Formula and preparation method of natural composite food for eliminating bromhidrosis and body odor

Abstract

The invention relates to the technical field of food science, and discloses a formula of a natural composite food for eliminating bromhidrosis and body odor and a preparation method of the natural composite food for eliminating bromhidrosis and body odor. Nattokinase: 150 to 550 parts of Mg; 10 to 80 parts of earthworm protein: Mg; a hawthorn leaf extract: 10 to 80 parts of Mg; and radix astragali: 10-80 parts of Mg. The hawthorn leaf extract comprises a flavonoid compound, an organic acid and a triterpenoid compound. In addition, the invention also provides a preparation method of the formula, which comprises the following steps: preparing raw materials; soaking raw materials; cooking the raw materials; a bacillus natto strain is inoculated; fermenting the inoculated natto; performing after-ripening treatment on the natto; extracting effective substances; purification of effective substances; concentrating and drying; and quality detection. After the food is eaten for 3-15 days, the armpit odor of a patient with bromhidrosis is obviously relieved, the sweating amount tends to be normal, the freshness of odor emitted by the body of the patient with body odor is obviously improved, and halitosis and bad breath odor of people are eliminated.

Description

Technical Field

[0001] The invention relates to the technical field of food science, in particular to a formula of a natural composite food for eliminating body odor and body odor and a preparation method thereof. Background Art

[0002] In today's society, body odor and body odor cause trouble to many people, not only affecting personal image, but also having a negative impact on social and mental health. There are many products and methods on the market to address this problem, but there are still many problems that need to be solved.

[0003] From the perspective of raw materials, the raw material selection of traditional products is relatively simple. Many products rely on only one ingredient. For example, common antiperspirants mostly use a single chemical ingredient such as aluminum chloride to reduce sweat secretion. Although it can alleviate odor to a certain extent, it cannot fundamentally solve the problem. Because the generation of body odor and body odor is not only related to sweat secretion, but also involves many factors such as bacterial growth and skin metabolism. It is difficult for a single ingredient to fully regulate these physiological processes, resulting in limited effects.

[0004] In terms of preparation process, the existing technology also has obvious defects. The traditional fermentation process does not accurately control conditions such as temperature and humidity. Take the preparation of nattokinase as an example. If the fermentation temperature is unstable, the growth and metabolism of Bacillus natto will be seriously affected, resulting in low nattokinase production and unstable activity.

[0005] From the perspective of product effects, existing products generally have the problem of single efficacy and short-term effects. Due to the limitations of raw materials and processes, these products can only temporarily suppress odors and cannot completely solve the problem; or they only work on a certain factor that produces odors.

[0006] In summary, the existing technologies for eliminating body odor and body odor have many deficiencies in terms of raw material selection, preparation process and product effect. Summary of the invention

[0007] In view of the deficiencies in the prior art, the present invention provides a formula of a natural composite food for eliminating body odor and body odor and a preparation method thereof, which solves the problem of single and non-persistent product effect in the preparation process of the natural composite food for eliminating body odor.

[0008] To achieve the above object, the present invention is implemented by the following technical scheme: a formula of a natural composite food for eliminating body odor and body odor, the formula comprising the following components of quality: Leeches: 200-600 mg; Nattokinase: 150~550Mg; Earthworm protein: 10~80Mg; Hawthorn leaf extract: 10-80 mg; Astragalus: 10-80mg.

[0009] Preferably, the hawthorn leaf extract comprises flavonoids, organic acids and triterpenoids.

[0010] In addition, the present invention also provides a method for preparing the formula, comprising the following steps: Raw material preparation; Soaking of raw materials; Steaming of raw materials; Inoculate with Bacillus natto; Fermentation of natto after inoculation; Post-ripening treatment of natto; Extraction of effective substances; Purification of effective substances; Concentration and drying; Quality inspection.

[0011] Preferably, the raw material cooking comprises: adopting one of normal pressure cooking or high pressure cooking, the normal pressure cooking time is 4 to 6 hours, and the high pressure cooking is cooking at a pressure of 0.1 to 0.15 MPa for 20 to 30 minutes.

[0012] Preferably, the inoculation of Bacillus subtilis natto comprises: cooling the cooked natto to 40-50° C., inoculating the Bacillus subtilis natto, and the inoculation amount being 1%-3% of the mass of the natto.

[0013] Preferably, the fermentation of the inoculated natto includes: placing the inoculated natto in a fermentation room or fermentation equipment for fermentation, the fermentation temperature is 37-42° C., the relative humidity is maintained at 80%-90%, and the fermentation time is 18-24 hours.

[0014] Preferably, the post-ripening treatment of the natto comprises: the fermented natto is moved to a low temperature environment of 0-5°C for post-ripening treatment for 12 to 24 hours.

[0015] Preferably, the extraction of the effective substances comprises: Crushing: Crushing the ripened natto by mechanical crushing to make natto slurry; Extraction: Add buffer solution to the natto homogenate and extract for 2 to 4 hours at 4 to 10°C with stirring; Centrifugal separation: centrifuge the mixed solution after extraction at a speed of 8000-12000 rpm for 20-30 minutes, take the supernatant and remove the residue and impurities.

[0016] Preferably, the purification of the effective substance comprises: Ultrafiltration: Use ultrafiltration membrane to ultrafilter the extract to remove macromolecular impurities such as proteins and polysaccharides, and concentrate the nattokinase solution; Chromatography: One of the ion exchange chromatography and gel filtration chromatography methods is selected to further purify nattokinase.

[0017] Preferably, the quality detection includes detection including enzyme activity determination, purity detection, microbial limit detection, and heavy metal content detection.

[0018] The present invention provides a formula of a natural composite food for eliminating body odor and body odor and a preparation method thereof. It has the following beneficial effects: 1. The present invention starts with the body's internal functions and treats both the symptoms and the root causes. After eating the food for 3-15 days, the armpit odor of patients with body odor is significantly reduced, the amount of sweating tends to be normal, the freshness of the body odor of people with body odor is significantly improved, bad breath and the odor of the mouth of the crowd are eliminated, the oral environment is improved, and people with sticky sweat constitution no longer sweat sticky, and the skin is refreshed. Compared with traditional external covering cleaning products, it provides a long-term, safe and brand-new solution for people troubled by odor.

[0019] 2. When purifying the effective substance of the present invention, ultrafiltration and chromatography techniques are used. When a mixed solution containing nattokinase and impurities flows through an ion exchange chromatography column, substances with different binding abilities to the medium ion groups will move at different speeds in the column, thereby achieving a separation effect. Gel filtration chromatography is separated based on the size of the molecules. Small molecules have a long diffusion path in the pores of the gel particles and move slowly; macromolecules can only flow in the gaps between the gel particles and move quickly. In this way, nattokinase is further purified, achieving the purpose of removing impurities and improving the purity of nattokinase. Compared with the conventional simple filtration purification method, the problem of low product purity, which affects the use effect and safety, is solved.

[0020] 3. The present invention accurately controls the fermentation temperature, humidity and time of natto fermentation after inoculation, thereby ensuring the good growth of Bacillus natto and efficiently producing nattokinase. Compared with fermentation without strict condition control, it solves the defects of low nattokinase production and unstable activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is a flow chart of the preparation method of the formulation in the present invention. DETAILED DESCRIPTION

[0022] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0023] Please refer to the attached Figure 1 The embodiment of the present invention provides a formula of a natural composite food for eliminating body odor and body odor, the formula comprising the following components of quality: Leeches: 200-600 mg; Nattokinase: 150~550Mg; Earthworm protein: 10~80Mg; Hawthorn leaf extract: 10-80 mg; Astragalus: 10-80mg.

[0024] Hawthorn leaf extract includes flavonoids, organic acids and triterpenoids.

[0025] In addition, the present invention also provides a method for preparing the formula, comprising the following steps: Raw material preparation; Soaking of raw materials; Steaming of raw materials; Inoculate with Bacillus natto; Fermentation of natto after inoculation; Post-ripening treatment of natto; Extraction of effective substances; Purification of effective substances; Concentration and drying; Quality inspection.

[0026] The raw material cooking includes: using one of normal pressure cooking or high pressure cooking, the normal pressure cooking time is 4 to 6 hours, and the high pressure cooking is cooking at a pressure of 0.1 to 0.15 MPa for 20 to 30 minutes to ensure that the soybeans are cooked thoroughly and have a soft texture.

[0027] The inoculation of Bacillus natto includes: cooling the cooked natto to 40-50° C., inoculating the Bacillus natto with an inoculation amount of 1%-3% of the mass of the natto, and fully stirring to evenly distribute the bacteria on the soybean surface.

[0028] The fermentation of natto after inoculation includes: placing the inoculated natto in a fermentation room or fermentation equipment for fermentation, the fermentation temperature is 37-42°C, the relative humidity is maintained at 80%-90%, and the fermentation time is 18-24 hours. During this period, natto Bacillus subtilis multiplies and secretes nattokinase metabolites, and a layer of white mucus-like substance will gradually form on the surface of soybeans, accompanied by a special ammonia smell.

[0029] The post-ripening treatment of natto includes: after the fermentation is completed, the natto is moved to a low temperature environment of 0 to 5°C for post-ripening treatment for 12 to 24 hours to further enhance the activity and flavor of nattokinase.

[0030] Extraction of effective substances includes: Crushing: Crushing the ripened natto by mechanical crushing to make natto slurry; Extraction: Add a buffer solution, such as phosphate buffer, to the natto homogenate, and extract for 2 to 4 hours at 4 to 10°C with stirring to fully dissolve the nattokinase in the solution; Centrifugal separation: centrifuge the mixed solution after extraction at a speed of 8000-12000 rpm for 20-30 minutes, take the supernatant and remove the residue and impurities.

[0031] Purification of active ingredients includes: Ultrafiltration: Use ultrafiltration membrane to ultrafilter the extract. Select the appropriate ultrafiltration membrane with a molecular weight cutoff according to the molecular weight of nattokinase to remove macromolecular impurities such as proteins and polysaccharides and concentrate the nattokinase solution. Chromatography: One of the ion exchange chromatography and gel filtration chromatography methods is used to further purify nattokinase. When the mixed solution containing nattokinase and impurities flows through the ion exchange chromatography column, substances with different binding abilities to the medium ion groups will move at different speeds in the column, thereby achieving a separation effect. Gel filtration chromatography is based on molecular volume separation. Small molecules have a long diffusion path in the pores of gel particles and move slowly; large molecules can only flow in the gaps between gel particles and move quickly. In this way, nattokinase can be further purified. Through the ion exchange chromatography column, the charge interaction between nattokinase and the chromatography medium is used for separation and purification.

[0032] The finished nattokinase products are quality tested, including enzyme activity determination (such as fibrin plate method), purity test (such as SDS-PAGE electrophoresis), microbial limit test, and heavy metal content test, to ensure that the products meet the relevant quality standards and requirements.

[0033] Example 1: Preparation of natural composite food for eliminating body odor and body odor Recipe composition: Leech: 450Mg; Nattokinase: 400Mg; Earthworm protein: 50Mg; Hawthorn leaf extract: 50Mg; Astragalus: 50Mg.

[0034] Preparation steps: Raw material preparation: soybean screening, select high-quality soybeans that are not moldy, not damaged, and have full grains, and remove impurities; Wash the soybeans thoroughly with clean water to remove dust and dirt on the surface; Soaking of raw materials: Place the washed soybeans in a specific container and add appropriate amount of clean water to soak for 16 hours to allow the soybeans to fully absorb water and expand. The soaking temperature is controlled at 20°C. Raw material cooking: high pressure cooking at 0.15MPa pressure for 30 minutes to ensure that the soybeans are cooked thoroughly and have a soft texture; Inoculate Bacillus natto: Cool the cooked natto to 45°C, inoculate Bacillus natto at an inoculation rate of 2% of the natto mass, and stir thoroughly to evenly distribute the bacteria on the soybean surface; Fermentation of inoculated natto: Place the inoculated natto in a fermentation room or fermentation equipment for fermentation at a temperature of 40°C and a relative humidity of 85% for 21 hours. During this period, natto Bacillus subtilis multiplies and secretes nattokinase metabolites, and a layer of white mucus-like substance gradually forms on the surface of soybeans, accompanied by a special ammonia smell. Post-ripening treatment of natto: After fermentation, the natto is moved to a low-temperature environment of 2°C for post-ripening treatment for 18 hours to further enhance the activity and flavor of nattokinase; Extraction of effective substances: crushing, crushing the ripened natto, using mechanical crushing to make natto homogenate; Extraction: add a buffer solution, such as phosphate buffer, to the natto homogenate, and extract for 3 hours at 7°C with stirring to fully dissolve the nattokinase in the solution; Centrifugal separation: centrifuge the mixed solution after extraction at a speed of 10,000 rpm for 25 minutes, take the supernatant and remove the residue and impurities; Purification of active ingredients: Ultrafiltration, use ultrafiltration membrane to ultrafilter the extract, select the appropriate ultrafiltration membrane with a molecular weight cutoff according to the molecular weight of nattokinase, remove macromolecular impurities such as proteins and polysaccharides, and concentrate the nattokinase solution; Chromatography, selecting one of ion exchange chromatography and gel filtration chromatography to further purify nattokinase, using an ion exchange chromatography column to separate and purify nattokinase by utilizing the charge interaction between nattokinase and the chromatography medium; Concentration and drying: Concentration: vacuum concentration is used to concentrate the purified nattokinase solution to reduce the water content and increase the concentration of the enzyme; Drying: drying the concentrated nattokinase solution, freeze drying or spray drying, to obtain a nattokinase finished product, which is a light yellow powdery substance that is easy to store and use; Quality testing: The finished nattokinase products are tested for quality, including enzyme activity determination (such as fibrin plate method), purity testing (such as SDS-PAGE electrophoresis), microbial limit testing, and heavy metal content testing to ensure that the products meet the relevant quality standards and requirements.

[0035] Example 2: Preparation of natural composite food by cooking at room temperature Recipe composition: Leech: 200Mg; Nattokinase: 550Mg; Earthworm protein: 30Mg; Hawthorn leaf extract: 80Mg; Astragalus: 80Mg.

[0036] Preparation steps: Raw material preparation: soybean screening, select high-quality soybeans that are not moldy, not damaged, and have full grains, and remove impurities; Wash the soybeans thoroughly with clean water to remove dust and dirt on the surface; Soaking of raw materials: Place the washed soybeans in a specific container and add appropriate amount of clean water to soak for 10 hours to allow the soybeans to fully absorb water and expand. The soaking temperature is controlled at 20°C. Raw material cooking: The normal pressure cooking time is 5 hours to ensure that the soybeans are cooked thoroughly and have a soft texture; Inoculate Bacillus natto: Cool the cooked natto to 41°C, inoculate with Bacillus natto at an inoculation rate of 1.5% of the natto mass, and stir thoroughly to evenly distribute the bacteria on the soybean surface; Fermentation of inoculated natto: Place the inoculated natto in a fermentation room or fermentation equipment for fermentation at a temperature of 37°C and a relative humidity of 82% for 22 hours. During this period, natto Bacillus subtilis multiplies and secretes nattokinase metabolites, and a layer of white mucus-like substance gradually forms on the surface of soybeans, accompanied by a special ammonia smell. Post-ripening treatment of natto: After fermentation, the natto is moved to a low-temperature environment of 0°C for post-ripening treatment for 20 hours to further enhance the activity and flavor of nattokinase; Extraction of effective substances: crushing, crushing the ripened natto, using mechanical crushing to make natto homogenate; Extraction: add a buffer solution, such as phosphate buffer, to the natto homogenate, extract for 4 hours at 4°C with stirring, so that the nattokinase is fully dissolved in the solution; Centrifugal separation: centrifuge the mixed solution after extraction at a speed of 11000 rpm for 23 minutes, take the supernatant and remove the residue and impurities; Purification of active ingredients: Ultrafiltration, use ultrafiltration membrane to ultrafilter the extract, select the appropriate ultrafiltration membrane with a molecular weight cutoff according to the molecular weight of nattokinase, remove macromolecular impurities such as proteins and polysaccharides, and concentrate the nattokinase solution; Chromatography, selecting one of ion exchange chromatography and gel filtration chromatography to further purify nattokinase, using an ion exchange chromatography column to separate and purify nattokinase by utilizing the charge interaction between nattokinase and the chromatography medium; Concentration and drying: Concentration: vacuum concentration is used to concentrate the purified nattokinase solution to reduce the water content and increase the concentration of the enzyme; Drying: drying the concentrated nattokinase solution, freeze drying or spray drying, to obtain a nattokinase finished product, which is a light yellow powdery substance that is easy to store and use; Quality testing: The finished nattokinase products are tested for quality, including enzyme activity determination (such as fibrin plate method), purity testing (such as SDS-PAGE electrophoresis), microbial limit testing, and heavy metal content testing to ensure that the products meet the relevant quality standards and requirements.

[0037] Example 3: Preparation of large-scale inoculated natural composite food Recipe composition: Leech: 500Mg; Nattokinase: 200Mg; Earthworm protein: 80Mg; Hawthorn leaf extract: 70Mg; Astragalus: 80Mg.

[0038] Preparation steps: Raw material preparation: soybean screening, select high-quality soybeans that are not moldy, not damaged, and have full grains, and remove impurities; Wash the soybeans thoroughly with clean water to remove dust and dirt on the surface; Soaking of raw materials: Place the washed soybeans in a specific container and add appropriate amount of clean water to soak for 8.5 hours to allow the soybeans to fully absorb water and expand. The soaking temperature is controlled at 20°C. Raw material cooking: high pressure cooking at 0.15MPa pressure for 20 minutes to ensure that the soybeans are cooked thoroughly and have a soft texture; Inoculate Bacillus natto: Cool the cooked natto to 48°C, inoculate Bacillus natto at an inoculation rate of 3% of the natto mass, and stir thoroughly to evenly distribute the bacteria on the soybean surface; Fermentation of inoculated natto: Place the inoculated natto in a fermentation room or fermentation equipment for fermentation at a temperature of 42°C and a relative humidity of 88% for 18 hours. During this period, Bacillus natto multiplies and secretes nattokinase metabolites, and a layer of white mucus-like substance gradually forms on the surface of soybeans, accompanied by a special ammonia smell. Post-ripening treatment of natto: After fermentation, the natto is moved to a low-temperature environment of 5°C for post-ripening treatment for 12 hours to further enhance the activity and flavor of nattokinase; Extraction of effective substances: crushing, crushing the ripened natto, using mechanical crushing to make natto homogenate; Extraction: add a buffer solution, such as phosphate buffer, to the natto homogenate, and extract for 2.5 hours at 8°C with stirring to fully dissolve the nattokinase in the solution; Centrifugal separation: centrifuge the mixed solution after extraction at a speed of 9000 rpm for 30 minutes, take the supernatant and remove the residue and impurities; Purification of active ingredients: Ultrafiltration, use ultrafiltration membrane to ultrafilter the extract, select the appropriate ultrafiltration membrane with a molecular weight cutoff according to the molecular weight of nattokinase, remove macromolecular impurities such as proteins and polysaccharides, and concentrate the nattokinase solution; Chromatography, selecting one of ion exchange chromatography and gel filtration chromatography to further purify nattokinase, using an ion exchange chromatography column to separate and purify nattokinase by utilizing the charge interaction between nattokinase and the chromatography medium; Concentration and drying: Concentration: vacuum concentration is used to concentrate the purified nattokinase solution to reduce the water content and increase the concentration of the enzyme; Drying: drying the concentrated nattokinase solution, freeze drying or spray drying, to obtain a nattokinase finished product, which is a light yellow powdery substance that is easy to store and use; Quality testing: The finished nattokinase products are tested for quality, including enzyme activity determination (such as fibrin plate method), purity testing (such as SDS-PAGE electrophoresis), microbial limit testing, and heavy metal content testing to ensure that the products meet the relevant quality standards and requirements.

[0039] Comparative Example 1: Adjusting the amount of raw materials (corresponding to Example 1) Recipe Adjustment: Leech: 200Mg; Nattokinase: 550Mg; Earthworm protein: 50Mg; Hawthorn leaf extract: 50Mg; Astragalus: 50Mg.

[0040] Preparation steps: The natural composite food for eliminating body odor and body odor was prepared according to the method of Example 1.

[0041] Comparative Example 2: Changing the fermentation temperature (corresponding to Example 2) Formula adjustment: keep all components of Example 2 unchanged Preparation steps: According to the method of Example 2, a natural composite food for eliminating body odor and body odor was prepared. In the fermentation step of the inoculated natto, the fermentation temperature was changed to 34°C, the humidity was still maintained at 82%, the fermentation time was still 22 hours, and the other preparation steps were the same as in Example 2.

[0042] Comparative Example 3: Simplified preparation process (corresponding to Example 3) Formula adjustment: keep all components of Example 3 unchanged Preparation steps: A natural composite food for eliminating body odor and body odor is prepared according to the method of Example 3. During the preparation process, the chromatography step in the effective substance purification step is omitted, and only ultrafiltration membrane is used for ultrafiltration treatment. The parameters of the raw material soaking, cooking, fermentation, post-ripening treatment and effective substance extraction steps are consistent with those in Example 3.

[0043] Comparative Example 4: Removal of key raw materials (corresponding to Example 1) Recipe Adjustment: Leech: 450Mg; Nattokinase: 400Mg; Earthworm protein: 50Mg; Astragalus: 50mg Preparation steps: When preparing the raw materials, the hawthorn leaf extract was removed, and the total amount of other raw materials, leech, nattokinase, earthworm protein, and astragalus, was made up to 100%. The other preparation steps were the same as those in Example 1.

[0044] Comparative experiment Experiment 1: Enzyme activity determination comparison experiment Experimental samples: Select the nattokinase finished products of Example 1 and Comparative Example 1.

[0045] Experimental steps: using the fibrin plate method, first, prepare an agarose gel plate containing fibrin, spread it evenly in a culture dish, then punch holes in the plate, add the nattokinase sample solutions of Example 1 and Comparative Example 1, respectively, set 3 parallel holes for each sample, place the plate in a 37°C constant temperature incubator and incubate for 18 to 24 hours. After the incubation, measure the diameter of the dissolution zone and calculate the enzyme activity according to the standard curve.

[0046] Experimental data Table 1: Experimental summary: It can be seen from the experimental data that the average diameter of the dissolution circle formed by the nattokinase finished product of Example 1 is significantly larger than that of Comparative Example 1. This is because in Example 1 of the present invention, the dosage of raw materials is reasonable and the components work synergistically to create favorable conditions for the production and activity maintenance of nattokinase. The leech and earthworm protein components may improve the microecology of the fermentation environment, promote the growth and metabolism of Bacillus natto, and produce more highly active nattokinase.

[0047] However, the change in the amount of raw materials in Comparative Example 1 broke this synergistic balance. For example, the reduction in the amount of leeches may lead to insufficient promotion factors in the fermentation process, affecting the growth and reproduction of Bacillus natto, thereby reducing the production and activity of nattokinase.

[0048] Nattokinase has the function of dissolving blood clots. The higher the enzyme activity, the stronger the ability to dissolve fibrin, and the larger the dissolution circle formed on the fibrin plate. The results of this experiment show that a reasonable raw material formula is crucial to improving the enzyme activity of nattokinase, which is directly related to the efficacy and quality of the product.

[0049] Experiment 2: Purity detection comparison experiment Experimental sample: select the nattokinase finished product of embodiment 2 and comparative example 2.

[0050] Experimental method: Use SDS-PAGE electrophoresis to prepare separation gel and concentration gel, mix the sample with loading buffer, heat and denature it and load it, set the appropriate voltage for electrophoresis to separate the protein in the gel, and after the electrophoresis, stain it with Coomassie Brilliant Blue staining solution, and then decolorize it with decolorizing solution until the bands are clear. By comparing it with the standard protein marker, analyze the purity of nattokinase in the sample.

[0051] Experimental data Table 2: Experimental summary: It can be seen from the experimental data that the purity of nattokinase in Example 2 is higher than that in Comparative Example 2. This is mainly due to the complete and precise preparation process of Example 2. In the fermentation process, appropriate temperature, humidity and time control allow Bacillus natto to grow stably, efficiently produce nattokinase, and reduce the production of impurities.

[0052] The fermentation temperature of Comparative Example 2 was changed, which affected the normal metabolism of Bacillus natto, resulting in the production of more impurities and reducing the purity of nattokinase. In the purification of effective substances, Example 2 adopted a two-step method of ultrafiltration and chromatography, in which ultrafiltration first removed macromolecular impurities and chromatography further purified the product, thereby improving the purity in a two-pronged manner.

[0053] The principle of SDS-PAGE electrophoresis is to separate based on the size and charge difference of protein molecules. Under the action of the electric field, proteins migrate in the gel, and proteins with small molecular weight and high charge migrate quickly. After Coomassie brilliant blue staining, the grayscale of the band is proportional to the protein content. Therefore, the experimental results intuitively reflect that the preparation process of Example 2 is better, and high-purity nattokinase can be obtained, which is of great significance to product quality and efficacy improvement.

[0054] Experiment 3: Comparative experiment of microbial limit detection Experimental samples: Take the nattokinase finished products of Example 3 and Comparative Example 3.

[0055] Experimental method: adopt plate count method, prepare nutrient agar medium, rose bengal sodium agar medium, bile salt lactose medium, weigh appropriate amount of samples of Example 3 and Comparative Example 3 respectively, add sterile physiological saline to make test solution. Dilute the test solution in a gradient manner, take the test solution of appropriate dilution and inoculate it into the above culture medium respectively, inoculate 2 to 3 plates for each dilution, and place the inoculated plate at a specified temperature for culture. Nutrient agar medium is used to detect the total number of aerobic bacteria, and cultured at 30 to 35°C for 3 to 5 days; rose bengal sodium agar medium is used to detect the total number of molds and yeasts, and cultured at 23 to 28°C for 5 to 7 days; bile salt lactose medium is used to detect Escherichia coli control bacteria, and cultured at 35 to 37°C for 18 to 24 hours. After the culture is completed, count the number of colonies on the plate to determine whether the microbial limit meets the regulations.

[0056] Experimental data Table 3: Experimental summary: From the experimental data, the total number of aerobic bacteria and the total number of molds and yeasts in Example 3 are lower than those in Comparative Example 3. This is because in the preparation process of Example 3, the environment and parameters of the fermentation, post-ripening and effective matter extraction links are strictly controlled, and the appropriate fermentation temperature and humidity are not only conducive to the growth of Bacillus natto, but also can inhibit the reproduction of miscellaneous bacteria, and the low temperature environment of the post-ripening treatment can also reduce the growth of microorganisms.

[0057] The change in fermentation temperature in Comparative Example 3 disrupts the balance of microbial growth, which may make it easier for some bacteria to grow, resulting in an increase in the number of microorganisms. Microbial limit detection is crucial. Excessive microorganisms may affect product quality and stability. Aerobic bacteria, molds and yeasts will decompose the ingredients in the product under suitable conditions, reducing the efficacy of the product. If pathogenic bacteria are present, it will also endanger human health. This experiment shows that optimizing the preparation process is of great significance to controlling microbial limits and can ensure product quality and safety.

[0058] Experiment 4: Heavy metal content detection Experimental samples: Select the nattokinase finished products of Example 1 and Comparative Example 4.

[0059] Experimental method: Atomic absorption spectrometry is used to digest the sample and convert it into a solution state suitable for detection. An atomic absorption spectrometer is used to set the detection wavelengths and conditions of heavy metal elements such as lead, mercury, cadmium, and arsenic. The digested sample solution is injected into the atomic absorption spectrometer, the absorbance is measured, and the content of heavy metals in the sample is calculated based on the standard curve.

[0060] Experimental data Table 4: Experimental summary: It can be seen from the data that the heavy metal content in Example 1 is significantly lower than that in Comparative Example 4. This is mainly due to the strict raw material screening and sophisticated preparation process of Example 1. In terms of raw material selection, leech and earthworm protein raw materials are carefully selected to control the risk of heavy metal introduction at the source. In the preparation process, multiple purification steps effectively remove impurities and reduce heavy metal residues.

[0061] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A formula of a natural composite food for eliminating body odor and body odor, characterized in that: The formulation includes the following qualities of components: Leeches: 200-600 mg; Nattokinase: 150~550Mg; Earthworm protein: 10~80Mg; Hawthorn leaf extract: 10-80 mg; Astragalus: 10-80mg.

2. The formula of the natural composite food for eliminating body odor and body odor according to claim 1, characterized in that: The hawthorn leaf extract comprises flavonoid compounds, organic acids and triterpenoid compounds.

3. A method for preparing a formulation, characterized in that: The formula of the natural composite food for eliminating body odor and body odor according to any one of claims 1 to 2 comprises the following steps: Raw material preparation; Soaking of raw materials; Steaming of raw materials; Inoculate with Bacillus natto; Fermentation of natto after inoculation; Post-ripening treatment of natto; Extraction of effective substances; Purification of effective substances; Concentration and drying; Quality inspection.

4. The method for preparing the formula according to claim 3, characterized in that: The raw material cooking comprises: adopting one of normal pressure cooking or high pressure cooking, the normal pressure cooking time is 4 to 6 hours, and the high pressure cooking is cooking at a pressure of 0.1 to 0.15 MPa for 20 to 30 minutes.

5. The method for preparing the formula according to claim 3, characterized in that: The inoculation of the natto Bacillus subtilis strain comprises: cooling the cooked natto to 40-50° C., inoculating the natto Bacillus subtilis strain, and the inoculation amount is 1%-3% of the mass of the natto.

6. The method for preparing the formulation according to claim 3, characterized in that: The fermentation of the inoculated natto includes: placing the inoculated natto in a fermentation room or fermentation equipment for fermentation, the fermentation temperature is 37-42° C., the relative humidity is maintained at 80%-90%, and the fermentation time is 18-24 hours.

7. The method for preparing the formulation according to claim 3, characterized in that: The post-ripening treatment of the natto includes: the fermented natto is moved to a low temperature environment of 0 to 5° C. for post-ripening treatment for 12 to 24 hours.

8. The method for preparing the formulation according to claim 3, characterized in that: The extraction of the effective substance comprises: Crushing: Crushing the ripened natto by mechanical crushing to make natto slurry; Extraction: Add buffer solution to the natto homogenate and extract for 2 to 4 hours at 4 to 10°C with stirring; Centrifugal separation: centrifuge the mixed solution after extraction at a speed of 8000-12000 rpm for 20-30 minutes, take the supernatant and remove the residue and impurities.

9. The method for preparing the formulation according to claim 3, characterized in that: The purification of the effective substance comprises: Ultrafiltration: Use ultrafiltration membrane to ultrafilter the extract to remove macromolecular impurities such as proteins and polysaccharides, and concentrate the nattokinase solution; Chromatography: One of the ion exchange chromatography and gel filtration chromatography methods is selected to further purify nattokinase.

10. The method for preparing the formulation according to claim 3, characterized in that: The quality inspection includes enzyme activity detection, purity detection, microbial limit detection, and heavy metal content detection.

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