Composition, preparation method of composition and application thereof

Abstract

The present invention discloses a composition, a preparation method of the composition and an application thereof. The composition comprises calcium citrate, natto powder, powdered Bacillus subtilis, earthworm powder, leech powder and red yeast rice powder in a specific compound ratio, and can effectively remove blood clots in blood vessels, reduce blood lipids, improve blood viscosity, enhance blood circulation function, increase bone marrow blood perfusion, restore bone cell nutritional metabolism function, improve calcium absorption and utilization rate, and enhance bone density. The composition can be used to prepare food, medicine or animal feed for treating and / or preventing osteoporosis.

Description

Technical Field

[0001] The present invention relates to the technical field of compositions, and in particular to a composition, a preparation method of the composition and applications thereof. Background Art

[0002] Osteoporosis is a chronic disease associated with aging and a common disease in the elderly. When it occurs, the microstructure of bones is damaged, bone density decreases, bone brittleness increases, and fractures are more likely to occur. Osteoporosis and cardiovascular disease are both common diseases in the elderly. Many studies have shown that these two diseases share common physiological, pathological, and genetic foundations. These evidences show that the metabolism of bone tissue is closely linked to the blood system. During bone metabolism, blood vessels play the important role of transporting nutrients and metabolic waste. As a functional entity, blood circulation plays an irreplaceable role in the body's nutrient metabolism. As a functional entity, changes in blood circulation function may affect bone metabolism and osteoporosis through certain mechanisms.

[0003] The mainstream osteoporosis prevention and treatment products on the market fall into two main categories: supplemental products, such as calcium supplements, vitamin D products, glucosamine products, chondroitin sulfate products, estrogen products, calcitonin products, and parathyroid hormone products; and regulatory products, such as selective estrogen receptor modulators and bisphosphonates. Both categories of products prevent and treat osteoporosis by regulating bone density. Currently, there are no osteoporosis prevention and treatment products developed specifically to promote blood health. Summary of the Invention

[0004] In view of the deficiencies in the prior art, the present invention provides a composition, a preparation method of the composition and applications thereof.

[0005] The technical solution of the present invention is as follows:

[0006] In a first aspect, the present invention provides a composition, which comprises, calculated by weight percentage, 25% to 45% calcium citrate, 0.0001% to 0.0008% vitamin D3, 0.01% to 0.05% vitamin K2, 1% to 3% powdered Bacillus subtilis, 10% to 30% natto powder, 10% to 30% earthworm powder, 10% to 30% leech powder, and 2% to 15% red yeast rice powder.

[0007] In a second aspect, the present invention provides a method for preparing a composition, for preparing the composition as described in the first aspect, the method for preparing the composition comprising the following steps:

[0008] Providing a first powder and a second powder, wherein the first powder comprises calcium citrate, vitamin D3, and vitamin K2, and the second powder comprises earthworm powder, leech powder, and red yeast rice powder; and

[0009] Powdered Bacillus subtilis and natto powder are provided according to a formula ratio, and the powdered Bacillus subtilis, the natto powder, the first powder and the second powder are mixed to obtain the composition.

[0010] In a third aspect, the present application provides the use of the composition as described in the first aspect, or the composition prepared by the preparation method of the composition as described in the second aspect, in the preparation of a drug or animal feed for treating and / or preventing osteoporosis.

[0011] In a fourth aspect, the present application provides the use of the composition as described in the first aspect, or the composition prepared by the preparation method of the composition as described in the second aspect, in the preparation of food that helps to improve bone density.

[0012] This application provides a composition, a preparation method of the composition, and its application, which produce the following technical effects:

[0013] The composition of the present application includes calcium citrate, natto powder, powdered Bacillus subtilis, earthworm powder, leech powder and red yeast rice powder in a specific compound ratio, which can effectively clear blood clots in blood vessels, reduce blood lipids, improve blood viscosity, enhance blood circulation function, increase bone marrow blood perfusion, restore bone cell nutritional metabolism function, improve calcium absorption and utilization rate, and enhance bone density. The composition can be used to prepare medicines or animal feeds for treating and / or preventing osteoporosis, and the composition can be used to prepare foods that help improve bone density. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The technical solutions and other beneficial effects of the present invention will be made apparent by describing in detail the specific embodiments of the present invention in conjunction with the accompanying drawings.

[0015] Figure 1 A schematic flow chart of a method for preparing a composition provided in an embodiment of the present application. DETAILED DESCRIPTION

[0016] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0017] Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those familiar to those skilled in the art. The raw materials and reagents used herein are commercially available or can be prepared by methods known in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the present invention. The preferred embodiments and materials described herein are for illustrative purposes only and do not limit the scope of this application.

[0018] It should be noted that the order of description of the following embodiments is not intended to limit the preferred order of the embodiments, and the various embodiments of the present invention may be presented in the form of a range. It should be understood that the description in the form of a range is merely for convenience and brevity and should not be understood as a rigid limitation on the scope of the present invention; therefore, it should be considered that the range description has specifically disclosed all possible sub-ranges and single numerical values ​​within the range. For example, the range description from 1 to 6 should be considered to have specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within the range, such as 1, 2, 3, 4, 5 and 6, which apply regardless of the range. In addition, whenever a numerical range is indicated herein, it is meant to include any cited numbers (fractions or integers) within the indicated range. Taking "25% to 45% calcium citrate" as an example, in the composition, the weight percentage of calcium citrate can be 25%, 28%, 30%, 32%, 35%, 38%, 40%, 43%, 45% or a value between any two of the foregoing values.

[0019] In the description of this application, the term "including" means "including but not limited to".

[0020] The term "plurality" means two or more.

[0021] The selection scope of the term "and / or" includes any one of two or more related listed items, and also includes any and all combinations of the related listed items, and the said any and all combinations include any two related listed items, any more related listed items, or the combination of all related listed items. For example, "A and / or B" includes three parallel solutions of A, B and A+B. For another example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the four-item combination of A, B, C, and D (that is, the technical solution connected by "logical AND").

[0022] An embodiment of the present application provides a composition, which includes, by weight percentage, 25% to 45% calcium citrate, 0.0001% to 0.0008% vitamin D3, 0.01% to 0.05% vitamin K2, 1% to 3% powdered Bacillus subtilis, 10% to 30% natto powder, 10% to 30% earthworm powder, 10% to 30% leech powder, and 2% to 15% red yeast rice powder.

[0023] In the composition of the embodiments of the present application, each component is formulated in a specific ratio, wherein calcium citrate is used to provide a calcium source; vitamin K2 has the function of promoting bone formation and inhibiting bone absorption; vitamin D3 is used to promote calcium absorption and the reabsorption of urinary calcium, promote the deposition of calcium salts in the bone matrix, and at the same time regulate the coordination of nerve and muscle tissue, and has the function of preventing falls and fractures. The combined use of calcium citrate, vitamin K2 and vitamin K2 can synergistically enhance the effect. In addition, powdered Bacillus subtilis can promote the reproduction of beneficial bacteria in the intestine, optimize the balance of intestinal flora, and improve the body's absorption of calcium; natto powder contains nattokinase, which has the effect of dissolving blood clots and unblocking blood vessels; earthworm powder has the pharmacological effects of anti-thrombosis and reducing blood viscosity; leech powder has the effects of breaking blood, removing blood stasis, and promoting menstruation; red yeast rice powder has the effects of clearing turbidity and lowering blood lipids. The combined use of the above-mentioned multiple ingredients can effectively clear blood clots in blood vessels, reduce blood lipids, improve blood viscosity, enhance blood circulation function, enhance bone marrow blood perfusion, restore bone cell nutritional metabolic function, improve calcium absorption and utilization rate, and enhance bone density. Therefore, the composition can be used to prepare drugs or animal feed for treating and / or preventing osteoporosis, and the composition can be used to prepare foods that help improve bone density.

[0024] To further enhance the calcium supplementation effect of the composition, in some embodiments of the present application, the powdered Bacillus subtilis strain is Bacillus subtilis CX01, which has a deposit number of CCTCC NO: M20221746 and a deposit date of November 9, 2022, with the China Center for Type Culture Collection. It is understood that Bacillus subtilis CX01 can be present in the composition in the form of pure Bacillus subtilis CX01 or a dried product of a fermentation product of Bacillus subtilis CX01.

[0025] In order to further improve the calcium supplementation effect of the composition, in some embodiments of the present application, the viable bacterial count of the powdered Bacillus subtilis is not less than 1×10 8 CFU / g.

[0026] In some embodiments of the present application, the composition further comprises 2% to 20% of excipients, calculated by weight percentage. Excipients refer to additional ingredients used in the production of compositions and the preparation of prescriptions, and have important functions such as excipients, protection of active ingredients, improvement of stability, solubilization, solubilization, and controlled release, so that the composition can achieve a certain shelf life and bioavailability, thereby improving the safety and effectiveness of the composition. Excipients include, but are not limited to, one or more of flavor enhancers, excipients, diluents, fillers, solvents, premixes, disintegrants, binders, lubricants, surfactants, and adsorption carriers, wherein fillers include, but are not limited to, one or more of starch, dextrin, lactose, compressible starch, and microcrystalline cellulose; binders include, but are not limited to, one or more of hydroxypropyl methylcellulose, starch slurry, cross-linked polyvinylpyrrolidone, and sodium carboxymethylcellulose; disintegrants include, but are not limited to, one or more of dry starch, sodium carboxymethyl starch, and cross-linked polyvinylpyrrolidone.

[0027] In some embodiments of the present application, the composition is a food, a medicine, or an animal feed.

[0028] In some embodiments of the present application, the composition further comprises one or more probiotic microorganisms, such as bifidobacteria, lactobacilli, etc.

[0029] In some embodiments of the present application, the composition is one or more of tablets, granules, powders, sprays, suspensions, emulsions, capsules or ointments.

[0030] In some embodiments of the present application, vitamin K2 is of the MK-7 type. Compared with MK-4, MK-8, MK-9 and MK-10 types, MK-7 has a long half-life in the body, and MK-7 is one of the components with better bioactivity and bioavailability among the aforementioned types of vitamin K2, thereby further enhancing the efficacy of the composition.

[0031] The present application also provides a method for preparing a composition as described above, such as Figure 1 As shown, the preparation method of the composition comprises the following steps:

[0032] S1. Providing a first powder and a second powder, wherein the first powder comprises calcium citrate, vitamin D3, and vitamin K2, and the second powder comprises earthworm powder, leech powder, and red yeast rice powder;

[0033] S2. providing powdered Bacillus subtilis and natto powder according to a formula ratio, and mixing the powdered Bacillus subtilis, natto powder, the first powder, and the second powder to obtain a composition.

[0034] The fineness of the first powder is, for example, 80-120 mesh, and / or the fineness of the second powder is, for example, 80-120 mesh. Calcium citrate, vitamin D3, vitamin K2, earthworm powder, leech powder, red yeast rice powder, and natto powder are all commercially available, or one or more of these can be prepared independently. It should be noted that when the composition further includes excipients, the powdered Bacillus subtilis, natto powder, the first powder, the second powder, and the excipients are mixed to prepare the composition.

[0035] In some embodiments of the present application, the method for preparing the first powder includes the following steps:

[0036] S11, providing vitamin D3 and vitamin K2 according to a formula ratio, and dissolving the vitamin D3 and vitamin K2 in a solvent to obtain a vitamin solution;

[0037] S12. Calcium citrate is provided according to the formula ratio, and the vitamin solution prepared in step S11 is sprayed onto the calcium citrate to obtain a first powder.

[0038] In step S11, the solvent includes an alcohol of the general formula R1-OH and water, where R1 is selected from a C1-C10 alkyl group or a C1-C10 alkyl group substituted with at least one hydroxyl group. R1 is selected from, for example, a C1-C3 alkyl group, a C1-C5 alkyl group, or a C1-C8 alkyl group. The solvent is exemplified as a mixture of ethanol and water. In at least one embodiment of the present application, the solvent is a mixture of ethanol and water, with the volume percentage of ethanol being 95%.

[0039] In some embodiments of the present application, a method for preparing vitamin K2 includes the steps of inoculating Bacillus subtilis CX01 into a first fermentation medium, fermenting and culturing the medium at 35°C to 37°C for 72 to 120 hours to obtain a first fermentation product, performing a first solid-liquid separation on the first fermentation product, collecting a first supernatant, and then purifying the first supernatant to obtain vitamin K2. It should be noted that the fermentation and culturing can be performed in a shake flask or a fermentor.

[0040] The first fermentation medium includes: 20 g / L to 60 g / L glucose, 20 g / L to 60 g / L soy peptone, 2 g / L to 6 g / L NaCl, and 10 g / L to 20 g / L yeast extract, wherein the glucose is, for example, anhydrous glucose.

[0041] In some embodiments of the present application, during the preparation of vitamin K2, the inoculation amount of Bacillus subtilis CX01 in the first fermentation medium is 1% to 5%.

[0042] During the preparation process of vitamin K2, the first solid-liquid separation treatment is used to remove the bacteria. The first solid-liquid separation treatment includes but is not limited to one or more of sedimentation, filtration and evaporation. Sedimentation includes but is not limited to one or more of gravity sedimentation, centrifugal sedimentation and electromagnetic sedimentation. Filtration separation includes but is not limited to one or more of reverse osmosis, membrane filtration, nanofiltration, ultrafiltration and microfiltration. An example of the first solid-liquid separation treatment is centrifugal sedimentation.

[0043] In some embodiments of the present application, the purification process includes the steps of ultrafiltration of the collected first supernatant, followed by concentration to obtain a concentrate, followed by extraction of the concentrate with vegetable oil, and then vacuum distillation of the oil layer obtained from the extraction to obtain a crude vitamin K2 extract, which is then filtered to remove impurities and dried to obtain solid vitamin K2. The ultrafiltration membrane used in the ultrafiltration is of a 5KD specification.

[0044] In some embodiments of the present application, a method for preparing natto powder includes the steps of inoculating Bacillus subtilis CX01 into a second fermentation medium, fermenting the culture at 35°C to 37°C for 16 to 20 hours to obtain a second fermentation product, performing a second solid-liquid separation on the second fermentation product, collecting a second supernatant, and sequentially performing a concentration step and a spray drying step on the second supernatant to obtain natto powder. The fineness of the natto powder is, for example, 80 to 120 mesh.

[0045] The second fermentation medium comprises: 20 g / L to 40 g / L of glucose, 10 g / L to 30 g / L of defatted soy flour, 4 g / L to 8 g / L of Na2HPO4·12H2O, 0.5 g / L to 2 g / L of NaH2PO4·H2O, 0.2 g / L to 1 g / L of MgSO4·7H2O, and 0.1 g / L to 0.5 g / L of CaCl2. The glucose is, for example, anhydrous glucose.

[0046] In some embodiments of the present application, during the preparation of natto powder, the inoculation amount of Bacillus subtilis CX01 in the second fermentation medium is 1% to 5%.

[0047] In the preparation process of natto powder, the second solid-liquid separation treatment is used to remove the bacteria. The second solid-liquid separation treatment includes but is not limited to one or more of sedimentation, filtration and evaporation. Sedimentation is but is not limited to one or more of gravity sedimentation, centrifugal sedimentation and electromagnetic sedimentation. Filtration separation is but is not limited to one or more of reverse osmosis, membrane filtration, nanofiltration, ultrafiltration and microfiltration. An example of the second solid-liquid separation treatment is centrifugal sedimentation.

[0048] In some embodiments of the present application, the sequential concentration and spray drying of the second supernatant comprises the steps of: ultrafiltration and concentration of the second supernatant, and then spray drying the concentrated solution obtained by the ultrafiltration and concentration, wherein the specification of the ultrafiltration membrane used in the ultrafiltration is 5KD.

[0049] The present application also provides an embodiment of the use of the composition as described above, or a composition prepared by the method for preparing the composition as described above, in the preparation of a drug or animal feed for treating and / or preventing osteoporosis.

[0050] The embodiments of the present application also provide the use of the composition as described above, or the composition prepared by the method for preparing the composition as described above, in the preparation of food that helps to improve bone density.

[0051] The technical solutions and technical effects of the present application are described in detail below through specific embodiments, comparative examples and experimental examples. The following embodiments are only some embodiments of the present application and do not specifically limit the present application.

[0052] Example 1

[0053] This embodiment provides a composition, which is composed of the following components, calculated by weight percentage: 40% calcium citrate, 0.0005% vitamin D3, 0.04% vitamin K2, 1% powdered Bacillus subtilis, 15% natto powder, 15% earthworm powder, 15% leech powder, 10% red yeast rice powder, 2.7% sodium carboxymethyl cellulose, and 1.2595% magnesium stearate.

[0054] Among them, earthworm powder was purchased from Shanxi Lisheng Biotechnology Co., Ltd., the product model is LS-0425-94, and the fineness of earthworm powder is 100 mesh. Leech powder was purchased from Shanxi Lisheng Biotechnology Co., Ltd., the product model is SZS, and the fineness of leech powder is 100 mesh. Red yeast rice powder was purchased from Shaanxi Saien Biotechnology Co., Ltd., the product model is SXSE59522, and the fineness of red yeast rice powder is 100 mesh. Vitamin D3 was purchased from Zhejiang Tianhecheng Biotechnology Co., Ltd., the product model is THC-VD01A. The strain of powdered Bacillus subtilis is Bacillus subtilis CX01, and the fineness of powdered Bacillus subtilis is 100 mesh. Vitamin K2 and natto powder are both prepared by ourselves, and vitamin K2 is MK-7 type.

[0055] The preparation method of vitamin K2 includes the following steps: sterilizing 300L of a first fermentation medium in a 500L fermentor, inoculating the first fermentation medium with a 3% inoculum of Bacillus subtilis CX01 seed solution, and aerobically fermenting the medium at 37°C for 72 hours to obtain a first fermentation product; then centrifuging the first fermentation product to remove bacterial cells and collecting a first supernatant; ultrafiltration of the collected first supernatant and concentration to obtain a concentrate, extracting the concentrate with vegetable oil, and then vacuum distilling the oil layer obtained by extraction to obtain a crude extract of vitamin K2, filtering to remove impurities, and drying to obtain solid vitamin K2. The formula of the first fermentation medium is: 30g / L anhydrous glucose, 30g / L soy peptone, 4g / L NaCl, and 15g / L yeast extract, with a pH of 7.0.

[0056] The preparation method of natto powder includes the following steps: placing 300 L of a second fermentation medium in a 500 L fermentation tank for sterilization, then inoculating Bacillus subtilis CX01 seed liquid into the second fermentation medium at an inoculum rate of 3%, and aerobically fermenting and culturing at 37°C for 18 hours to obtain a second fermentation product; then, centrifuging the second fermentation product to remove bacterial cells and collecting a second supernatant; then, ultrafiltration and concentration of the second supernatant, and then spray drying the concentrated liquid obtained by the ultrafiltration and concentration to obtain natto powder with a fineness of 100 mesh.

[0057] The preparation method of the composition comprises the following steps:

[0058] S1.1. Preparing a first powder: Weigh vitamin D3 and vitamin K2 according to a formula ratio, dissolve the vitamin D3 and vitamin K2 in an ethanol aqueous solution (ethanol volume percentage is 95%) to obtain a vitamin solution; weigh calcium citrate according to a formula ratio, and spray the obtained vitamin solution onto the calcium citrate to obtain a first powder with a fineness of 100 mesh;

[0059] S1.2. Preparation of a second powder: earthworm powder, leech powder, and red yeast rice powder are prepared according to a formula ratio, dispersed in water to prepare a suspension, and then spray-dried to obtain a second powder with a fineness of 100 mesh;

[0060] S1.3. Weigh powdered Bacillus subtilis, natto powder, sodium carboxymethyl cellulose, and magnesium stearate according to the formula ratio, stir the first powder, the second powder, powdered Bacillus subtilis, natto powder, sodium carboxymethyl cellulose, and magnesium stearate, mix well, and tabletize to obtain a tablet composition.

[0061] Example 2

[0062] This embodiment provides a composition. Compared with the composition in Example 1, the difference between the composition in this embodiment and the composition in Example 1 is that the compounding ratio of each component is different. In this embodiment, the composition is composed of the following components, calculated by weight percentage: 30% calcium citrate, 0.0005% vitamin D3, 0.05% vitamin K2, 1.5% powdered Bacillus subtilis, 18% natto powder, 18% earthworm powder, 18% leech powder, 10% red yeast rice powder, 3.1% sodium carboxymethyl cellulose, and 1.3495% magnesium stearate. The sources of each component remain the same as in Example 1.

[0063] The preparation method of the composition in this example is carried out with reference to the preparation method of the composition in Example 1.

[0064] Example 3

[0065] This embodiment provides a composition. Compared with the composition in Example 1, the difference between the composition in this embodiment and the composition in Example 1 is that the compounding ratio of each component is different. In this embodiment, the composition is composed of the following components, calculated by weight percentage: 25% calcium citrate, 0.0005% vitamin D3, 0.05% vitamin K2, 2% powdered Bacillus subtilis, 20% natto powder, 20% earthworm powder, 20% leech powder, 10% red yeast rice powder, 2% sodium carboxymethyl cellulose, and 0.9495% magnesium stearate. The sources of each component remain the same as in Example 1.

[0066] The preparation method of the composition in this example is carried out with reference to the preparation method of the composition in Example 1.

[0067] Example 4

[0068] This example provides a composition. Compared to the composition in Example 1, the composition in this example differs in that the strain of powdered Bacillus subtilis is different. In this example, the powdered Bacillus subtilis strain is Bacillus subtilis 168, purchased from the China Center for Type Culture Collection, with strain accession number CCTCCAB 130001. Aside from the powdered Bacillus subtilis, the sources and blend ratios of the other components remain the same as in Example 1.

[0069] The preparation method of the composition in this example is carried out with reference to the preparation method of the composition in Example 1.

[0070] Example 5

[0071] This example provides a composition. Compared to the composition in Example 1, the composition in this example differs in the type of vitamin K2. In this example, the vitamin K2 is MK-4, purchased from Native Peptide Biotechnology (Tianjin) Co., Ltd., product model YST-XSSK. Aside from vitamin K2, the sources and compounding ratios of the other ingredients remain the same as in Example 1.

[0072] The preparation method of the composition in this example is carried out with reference to the preparation method of the composition in Example 1.

[0073] Example 6

[0074] This example provides a composition. Compared to the composition in Example 1, the composition in this example differs in that the source of natto powder is different. In this example, natto powder was purchased from Xi'an Ao Biotechnology Co., Ltd., product model QA062. Aside from the natto powder, the sources and compounding ratios of the other ingredients remained the same as in Example 1.

[0075] The preparation method of the composition in this example is carried out with reference to the preparation method of the composition in Example 1.

[0076] Comparative Example 1

[0077] This comparative example provides a composition comprising the following components, calculated by weight: 70% calcium citrate, 0.0005% vitamin D3, 0.04% vitamin K2, 15% sodium carboxymethylcellulose, and 14.9595% magnesium stearate. The sources of the components are consistent with those in Example 1.

[0078] The preparation method of the composition in this comparative example includes the following steps: weighing vitamin D3 and vitamin K2 according to a formula ratio, dissolving the vitamin D3 and vitamin K2 in an ethanol aqueous solution (95% by volume of ethanol) to obtain a vitamin solution; weighing calcium citrate according to a formula ratio, spraying the obtained vitamin solution onto the calcium citrate to obtain a first powder having a fineness of 100 mesh; and weighing sodium carboxymethyl cellulose and magnesium stearate according to a formula ratio, stirring the first powder, sodium carboxymethyl cellulose, and magnesium stearate until uniformly mixed, and tableting to obtain a tablet composition. The specifications of the tablet composition in this comparative example are consistent with those in Example 1.

[0079] Comparative Example 2

[0080] This comparative example provides a composition comprising the following components, calculated by weight: 50% calcium citrate, 0.0005% vitamin D3, 0.04% vitamin K2, 1% powdered Bacillus subtilis, 20% natto powder, 15% sodium carboxymethyl cellulose, and 13.9595% magnesium stearate. The sources of the ingredients are consistent with those in Example 1.

[0081] The preparation method of the composition in this comparative example includes the steps of: weighing vitamin D3 and vitamin K2 according to a formula ratio, dissolving the vitamin D3 and vitamin K2 in an ethanol aqueous solution (ethanol volume percentage is 95%) to obtain a vitamin solution; weighing calcium citrate according to a formula ratio, spraying the obtained vitamin solution onto the calcium citrate to obtain a first powder with a fineness of 100 mesh; and weighing powdered Bacillus subtilis, natto powder, sodium carboxymethyl cellulose, and magnesium stearate according to a formula ratio, stirring the first powder, powdered Bacillus subtilis, natto powder, sodium carboxymethyl cellulose, and magnesium stearate, mixing them uniformly, and tableting to obtain a tablet composition. The specifications of the tablet composition in this comparative example are consistent with those in Example 1.

[0082] Comparative Example 3

[0083] This comparative example provides a composition comprising the following components, calculated by weight: 50% calcium citrate, 0.0005% vitamin D3, 0.04% vitamin K2, 10% red yeast rice powder, 25% sodium carboxymethyl cellulose, and 14.9595% magnesium stearate. The sources of the components are consistent with those in Example 1.

[0084] The preparation method of the composition in this comparative example includes the steps of: weighing vitamin D3 and vitamin K2 according to a formula ratio, dissolving the vitamin D3 and vitamin K2 in an ethanol aqueous solution (ethanol volume percentage is 95%) to obtain a vitamin solution; weighing calcium citrate according to a formula ratio, spraying the obtained vitamin solution onto the calcium citrate to obtain a first powder with a fineness of 100 mesh; and weighing red yeast rice powder, sodium carboxymethyl cellulose, and magnesium stearate according to a formula ratio, stirring the first powder, red yeast rice powder, sodium carboxymethyl cellulose, and magnesium stearate, mixing them uniformly, and tableting to obtain a tablet composition. The specifications of the tablet composition in this comparative example are consistent with those in Example 1.

[0085] Comparative Example 4

[0086] This comparative example provides a composition comprising, by weight percentage, 50% calcium citrate, 0.0005% vitamin D3, 0.04% vitamin K2, 15% earthworm powder, 15% leech powder, 9% sodium carboxymethylcellulose, and 10.9595% magnesium stearate. The sources of the ingredients are consistent with those in Example 1.

[0087] The preparation method of the composition in this comparative example includes the steps of: weighing vitamin D3 and vitamin K2 according to a formula ratio, dissolving the vitamin D3 and vitamin K2 in an ethanol aqueous solution (ethanol content is 95% by volume) to obtain a vitamin solution; weighing calcium citrate according to a formula ratio, spraying the obtained vitamin solution onto the calcium citrate to obtain a first powder having a fineness of 100 mesh; and weighing earthworm powder, leech powder, sodium carboxymethyl cellulose, and magnesium stearate according to a formula ratio, stirring the first powder, earthworm powder, leech powder, sodium carboxymethyl cellulose, and magnesium stearate until uniformly mixed, and tableting to obtain a tablet composition. The specifications of the tablet composition in this comparative example remain the same as those in Example 1.

[0088] Comparative Example 5

[0089] This comparative example provides a composition comprising the following components, calculated by weight: 2% powdered Bacillus subtilis, 20% natto powder, 20% earthworm powder, 20% leech powder, 10% red yeast rice powder, 14% sodium carboxymethyl cellulose, and 14% magnesium stearate. The sources of the ingredients are consistent with those in Example 1.

[0090] The preparation method of the composition in this comparative example includes the steps of: taking earthworm powder, leech powder, and red yeast rice powder according to a formula ratio, dispersing the obtained earthworm powder, leech powder, and red yeast rice powder in water to prepare a suspension, and then spray drying to obtain a second powder with a fineness of 100 mesh; and respectively weighing powdered Bacillus subtilis, natto powder, sodium carboxymethyl cellulose, and magnesium stearate according to the formula ratio, stirring the second powder, powdered Bacillus subtilis, natto powder, sodium carboxymethyl cellulose, and magnesium stearate, mixing them uniformly, and tableting to obtain a tablet composition. The specifications of the tablet composition in this comparative example are consistent with those in Example 1.

[0091] Experimental example

[0092] 140 SPF male rats weighing 200-220 g were fed a basal diet for seven days and then randomly divided into the following 14 groups based on body weight: a low calcium control group, a blood stasis low calcium group, a calcium citrate group, Example 1 to Example 6 groups, and Comparative Example 1 to Comparative Example 5 groups, with 10 rats in each group. Except for the low calcium control group, each rat in the remaining 13 groups received an intraperitoneal injection of 10 mg / kg of guanidine. The low calcium control group received an intraperitoneal injection of a 0.9% sodium chloride solution equal in volume to the guanidine.

[0093] For the calcium citrate group, Example 1 to Example 6 groups, and Comparative Example 1 to Comparative Example 5 groups, the corresponding drugs were gavaged daily according to the dosage in Table 1 below. The low calcium control group and the blood stasis low calcium group were gavaged with an equal volume of normal saline once a day for 12 consecutive weeks. During the experiment, the low calcium control group was fed with low calcium feed (purchased from Jiangsu Collaborative Pharmaceutical Bioengineering Co., Ltd., product model 1019025-9, calcium content 0.1%) and deionized water, and the remaining groups were fed with low calcium high fat feed (purchased from Jiangsu Collaborative Pharmaceutical Bioengineering Co., Ltd., customized product: fat content 60%, calcium content 0.1%) and deionized water.

[0094] Table 1

[0095]

[0096] During the oral gavage period, the animals were weighed once a week. After the experiment, 5 mL of blood was collected from each group of rats via the abdominal aorta into heparinized tubes. Low-, medium-, and high-cut whole blood viscosity values ​​were measured using a SA6600 fully automatic hemorheology tester. 2 mL of blood was collected into a standard test tube, centrifuged, and then tested for four lipid profiles using a kit from the Nanjing Jiancheng Bioengineering Institute. The rats were then sacrificed by cervical dislocation. Bone density at the distal and central ends of the femur was measured using a bone densitometer. The right femur was oven-baked at 105°C until constant weight, and the calcium content was determined using atomic absorption spectrophotometry.

[0097] The body weight changes of the rats in each group are shown in Table 2 below:

[0098] Table 2

[0099]

[0100] As can be seen from Table 2, after 12 weeks of feeding, the average weight of rats in the calcium citrate group, the blood stasis and high fat model group, the Example 1 to Example 6 groups, and the Comparative Example 1 to Comparative Example 5 groups increased significantly compared with the low calcium control group, indicating that the high-fat diet has a significant effect on the weight of rats. However, compared with the blood stasis and high fat model group, the calcium citrate group, and the Comparative Example 1 group, the average weight gain of rats in the Example 1 to Example 6 groups and the Comparative Example 2 to Comparative Example 5 groups was lower, indicating that the compositions in Examples 1 to Example 6 and Comparative Examples 2 to Comparative Example 5 have a certain control effect on the weight of rats.

[0101] The four blood lipid test indicators of rats in each group are shown in Table 3 below:

[0102] Table 3

[0103]

[0104]

[0105] As shown in Table 3, compared with the low calcium control group, the TG, TC, and LDL-C in the blood stasis and hyperlipidemia model group increased significantly, and the HDL-C decreased significantly, indicating that the hyperlipidemia model was successfully established. Compared with the blood stasis and hyperlipidemia model group, the four blood lipid indicators of the rats in Example 1 to Example 6 and Comparative Example 2 to Comparative Example 5 groups improved significantly, and the improvement of the four blood lipid indicators of the rats in Example 1 to Example 6 groups was particularly significant, indicating that the combination of the present invention is more effective in lowering blood lipids than a single component.

[0106] The whole blood viscosity index test results of rats in each group are shown in Table 4 below:

[0107] Table 4

[0108]

[0109]

[0110] As shown in Table 3, compared with the low calcium control group, the whole blood viscosity indicators of the blood stasis and high lipid model group were significantly different in low shear, medium shear and high shear viscosity. The blood stasis and high lipid model group was significantly higher than the low calcium control group, indicating that the blood stasis model was successfully established.

[0111] Compared with the blood stasis and high lipid model group, the blood viscosity of the rats in Example 1 to Example 6 and Comparative Example 2 to Comparative Example 5 groups was significantly different, and the blood viscosity of the rats in Example 1 to Example 6 groups decreased more significantly, especially in Example 1 to Example 3 groups, indicating that the composition of the embodiment of the present invention has the effect of reducing blood viscosity and can significantly enhance blood circulation function.

[0112] The results of bone density, femoral mass and bone calcium content tests of rats in each group are shown in Table 5 below:

[0113] Table 5

[0114]

[0115] As shown in Table 5, compared with the blood stasis and high lipid model group, the calcium citrate group showed significant increases in femoral bone density, femoral mass, and bone calcium content, indicating that the low bone density rat model was successfully established. Under conditions of high blood viscosity, elevated blood lipids, and impaired blood circulation, the active ingredients of the composition in Comparative Example 1, including vitamin D3, vitamin K2, and calcium citrate, were more effective in enhancing bone density than calcium citrate alone, indicating that calcium citrate, combined with vitamin D3 and vitamin K2, has a synergistic effect on calcium supplementation.

[0116] The composition in Comparative Example 2 is based on the composition in Comparative Example 1, in which natto powder and powdered Bacillus subtilis are added. The blood viscosity and blood lipid indicators of the rats in the Comparative Example 2 group are better than those in the Comparative Example 1 group. The rats in the Comparative Example 2 group have better bone density than the rats in the Comparative Example 1 group. The reason is that: powdered Bacillus subtilis can colonize in the intestine to promote intestinal health, and can also produce vitamin K2 in the intestine, which has an anticoagulant effect. After being absorbed by the intestine, it works together with the functional substance nattokinase in the natto powder to exert the effect of promoting blood circulation and thrombolysis. This shows that the addition of blood-activating and thrombolytic functional substances and intestinal probiotics can improve the calcium supplementation effect and enhance bone density.

[0117] The compositions in Comparative Examples 3 and 4 were based on the composition in Comparative Example 1, with the addition of one or more of red yeast rice powder, earthworm powder, and leech powder. The bone density, femoral mass, and bone calcium effects of the rats in Comparative Example 3 and 4 were superior to those in Comparative Example 1, indicating that the addition of one or more of red yeast rice powder, earthworm powder, and leech powder to calcium supplementation alone can promote calcium absorption and improve bone density. Comparative Example 5, which did not contain calcium citrate but primarily contained the functional ingredients of natto powder, powdered Bacillus subtilis, earthworm powder, leech powder, and red yeast rice powder, was able to reduce blood lipids and blood viscosity, significantly promoting blood health, but had limited calcium supplementation effects.

[0118] Compared with the comparative example 1 group to the comparative example 5 group, the comprehensive levels of the four blood lipid indicators, blood viscosity index and bone density index of the rats in the embodiment 1 group to the embodiment 6 group were better, indicating that the combined use of calcium supplements (such as calcium citrate), natto powder, powdered Bacillus subtilis, earthworm powder, leech powder and red yeast rice powder can promote blood circulation, improve blood health, and is beneficial to the absorption and utilization of calcium, and the calcium supplementation effect is better.

[0119] In the above embodiments, the description of each embodiment has its own focus. For parts not described in detail in a certain embodiment / comparative example / experimental example, reference can be made to the relevant description of other embodiments.

[0120] The above is a detailed introduction to a composition, a preparation method of the composition, and an application thereof provided in an embodiment of the present invention. Specific examples are used herein to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only used to help understand the technical solutions and core ideas of the present invention. Those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A composition, characterized in that Calculated by weight percentage, the composition consists of 25% to 45% of calcium citrate, 0.0001% to 0.0008% of vitamin D3, 0.01% to 0.05% of vitamin K2, 1% to 3% of powdered Bacillus subtilis, 10% to 30% of natto powder, 10% to 30% of earthworm powder, 10% to 30% of leech powder, 2% to 15% of red yeast rice powder, and 2% to 20% of auxiliary materials; The powdered Bacillus subtilis strain is Bacillus subtilis CX01, the deposit number of the Bacillus subtilis CX01 is CCTCC NO: M20221746, the deposit date is November 9, 2022, and the deposit unit is China Center for Type Culture Collection; the viable cell count of the powdered Bacillus subtilis is not less than 1×10 8 CFU / g; The vitamin K2 is of MK-7 type, and the preparation method of the vitamin K2 comprises the steps of: inoculating the Bacillus subtilis CX01 into a first fermentation medium, fermenting and culturing at 35°C to 37°C for 72 hours to 120 hours to obtain a first fermentation product, performing a first solid-liquid separation treatment on the first fermentation product to collect a first supernatant, and then purifying the first supernatant to obtain the vitamin K2; the first fermentation medium comprises: 20 g / L to 60 g / L of glucose, 20 g / L to 60 g / L of soy peptone, 2 g / L to 6 g / L of NaCl, and 10 g / L to 20 g / L of yeast extract; The preparation method of natto powder comprises the following steps: inoculating Bacillus subtilis CX01 into a second fermentation medium, fermenting and culturing at 35°C to 37°C for 16 hours to 20 hours to obtain a second fermentation product, performing a second solid-liquid separation process on the second fermentation product to collect a second supernatant, and then sequentially performing a concentration process and a spray drying process on the second supernatant to obtain the natto powder; the second fermentation medium comprises: 20 g / L to 40 g / L of glucose, 10 g / L to 30 g / L of defatted soy flour, 4 g / L to 8 g / L of Na2HPO4·12H2O, 0.5 g / L to 2 g / L of NaH2PO4·H2O, 0.2 g / L to 1 g / L of MgSO4·7H2O, and 0.1 g / L to 0.5 g / L of CaCl2.

2. The composition according to claim 1, characterized in that The composition is a food, a medicine or an animal feed; And / or, the composition is one or more of tablets, granules, powders, sprays, suspensions, emulsions, capsules or pastes.

3. A method for preparing a composition, characterized in that: For preparing the composition according to claim 1 or 2, the preparation method of the composition comprises the following steps: Providing a first powder and a second powder, wherein the first powder comprises calcium citrate, vitamin D3, and vitamin K2, and the second powder comprises earthworm powder, leech powder, and red yeast rice powder; as well as Powdered Bacillus subtilis and natto powder are provided according to a formula ratio, and the powdered Bacillus subtilis, the natto powder, the first powder and the second powder are mixed to obtain the composition.

4. The method for preparing the composition according to claim 3, wherein: The preparation method of the first powder comprises the following steps: Providing vitamin D3 and vitamin K2 according to a formula ratio, and dissolving the vitamin D3 and vitamin K2 in a solvent to obtain a vitamin solution; as well as providing calcium citrate according to a formula ratio, and spraying the vitamin solution onto the calcium citrate to obtain the first powder; The solvent comprises an alcohol with a general formula of R1-OH and water, and R1 is selected from a C1-C10 alkyl group or a C1-C10 alkyl group substituted with at least one hydroxyl group.

5. The method for preparing the composition according to claim 3, wherein: The preparation method of the second powder includes the steps of: taking earthworm powder, leech powder and red yeast rice powder according to a formula ratio, dispersing the obtained earthworm powder, leech powder and red yeast rice powder in water to prepare a suspension, and then spray drying to obtain the second powder.

6. Use of the composition according to claim 1 or 2, or the composition prepared by the method for preparing the composition according to any one of claims 3 to 5, in the preparation of a medicament or animal feed for treating and / or preventing osteoporosis.

7. Use of the composition according to claim 1 or 2, or the composition prepared by the method for preparing the composition according to any one of claims 3 to 5, in preparing a food that helps to improve bone density.

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