Extraction method and application of eggshell membrane active ingredient composition for improving bone joint health

Abstract

The invention relates to the technical field of preparation of functional food raw materials, and particularly discloses an extraction method and application of an eggshell membrane active ingredient composition for improving bone joint health. The extraction method comprises the following steps: S1, preparing raw materials; s2, performing low-temperature physical extraction, namely performing low-temperature extraction on the crude eggshell membrane in an environment of 5-15 DEG C by taking pure water or a NaCl solution as a solvent; s3, separation and purification: carrying out preliminary separation on the leach liquor by using an ultrafiltration membrane, then carrying out enzymolysis treatment by using a compound enzyme, and finally, further filtering enzymatic hydrolysate by using a refined filtration membrane; s4, performing micronization treatment to obtain the eggshell membrane active ingredient composition. According to the method, low-temperature physical extraction, efficient membrane separation and enzymolysis treatment are adopted, active ingredients can be efficiently extracted from eggshell membranes, the process conditions are mild, efficient and low in cost, and the method has wide market application prospects, especially in the fields of joint health, skeleton strengthening and sports health care.

Description

Technical Field

[0001] The invention belongs to the technical field of preparation of functional food raw materials, and specifically relates to an extraction method and application of an eggshell membrane active ingredient composition for improving bone and joint health. Background Art

[0002] As people's lifestyles change and they age, joint problems are increasingly becoming an important factor affecting their quality of life. In particular, problems such as arthritis and joint cartilage degeneration bring great pain and burden to patients. There are many health foods on the market for joint health, including chondroitin, glucosamine, fish oil, etc., but their effectiveness and safety are often questioned by consumers.

[0003] Among these health foods, eggshell membrane (ESM) has gradually attracted attention as a natural material rich in collagen, hyaluronic acid, elastin, glycosaminoglycans and other active ingredients. The active ingredients in eggshell membrane have good biocompatibility and can promote cartilage repair, relieve joint inflammation and improve joint flexibility.

[0004] However, the existing methods for preparing active ingredients from eggshell membranes have the following shortcomings: 1. Complex extraction process: The existing extraction methods of active ingredients from eggshell membrane are generally complicated, often using high temperature or chemical reagents, which may destroy its natural structure and active ingredients, thus affecting the efficacy of the final product; 2. Low extraction efficiency: The existing extraction methods are inefficient and cannot fully extract the active ingredients in the eggshell membrane, resulting in high product costs; 3. Low purity: The active ingredients of eggshell membranes prepared by existing extraction methods have low purity and are usually accompanied by other eggshell components, such as protein and calcium. These impurities may affect the quality and efficacy of the product; 4. Poor taste and bioavailability: The active ingredients of eggshell membrane prepared by existing extraction methods are mostly in the form of coarse powder, with poor solubility, easy to form precipitation, low bioavailability, and the taste is not suitable for consumers to use for a long time.

[0005] Therefore, developing an extraction method with mild process conditions, high efficiency, low cost, and the ability to effectively retain the active ingredients of eggshell membrane is an urgent problem to be solved in this field. Summary of the invention

[0006] In view of the above-mentioned problems existing in the existing methods for preparing active ingredients of eggshell membranes, the present invention provides a method for extracting an eggshell membrane active ingredient composition for improving bone and joint health and its application. The method adopts low-temperature physical extraction, high-efficiency membrane separation and enzymatic hydrolysis to efficiently extract active ingredients from eggshell membranes. The process conditions are mild, efficient and low-cost, thus overcoming the technical difficulties existing in the prior art.

[0007] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical solutions: In a first aspect, the present invention provides a method for extracting an eggshell membrane active ingredient composition for improving bone and joint health, comprising the following steps: S1. Raw material preparation: removing the eggshells of fresh poultry eggs, washing the shelled eggshell membranes with water and preliminarily removing impurities to obtain crude eggshell membranes; S2, low temperature physical extraction: using pure water or NaCl solution as solvent, placing the crude eggshell membrane in an environment of 5°C to 15°C for low temperature extraction to obtain an extract; S3, separation and purification: firstly perform preliminary separation on the extract using an ultrafiltration membrane to obtain a first filtrate, then add a composite enzyme to the first filtrate for enzymolysis to obtain an enzymolysis solution, and finally further filter the enzymolysis solution using a fine filtration membrane to obtain a second filtrate; S4, micronization treatment: micronizing the second filtrate to obtain the eggshell membrane active ingredient composition.

[0008] Compared with the prior art, the extraction method provided by the present invention has at least the following beneficial effects: 1. Low-temperature physical extraction: The present invention adopts a low-temperature physical extraction method, in a low-temperature environment of 5°C to 15°C, using pure water or NaCl solution as a solvent to efficiently extract active ingredients from eggshell membranes. This method can effectively avoid the use of high temperature or chemical reagents, and retain the natural components in the eggshell membranes, such as collagen, hyaluronic acid, elastin and glycosaminoglycans, to the maximum extent, ensuring the biological activity and safety of the product.

[0009] 2. High-efficiency separation and purification technology: The present invention combines optimized membrane separation technology with enzymatic treatment to efficiently separate impurities in eggshell membranes, ensuring that the purity of the final extract reaches more than 90%, effectively improving the quality and stability of the product.

[0010] 3. Improved bioavailability: The present invention also combines micronization technology to ultra-finely powder the active ingredients of eggshell membranes, making them easier to be absorbed by the human body, significantly improving their bioavailability and efficacy absorption rate. Through this treatment, ingredients such as collagen can more efficiently play a role in human joints, thereby improving the dissolution rate and absorption efficiency of the eggshell membrane active ingredient composition in the digestive tract.

[0011] 4. The present invention adopts low-temperature physical extraction, high-efficiency membrane separation and enzymatic treatment technology to produce a high-purity, high-activity, low-cost eggshell membrane active ingredient composition, which has broad market application prospects, especially in the fields of joint health, bone strengthening and sports health care.

[0012] In one embodiment, in step S1, the fresh poultry eggs come from a healthy farm and the egg-laying time is within 24 hours.

[0013] In order to ensure that the eggshell membrane is not contaminated by external substances, eggs are selected from healthy farms. Healthy farms have strict environmental management and the poultry and livestock are healthy and disease-free. After the eggs are laid, they will go through a professional screening process. Those with surface defects or suspected contamination will be removed first. When handling these eggs, the staff wears disinfected work clothes and gloves to avoid bringing external germs to the eggs.

[0014] In order to prevent the eggshell membrane from drying out or deteriorating, fresh eggs are preferred, preferably those laid within 24 hours, including chicken eggs, duck eggs, goose eggs, etc. During transportation, the eggs are placed in special containers, and the temperature and humidity inside the containers are maintained to maintain the original state of the eggshell membrane to the greatest extent. After arriving at the special processing workshop, the eggs must undergo multiple testing procedures, from the integrity of the eggshell to the state of the eggshell membrane, to ensure the integrity of the eggshell membrane. Only eggs that fully meet the standards will be used for further eggshell membrane extraction and other related operations, so as to ensure that the eggshell membrane obtained from these eggs is pure and pollution-free, providing high-quality raw materials for subsequent research and development or production applications.

[0015] In one embodiment, in step S1, the water temperature for washing the eggshell membranes is controlled between 10°C and 15°C.

[0016] The shelled eggshell membrane is rinsed with clean water to remove impurities on the surface of the eggshell membrane, such as egg white residue. The water temperature is controlled between 10°C and 15°C to avoid high temperature damaging the natural structure of the eggshell membrane. After washing, the impurities in the eggshell membrane are further removed by meticulous manual or mechanical screening to ensure the purity of the extract.

[0017] In one embodiment, in step S2, the mass concentration of the NaCl solution is 0.1%~0.5%.

[0018] During the low-temperature physical extraction process, low-concentration salt water or pure water is usually used as a solvent to prevent the active ingredients in the eggshell membrane from being destroyed.

[0019] In one embodiment, in step S2, low-temperature extraction is performed by cold pressing or soaking.

[0020] Specifically, the pressure of cold pressing and low-temperature extraction is set between 2MPa and 5MPa, and the time is 20min to 30min; the time of immersion and low-temperature extraction is 6h to 8h.

[0021] The present invention uses cold pressing or soaking for low-temperature extraction. The control of temperature is very critical and should not exceed 15°C, so as to avoid destroying active ingredients such as natural collagen, and can retain the biological activity of the active ingredients to the greatest extent, which is conducive to subsequent efficient extraction. In addition, by accurately controlling the cold pressing low-temperature extraction pressure and extraction time, the target component can be extracted more effectively. The concentration of the extract in the extract (mass percentage of the extracted solids) is 5% to 10%. If the concentration is too low, the active ingredient is not completely extracted; if the concentration is too high, it may affect the subsequent purification process.

[0022] In one embodiment, in step S3, the molecular weight cutoff of the ultrafiltration membrane is 5 kDa~10 kDa.

[0023] In one embodiment, in step S3, the pore size of the fine filtration membrane is 0.2 μm to 0.5 μm.

[0024] In one embodiment, in step S3, the complex enzyme includes collagenase and hyaluronidase.

[0025] Preferably, the added amount of the complex enzyme is 0.5%-3% of the mass of the first filtrate, wherein the mass ratio of the collagenase to the hyaluronidase is (0.2-1.5):1.

[0026] In one embodiment, in step S3, the temperature of the enzymatic hydrolysis treatment is controlled at 30°C to 40°C, the time is 3h to 5h, and the pH is 5.0 to 8.0.

[0027] During the separation and purification process, an ultrafiltration membrane with a molecular weight cutoff of 5kDa~10kDa is first used for preliminary separation. This pore size is suitable for filtering out larger molecular impurities, such as calcium and protein, and retaining the effective active ingredients in the eggshell membrane. Then, collagenase and hyaluronidase are used for enzymatic hydrolysis at 30℃~40℃ and pH 5.0~8.0 for 3h~5h to further release the active ingredients. Finally, a fine filter membrane with a pore size of 0.2μm~0.5μm is used for fine filtration to remove particulate impurities, thereby obtaining a relatively pure filtrate containing active ingredients. The purity of the eggshell membrane extract in the second filtrate reaches more than 90%, and the target components are collagen, hyaluronic acid, elastin and glycosaminoglycans.

[0028] Compared with trypsin and neutral protease used in the prior art, the collagenase of the present invention can specifically degrade the triple helix structure in the eggshell membrane and improve the release efficiency of collagen, while hyaluronidase can efficiently decompose the macromolecular hyaluronic acid, making it low molecular weight and enhancing its bioavailability. The combined use of the two enzymes can significantly improve the extraction rate, purity and biological activity of the active ingredients, while reducing by-products and optimizing the functional properties of the final product.

[0029] The amount of the compound enzyme added is 0.5% to 3% of the mass of the first filtrate. Within this range, the enzymatic reaction can be effectively carried out. At the same time, the mass ratio of collagenase to hyaluronidase is (0.2 to 1.5): 1. The pH value during the enzymatic treatment also needs to be controlled within a suitable range. Different pH values ​​will affect the activity of the enzyme, and thus affect the enzymatic effect. Generally speaking, it needs to be determined according to the optimal pH range of the activity of the compound enzyme to ensure that the active ingredients in the eggshell membrane are released to the maximum extent during the enzymatic treatment process at 30°C to 40°C and 3h to 5h. The present invention is limited to pH 5.0 to 8.0, preferably pH 6.5 to 8.0.

[0030] In one embodiment, in step S4, spray drying, ball milling or supercritical fluid drying is used for micronization.

[0031] Specifically, the experimental parameters for the micronization treatment using the spray drying method are: temperature 40°C~60°C, spray rate 5mL / min~10mL / min, and the particle size of the eggshell membrane active ingredient composition obtained is 50μm~100μm; the experimental parameters for the micronization treatment using the ball milling method are: rotation speed 200rpm~500rpm, time 1h~3h, and the particle size of the eggshell membrane active ingredient composition obtained is 10μm~50μm; the experimental parameters for the micronization treatment using the supercritical fluid drying method are: pressure 10MPa~30MPa, temperature 35°C~50°C, and the particle size of the eggshell membrane active ingredient composition obtained is 5μm~20μm.

[0032] The eggshell membrane active ingredient composition obtained by traditional extraction methods has poor solubility and is prone to precipitation, which affects taste and absorption. After micronization, the powder particles become smaller, the specific surface area increases, and the surface energy also increases, making it easier to disperse in water or other liquids, improving solubility, stability and taste, making it more suitable for use in beverages, granules and other product forms. In addition, eggshell membrane extracts mainly contain macromolecular substances such as collagen, hyaluronic acid, and elastin, which are usually difficult to be directly absorbed by the human body. Through micronization (such as ball milling, spray drying or supercritical fluid drying), the particle size of the eggshell membrane extract can be reduced to 5μm~100μm, increasing the specific surface area, thereby improving its dissolution rate and absorption efficiency in the digestive tract.

[0033] In a second aspect, the present invention provides an eggshell membrane active ingredient composition prepared by the above-mentioned extraction method of the eggshell membrane active ingredient composition for improving bone and joint health.

[0034] In a third aspect, the present invention provides use of the eggshell membrane active ingredient composition in the preparation of a composition for improving bone and joint health.

[0035] The eggshell membrane active ingredient composition extracted by the process contains natural collagen, hyaluronic acid and glucosamine, all of which can help repair joint cartilage, enhance joint lubrication, reduce joint friction, and relieve joint pain, and can be used to prepare a composition for improving bone and joint health.

[0036] In one embodiment, flavors and sweeteners are added to the eggshell membrane active ingredient composition to prepare the composition for improving bone and joint health; the flavors include peppermint essential oil, lemon essential oil, and orange peel essential oil; and the sweeteners include erythritol and stevia extract.

[0037] Preferably, the added amount of the flavor is 0.1% to 1.0% by weight of the eggshell membrane active ingredient composition, and the added amount of the sweetener is 0.2% to 1.0% by weight of the eggshell membrane active ingredient composition.

[0038] To improve the taste of the eggshell membrane active ingredient composition, an appropriate amount of natural flavors (such as peppermint essential oil, lemon essential oil, orange peel essential oil, etc.) and natural sweeteners (such as erythritol, stevia extract) can be added to it to make the product taste fresher without affecting the effect of the active ingredients. The amount of flavor added is generally 0.1%~1.0%, and the amount of sweetener added is 0.2%~1.0%, which is usually adjusted according to the sweetness requirements of the final product.

[0039] Through taste improvement technology, the final product tastes fresh, is more easily accepted by consumers, and meets market demand.

[0040] In one embodiment, the composition for improving bone and joint health is in the form of powder, capsule, oral liquid or granules.

[0041] Powder: The extracted eggshell membrane active ingredient composition is in powder form, which is directly packaged as a composition product for improving bone and joint health, which can be dissolved in water or drinks, making it convenient for consumers to take. The solubility of the powder in water is more than 90%.

[0042] Capsules: Highly transparent plant capsules are used to fill the extracted eggshell membrane active ingredient composition, which is convenient for taking according to dosage. For example, each capsule contains 250mg~500mg of the eggshell membrane active ingredient composition.

[0043] Oral liquid: After the extracted eggshell membrane active ingredient composition is concentrated, it is mixed with liquids such as water or juice to form a liquid product. The liquid product can be packaged in glass bottles or PET bottles. For example: the capacity of each bottle is 100mL~500mL.

[0044] Granules: The extracted eggshell membrane active ingredient composition and conventional excipients are subjected to conventional granulation treatment in the art to form a granular product that is easy to carry. For example, each granular product contains 50mg~100mg of active ingredients, which is suitable for consumers of different ages.

[0045] The eggshell membrane active ingredient composition of the present invention can be adapted to a variety of product forms, such as powder, capsule, liquid and the like, and can provide different ways of taking according to the preferences and needs of consumers. The eggshell membrane active ingredient composition of the present invention and the composition for improving bone and joint health prepared therefrom are not only suitable for bone and joint health care of the elderly population, but also suitable for athletes, teenagers and middle-aged populations, helping to alleviate sports injuries and maintain the health of bone and joints. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 : Evaluation curve of dog lameness degree in the test example of the present invention.

[0047] Figure 2 : Evaluation curve of knee joint pain in dogs in the test example of the present invention.

[0048] Figure 3 : Evaluation curve of hip joint pain in dogs in the test example of the present invention.

[0049] Figure 4 : Evaluation curve of dog weight-bearing degree in the test example of the present invention. DETAILED DESCRIPTION

[0050] The present invention is further described below in conjunction with specific embodiments. It should be noted that the following embodiments are only used to illustrate the present invention rather than to limit the technical solutions described in the present invention, and all technical solutions and improvements that do not depart from the present invention should be included in the scope of the claims of the present invention.

[0051] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of the features. In the description of the invention, the meaning of "multiple" is at least two, such as two, three, etc., unless otherwise clearly and specifically defined. In the description of the present invention, the meaning of "several" is at least one, such as one, two, etc., unless otherwise clearly and specifically defined.

[0052] The words "preferably", "more preferably", etc. in the present invention refer to embodiments of the present invention that may provide certain beneficial effects in certain circumstances. However, other embodiments may also be preferred under the same circumstances or other circumstances. In addition, the description of one or more preferred embodiments does not imply that other embodiments are not applicable, nor is it intended to exclude other embodiments from the scope of the present invention.

[0053] When a numerical range is disclosed herein, the above range is considered to be continuous and includes the minimum and maximum values ​​of the range, as well as every value between such minimum and maximum values. Further, when a range refers to an integer, every integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be combined. In other words, unless otherwise indicated, all ranges disclosed herein should be understood to include any and all subranges included therein.

[0054] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0055] The following are specific examples. Unless otherwise specified, the raw materials used in the examples are all commercially available products.

[0056] The embodiment of the present invention provides a method for extracting an eggshell membrane active ingredient composition, comprising the following steps: S1. Raw material preparation: fresh poultry eggs from healthy farms are selected, and the egg-laying time is within 24 hours; the eggshells of fresh poultry eggs are removed, and the eggshell membranes after shelling are washed with clean water to remove surface impurities. The water temperature is controlled between 10°C and 15°C, so as to obtain crude eggshell membranes.

[0057] S2. Low-temperature physical extraction: Using pure water or a NaCl solution with a mass concentration of 0.1% to 0.5% as a solvent, the crude eggshell membrane is placed in an environment of 5°C to 15°C, and low-temperature extraction is performed by cold pressing (the pressure is set between 2MPa and 5MPa, and the time is 20min to 30min) or soaking (immersion time is 6h to 8h) to obtain an extract, and the concentration of the extract in the extract is 5% to 10%.

[0058] S3, separation and purification: firstly, the extract is preliminarily separated by an ultrafiltration membrane with a molecular weight cutoff of 5kDa~10kDa, the macromolecular impurities are filtered out, and the main components of the eggshell membrane are retained to obtain a first filtrate; then, collagenase and hyaluronidase are added to the first filtrate, and enzymatic hydrolysis is carried out for 3h~5h in an environment of 30℃~40℃ and pH 5.0~8.0 to obtain an enzymatic solution; the amount of the complex enzyme added is 0.5%~3% of the mass of the first filtrate, and the mass ratio of collagenase to hyaluronidase is (0.2~1.5):1; finally, the enzymatic solution is further filtered by a fine filter membrane with a pore size of 0.2μm~0.5μm to remove particulate impurities to obtain a second filtrate, and the purity of the eggshell membrane extract in the second filtrate reaches more than 90%.

[0059] S4. Micro-particle treatment: The second filtrate is micro-particle treated by spray drying (temperature controlled at 40°C~60°C, spray rate of 5mL / min~10mL / min), supercritical fluid drying (pressure 10MPa~30MPa, temperature 35°C~50°C) or ball milling (rotation speed 200rpm~500rpm, time 1h~3h) to obtain an eggshell membrane active ingredient composition with a particle size of 5μm~100μm.

[0060] Flavors and sweeteners are added to the eggshell membrane active ingredient composition to prepare a composition for improving bone and joint health. The flavors include peppermint essential oil, lemon essential oil, and orange peel essential oil; the sweeteners include erythritol and stevia extract. The amount of the flavor added is 0.1% to 1.0% of the mass of the eggshell membrane active ingredient composition, and the amount of the sweetener added is 0.2% to 1.0% of the mass of the eggshell membrane active ingredient composition. Example

[0061] This embodiment provides a method for extracting an eggshell membrane active ingredient composition, comprising the following steps: S1. Raw material preparation: fresh eggs from healthy farms are selected, and the egg-laying time is within 24 hours; the eggshells of the fresh eggs are removed, and the eggshell membranes after shelling are washed with clean water to remove surface impurities, and the water temperature is controlled at 10°C, thereby obtaining crude eggshell membranes.

[0062] S2. Low-temperature physical extraction: Using pure water as solvent, the crude eggshell membrane was placed in an environment of 5°C and subjected to low-temperature extraction by cold pressing (the pressure was set at 2 MPa for 30 min) to obtain an extract having an extract concentration of 10%.

[0063] S3, separation and purification: first, the extract is preliminarily separated by an ultrafiltration membrane with a molecular weight cutoff of 5 kDa to filter out macromolecular impurities and retain the main components of the eggshell membrane to obtain a first filtrate; then, collagenase and hyaluronidase are added to the first filtrate, and enzymatic hydrolysis is performed for 5 hours at 30° C. and pH 5.0 to obtain an enzymatic hydrolyzate; the amount of the complex enzyme added is 0.5% of the mass of the first filtrate, and the mass ratio of collagenase to hyaluronidase is 0.2:1; finally, the enzymatic hydrolyzate is further filtered with a fine filtration membrane with a pore size of 0.2 μm to remove particulate impurities to obtain a second filtrate, and the purity of the eggshell membrane extract in the second filtrate reaches more than 90%.

[0064] S4. Micro-particle treatment: The second filtrate was micro-particle treated by spray drying (temperature: 40° C., spray rate: 10 mL / min) to obtain an eggshell membrane active ingredient composition having a particle size of 50 μm.

[0065] An appropriate amount of peppermint essential oil and erythritol are added to a powdered eggshell membrane active ingredient composition, and the composition for improving bone and joint health is directly vacuum-packaged; wherein the amount of peppermint essential oil added is 0.1% of the mass of the eggshell membrane active ingredient composition, and the amount of erythritol added is 1.0% of the mass of the eggshell membrane active ingredient composition. Example

[0066] This embodiment provides a method for extracting an eggshell membrane active ingredient composition, comprising the following steps: S1. Raw material preparation: fresh eggs from healthy farms are selected, and the egg-laying time is within 24 hours; the eggshells of the fresh eggs are removed, and the eggshell membranes after shelling are washed with clean water to remove surface impurities, and the water temperature is controlled at 12°C, thereby obtaining crude eggshell membranes.

[0067] S2. Low-temperature physical extraction: Using a 0.1% mass concentration of NaCl solution as a solvent, the crude eggshell membrane was placed in an environment of 10°C and subjected to low-temperature extraction by cold pressing (the pressure was set at 3 MPa for 24 min) to obtain an extract having an extract concentration of 8%.

[0068] S3, separation and purification: first, the extract is preliminarily separated by an ultrafiltration membrane with a molecular weight cutoff of 7 kDa to filter out macromolecular impurities and retain the main components of the eggshell membrane to obtain a first filtrate; then, collagenase and hyaluronidase are added to the first filtrate, and enzymatic hydrolysis is carried out for 3.5 hours at 38° C. and pH 6.5 to obtain an enzymatic solution; the amount of the complex enzyme added is 1.2% of the mass of the first filtrate, and the mass ratio of collagenase to hyaluronidase is 0.6:1; finally, the enzymatic solution is further filtered with a fine filter membrane with a pore size of 0.3 μm to remove particulate impurities to obtain a second filtrate, and the purity of the eggshell membrane extract in the second filtrate reaches more than 90%.

[0069] S4. Micro-particle treatment: The second filtrate was micro-particle treated by supercritical fluid drying (temperature: 45° C., pressure: 15 MPa) to obtain an eggshell membrane active ingredient composition having a particle size of 18 μm.

[0070] An appropriate amount of orange peel essential oil and erythritol are added to a powdered eggshell membrane active ingredient composition, and a high-transparency plant capsule is used to fill the eggshell membrane active ingredient composition with added flavors and sweeteners to prepare a composition for improving bone and joint health, wherein each capsule contains 250 mg of the eggshell membrane active ingredient composition; wherein the amount of orange peel essential oil added is 0.6% of the mass of the eggshell membrane active ingredient composition, and the amount of erythritol added is 0.4% of the mass of the eggshell membrane active ingredient composition. Example

[0071] This embodiment provides a method for extracting an eggshell membrane active ingredient composition, comprising the following steps: S1. Raw material preparation: fresh duck eggs from healthy farms are selected, and the egg-laying time is within 24 hours; the eggshells of the fresh duck eggs are removed, and the shell membranes after shelling are washed with clean water to remove surface impurities, and the water temperature is controlled at 15°C, thereby obtaining crude eggshell membranes.

[0072] S2. Low-temperature physical extraction: Using a 0.2% mass concentration of NaCl solution as a solvent, the crude eggshell membrane is placed in an environment of 15°C and subjected to low-temperature extraction by cold pressing (the pressure is set at 5 MPa for 20 min) to obtain an extract having an extract concentration of 10%.

[0073] S3, separation and purification: first, the extract is preliminarily separated by an ultrafiltration membrane with a molecular weight cutoff of 10 kDa to filter out macromolecular impurities and retain the main components of the eggshell membrane to obtain a first filtrate; then, collagenase and hyaluronidase are added to the first filtrate, and enzymatic hydrolysis is performed for 3 hours at 40° C. and pH 7.5 to obtain an enzymatic solution; the amount of the complex enzyme added is 3% of the mass of the first filtrate, and the mass ratio of collagenase to hyaluronidase is 1.2:1; finally, the enzymatic solution is further filtered with a fine filter membrane with a pore size of 0.5 μm to remove particulate impurities to obtain a second filtrate, and the purity of the eggshell membrane extract in the second filtrate reaches more than 90%.

[0074] S4. Micro-particle treatment: The second filtrate was micro-particle treated by supercritical fluid drying (temperature: 35° C., pressure: 28 MPa) to obtain an eggshell membrane active ingredient composition having a particle size of 5 μm.

[0075] An appropriate amount of lemon essential oil and stevia extract is added to the powdered eggshell membrane active ingredient composition, mixed with fruit juice, and packaged in glass bottles, each bottle having a capacity of 100 mL; wherein the added amount of lemon essential oil is 1.0% of the mass of the eggshell membrane active ingredient composition, and the added amount of stevia extract is 0.2% of the mass of the eggshell membrane active ingredient composition. Example

[0076] This embodiment provides a method for extracting an eggshell membrane active ingredient composition, comprising the following steps: S1. Raw material preparation: fresh goose eggs from healthy farms are selected, and the egg-laying time is within 24 hours; the eggshells of the fresh goose eggs are removed, and the eggshell membranes after shelling are washed with clean water to remove surface impurities, and the water temperature is controlled at 10°C, thereby obtaining crude eggshell membranes.

[0077] S2. Low-temperature physical extraction: Using a 0.5% mass concentration of NaCl solution as a solvent, the crude eggshell membrane was placed in an environment of 10°C and subjected to low-temperature extraction by immersion (immersion time 6 hours) to obtain an extract having an extract concentration of 5%.

[0078] S3, separation and purification: first, the extract is preliminarily separated by an ultrafiltration membrane with a molecular weight cutoff of 10 kDa to filter out macromolecular impurities and retain the main components of the eggshell membrane to obtain a first filtrate; then, collagenase and hyaluronidase are added to the first filtrate, and enzymatic hydrolysis is performed at 30° C. and pH 8.0 for 4 hours to obtain an enzymatic solution; the amount of the complex enzyme added is 1.5% of the mass of the first filtrate, and the mass ratio of collagenase to hyaluronidase is 1.5:1; finally, the enzymatic solution is further filtered with a fine filter membrane with a pore size of 0.2 μm to remove particulate impurities to obtain a second filtrate, and the purity of the eggshell membrane extract in the second filtrate reaches more than 90%.

[0079] S4. Micro-particle treatment: The second filtrate was micro-particle treated by ball milling (rotation speed 300 rpm) for 1.5 hours to obtain an eggshell membrane active ingredient composition with a particle size of 20 μm.

[0080] An appropriate amount of orange peel essential oil and erythritol are added to a powdered eggshell membrane active ingredient composition, and the mixture is granulated to prepare a granular product that is easy to carry. Each granular product contains 100 mg of active ingredients. The amount of orange peel essential oil added is 0.3% of the mass of the eggshell membrane active ingredient composition, and the amount of erythritol added is 0.8% of the mass of the eggshell membrane active ingredient composition. Comparative Example 1

[0081] The method of Comparative Example 1 is substantially the same as that of Example 2, except that in step S2, the crude eggshell membrane is treated with an organic acid. Comparative Example 2

[0082] The method of this comparative example 2 is basically the same as that of Example 2, except that in step S2, the extraction temperature is 50°C. Comparative Example 3

[0083] The method of Comparative Example 3 is basically the same as that of Example 2, except that in step S3, the enzyme added in the enzymatic treatment is collagenase. Comparative Example 4

[0084] The method of Comparative Example 4 is basically the same as that of Example 2, except that in step S3, the enzyme added in the enzymatic treatment is hyaluronidase. Comparative Example 5

[0085] The method of this comparative example 5 is basically the same as that of Example 2, except that in step S3, the enzymes added for the enzymatic treatment are trypsin and neutral protease. Comparative Example 6

[0086] The method of Comparative Example 6 is basically the same as that of Example 2, except that in step S4, no micronization process is performed.

[0087] The contents of active ingredients in the eggshell membranes obtained by the preparation processes of Examples 1 to 4 of the present invention and Comparative Examples 1 to 6 are shown in Table 1.

[0088] Test example

[0089] The eggshell membrane active ingredient composition prepared in the embodiment of the present invention is verified to have the effect of repairing articular cartilage, enhancing joint lubrication, reducing joint friction, and relieving joint pain.

[0090] During the four-week (28-day) trial period, 20 adult pet dogs (weight 5.82±0.36kg) were randomly divided into two groups, with 10 pet dogs in each group. One group (experimental group) was fed with basal diet and supplemented with 180mg / day or more eggshell membrane capsules, and the other group (control group) was only fed with basal diet. The degree of lameness, knee pain, hip pain, knee joint activity angle and weight-bearing degree of the dogs were evaluated after one, two, three and four weeks of feeding, respectively. See Table 5. The specific evaluation criteria are as follows: Evaluation of dog lameness: The dogs were observed to walk in a natural state and the degree of lameness was scored according to the evaluation criteria shown in Table 2. The dog lameness evaluation curve is shown in Figure 1 shown.

[0091] Table 2 Evaluation criteria for dog lameness score Walking status 1 Normal walking 2 Slight limp when walking, normal posture 3 Moderate limp when walking, normal posture 4 Severe limp and abnormal posture when walking 5 Reluctance to walk, no more than 5 steps Evaluation of knee and hip joint pain in dogs: Each dog was carefully palpated by a professional at the knee and hip joints, and pain was assessed according to the evaluation criteria shown in Table 3. The evaluation curves of knee and hip joint pain in dogs are as follows: Figure 2 , 3 shown.

[0092] Table 3 Evaluation criteria for knee and hip pain in dogs score state 0 No pain 1 Mild pain: Turning head, slight reluctance to palpation 2 Moderate pain: The patient will withdraw the limb being palpated. The patient will show obvious discomfort and slight whimpering. 3 Severe pain: biting or hissing, no physical contact allowed Evaluation of the dog's weight-bearing capacity: The dogs stand in a natural state, and the balance of the dogs when standing is observed. The dogs in each group are scored according to the evaluation criteria shown in Table 4. The dog weight-bearing capacity evaluation curve is shown in Table 4. Figure 4 shown.

[0093] Table 4 Evaluation criteria for dog weight bearing capacity score Balance 1 normal 2 Slightly unbalanced 3 Moderate imbalance 4 Serious imbalance 5 Extreme imbalance Evaluation of the dog's knee joint motion angle: Measure the dog's knee flexion and extension range. A knee flexion and extension range of 120°~140° is normal, and less than 120° indicates limited motion.

[0094] Table 5 Effect of the eggshell membrane active ingredient composition on subjective evaluation of joints Experimental Group Control group P-value Lameness degree (7 days) 1.80±0.79 2.10±0.74 0.391 Lameness degree (14 days) 1.70±0.67 2.10±0.74 0.222 Lameness degree (21 days) 1.20±0.92 2.00±0.82 0.054 Lameness degree (28 days) 0.90±1.10 2.00±0.82 0.021 Knee pain evaluation (7 days) 1.40±0.52 1.50±1.18 0.809 Knee pain evaluation (14 days) 1.30±0.67 1.50±1.18 0.647 Knee pain evaluation (21 days) 1.00±0.82 1.40±1.26 0.412 Knee pain evaluation (28 days) 0.80±0.92 1.40±1.26 0.241 Hip pain evaluation (7 days) 1.00±1.25 1.50±1.18 0.369 Hip pain evaluation (14 days) 1.00±1.25 1.60±1.07 0.264 Hip pain evaluation (21 days) 0.90±1.29 1.50±1.18 0.291 Hip pain evaluation (28 days) 0.70±1.16 1.50±1.18 0.143 Knee joint range of motion / °(7 days) 128.40±17.39 127.30±18.35 0.892 Knee joint range of motion / °(14 days) 124.40±17.51 125.90±18.19 0.853 Knee joint range of motion / °(21 days) 122.40±15.04 127.00±17.19 0.532 Knee joint range of motion / °(28 days) 122.40±13.20 127.00±17.19 0.511 Weight bearing level (7 days) 1.40±0.97 1.60±0.97 0.649 Weight bearing level (14 days) 1.30±1.06 1.60±0.97 0.517 Weight bearing level (21 days) 1.00±1.15 1.60±0.97 0.224 Weight bearing level (28 days) 0.70±1.25 1.60±0.97 0.089 According to the above data combined Figures 1 to 4 It can be seen that compared with dogs fed only with basic food, dogs fed with the eggshell membrane active ingredient composition had reduced joint pain and were healthier overall, indicating that the eggshell membrane active ingredient composition produced by this process can reduce the degree of lameness and joint pain in pet dogs, increase the weight-bearing capacity of pet dogs, and significantly improve bone and joint health.

[0095] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0096] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modification, equivalent substitution or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for extracting an eggshell membrane active ingredient composition for improving bone and joint health, characterized in that: The following steps are involved: S1. Raw material preparation: removing the eggshells of fresh poultry eggs, washing the shelled eggshell membranes with water and preliminarily removing impurities to obtain crude eggshell membranes; S2, low temperature physical extraction: using pure water or NaCl solution as solvent, placing the crude eggshell membrane in an environment of 5°C to 15°C for low temperature extraction to obtain an extract; S3, separation and purification: firstly perform preliminary separation on the extract using an ultrafiltration membrane to obtain a first filtrate, then add a composite enzyme to the first filtrate for enzymolysis to obtain an enzymolysis solution, and finally further filter the enzymolysis solution using a fine filtration membrane to obtain a second filtrate; S4, micronization treatment: micronizing the second filtrate to obtain the eggshell membrane active ingredient composition.

2. The method for extracting the eggshell membrane active ingredient composition for improving bone and joint health according to claim 1, characterized in that: In step S1, the fresh poultry eggs are from healthy farms and the egg-laying time is within 24 hours; and / or In step S1, the water temperature for washing the eggshell membranes is controlled between 10°C and 15°C; and / or In step S2, the mass concentration of the NaCl solution is 0.1% to 0.5%; and / or In step S2, low-temperature extraction is performed by cold pressing or soaking.

3. The method for extracting the eggshell membrane active ingredient composition for improving bone and joint health according to claim 2, characterized in that: In step S2, the pressure of cold pressing and low temperature extraction is set between 2MPa and 5MPa, and the time is 20min to 30min; or In step S2, the soaking and low-temperature extraction time is 6h~8h.

4. The method for extracting the eggshell membrane active ingredient composition for improving bone and joint health according to claim 1, characterized in that: In step S3, the molecular weight cut-off of the ultrafiltration membrane is 5 kDa to 10 kDa; and / or In step S3, the complex enzyme includes collagenase and hyaluronidase; and / or In step S3, the temperature of the enzymatic hydrolysis treatment is controlled at 30°C to 40°C, the time is 3h to 5h, and the pH is 5.0 to 8.0; and / or In step S3, the pore size of the fine filtration membrane is 0.2 μm to 0.5 μm; and / or In step S4, spray drying, ball milling or supercritical fluid drying is used to perform micronization.

5. The method for extracting the eggshell membrane active ingredient composition for improving bone and joint health according to claim 4, characterized in that: In step S3, the amount of the complex enzyme added is 0.5% to 3% of the mass of the first filtrate; and / or In step S3, the mass ratio of the collagenase to the hyaluronidase is (0.2-1.5):

1.

6. The method for extracting the eggshell membrane active ingredient composition for improving bone and joint health according to claim 4, characterized in that: In step S4, the experimental parameters of the micronization treatment by spray drying are: temperature 40°C to 60°C, spray rate 5mL / min to 10mL / min, and the particle size of the prepared eggshell membrane active ingredient composition is 50μm to 100μm; or In step S4, the experimental parameters of the micronization treatment by ball milling are: rotation speed 200 rpm~500 rpm, time 1h~3h, and the particle size of the prepared eggshell membrane active ingredient composition is 10μm~50μm; or In step S4, the experimental parameters of the micronization treatment using the supercritical fluid drying method are: pressure 10MPa~30MPa, temperature 35°C~50°C, and the particle size of the prepared eggshell membrane active ingredient composition is 5μm~20μm.

7. An eggshell membrane active ingredient composition prepared by the method for extracting an eggshell membrane active ingredient composition for improving bone and joint health according to any one of claims 1 to 6.

8. Use of the eggshell membrane active ingredient composition as claimed in claim 7 in the preparation of a composition for improving bone and joint health.

9. The use according to claim 8, characterized in that: Flavors and sweeteners are added to the eggshell membrane active ingredient composition; the flavors include peppermint essential oil, lemon essential oil, and orange peel essential oil; and the sweeteners include erythritol and stevia extract.

10. The use according to claim 8 or 9, characterized in that: The composition for improving bone and joint health is in the form of powder, capsule, oral liquid or granule.

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