A method for preparing and applying solid-state fermented natto with lipid-lowering and thrombolytic functions.

Abstract

This invention relates to the field of bio-food fermentation technology, specifically a method for preparing solid-state fermented natto with lipid-lowering and thrombolytic functions. The method involves preparing a composite fermentation substrate using a mixture of soybeans, chickpeas, black beans, and red yeast rice. A specific combination of Bacillus subtilis, Monascus purpureus, and Aspergillus oryzae is then quantitatively introduced into this substrate for co-fermentation. Leveraging the symbiotic interaction of multiple microorganisms and their metabolic potential for deep degradation of macromolecules, this preparation pathway successfully promotes the simultaneous high-level expression of multiple targeted functional components. The resulting mass-produced solid-state fermented natto complex stably accumulates highly active nattokinase, monacolin K, and angiotensin-converting enzyme inhibitory small molecule proteins. The superposition of multiple core active substances enables the complex to exhibit synergistic effects in lowering blood lipids, efficiently dissolving thrombi, and assisting in lowering blood pressure.

Description

Technical Field

[0001] This invention relates to the field of bio-food fermentation technology, specifically to a method and application for preparing solid-state fermented natto with lipid-lowering and thrombolytic functions. Background Technology

[0002] Cardiovascular and cerebrovascular diseases are among the most prevalent and deadliest diseases globally, posing a significant public health threat to human quality of life. Within the complex pathological mechanisms of these diseases, hyperlipidemia, leading to abnormally elevated levels of total cholesterol, triglycerides, and low-density lipoprotein cholesterol in the blood, easily causes atherosclerosis and irreversibly reduces the elasticity of blood vessel walls. This narrowing of the blood vessel lumen further provides an extremely adverse pathological basis for thrombus adhesion and formation. Simultaneously, hypertension, as an independent risk factor for cardiovascular and cerebrovascular events, accelerates the progression of atherosclerosis by continuously damaging vascular endothelial cells and significantly increasing intravascular wall pressure. These three factors intertwine to form a highly destructive vicious cycle.

[0003] In response to the complex pathological conditions described above, current conventional clinical intervention pathways heavily rely on targeted treatment with chemically synthesized drugs that target specific cholesterol pathways. Clinically, statins such as simvastatin or atorvastatin are commonly used to block cholesterol synthesis pathways to lower lipids, while anticoagulants like warfarin or thrombolytic drugs like urokinase are used to manage thrombotic crises. However, in the field of nutrition for prevention and adjunctive intervention, existing fermented foods are mostly limited to the superficial transformation of a single substrate by a single microbial strain. Typical examples include conventional natto products made solely from soybeans fermented with Bacillus subtilis, whose core metabolites focus only on fibrinolytic activity; or red yeast rice made solely from rice fermented with Monascus purpureus, whose effective components are essentially confined to the lipid-lowering category.

[0004] Existing single-target chemical drugs or single-function fermented foods are clearly insufficient to comprehensively mitigate the complex pathological risks posed by hyperlipidemia, thrombosis, and hypertension. Long-term use of statins or thrombolytic drugs can easily lead to severe adverse reactions such as liver damage, rhabdomyolysis, and even systemic bleeding. Current single-fermentation systems, failing to fully exploit the metabolic potential of multi-strain interactions, significantly limit the conversion rate of macromolecular nutrients in the matrix into various highly active small-molecule functional components. The current state of research in this field urgently requires a novel intervention product that combines natural high safety with multiple targeted effects to address the significant deficiencies in current cardiovascular disease prevention and daily health management programs. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a method for preparing and applying solid-state fermented natto with lipid-lowering and thrombolytic functions, solving the problems of single function, low yield of core active substances, and lack of multi-functional synergistic effects in existing fermented foods.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a method for preparing solid-state fermented natto with lipid-lowering and thrombolytic functions, comprising the following steps: Step 1: Matrix Construction Mixed beans and red yeast rice are pretreated separately and then thoroughly mixed to construct a composite fermentation substrate that serves as a nutrient source for microorganisms. Step 2: Inoculation and Co-fermentation Solid-state fermentation is performed by introducing a specific combination of production strains into the composite fermentation substrate. The production strain system consists of Bacillus subtilis, Monascus purpureus, and Aspergillus oryzae. Based on the total mass of the mixed substrate, the corresponding physical inoculum amounts are set as follows: Bacillus subtilis 2%, Monascus purpureus 1.5%, and Aspergillus oryzae 1%. Step 3: Product Post-processing The metabolites that have undergone a three-strain co-fermentation cycle are subjected to low-temperature drying and mechanical pulverization to finally obtain a solid fermented natto complex in a uniform powder state.

[0007] Preferably, the mixed beans include soybeans, chickpeas and black beans, and the mass ratio of soybeans, chickpeas and black beans is 5:3:2, and the amount of red yeast rice added is 20% of the total mass of the soybeans, chickpeas and black beans.

[0008] Preferably, the matrix construction process in step one is as follows: Soybeans, chickpeas, and black beans, weighed according to a predetermined ratio, are soaked in water for 12 hours. Then, they are placed in a high-temperature and high-pressure equipment at 121°C and steamed for 30 minutes. After the temperature of the mixed bean material system naturally and evenly drops to 40°C, it is ready for use. The red yeast rice undergoes a separate sterilization and inactivation process. After this operation, the sterile red yeast rice is directly and evenly mixed with the aforementioned cooled mixed bean matrix.

[0009] Preferably, in the second step of the co-fermentation stage, the overall operating temperature of the fermentation system is maintained between 30°C and 37°C, and the relative humidity of the space is maintained between 70% and 80% through an environmental control device. The duration of this solid-state co-fermentation cycle is between 48 hours and 72 hours.

[0010] Preferably, the post-processing procedure for the product in step three is as follows: The fermented solid material is placed in a 40°C constant temperature drying chamber and continuously dehydrated until the product moisture content reaches the standard. The dried agglomerated product is physically crushed using an ultra-fine pulverizing device. The sieved material, which has been thoroughly filtered through an 80-mesh standard sieve, is collected to obtain a fine-textured composite powder product.

[0011] Preferably, the Bacillus subtilis strain is selected from the strain with accession number CGMCC No.1.1086 from the China General Microbiological Culture Collection Center; the Monascus purpureus strain is selected from the strain with accession number CICC No.5039 from the China Industrial Microbiological Culture Collection Center; and the Aspergillus oryzae strain is selected from the strain with accession number CGMCC No.3.042 from the China General Microbiological Culture Collection Center.

[0012] Preferably, the solid-state fermented natto contains a stable enrichment of multiple core bioactive components with synergistic metabolic effects. According to standard quantitative detection procedures, the nattokinase activity index is ≥15000FU / g, the absolute content of Monacolin K (lovastatin in conventional chemical name) is ≥5mg / g, and the angiotensin-converting enzyme inhibitory small molecule protein extracted during the multi-strain fermentation metabolic process shows an effective biological inhibition rate of ≥70%.

[0013] Preferably, solid-state fermented natto is used in the preparation of functional health foods and special medical purpose formula foods with the effects of lowering blood lipids, dissolving thrombi, or assisting in lowering blood pressure.

[0014] This invention provides a method for preparing and applying solid-state fermented natto with lipid-lowering and thrombolytic functions. It possesses the following beneficial effects: 1. This invention overcomes the metabolic limitations of a single fermentation pathway by using Bacillus subtilis, Monascus purpureus, and Aspergillus oryzae with specific preservation numbers to co-ferment a specific ratio of legumes and red yeast rice composite substrate. This allows for the deep enzymatic transformation of large protein and carbohydrate molecules within the system, achieving the technical effect of simultaneously promoting the high-level expression of small molecule proteins by nattokinase, monacolin K, and angiotensin-converting enzyme within a single preparation pathway.

[0015] 2. This invention utilizes Aspergillus oryzae, which has a strong protein hydrolysis ability, to assist in the degradation of macromolecular proteins in the fermentation substrate, providing a rich and easily absorbed small molecule nutrient source for the rapid proliferation of Bacillus subtilis and Monascus purpureus. This achieves excellent yield results by significantly increasing nattokinase activity to ≥15000FU / g and boosting Monascus K content to ≥5mg / g.

[0016] 3. This invention avoids the risks of clinical side effects such as liver and kidney damage or coagulation dysfunction associated with chemically synthesized drugs by relying on clearly defined proportions of natural plant materials that are both food and medicine as fermentation substrates throughout the entire fermentation cycle. It achieves a high-safety application effect of reducing serum total cholesterol by 35% in hyperlipidemic model rats with no obvious toxicological reactions throughout the entire fermentation cycle.

[0017] 4. This invention eliminates the risk of bio-enzyme and small molecule protein activity decay caused by traditional high-temperature extraction and processing through solid-state co-fermentation in the medium temperature range of 30℃ to 37℃ and low-temperature controlled drying at 40℃. It achieves the green manufacturing technology effect of extremely low energy consumption throughout the process, significantly shortened production cycle, and seamless connection of fermentation residue to the bio-organic fertilizer resource conversion path. Attached Figure Description

[0018] Figure 1 This is a process flow diagram for preparing the solid-state fermented natto complex of the present invention; Figure 2 This is a fibrin plate lysate diagram used for the determination of nattokinase activity in this invention. Figure 3 This is the HPLC chromatogram for the determination of monacolin K content in this invention; Figure 4 This is a bar chart showing the effect of the complex of the present invention on serum TC levels in hyperlipidemic rats; Figure 5 This is a bar chart showing the effect of the compound of the present invention on the in vitro thrombolysis rate in rats. Detailed Implementation

[0019] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Example

[0020] like Figure 1 As shown, this embodiment of the invention provides a method for preparing solid-state fermented natto with lipid-lowering and thrombolytic functions, comprising the following steps: S1. Activation of microorganisms and preparation of seed culture: Bacillus subtilis (CGMCC No. 1.1086) was extracted and aseptically inoculated onto the surface of a standard LB slant agar medium. The medium was incubated at 37°C for 24 hours to obtain activated strains in the logarithmic growth phase. Monascus purpureus (CICC No. 5039) and Aspergillus oryzae (CGMCC No. 3.042) were inoculated in parallel onto PDA slant agar medium and incubated at 30°C for 72 hours and 48 hours, respectively. After activation, all three strains were transferred to their respective liquid media and cultured with shaking. Bacillus subtilis was cultured at 37°C and 180 rpm for 12 hours. Monascus purpureus and Aspergillus oryzae were cultured at 30°C and 150 rpm for 48 and 36 hours, respectively, to accurately obtain three standardized seed solutions with viable cell concentrations of 10⁸ CFU / mL.

[0021] S2. Matrix preparation and maturation: Accurately weigh 500g of soybeans, 300g of chickpeas, and 200g of black beans, and place them in a large mixing container for mechanical mixing. Pour 3000mL of high-purity deionized water into the mixture and allow it to soak for up to 12 hours. Using a porous filter, remove the swollen mixed beans and transfer them to a high-pressure steam sterilizer for deep cooking at a peak temperature of 121℃ for 30 minutes. After depressurization, remove the cooked beans and spread them out to cool naturally to the safe inoculation temperature threshold of 40℃. Weigh 200g of red yeast rice and place it in a separate steam sterilizer for inactivation treatment at 121℃ for 20 minutes. After cooling, pour it into the cooled cooked bean system and repeatedly stir to obtain a homogeneous composite fermentation substrate with a total weight of approximately 1200g.

[0022] S3. Combined inoculation with three strains and fermentation control: A sterile operating environment was established, and 24g of Bacillus subtilis seed solution, 18g of Monascus purpureus seed solution, and 12g of Aspergillus oryzae seed solution were sequentially pumped into the aforementioned 1200g composite fermentation substrate. A sterile scraper was used to continuously agitate the substrate to ensure that the microbial cell clusters were evenly attached to the surface of each fermentation substrate particle. The substrate carrying the bacteria was then loaded into an industrial-grade solid-state fermentation tank. The fermentation chamber temperature was set at 35°C, and the humidifier was instructed to maintain the relative humidity inside the chamber at 75%. The total fermentation cycle was set to 60 hours. During the fermentation operation, the mechanical rotary arm was activated once daily for 10 minutes each time to break up localized anaerobic heat spots and ensure overall ventilation efficiency.

[0023] S4. Low-temperature curing and grinding of the product: After the system indicates the end of the fermentation cycle, immediately remove all the wet fermentation product covered with mycelium from the tank. Spread it evenly on a tray and push it into a low-temperature circulating air drying chamber set at 40℃. Perform a gentle dehydration program for 24 hours, monitoring in real time until the absolute moisture content of the product drops back to 6%. The operator feeds the dried, crisp product into the feed hopper of an ultra-fine pulverizer for high-speed physical crushing. Use a powder collector to collect the particles that successfully penetrate an 80-mesh standard sieve. This batch yielded 950g of a pale yellow composite powder with a rich fermented aroma, thus successfully producing the solid-state fermented natto complex product as defined in this invention.

[0024] Example 2: This embodiment of the invention provides the detection of active ingredients in a solid-state fermented natto complex: 1. Nattokinase activity assay Reagent preparation: fibrinogen (bovine plasma source), thrombin (bovine plasma source), agarose, Tris-HCl buffer (pH 7.4).

[0025] Preparation of fibrin plates: Weigh 1g of agarose, add 100mL of Tris-HCl buffer, heat to dissolve, cool to 50℃, add 10mg of fibrinogen, stir to dissolve, then add 0.1mL of thrombin (100U / mL), mix quickly, pour into a culture dish, and solidify at room temperature to prepare fibrin plates.

[0026] Sample preparation: Accurately weigh 1g of the composite powder prepared in Example 1, add 10mL of Tris-HCl buffer, shake to extract for 30 minutes, centrifuge at 8000r / min for 10 minutes, and take the supernatant as the sample solution.

[0027] Assay: 3 mm diameter wells were punched in fibrin plates, and 20 μL of sample solution was added to each well. The plates were incubated at 37°C for 18 hours. Results are as follows: Figure 2 As shown, the diameter of the lysate zone was measured. A standard curve was plotted using standard nattokinase (with known activity), and the nattokinase activity in the sample was calculated based on the lysate zone diameter.

[0028] Results: The nattokinase activity of the complex was measured to be 16200 FU / g, which meets the design requirement of ≥15000 FU / g.

[0029] 2. Determination of Monacoline K content Instruments and reagents: High performance liquid chromatograph (equipped with ultraviolet detector), Monacoline K standard, methanol (chromatographic grade), potassium dihydrogen phosphate (analytical grade), phosphoric acid (analytical grade).

[0030] Chromatographic conditions: C18 column (4.6 mm × 250 mm, 5 μm); mobile phase: methanol-0.02 mol / L potassium dihydrogen phosphate solution (pH 4.0) = 75:25 (v / v); flow rate: 1.0 mL / min; detection wavelength: 238 nm; column temperature: 30 ℃; injection volume: 20 μL.

[0031] Sample preparation: Accurately weigh 0.5g of the composite powder prepared in Example 1, add 10mL of methanol, extract by ultrasonication for 30 minutes, centrifuge at 10000r / min for 10 minutes, take the supernatant and filter it through a 0.45μm organic phase filter membrane to obtain the sample test solution.

[0032] Standard curve preparation: Accurately weigh an appropriate amount of Monacoline K standard, dissolve and dilute it with methanol to prepare a series of standard solutions of different concentrations, and inject and determine the solutions according to the above chromatographic conditions. Plot the standard curve with peak area as the ordinate and concentration as the abscissa.

[0033] Sample determination: Inject the sample solution into the high-performance liquid chromatograph (HPLC), and calculate the monacolin K content based on the peak area substituted into the standard curve. The results are as follows: Figure 3 As shown.

[0034] Results: The monacolin K content of the complex was found to be 5.3 mg / g, which meets the design requirement of ≥5 mg / g.

[0035] 3. ACE inhibition rate measurement Reagent preparation: Angiotensin-converting enzyme (ACE, rabbit lung source), hippuryl-histyl-leucine (HHL), trichloroacetic acid (TCA), ethyl acetate, sodium hydroxide solution.

[0036] Reaction system: Add 0.1 mL ACE solution (0.1 U / mL), 0.2 mL HHL solution (5 mmol / L), and 0.1 mL sample solution (same as the sample extract for the nattokinase activity assay in Example 2) to a centrifuge tube in sequence, and react in a water bath at 37°C for 60 minutes.

[0037] To terminate the reaction: Add 0.2 mL of trichloroacetic acid (10%), shake to mix, terminate the reaction, and centrifuge at 10000 r / min for 10 minutes.

[0038] Product extraction: Take 0.4 mL of the supernatant, add 0.8 mL of ethyl acetate, shake and extract for 10 minutes, centrifuge at 8000 r / min for 5 minutes, take 0.6 mL of the upper ethyl acetate phase, place it in a 70℃ water bath to evaporate the solvent, and add 0.5 mL of distilled water to dissolve the residue.

[0039] Detection: Add 0.5 mL of sodium hydroxide solution (0.5 mol / L), shake well, and measure the absorbance value at a wavelength of 228 nm (sample A). Simultaneously, set up a blank control group (using distilled water instead of the sample solution) and a negative control group (using inactivated ACE solution instead of the ACE solution), and measure the absorbance values ​​of each (blank A, negative A).

[0040] ACE inhibition rate calculation: ACE inhibition rate (%) = [1 - (A sample - A negative) / (A blank - A negative)] × 100%.

[0041] Results: The ACE inhibition rate of the complex was measured to be 72.5%, which meets the design requirement of ≥70%.

[0042] Example 3: This example provides an animal experimental study of solid-state fermented natto complex: 1. Experimental Materials Laboratory animals: 60 male SPF-grade SD rats, weighing 200-220g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0043] Experimental sample: Solid-state fermented natto complex prepared in Example 1.

[0044] Positive drug: Simvastatin tablets (20mg / tablet), purchased from Sinopharm Industrial Co., Ltd.

[0045] Feed: ordinary basic feed, high-fat feed (basic feed + 10% lard + 1.5% cholesterol + 0.2% sodium cholate).

[0046] Test kits: Total cholesterol (TC) test kit, triglyceride (TG) test kit, low-density lipoprotein cholesterol (LDL-C) test kit, and high-density lipoprotein cholesterol (HDL-C) test kit were all purchased from Nanjing Jiancheng Bioengineering Institute.

[0047] 2. Experimental Methods Establishment of a hyperlipidemia model: Sixty SD rats were acclimatized for one week and then randomly divided into a normal control group (n=10) and a model group (n=50). The normal control group was fed a standard basal diet, while the model group was fed a high-fat diet for four consecutive weeks to establish a hyperlipidemia rat model. After successful model establishment, the rats in the model group were randomly divided into a model control group, a positive drug group, a low-dose complex group, a medium-dose complex group, and a high-dose complex group, with 10 rats in each group.

[0048] Administration regimen: The normal control group and the model control group were administered an equal volume of physiological saline by gavage daily; the positive control group was administered simvastatin solution (10 mg / kg) by gavage daily; the low, medium, and high dose groups of the complex were administered 150, 300, and 600 mg / kg of solid fermented natto complex suspension (prepared with physiological saline) by gavage daily, respectively. All rats were continued to be fed a high-fat diet for 28 consecutive days, during which the rats' diet, water intake, and weight changes were recorded.

[0049] Sample collection: After administration, rats were fasted for 12 hours and anesthetized by intraperitoneal injection of 10% chloral hydrate (3 mL / kg). Blood was collected from the abdominal aorta, placed in centrifuge tubes, allowed to stand at room temperature for 30 minutes, and centrifuged at 3000 r / min for 15 minutes to separate serum for the detection of lipid indicators and ACE activity. At the same time, blood was collected from the inferior vena cava of rats for the thrombosis experiment.

[0050] Detection indicators and methods: Lipid indicators: Serum levels of total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) were measured using a fully automated biochemical analyzer, following the instructions of the test kit. The effect of solid-state fermented natto complex on serum TC levels in hyperlipidemic rats was investigated. Figure 4 As shown.

[0051] Thrombolysis experiment: An in vitro thrombosis method was used. 0.5 mL of blood from the inferior vena cava of rats was injected into a siliconized test tube and incubated at 37°C for 30 minutes. After thrombus formation, the length and weight of the thrombus were measured. The thrombus was divided into two equal parts. One part was added to 0.5 mL of physiological saline (control group), and the other part was added to 0.5 mL of complex extract (concentration of 100 mg / mL). The two parts were incubated at 37°C for 24 hours, and the weight of the remaining thrombus was measured to calculate the thrombolysis rate.

[0052] ACE activity and blood pressure measurement: The ACE activity in serum was detected using the method for ACE inhibition rate determination in Example 2; the systolic and diastolic blood pressure of rats were measured using a non-invasive blood pressure measuring instrument (rat tail sheath method) before and after drug administration.

[0053] 3. Experimental Results Effects on rat body weight: During the experiment, all rats in each group had normal diet and water intake, and their body weight showed a slow growth trend. There was no significant difference in body weight growth among the groups, indicating that the complex had no adverse effects on the growth and development of rats.

[0054] Effects on lipid levels: Compared with the model control group, the positive control group and all dose groups of the complex significantly reduced serum TC, TG, and LDL-C levels in rats, and increased HDL-C levels (P<0.05 or P<0.01). Among them, the medium dose group of the complex (300 mg / kg) showed the most significant effect, with TC decreasing by 35%, TG decreasing by 28%, LDL-C decreasing by 40%, and HDL-C increasing by 18%. Its lipid-lowering effect was comparable to that of the positive control group (simvastatin 10 mg / kg). The specific results are shown in Table 1.

[0055] Table 1. Effects of the complex on lipid parameters in hyperlipidemic rats

[0056] Effect on thrombolysis: Compared with the control group thrombi (containing physiological saline), the thrombi containing the complex extract showed a significant reduction in weight and a 45% increase in thrombolysis rate, indicating that the complex has a significant thrombolytic effect. Specific results are shown in Table 2 and... Figure 5 .

[0057] Table 2. Effects of the complex on in vitro thrombolysis rate in rats.

[0058] Effects on ACE activity and blood pressure: Compared with the model control group, all dose groups of the complex significantly inhibited serum ACE activity in rats (P<0.05 or P<0.01), with the medium dose group showing an ACE activity inhibition rate of 65%. At the same time, the systolic and diastolic blood pressure of rats in the medium and high dose groups of the complex decreased slightly, with an average decrease of 10-15 mmHg in systolic blood pressure and an average decrease of 5-8 mmHg in diastolic blood pressure, and the differences were statistically significant. The specific results are shown in Table 3.

[0059] Table 3 Effects of the complex on serum ACE activity and blood pressure in rats

[0060] Safety evaluation: During the experiment, no rats in any group died, and no abnormal symptoms such as diarrhea, vomiting, or lethargy were observed. After the administration was completed, the rats were dissected, and the major organs such as the heart, liver, spleen, lungs, and kidneys were observed. No pathological damage was visible to the naked eye. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (Cr), and blood urea nitrogen (BUN) levels showed no significant differences compared with the normal control group, indicating that the complex is highly safe within the experimental dosage range and has no obvious toxic side effects.

[0061] Example 4: This embodiment of the invention provides formulation applications of the complex in diversified dosage forms: Application Scenario 1: Formulation and preparation of natto complex chewable tablets in the health food category The ingredient list includes 200g of solid-state fermented natto complex, 600g of mannitol as a filler, 150g of microcrystalline cellulose, 5g of magnesium stearate as a flow aid, and 5g of steviol glycosides as a flavoring agent. All excipients and main components, except for magnesium stearate, are pulverized to a fineness suitable for passing through a 100-mesh sieve and then injected into a three-dimensional mixer. A suitable amount of 70% ethanol solution is sprayed as a wetting binder and kneaded to form a soft mass, which is then extruded through a 20-mesh stainless steel sieve to obtain wet granules. The wet granules are placed in a 60℃ oven to dehydrate to a moisture content ≤5% and then granulated through an 18-mesh sieve. Weighed magnesium stearate lubricating powder is added to the dry granules, and the mixture is then fed to a rotary tablet press to complete the tableting process. Each round tablet produced in this batch weighs 1g and contains 200mg of the complex. Consumers can easily achieve the intervention dose by chewing and swallowing 1 to 2 tablets at a time, 1 to 2 times a day.

[0062] Application Scenario 2: Processing of lipid-lowering powder formulations for special medical purposes The core components of the formula consist of 100g of the complex, synergistically blended with 300g of whole milk powder, 400g of maltodextrin, 150g of high-quality soy protein isolate, 30g of fructooligosaccharides, and trace amounts of complex vitamins and trace elements. All solid materials undergo rigorous 80-mesh sieving before being placed in a V-shaped mixing chamber and continuously tumbled for 30 minutes. High-pressure homogenization is used to guide the mixture to a spray drying tower, with the inlet air temperature set at 180℃ and the outlet air temperature controlled at 80℃. This instantaneous powdering technology not only locks in activity but also greatly optimizes water solubility. The aseptic filling line seals each bag at 10g per individual package, precisely locking in 1g of the complex content per bag. Patients only need to add warm water for rapid reconstitution and administration.

[0063] Application Scenario 3: Development of lipid-lowering drug composition tablets in the form of pharmaceutical excipients Following the clinical concept of reducing drug toxicity, 300g of solid-state fermented natto complex and 10g of the chemical raw material simvastatin were accurately weighed. Simultaneously, 500g of conventional pharmaceutical excipients, 150g of starch, 30g of disintegrant sodium carboxymethyl starch, and 10g of magnesium stearate were prepared. The first four main raw materials were pulverized, sieved, and then dry-mixed evenly. A 5% concentration of povidone K30 ethanol binder was slowly added dropwise to form a soft mass. Wet granules were obtained through an 18-mesh sieve and dried with hot air at 60℃. After the moisture content of the dry granules was verified to be acceptable, the remaining excipients were added. Finally, a tableting mechanism was used to obtain micro-tablets with a total weight of 0.5g / tablet. In this combination formulation, the natto complex and simvastatin work together to exert a targeted effect, significantly reducing the initial dose limit that chemical drugs must introduce to achieve equivalent efficacy.

[0064] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for preparing solid-state fermented natto with lipid-lowering and thrombolytic functions, characterized in that, Includes the following steps: Step 1: Matrix Construction Mixed beans and red yeast rice are pretreated separately and then thoroughly mixed to construct a composite fermentation substrate that serves as a nutrient source for microorganisms. Step 2: Inoculation and Co-fermentation Solid-state fermentation is performed by introducing a specific combination of production strains into the composite fermentation substrate. The production strain system consists of Bacillus subtilis, Monascus purpureus, and Aspergillus oryzae. Based on the total mass of the mixed substrate, the corresponding physical inoculum amounts are set as follows: Bacillus subtilis 2%, Monascus purpureus 1.5%, and Aspergillus oryzae 1%. Step 3: Product Post-processing The metabolites that have undergone a three-strain co-fermentation cycle are subjected to low-temperature drying and mechanical pulverization to finally obtain a solid fermented natto complex in a uniform powder state.

2. The method for preparing solid-state fermented natto with lipid-lowering and thrombolytic functions according to claim 1, characterized in that, The mixed beans include soybeans, chickpeas and black beans, and the mass ratio of soybeans, chickpeas and black beans is 5:3:

2. The amount of red yeast rice added is 20% of the total mass of the soybeans, chickpeas and black beans.

3. The method for preparing solid-state fermented natto with lipid-lowering and thrombolytic functions according to claim 1, characterized in that, The specific process for matrix construction in step one is as follows: Soybeans, chickpeas, and black beans, weighed according to a predetermined ratio, are soaked in water for 12 hours. Then, they are placed in a high-temperature and high-pressure equipment at 121°C and steamed for 30 minutes. After the temperature of the mixed bean material system naturally and evenly drops to 40°C, it is ready for use. The red yeast rice undergoes a separate sterilization and inactivation process. After this operation, the sterile red yeast rice is directly and evenly mixed with the aforementioned cooled mixed bean matrix.

4. The method for preparing solid-state fermented natto with lipid-lowering and thrombolytic functions according to claim 1, characterized in that, In the second step of the co-fermentation stage, the overall operating temperature of the fermentation system is maintained between 30°C and 37°C, and the relative humidity of the space is maintained between 70% and 80% through an environmental control device. The duration of this solid co-fermentation cycle is between 48 hours and 72 hours.

5. The method for preparing solid-state fermented natto with lipid-lowering and thrombolytic functions according to claim 1, characterized in that, The specific post-processing procedure for the product in step three is as follows: The fermented solid material is placed in a 40°C constant temperature drying chamber and continuously dehydrated until the product moisture content reaches the standard. The dried agglomerated product is physically crushed using an ultra-fine pulverizing device. The sieved material, which has been thoroughly filtered through an 80-mesh standard sieve, is collected to obtain a fine-textured composite powder product.

6. The method for preparing solid-state fermented natto with lipid-lowering and thrombolytic functions according to claim 1, characterized in that, The Bacillus subtilis strain was selected from the strain with accession number CGMCC No.1.1086 from the China General Microbiological Culture Collection Center; the Monascus purpureus strain was selected from the strain with accession number CICC No.5039 from the China Industrial Microbiological Culture Collection Center; and the Aspergillus oryzae strain was selected from the strain with accession number CGMCC No.3.042 from the China General Microbiological Culture Collection Center.

7. The solid-state fermented natto prepared by the method according to any one of claims 1-6, characterized in that, The solid-state fermented natto contains a stable accumulation of multiple core bioactive components with synergistic metabolic effects. According to standard quantitative detection procedures, the nattokinase activity index is ≥15000FU / g, the absolute content of Monacolin K (lovastatin in conventional chemical name) is ≥5mg / g, and the angiotensin-converting enzyme inhibitory small molecule protein extracted during the multi-strain fermentation and metabolism process shows an effective biological inhibition rate of ≥70%.

8. The application of the solid-state fermented natto according to claim 7 in the preparation of functional health foods and special medical purpose formula foods with the effects of lowering blood lipids, dissolving thrombi, or assisting in lowering blood pressure.

9. The application of the solid-state fermented natto according to claim 7 in the preparation of lipid-lowering drugs, thrombolytic drugs, and adjuvant antihypertensive drug excipient compositions for cardiovascular and cerebrovascular diseases.

10. The application according to claim 7 or 8, characterized in that, The solid-state fermented natto used as the core active ingredient has a set safe daily intake physical dose range of 1g to 3g for adults.

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