disclose the present invention so that those skilled in the art can implement the present invention. The preferred embodiments described below are only examples, and those skilled in the art can think of other obvious variations. The basic principles of the present invention defined in the following description can be applied to other embodiments, variations, improvements, equivalents, and other technical solutions that do not deviate from the spirit and scope of the present invention.

[0019] Experiment 1 Preparation method of Bacillus subtilis fibrinolytic enzyme

[0020] One embodiment of the present invention provides a method for preparing Bacillus subtilis fibrinolytic enzyme, which is as follows:

[0021] (1) Activation of bacterial strains: Take out the Bacillus subtilis strains stored in a -80°C freezer and activate them on a fresh slant for 18 to 24 hours. Take a loopful of bacterial cells from the slant and inoculate them into a conical flask containing LB liquid culture medium. Incubate the flask at 37°C and 200 rpm for 18 hours.

[0022] (2) Culture medium: 300 L of fermentation tank culture medium was prepared, containing the following nutrients in percentage by weight: 2% peptone, 2% glucose, 1% disodium hydrogen phosphate, 0.1% sodium dihydrogen phosphate, 0.05% magnesium sulfate, 0.02% anhydrous calcium chloride, 0.1% defoaming agent, and the balance water.

[0023] (3) Fermentation: The prepared fermentation tank culture medium is sterilized at 115° C. for 30 min, cooled to room temperature, and the cultured bacteria in step (1) are inoculated into the fermentation tank at 1 wt % for 24 h, and the temperature in the fermentation tank is controlled at 37° C. and the dissolved oxygen is 20-50%; the fermentation broth is concentrated by centrifugation, microfiltration, and ultrafiltration to obtain a concentrated solution.

[0024] (4) Ethanol precipitation: Add a certain volume of 95% ethanol to the concentrate to make the final ethanol concentration reach 60%. Stir for 5 minutes and then let it stand for 30 minutes. After the Bacillus subtilis fibrinolytic enzyme is fully precipitated, centrifuge at 5000 rpm for 3 minutes. Remove the supernatant and add water to re-dissolve to obtain a reconstituted solution.

[0025] (5) Sepharose Fast Flow ion exchange chromatography: The complex solution was loaded at a flow rate of 2.5 ml / min, the column was washed with 0.02 M phosphate buffer, and then gradient eluted with phosphate buffer containing 1.0 mol / L NaCl. The eluate was collected to obtain the Bacillus subtilis plasmin solution.

[0026] (6) Spray drying: 7% maltodextrin is added to the Bacillus subtilis fibrinolytic enzyme solution and spray dried with an inlet air temperature of 140°C and an outlet air temperature of 63°C-68°C to obtain the Bacillus subtilis fibrinolytic enzyme.

[0027] Experiment 2 Effects of Bacillus subtilis fibrinolytic enzyme on Caenorhabditis elegans

[0028] Cultivation of nematodes: Feed with NGM plates containing OP 50 E. coli at 20°C. Synchronize the culture when 80% of the worms on the NGM plates are in the egg-laying stage. The day after the egg-laying stage of the nematodes is marked as day 0; keep 30 nematodes on each plate, and keep changing new plates every 2 days.

[0029] Grouping and dosing method: 21 plates were randomly divided into 7 groups. Starting from the adult stage, different groups were fed with Bacillus subtilis fibrinolytic enzyme 0.1 mg / ml + OP 50 (low concentration), Bacillus subtilis fibrinolytic enzyme 0.2 mg / ml + OP 50 (medium concentration), Bacillus subtilis fibrinolytic enzyme 0.3 mg / ml + OP 50 (high concentration), Bacillus subtilis fibrinolytic enzyme 0.2 mg / ml + spermidine 5 ug / ml + OP 50 (mixed group 1), Bacillus subtilis fibrinolytic enzyme 0.2 mg / ml + taurine 5 mg / ml + OP 50 (mixed group 2), Bacillus subtilis fibrinolytic enzyme 0.2 mg / ml + spermidine 5 ug / ml + taurine 5 ug / ml + OP 50 (mixed group 3), and the group containing only OP 50 bacterial solution was used as the blank control.

[0030] Determination of nematode lifespan: The number of dead, escaped, and cystic nematodes was recorded every day. When the nematode did not move and did not move when the platinum wire touched its head, it was recorded as dead. Based on its daily mortality rate, a lifespan curve was drawn.

[0031] Determination of nematode movement ability: The movement ability of nematodes is evaluated by the number of times the nematode bends. Place the nematode on NGM medium without food and observe its bending. One bend is when the nematode completes an S-shaped movement. Allow the nematode to move freely for 1 minute before the experiment, and then observe the number of times the nematode bends within 1 minute as the experimental indicator.

[0032] Table 1 Effects of Bacillus subtilis fibrinolytic enzyme on the motility of Caenorhabditis elegans (n=30)

[0033]

[0034]

[0035] Note: Compared with the blank group, *p<0.05, **p<0.01.

[0036] from Figure 1 As shown in Table 1, compared with the blank control group, Bacillus subtilis fibrinolytic enzyme has a certain promoting effect on the life span and movement ability of nematodes, and when the concentration is 0.3 mg / ml, it can significantly prolong the average life span of nematodes, with the average life span extended by 18.24%; the movement frequency increased by 60.53% (P < 0.01); and when Bacillus subtilis fibrinolytic enzyme is used in combination with spermidine or taurine, it can synergistically increase the movement frequency of nematodes and prolong the average life span.

[0037] Experiment 3 Effect of Bacillus subtilis fibrinolytic enzyme on anti-aging of D-galactose-induced aging mice

[0038] Animal grouping and administration method: Mice were randomly divided into 6 groups according to their weight, with 10 mice in each group, half male and half female, and the breeding environment met the requirements of SPF experimental conditions. The blank control group ate and drank water normally without treatment. The model group received subcutaneous injection of D-galactose (D-gal) in the neck. In addition to subcutaneous injection of D-gal in the neck, the drug-treated group was given Bacillus subtilis fibrinolytic enzyme by gavage every day, with a dose of 300IU / 100g, 200IU / 100g, and 100IU / 100g. In addition to subcutaneous injection of D-gal in the neck, the vitamin C group was given vitamin C 20mg / 100g by gavage every day. The drug was administered continuously for 60 days. After the experiment, the liver was taken for the determination of antioxidant enzymes (SOD, GSH-Px), CAT activity and MDA content, and HE staining section analysis.

[0039] Table 2 Effects of Bacillus subtilis fibrinolytic enzyme on antioxidant enzyme activity and MDA content in liver tissue of induced aging mice

[0040]

[0041] Note: Compared with the model group, *p<0.05, **p<0.01; Compared with the control group, # p<0.05, ## p<0.01.

[0042] As shown in Table 2, the activities of SOD, GSH-Px, and CAT in the liver of the model group mice were significantly lower than those in the control group, and the MDA content increased. Oral administration of D-gal caused a decrease in the activity of antioxidant enzymes in the liver tissue of mice, while lipid peroxides increased, which led to oxidative damage and aging, indicating that the model was successfully established. Oral administration of D-gal and different doses of Bacillus subtilis plasmin can reduce the decrease in SOD, GSH-Px, and CAT activities in the liver and the increase in MDA levels, effectively preventing the oxidative damage caused by D-gal, and the prevention effect increases with increasing doses. Compared with the model group, the activities of the three antioxidant enzymes in the high-dose group were significantly increased (P < 0.05), and the MDA level was significantly reduced (P < 0.05). It is close to the Vc group with obvious antioxidant effect (P < 0.05).

[0043] When the liver is damaged, the normal metabolic function of the liver will be hindered, resulting in aging. Figure 2 HE staining sections of liver tissues in each group showed that A: control group; B: model group; C: low-dose group; D: medium-dose group; E: high-dose group; F: Vc group. The liver boundary of group A in the control group was clear, the cell morphology was normal, the liver fat was not degenerated, the central vein was regular and round, and the intercellular space was small. The liver tissue of group B in the model group underwent pathological morphological changes, the cell volume and intercellular space became larger, the morphology was incomplete, and the cells were filled with lipid droplets and vacuoles of different sizes. After intragastric administration of low-dose Bacillus subtilis plasmin in group C, the degree of damage to the liver lobule structure was small, the lipid droplet vacuoles were reduced, but the hepatic cords and hepatic sinusoids were blurred; in the high-dose group E, the hepatocytes were neatly arranged, the morphology was complete, the size was uniform, the hepatic cords and hepatic sinusoids were basically visible, and the lipid droplet vacuoles were basically absent. The morphology of liver tissue was close to that of the normal control group.

[0044] This shows that Bacillus subtilis fibrinolytic enzyme has antioxidant capacity, which protects the liver, reduces oxidative damage to liver tissue, and thus delays aging.

[0045] Experiment 4 Effect of Bacillus subtilis fibrinolytic enzyme on anti-aging and life-span extension in naturally aged mice

[0046] Animal grouping and administration method: 100 15-month-old C57BL / 6J mice were selected and randomly divided into 5 groups, with 20 mice in each group, half male and half female, and the breeding environment met the requirements of SPF experimental conditions. The blank control group ate and drank water normally without treatment. The other 4 groups were given Bacillus subtilis fibrinolytic enzyme, Bacillus subtilis fibrinolytic enzyme and spermidine (mixed group 1), Bacillus subtilis fibrinolytic enzyme and taurine (mixed group 2), and Bacillus subtilis fibrinolytic enzyme and spermidine and taurine (mixed group 3) by gavage every day, among which the dose of Bacillus subtilis fibrinolytic enzyme was 200IU / 100g, the dose of spermidine was 9ug / 100g, and the dose of taurine was 10mg / 100g. After 60 days of continuous feeding, 10 mice in each group were taken for behavioral experiments including hanging test, rotating rod fatigue test, forced running test, and Barnes maze test. Liver tissues were taken to measure the content of tumor necrosis factor-α (TNF-α), pro-inflammatory factor interleukin-1β (IL-1β), interleukin-6 (IL-6), and anti-inflammatory factor interleukin-10 (IL-10). The anti-inflammatory effect in vivo on naturally aged mice was evaluated. The remaining 10 mice in each group continued to be divided into groups and fed until natural death, and the life span of mice in each group was recorded.

[0047] The specific operation of the behavioral experiment is as follows:

[0048] Suspension experiment: The mice were hung on a wire, and the time from the start of hanging to the fall of the mice was recorded. The test was performed 3 times in total and the average value was taken.

[0049] Rotarod fatigue test: A rotarod fatigue test apparatus for mice and rats was used, with an initial rotation speed of 4 r / min, which was accelerated to 40 r / min in 30 s and lasted for 5 min. The time from the start of rotation to the fall of the mouse was recorded, and the test was performed 3 times to take the average value.

[0050] Forced running experiment: Use DB030 animal treadmill, running time is 30min, electrical stimulation intensity is 0.8mA, stimulation frequency is 5 times / 8s, exhaustion standard is that the mouse does not continue to run after 5 consecutive electrical stimulations, and the time of fast running to exhaustion of the mouse is recorded.

[0051] Barnes maze test: Using the Barnes maze, the distance and time taken by the mice to explore from the beginning to the time they enter the dark box were recorded. The mice were trained three times before the formal test, with an interval of 24 hours each time. If the mice still failed to find the dark box by the end of the experiment, the longest time was recorded.

[0052] Table 3 Effects of Bacillus subtilis fibrinolytic enzyme on physical performance of naturally aged mice (n=10)

[0053] Group Suspension time / s Rotating rod time / s Running time / s Blank control 61.43±21.71 23.67±5.20 114.48±21.82 Bacillus subtilis fibrinolytic enzyme group 106.10±26.19* 26.48±5.46 165.89±41.53** Mixed Group 1 112.60±25.57* 29.61±5.47 172.91±45.38** Mixed Group 2 113.25±23.69* 33.58±5.31* 169.45±51.21** Mixed Group 3 125.07±33.72** 37.30±5.77* 213.95±53.73**

[0054] Note: Compared with the blank group, *p<0.05, **p<0.01.

[0055] Table 4 Effect of Bacillus subtilis fibrinolytic enzyme on memory of naturally aged mice (n=10)

[0056] Group Time / s Distance / mm Blank control 150.67±44.84 6577.78±2059.00 Bacillus subtilis fibrinolytic enzyme group 36.22±12.29** 1629.45±639.87** Mixed Group 1 34.22±14.98** 1184.58±770.82** Mixed Group 2 32.33±13.12** 1293.36±533.72** Mixed Group 3 30.37±11.55** 1069.38±549.63**

[0057] Note: **p<0.01 compared with the blank group.

[0058] The behavioral experiment explored the motor ability and memory of each group of mice. As shown in Tables 3 and 4, compared with the blank control group, the hanging time and running time of mice in each drug-treated group increased significantly, the distance and time spent exploring during the dark box were significantly shortened, and the memory increased significantly; this indicates that Bacillus subtilis fibrinolytic enzyme significantly improved the physical fitness and memory of naturally aged mice, and the effect was better when taken with taurine and spermidine. From the data comparison of mixed group 3 with mixed group 1 and mixed group 2, it can be seen that compared with the combination of Bacillus subtilis fibrinolytic enzyme with taurine and Bacillus subtilis fibrinolytic enzyme with spermidine, the combination of Bacillus subtilis fibrinolytic enzyme with taurine and spermidine significantly increased the hanging time and running time of mice, indicating that the synergistic effect between Bacillus subtilis fibrinolytic enzyme and taurine and spermidine can significantly increase the anti-aging effect.

[0059] Table 5 Effects of Bacillus subtilis fibrinolytic enzyme on inflammatory factors in liver tissue of naturally aged mice (n=10)

[0060]

[0061] Note: Compared with the blank group, *p<0.05, **p<0.01.

[0062] Inflammation is a protective response to various cell and tissue injuries to destroy and remove harmful substances and damaged tissues, thereby promoting tissue repair. Therefore, the level of inflammatory factors increases with age. Among them, proinflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6), play an important role in many physiological processes, including the regulation of immune and inflammatory responses. As shown in Table 5, after drug intervention, the levels of three proinflammatory factors, TNF-α, IL-1β and IL-6 in the liver tissues of the four groups of mice were significantly lower than those in the blank group, and the mixed intervention of taurine, spermidine and Bacillus subtilis plasmin had the best effect. The levels of TNF-α, IL-1β and IL-6 were reduced by 52.42%, 53.75% and 34.56% respectively compared with the blank group, and the level of anti-inflammatory factor IL-10 was increased, which can effectively prevent excessive damage to the body during inflammatory response. This indicates that Bacillus subtilis fibrinolytic enzyme used alone or in combination with taurine and spermidine can significantly inhibit the production of pro-inflammatory factors and effectively promote the production of anti-inflammatory factors, thus playing a protective role on the liver of aging mice. And from the data comparison between mixed group 3 and mixed group 1 and mixed group 2, it can be seen that: compared with the combination of Bacillus subtilis fibrinolytic enzyme with taurine and Bacillus subtilis fibrinolytic enzyme with spermidine, the combination of Bacillus subtilis fibrinolytic enzyme with taurine and spermidine has the best recovery effect on mice, indicating that the combination of Bacillus subtilis fibrinolytic enzyme with taurine and spermidine has a synergistic effect, significantly increasing the anti-aging effect.

[0063] Table 6 Effect of Bacillus subtilis fibrinolytic enzyme on the life span of naturally aged mice (n=10)

[0064] Group Natural age of death Blank control 23.34±2.23 Bacillus subtilis fibrinolytic enzyme 26.15±1.96 Mixed Group 1 28.25±1.72* Mixed Group 2 28.54±1.88* Mixed Group 3 31.94±2.18**

[0065] Note: Compared with the blank group, *p<0.05, **p<0.01.

[0066] Through the exploration of the life span of naturally aged mice, it can be seen from Table 6 that the life span of the group taking Bacillus subtilis fibrinolytic enzyme was extended by 22.56% compared with the control group. And the effect was more significant when used in conjunction with taurine and spermidine (p < 0.01).

[0067] Experiment 5 Effect of Bacillus subtilis fibrinolytic enzyme on anti-aging of elderly volunteers

[0068] 60 elderly people aged 60-70 were selected, half of them were male and half were female. Elderly people who had taken antihypertensive, lipid-lowering, immune preparations and other drugs within 1 month were excluded. The 60 subjects were randomly divided into 3 groups, 20 in each group. The control group took 500 mg of taurine with warm water every day. The treatment group took Bacillus subtilis plasmin with warm water twice a day, 5000 IU each time. The enhanced group took 500 mg of taurine and 10000 IU of Bacillus subtilis plasmin every day for 60 consecutive days.

[0069] The improvement of 12 aging characteristics in each group before and after taking the medicine was observed, including facial wrinkles, age spots, memory, appetite, sleep, language ability, blood lipids, blood pressure, bowel movements, recovery ability after exercise, self-resistance, and body coordination. The scores were recorded in the form of cumulative points, with 3 points for obvious, 2 points for moderate, 1 point for mild, and 0 points for not obvious. Blood was collected before and after taking the medicine to compare the immune indicators.

[0070] After investigating the improvement of the subjects' aging characteristics, it was found that due to the different physical conditions of each subject, the improvements in the 12 items were also different. Among them, Bacillus subtilis plasmin had obvious improvements on blood lipids, blood pressure, bowel movements, and self-resistance.

[0071] Table 7 Proportion of subjects' improvement scores of aging characteristics

[0072] Group Less than 12 points 13-24 points 25-36 points Control group 40％ 55％ 5％ Drug group 15％ 70％ 15％ Strengthening Group 0％ 65％ 35％

[0073] From the score proportions in Table 7, it was found that all three groups of elderly subjects showed improvement. Taking Bacillus subtilis plasmin alone improved the aging characteristics more significantly than taking taurine alone, and the synergistic use of taurine and Bacillus subtilis plasmin had the best effect.

[0074] The levels of peripheral blood T lymphocyte subsets, CD3 + 、CD4 + 、CD4 + / CD8 + The ratio is an important indicator of the stability of the internal environment of the human immune system.

[0075] Table 8 Comparison of immune indicators before and after taking the medicine (n=20)

[0076]

[0077] Note: *p<0.05 compared with before administration.

[0078] From Table 8, we can see that the CD3 + 、CD4 + 、CD4 + / CD 8 + All of them were significantly increased (P<0.05), indicating that Bacillus subtilis plasmin can regulate the cellular immune function of the elderly, thereby achieving the effect of anti-immune aging.

[0079] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes, and they can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and the illustrations shown and described herein.

Claims

1. Application of Bacillus subtilis fibrinolytic enzyme in the preparation of anti-aging and life-extending drugs.

2. The use of the Bacillus subtilis fibrinolytic enzyme according to claim 1 in the preparation of anti-aging and life-extending drugs, characterized in that: The Bacillus subtilis fibrinolytic enzyme is obtained by fermenting Bacillus subtilis QK02 strain, which has been preserved in the National Center for Type Culture Collection, with strain number CCTCCNO: M203078.

3. The use of the Bacillus subtilis fibrinolytic enzyme according to claim 1 in the preparation of anti-aging and life-extending drugs, characterized in that: The preparation process of the Bacillus subtilis fibrinolytic enzyme is as follows: activating the Bacillus subtilis strain with a fresh slant for 18 hours to 24 hours, taking a loop of bacterial cells from the slant and inoculating them into a triangular flask filled with LB liquid culture medium, and culturing them on a shaker at 37°C and 200r / min for 18 hours; inoculating the cultured strain into a fermenter at 1wt% and fermenting for 24 hours, controlling the temperature in the fermenter to 37°C and the dissolved oxygen to 20-50%; and concentrating the fermentation liquid through centrifugation, microfiltration, and ultrafiltration, and then ethanol precipitation, purification by ion exchange chromatography, and spray drying to obtain the Bacillus subtilis fibrinolytic enzyme.

4. The use of the Bacillus subtilis fibrinolytic enzyme according to claim 1 in the preparation of anti-aging and life-extending drugs, characterized in that: The Bacillus subtilis fibrinolytic enzyme can be combined with pharmaceutically acceptable auxiliary materials to prepare liquid medicine, capsules or tablets.

5. The use of the Bacillus subtilis fibrinolytic enzyme according to claim 1 in the preparation of anti-aging and life-extending drugs, characterized in that: The Bacillus subtilis fibrinolytic enzyme is combined with spermidine and taurine and pharmaceutically acceptable auxiliary materials to prepare liquid medicine, capsule or tablet.

Images