N-acetylneuraminic acid tablet and preparation method thereof

Abstract

The invention provides an N-acetylneuraminic acid tablet and a preparation method thereof. The N-acetylneuraminic acid tablet is prepared from chitosan-neuraminic acid composite powder, vitamin C, vitamin E, sodium hyaluronate, microcrystalline cellulose, lactose, sorbitol, magnesium stearate, silicon dioxide, inulin and natural essence. The chitosan-neuraminic acid composite powder is prepared through low-temperature freezing, freeze-drying, crushing and screening, and the N-acetylneuraminic acid is obtained through egg white enzymolysis, centrifugation, dialysis, freeze-drying and high performance liquid chromatography purification. By optimizing a formula and a process, the formability and purity of the tablet are remarkably improved, meanwhile, the tablet has good stability and functionality and is suitable for the fields of immunoregulation, oxidation resistance and the like, the problems that N-acetylneuraminic acid is poor in stability and difficult to efficiently utilize in a complex system are solved, the market demand of functional health products is met, and the market prospect is wide. And the method has wide application value.

Description

Technical Field

[0001] The invention relates to the technical field of health care products, and in particular to an N-acetylneuraminic acid tablet and a preparation method thereof. Background Art

[0002] N-acetylneuraminic acid (Neu5Ac), also known as sialic acid, is an important carbohydrate derivative widely present in organisms. It is widely used in the fields of health foods, pharmaceutical preparations and functional foods because of its significant biological activity in enhancing immune function, anti-inflammation, promoting neural development and anti-oxidation. In these application scenarios, the stability, active release, absorption efficiency and preparation processing performance of N-acetylneuraminic acid are key factors affecting its product quality and market competitiveness. For example, in tablet preparations, N-acetylneuraminic acid is required to have good chemical stability to avoid degradation during preparation, storage and administration; at the same time, tablets need to have good disintegration and controlled release properties to ensure the effective release and absorption of N-acetylneuraminic acid in the gastrointestinal tract. In addition, the mechanical strength, wear resistance and appearance quality of tablets are also important indicators that affect user experience. In practical applications, the performance optimization of N-acetylneuraminic acid tablets can not only significantly improve its bioavailability and ease of use, but also further broaden its application scope in the fields of immune regulation, anti-aging, etc., and promote technological progress and market development in related industries. Therefore, developing an N-acetylneuraminic acid tablet with high stability, high biological activity and excellent formulation performance has important practical significance for improving product performance and promoting industry upgrading.

[0003] Although N-acetylneuraminic acid has a wide range of application prospects, its stability and utilization efficiency in practical applications have always been the key problems that restrict its development. For example, the Chinese patent with publication number CN103284156A discloses a health care product composition and its application. Although the initial forming of the composition is achieved, during the storage process, N-acetylneuraminic acid is susceptible to environmental factors (such as temperature, humidity and light), resulting in degradation of its chemical structure and poor stability. In addition, the method fails to effectively solve the problem of in vivo absorption efficiency of N-acetylneuraminic acid, resulting in low bioavailability, which limits its actual effect in the field of health food and medicine. The main reason for these problems is that the hydroxyl and carboxyl groups contained in the molecular structure of N-acetylneuraminic acid have high activity and are easy to react with water molecules or oxygen in the external environment; at the same time, the existing formulation technology is insufficient in giving tablets controlled release properties and protecting the stability of active substances, and fails to form an effective barrier protection structure. Therefore, in view of the shortcomings of the prior art, it is urgent to develop a technical solution that can significantly improve the stability of N-acetylneuraminic acid, improve its bioavailability and optimize the tablet preparation performance to meet the actual needs of industry development. Summary of the invention

[0004] (1) Technical issues solved The purpose of the present invention is to provide an N-acetylneuraminic acid tablet and a preparation method thereof, so as to solve the problems of poor stability and low bioavailability of the current N-acetylneuraminic acid.

[0005] (2) Technical solution In order to achieve the above object, the present invention provides the following technical solutions: An N-acetylneuraminic acid tablet comprises the following components in parts by weight: 10-20 parts of chitosan-neuraminic acid composite powder, 2.0-5.0 parts of vitamin C, 1.0-3.0 parts of vitamin E, 5.0-10.0 parts of sodium hyaluronate, 30-50 parts of microcrystalline cellulose, 10-20 parts of lactose, 2.0-5.0 parts of sorbitol, 0.5-1.0 part of magnesium stearate, 0.5-1.0 part of silicon dioxide, 5-10 parts of inulin and 0.1-0.5 part of natural flavor.

[0006] Furthermore, the preparation method of the chitosan-neuraminic acid composite powder is as follows: by weight, N-acetylneuraminic acid gel is placed in a low-temperature freezing environment of −50~−60 °C for pre-freezing, the pre-freezing time is 120~180min, then the frozen hydrogel is placed in a freeze dryer, the vacuum degree is set to 10~20 Pa, the temperature is controlled at −50~−45 °C, and main drying is performed for 24~36 h. After the main drying is completed, the temperature of the freeze dryer plate layer is gradually increased to 25~30 °C, the vacuum degree is controlled at 10~15 Pa, and analytical drying is performed to remove residual bound water. The analytical drying time is 240~300min, and the loose porous block after freeze drying is then mechanically crushed into fine powder, sieved using an 80~100 mesh sieve, and finally the chitosan-neuraminic acid composite powder is obtained.

[0007] Further, the preparation method of the N-acetylneuraminic acid gel is as follows: first, by weight, 3.0-6.0 parts of chitosan are weighed and dissolved in 100-150 parts of 1.5 vol.% glacial acetic acid aqueous solution, stirred at a stirring rate of 400-500 rpm for 30-45 min to obtain a chitosan solution, then 10-16 parts of N-acetylneuraminic acid, 0.1-0.5 parts of transglutaminase and 1.0-3.0 parts of soy protein are added to the chitosan solution, the mixed solution is heated to 60-65°C, stirred at a stirring rate of 400-500 rpm for 30-45 min to obtain a uniform solution, then the uniform solution is subjected to ultrasonic treatment, the ultrasonic treatment power is set to 100-120 W, the time is 5-10 min, after the completion of the ultrasonic treatment, the obtained uniform solution is divided into a mold, and subjected to freeze-thaw treatment, the freeze-thaw treatment temperature is set to -20--25°C, and the freezing time is 12-20 h, and then thawed at room temperature for 8-12 h, and the freeze-thaw-thaw process was repeated 3 times to achieve the initial physical cross-linking of chitosan and N-acetylneuraminic acid, thereby forming N-acetylneuraminic acid gel.

[0008] The present invention adopts the design of chitosan-neuraminic acid composite powder mainly for enhancing the stability and functional release performance of N-acetylneuraminic acid in complex systems, and realizes a composite material with high stability, high biological activity and excellent processing performance by optimizing the preparation process and material synergy. In the preparation process, first, by the synergistic effect of chitosan, N-acetylneuraminic acid, transglutaminase and soy protein, a uniform and stable precursor solution is formed in a solution state, wherein chitosan provides good film-forming property and molecular chain structure, N-acetylneuraminic acid forms preliminary crosslinking with chitosan as an active ingredient, and the introduction of transglutaminase and soy protein further enhances the physical crosslinking strength and intermolecular force of the system. Subsequently, by freeze-thaw cycle treatment, the intermolecular physical crosslinking effect is further strengthened to form an N-acetylneuraminic acid gel with a porous structure, which can not only provide a large specific surface area to protect the active ingredient, but also lays the foundation for subsequent freeze-drying process. In the freeze-drying process, by accurately controlling the process parameters of low-temperature pre-freezing, main drying and analytical drying, the microstructural stability of the composite powder and the uniformity of the internal pores are further improved. The low-temperature pre-freezing stage makes the water in the gel form regular and uniform ice crystals, reducing the risk of pore collapse during the drying process; the main drying stage removes most of the free water by sublimation under vacuum conditions, maintaining the porous structure of the material; the analytical drying stage removes the residual bound water by gradually increasing the plate temperature, ensuring the completeness and stability of the drying of the material. Finally, by mechanical crushing and screening, a composite powder material with uniform particle size is obtained, and the porous network structure of chitosan effectively wraps and protects N-acetylneuraminic acid, significantly improving its stability during storage and processing. In addition, the interaction between chitosan and N-acetylneuraminic acid at the molecular and structural levels also gives the composite powder good sustained-release properties, and the directional release of active ingredients can be achieved in subsequent preparation processing, thereby meeting the needs of various application scenarios for biological activity and functional stability. The present invention not only improves the functionality and environmental adaptability of the material through the synergistic effect of multiple components and refined process design, but also provides a solid technical foundation for its wide application in the fields of food, medicine, etc.

[0009] Further, the preparation method of described N-acetylneuraminic acid comprises the following steps: A1. Egg white pretreatment: fresh egg white is diluted, filtered through a 200-mesh sieve, allowed to stand, and pH adjusted to obtain an egg white solution suitable for enzymatic hydrolysis; A2. Enzymatic hydrolysis reaction: the pretreated egg white solution is added with papain, and enzymatic hydrolysis is performed under suitable temperature and stirring conditions, and the enzyme activity is inactivated by heating to obtain an enzymatic solution; A3. Centrifugation and preliminary purification: The enzymatic hydrolysate is subjected to high-speed centrifugation, ethanol precipitation and filtration to obtain a preliminary purified N-acetylneuraminic acid solution; A4. Dialysis and lyophilization: The preliminarily purified solution was dialyzed through a dialysis bag and freeze-dried to obtain a crude N-acetylneuraminic acid powder; A5. HPLC purification: The crude powder is dissolved and purified by HPLC, the target component is collected and the solvent is removed by rotary evaporation to obtain N-acetylneuraminic acid powder.

[0010] Furthermore, the detailed process of step A1 is as follows: using egg white as a raw material, mixing fresh egg white and deionized water in a volume ratio of 1: (1.5-2), filtering through a 200-mesh sieve to remove impurities and unevenly distributed protein particles; placing the filtered egg white solution at 4-6 °C for 60-90 minutes, and adjusting the pH of the solution to 6.0-6.5 using 1 mol / L hydrochloric acid or 1 mol / L sodium hydroxide to obtain a pretreated egg white solution.

[0011] Furthermore, the detailed process of step A2 is as follows: placing the pretreated egg white solution in a reaction vessel, adding 0.8-1.2% volume of α-(2-3, 6, 8, 9) neuraminidase to the pretreated egg white solution, setting the stirring rate to 180-220 rpm, adjusting the reaction temperature to 45-50 ° C, and continuously stirring for 1.5-2.5 h. After the enzymatic hydrolysis reaction is completed, the reaction solution is quickly heated to 90-95 ° C and maintained for 8-12 min, and then cooled to obtain an enzymatic hydrolyzate.

[0012] Further, the detailed process of step A3 is as follows: transferring the cooled enzymatic hydrolysate to a high-speed centrifuge, centrifuging at 4-6 °C and 20000-30000 rpm for 20-25 min, and collecting the supernatant obtained by centrifugation as a crude extract of N-acetylneuraminic acid; slowly adding ethanol to the crude extract, the volume ratio of the crude extract to ethanol is 1:(3.0-5.0), the addition rate is controlled at 2-3 mL / min, and the stirring rate is maintained at 200-250 rpm until the solution is fully mixed; placing the mixed solution in an ice bath and standing for 25-35 min to precipitate protein and other high molecular impurities; then placing the solution in a high-speed centrifuge again, centrifuging at 4-6 °C and 20000-30000 rpm for 20-25 min, collecting the obtained supernatant, and filtering through a filter membrane with a pore size of 0.22-0.45 μm to remove suspended impurities to obtain a preliminarily purified N-acetylneuraminic acid solution.

[0013] Further, the detailed process of step A4 is as follows: the obtained preliminary purified solution is transferred to a dialysis bag with a molecular weight cutoff range of 3000~3500 Da, and is placed in deionized water at 4~6 ° C for dialysis. The dialysis time is 20~24 h, during which the dialysate is replaced every 3~5 h, and the volume of the dialysate replaced each time is 3 times the volume of the dialysis solution to ensure that small molecule impurities and salt ions are fully removed; after the dialysis is completed, the dialysate is transferred to a freeze dryer and freeze-dried at -45~-50 ° C for 24~30 h until the liquid is completely evaporated to obtain a crude N-acetylneuraminic acid powder.

[0014] Further, the detailed process of step A5 is as follows: dissolving the obtained crude N-acetylneuraminic acid powder in deionized water, the mass ratio of N-acetylneuraminic acid powder to deionized water is 1:(10~15), the stirring rate is controlled at 200~250rpm, and the stirring time is 15~20 min to ensure that the powder is completely dissolved; the dissolved solution is filtered through a filter membrane with a pore size of 0.22 μm and placed in a high performance liquid chromatography device for further purification; an Aminex HPX-87H chromatographic column is used, and the chromatographic conditions are set to a mobile phase concentration of 4~6 mM / L sulfuric acid, a flow rate of 0.5~0.7 mL / min, a detection wavelength of 205~215 nm, and a column temperature of 30~40 °C; the components corresponding to the target peak are collected, and the solvent is removed by a rotary evaporator at a water bath temperature of 40~50 °C, and the rotary evaporation time is 30~60 min, and N-acetylneuraminic acid powder is finally obtained.

[0015] The present invention adopts optimized multi-step preparation process design, which is mainly used to enhance the purity, stability and functionality of N-acetylneuraminic acid to meet its application requirements in biomedicine and food fields. Through the synergistic effect of egg white pretreatment and enzymolysis reaction, the efficient release of N-acetylneuraminic acid is achieved, wherein filtration, pH adjustment and specific enzymolysis effectively remove impurities and improve product selectivity. Subsequently, in combination with high-speed centrifugation, ethanol precipitation and dialysis operations, protein and small molecule impurities are gradually removed, while the chemical stability of the target molecule is maintained, and freeze drying further forms a powder with good activity retention. The present invention improves the purity and quality of N-acetylneuraminic acid through high-performance liquid chromatography purification depth, and through the synergistic optimization of each process link, ensures the high stability and high functionality of the final product, and provides reliable technical guarantee for its storage and application.

[0016] The present invention adopts a well-designed multi-component system mainly for enhancing the biostability, functionality and processing performance of N-acetylneuraminic acid tablets. Through the synergistic effect of each component, the comprehensive performance optimization of the tablets during preparation, storage and use is achieved. As the core active ingredient, chitosan-neuraminic acid composite powder has both high biological activity and excellent stability. Its combination with vitamin C and vitamin E further enhances the antioxidant capacity of the tablets, thereby effectively inhibiting the degradation of the active ingredients during storage and use. Sodium hyaluronate not only improves the additional functionality of the tablets by providing moisturizing and cell metabolism support functions, but also enhances the bioavailability and absorption efficiency of the active ingredients. Microcrystalline cellulose and lactose are the matrix components of the tablets, giving the tablets good mechanical strength and formability, while providing uniform drug distribution, ensuring the stability of dosage accuracy and tablet quality. Magnesium stearate and silicon dioxide, as lubricants and glidants, optimize the production process of the tablets and avoid the occurrence of adhesion and clogging. The addition of inulin and natural flavors not only improves the taste and user experience of the tablets, but also further enhances its market competitiveness as a functional food. The present invention achieves comprehensive improvement in functionality, stability and processing performance of N-acetylneuraminic acid tablets through reasonable matching and synergistic design of various components, and provides solid technical support for its wide application in the fields of medicine and health care.

[0017] The present invention also provides a method for preparing an N-acetylneuraminic acid tablet, comprising the following steps: S1. Put chitosan-neuraminic acid composite powder, vitamin C, vitamin E, sodium hyaluronate, microcrystalline cellulose, lactose, magnesium stearate, silicon dioxide, natural flavor and inulin into a three-dimensional mixer in sequence according to the proportion, set the mixing speed to 15-20 rpm, and the mixing time to 15-20 min to obtain a uniformly mixed dry powder.

[0018] S2. Transfer the mixed dry powder to the granulation equipment, slowly add the mixed solution of sorbitol and deionized water as a binder, control the stirring rate at 100~150 rpm, and stir for 5~10 min until uniform wet granules are formed.

[0019] S3. Transfer the wet granules to a hot air circulation drying oven, set the drying temperature to 50-60 °C, and the drying time to 2-4 h, until the moisture content of the granules is less than 3%. After drying, pass the dry granules through a 16-20 mesh sieve to obtain smooth and uniform granules.

[0020] S4. Transfer the evenly lubricated granules to the tablet press, set the tableting pressure to 15~20 kN, the tableting speed to 30~50 tablets / min, the tablet weight to 0.3~0.5g, and the tablet hardness to 60~80N. After the finished tablets pass the quality inspection, put them into aluminum foil bags or plastic bottles, and seal them for storage.

[0021] The present invention adopts the multi-step preparation method design of refinement to be mainly used for enhancing the uniformity, stability and functional release performance of N-acetylneuraminic acid tablets, and through the optimization of each stage process and the synergistic effect of components, the high quality and excellent performance of tablets are ensured. In step S1, by fully mixing the functional components such as chitosan-neuraminic acid composite powder and vitamin C, vitamin E, sodium hyaluronate with excipients such as microcrystalline cellulose and lactose in a three-dimensional mixer, a uniformly distributed dry powder base is formed, which lays a foundation for the uniform distribution of the functional components of tablets, while avoiding the separation or segregation of active ingredients. In step S2, the adhesive prepared by mixing sorbitol with deionized water not only improves the formability and bonding strength of the particles, but also avoids the degradation of the active components caused by excessive wetting, and the preparation of wet particles further optimizes the compression performance of subsequent tablets. In step S3, the wet granules are dried at low temperature by a hot air circulation drying oven, and a reasonable temperature and time are set to ensure that the moisture content of the granules is reduced to an ideal range, while avoiding the inactivation of the active ingredients or the destruction of the physical structure caused by high temperature. Subsequently, dry granules with uniform particle size are obtained by a whole granulation process, which improves the fluidity and tableting adaptability of the granules. Finally, in step S4, by setting a reasonable tableting pressure and speed, it is ensured that the hardness and weight of the tablets meet the standard requirements, and the tablets form a tight structure during the tableting process, so that the active ingredients can be quickly released and absorbed in the gastrointestinal tract. The comprehensive improvement of N-acetylneuraminic acid tablets in functionality, stability and processing performance is achieved, providing reliable technical support for its application in the fields of medicine and health food.

[0022] (3) Beneficial technical effects 1. The present invention forms a stable porous structure through the synergistic effect of chitosan, N-acetylneuraminic acid, transglutaminase and soy protein, significantly improves the stability and sustained-release performance of N-acetylneuraminic acid, and solves the problem of degradation and uneven release of active ingredients in the prior art. The optimized freeze-drying process and precise process parameter control ensure the stability of the material microstructure, improve the processing performance and adapt to a variety of application scenarios. The film-forming property and cross-linking effect of chitosan enhance the stability of the overall system, the proportion of each group is reasonable, and the synergy is better than the prior art, providing a more efficient and environmentally friendly solution for the food and pharmaceutical fields.

[0023] 2. The present invention significantly improves the purity, stability and functionality of N-acetylneuraminic acid by optimizing egg white pretreatment, enzymolysis and purification processes, achieves efficient release and impurity removal, and solves the problems of insufficient purity and reduced activity in the prior art. Combined with high-speed centrifugation, dialysis and freeze drying, the activity is maintained to the greatest extent, and the product quality is further improved by high-performance liquid chromatography. The synergistic effect of each process link ensures that the stability and functional release between components are better than the prior art, providing an efficient, safe and environmentally friendly solution for the biomedicine and food fields.

[0024] 3. The present invention not only enhances the antioxidant, moisturizing and metabolic support functions of the tablets through the synergistic effect of chitosan-neuraminic acid composite powder with vitamin C, vitamin E and sodium hyaluronate, but also significantly improves the stability and bioavailability of the active ingredients. Matrix components such as microcrystalline cellulose and lactose give the tablets good mechanical strength and uniformity, the optimized combination of lubricants improves the processing performance and release efficiency of the tablets, and inulin and natural flavors improve the user experience. Compared with the prior art, the present invention achieves comprehensive optimization of functionality, stability and processing performance, providing an efficient and reliable solution for the fields of medicine and health care.

[0025] 4. The present invention achieves a comprehensive improvement in the uniformity, stability and functional release performance of N-acetylneuraminic acid tablets by optimizing the multi-step preparation process. The chitosan-neuraminic acid composite powder works synergistically with components such as vitamin C and vitamin E to enhance the antioxidant and active stability of the tablets; the excipients and binders ensure the particle formability and fluidity, while ensuring the optimization of the tablet hardness performance through precise drying and tableting processes. The present invention solves the pain points of uneven active ingredients, degradation and poor processing adaptability in the prior art, and provides an efficient, safe and adaptable technical solution for the fields of medicine and health food. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a morphology picture of the chitosan-neuraminic acid composite powder prepared in Example 1 of the present invention.

[0027] Figure 2 This is a morphology picture of the N-acetylneuraminic acid gel prepared in Example 1 of the present invention. DETAILED DESCRIPTION

[0028] To make the purpose, technical solution and advantages of the embodiments of the present invention more clear, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention.

[0029] Example 1 An N-acetylneuraminic acid tablet comprises the following components, measured by weight: 10 parts of chitosan-neuraminic acid composite powder, 2.0 parts of vitamin C, 1.0 parts of vitamin E, 5.0 parts of sodium hyaluronate, 30 parts of microcrystalline cellulose, 10 parts of lactose, 2.0 parts of sorbitol, 0.5 parts of magnesium stearate, 0.5 parts of silicon dioxide, 5 parts of inulin and 0.1 parts of natural flavor.

[0030] The preparation method of the chitosan-neuraminic acid composite powder of the present invention comprises the following steps: placing N-acetylneuraminic acid gel in a low-temperature freezing environment of −50° C. for pre-freezing, the pre-freezing time being 120 minutes, then placing the frozen hydrogel in a freeze dryer, setting the vacuum degree to 10 Pa, controlling the temperature at −50° C., performing main drying, the main drying time being 24 hours, gradually increasing the freeze dryer plate temperature to 25° C., controlling the vacuum degree at 10 Pa, performing analytical drying to remove residual bound water, the analytical drying time being 240 minutes, and forming a loose porous block after freeze drying, then mechanically crushing the loose porous block into fine powder, sieving with an 80-mesh sieve, and finally obtaining the chitosan-neuraminic acid composite powder.

[0031] The preparation method of N-acetylneuraminic acid gel of the present invention is as follows: first, by weight, 3.0 parts of chitosan are weighed and dissolved in 100 parts of 1.5 vol.% glacial acetic acid aqueous solution, stirred at a stirring rate of 400 rpm for 30 minutes to obtain a chitosan solution, then 10 parts of N-acetylneuraminic acid, 0.1 parts of transglutaminase and 1.0 parts of soy protein are added to the chitosan solution, the mixed solution is heated to 60°C, stirred at a stirring rate of 400 rpm for 30 minutes to obtain a uniform solution, then the uniform solution is subjected to ultrasonic treatment, the ultrasonic treatment power is set to 100 W, the time is 5 minutes, after the completion of the ultrasonic treatment, the obtained uniform solution is divided into a mold, and a freeze-thaw treatment is performed, the freeze-thaw treatment temperature is set to -20°C, the freezing time is 12 hours, and then thawed at room temperature for 8 hours, and the above freeze-thaw-thaw process is repeated 3 times in total to achieve preliminary physical crosslinking of chitosan and N-acetylneuraminic acid, thereby forming N-acetylneuraminic acid gel.

[0032] The preparation method of N-acetylneuraminic acid of the present invention comprises the following steps: A1. Egg white pretreatment: fresh egg white is diluted, filtered through a 200-mesh sieve, allowed to stand, and pH adjusted to obtain an egg white solution suitable for enzymatic hydrolysis; A2. Enzymatic hydrolysis reaction: the pretreated egg white solution is added with papain, and enzymatic hydrolysis is performed under suitable temperature and stirring conditions, and the enzyme activity is inactivated by heating to obtain an enzymatic solution; A3. Centrifugation and preliminary purification: The enzymatic hydrolysate is subjected to high-speed centrifugation, ethanol precipitation and filtration to obtain a preliminary purified N-acetylneuraminic acid solution; A4. Dialysis and lyophilization: The preliminarily purified solution was dialyzed through a dialysis bag and freeze-dried to obtain a crude N-acetylneuraminic acid powder; A5. HPLC purification: The crude powder is dissolved and purified by HPLC, the target component is collected and the solvent is removed by rotary evaporation to obtain N-acetylneuraminic acid powder.

[0033] The detailed process of step A1 of the present invention is: using egg white as a raw material, mixing fresh egg white and deionized water in a volume ratio of 1:1.5, filtering through a 200-mesh sieve to remove impurities and unevenly distributed protein particles; placing the filtered egg white solution at 4 ° C for 60 minutes, and adjusting the pH of the solution to 6.0 with 1 mol / L hydrochloric acid or 1 mol / L sodium hydroxide to obtain a pretreated egg white solution.

[0034] The detailed process of step A2 of the present invention is as follows: placing the pretreated egg white solution in a reaction container, adding 0.8% volume of α-(2-3,6,8,9) neuraminidase of the pretreated egg white solution, setting the stirring rate to 180 rpm, adjusting the reaction temperature to 45 ° C, and continuously stirring for 1.5 h. After the enzymatic hydrolysis reaction is completed, the reaction solution is quickly heated to 90 ° C and maintained for 8 min, and then cooled to obtain an enzymatic hydrolyzate.

[0035] The detailed process of step A3 of the present invention is as follows: transferring the cooled enzymatic hydrolyzate to a high-speed centrifuge, centrifuging at 4 ° C and 20000 rpm for 20 min, collecting the supernatant obtained by centrifugation as a crude extract of N-acetylneuraminic acid; slowly adding ethanol to the crude extract, the volume ratio of the crude extract to ethanol is 1:3.0, the addition rate is controlled at 2 mL / min, and the stirring rate is maintained at 200 rpm until the solution is fully mixed; placing the mixed solution in an ice bath and standing for 25 min to precipitate protein and other high molecular impurities; then placing the solution in a high-speed centrifuge again, centrifuging at 4 ° C and 20000 rpm for 20 min, collecting the obtained supernatant, filtering through a filter membrane with a pore size of 0.22 μm to remove suspended impurities, and obtaining a preliminarily purified N-acetylneuraminic acid solution.

[0036] The detailed process of step A4 of the present invention is as follows: the obtained preliminary purified solution is transferred to a dialysis bag with a molecular weight cutoff range of 3000Da, and is placed in deionized water at 4 ° C for dialysis. The dialysis time is 20 h, during which the dialysate is replaced every 3 h, and the volume of the dialysate replaced each time is 3 times the volume of the dialysis solution to ensure that small molecule impurities and salt ions are fully removed; after the dialysis is completed, the dialysate is transferred to a freeze dryer and freeze-dried at -45 ° C for 24 h until the liquid is completely evaporated to obtain a crude N-acetylneuraminic acid powder.

[0037] The detailed process of step A5 of the present invention is as follows: dissolving the obtained crude N-acetylneuraminic acid powder in deionized water, wherein the mass ratio of the N-acetylneuraminic acid powder to the deionized water is 1:10, the stirring rate is controlled at 200 rpm, and the stirring time is 15 min, so as to ensure that the powder is completely dissolved; the dissolved solution is filtered through a filter membrane with a pore size of 0.22 μm and placed in a high performance liquid chromatography device for further purification; an Aminex HPX-87H chromatographic column is used, and the chromatographic conditions are set to a mobile phase concentration of 4 mM / L sulfuric acid, a flow rate of 0.5 mL / min, a detection wavelength of 205 nm, and a column temperature of 30 ° C; the components corresponding to the target peak are collected, and the solvent is removed by a rotary evaporator at a water bath temperature of 40 ° C, and the rotary evaporation time is 30 min, and finally the N-acetylneuraminic acid powder is obtained.

[0038] A method for preparing an N-acetylneuraminic acid tablet of the present invention comprises the following steps: S1. Put chitosan-neuraminic acid composite powder, vitamin C, vitamin E, sodium hyaluronate, microcrystalline cellulose, lactose, magnesium stearate, silicon dioxide, natural flavor and inulin into a three-dimensional mixer in sequence according to the proportion, set the mixing speed to 15 rpm, and the mixing time to 15 min to obtain a uniformly mixed dry powder.

[0039] S2. Transfer the mixed dry powder to the granulation equipment, slowly add the mixed solution of sorbitol and deionized water as a binder, control the stirring rate at 100 rpm, and stir for 5 min until uniform wet granules are formed.

[0040] S3. The wet granules were transferred to a hot air circulation drying oven, and the drying temperature was set to 50 °C and the drying time was set to 2 h, until the moisture content of the granules was less than 3%. After drying, the dry granules were sieved through a 16-mesh screen to obtain smooth and uniform granules.

[0041] S4. Transfer the evenly lubricated granules to the tablet press, set the tableting pressure to 15 kN, the tableting speed to 30 tablets / min, the tablet weight to 0.3g, and the tablet hardness to 60N. After the finished tablets pass the quality inspection, put them into aluminum foil bags or plastic bottles, and seal them for storage.

[0042] pass Figure 1 and Figure 2 The preparation effect of the present invention can be verified intuitively, wherein Figure 1 The morphology of chitosan-neuraminic acid composite powder is shown, indicating that chitosan successfully encapsulates N-acetylneuraminic acid to form a uniformly distributed composite structure; Figure 2 The morphology of N-acetylneuraminic acid gel was demonstrated, further proving that the preparation method of the present invention can effectively construct a gel network structure, thereby providing a solid basic support for improving stability and functionality.

[0043] Example 2 An N-acetylneuraminic acid tablet comprises the following components, measured by weight: 13 parts of chitosan-neuraminic acid composite powder, 3.0 parts of vitamin C, 2.0 parts of vitamin E, 6.5 parts of sodium hyaluronate, 36 parts of microcrystalline cellulose, 13 parts of lactose, 3.0 parts of sorbitol, 0.7 parts of magnesium stearate, 0.7 parts of silicon dioxide, 6.5 parts of inulin and 0.2 parts of natural flavor.

[0044] The preparation method of the chitosan-neuraminic acid composite powder of the present invention comprises the following steps: placing N-acetylneuraminic acid gel in a low-temperature freezing environment of −53° C. for pre-freezing, the pre-freezing time being 138 minutes, then placing the frozen hydrogel in a freeze dryer, setting the vacuum degree to 13 Pa, controlling the temperature at −47° C., performing main drying, the main drying time being 28 hours, gradually increasing the freeze dryer plate temperature to 27° C., controlling the vacuum degree at 12 Pa, performing analytical drying to remove residual bound water, the analytical drying time being 258 minutes, and forming a loose porous block after freeze drying; then mechanically crushing the loose porous block into fine powder, sieving with an 86-mesh sieve, and finally obtaining the chitosan-neuraminic acid composite powder.

[0045] The preparation method of N-acetylneuraminic acid gel of the present invention comprises the following steps: first, by weight, 4.0 parts of chitosan are weighed and dissolved in 115 parts of 1.5 vol.% glacial acetic acid aqueous solution, and the mixture is stirred at a stirring rate of 430 rpm for 35 minutes to obtain a chitosan solution, then 12 parts of N-acetylneuraminic acid, 0.2 parts of transglutaminase and 2.0 parts of soy protein are added to the chitosan solution, the mixed solution is heated to 62°C, and the mixture is stirred at a stirring rate of 430 rpm for 35 minutes to obtain a uniform solution, and then the uniform solution is subjected to ultrasonic treatment, the ultrasonic treatment power is set to 106 W, and the time is 7 minutes. After the ultrasonic treatment is completed, the obtained uniform solution is divided into a mold, and a freeze-thaw treatment is performed, the freeze-thaw treatment temperature is set to -22°C, the freezing time is 14 hours, and then the mixture is thawed at room temperature for 9 hours, and the freeze-thaw-thaw process is repeated 3 times in total to achieve preliminary physical crosslinking of chitosan and N-acetylneuraminic acid, thereby forming N-acetylneuraminic acid gel.

[0046] The preparation method of N-acetylneuraminic acid of the present invention comprises the following steps: A1. Egg white pretreatment: fresh egg white is diluted, filtered through a 200-mesh sieve, allowed to stand, and pH adjusted to obtain an egg white solution suitable for enzymatic hydrolysis; A2. Enzymatic hydrolysis reaction: the pretreated egg white solution is added with papain, and enzymatic hydrolysis is performed under suitable temperature and stirring conditions, and the enzyme activity is inactivated by heating to obtain an enzymatic solution; A3. Centrifugation and preliminary purification: The enzymatic hydrolysate is subjected to high-speed centrifugation, ethanol precipitation and filtration to obtain a preliminary purified N-acetylneuraminic acid solution; A4. Dialysis and lyophilization: The preliminarily purified solution was dialyzed through a dialysis bag and freeze-dried to obtain a crude N-acetylneuraminic acid powder; A5. HPLC purification: The crude powder is dissolved and purified by HPLC, the target component is collected and the solvent is removed by rotary evaporation to obtain N-acetylneuraminic acid powder.

[0047] The detailed process of step A1 of the present invention is: using egg white as a raw material, mixing fresh egg white and deionized water in a volume ratio of 1:1.7, filtering through a 200-mesh sieve to remove impurities and unevenly distributed protein particles; placing the filtered egg white solution at 5 ° C for 69 minutes, and adjusting the pH of the solution to 6.2 with 1 mol / L hydrochloric acid or 1 mol / L sodium hydroxide to obtain a pretreated egg white solution.

[0048] The detailed process of step A2 of the present invention is as follows: placing the pretreated egg white solution in a reaction container, adding 0.9% volume of α-(2-3, 6, 8, 9) neuraminidase to the pretreated egg white solution, setting the stirring rate to 192 rpm, adjusting the reaction temperature to 46 ° C, and continuously stirring for 1.8 h. After the enzymatic hydrolysis reaction is completed, the reaction solution is quickly heated to 92 ° C and maintained for 9 minutes, and then cooled to obtain an enzymatic hydrolyzate.

[0049] The detailed process of step A3 of the present invention is as follows: transferring the cooled enzymatic hydrolyzate to a high-speed centrifuge, centrifuging at 5 ° C and 23000 rpm for 22 min, collecting the supernatant obtained by centrifugation as a crude extract of N-acetylneuraminic acid; slowly adding ethanol to the crude extract, the volume ratio of the crude extract to ethanol is 1:3.6, the addition rate is controlled at 2.3 mL / min, and the stirring rate is maintained at 215 rpm until the solution is fully mixed; placing the mixed solution in an ice bath and standing for 28 min to precipitate protein and other high molecular impurities; then placing the solution in a high-speed centrifuge again, centrifuging at 5 ° C and 23000 rpm for 22 min, collecting the obtained supernatant, and filtering through a filter membrane with a pore size of 0.3 μm to remove suspended impurities to obtain a preliminarily purified N-acetylneuraminic acid solution.

[0050] The detailed process of step A4 of the present invention is as follows: the obtained preliminary purified solution is transferred to a dialysis bag with a molecular weight cutoff range of 3200Da, and is placed in deionized water at 5 °C for dialysis. The dialysis time is 21 h, during which the dialysate is replaced every 4 h, and the volume of the dialysate replaced each time is 3 times the volume of the dialysis solution to ensure that small molecule impurities and salt ions are fully removed; after the dialysis is completed, the dialysate is transferred to a freeze dryer and freeze-dried at -47 °C for 26 h until the liquid is completely evaporated to obtain a crude N-acetylneuraminic acid powder.

[0051] The detailed process of step A5 of the present invention is as follows: dissolving the obtained crude N-acetylneuraminic acid powder in deionized water, wherein the mass ratio of the N-acetylneuraminic acid powder to the deionized water is 1:12, the stirring rate is controlled at 215 rpm, and the stirring time is 17 min, so as to ensure that the powder is completely dissolved; the dissolved solution is filtered through a filter membrane with a pore size of 0.22 μm and placed in a high performance liquid chromatography device for further purification; an Aminex HPX-87H chromatographic column is used, and the chromatographic conditions are set to a mobile phase concentration of 4.6 mM / L sulfuric acid, a flow rate of 0.6 mL / min, a detection wavelength of 208 nm, and a column temperature of 33 ° C; the components corresponding to the target peak are collected, and the solvent is removed by a rotary evaporator at a water bath temperature of 43 ° C, and the rotary evaporation time is 39 min, and finally the N-acetylneuraminic acid powder is obtained.

[0052] A method for preparing an N-acetylneuraminic acid tablet of the present invention comprises the following steps: S1. Put chitosan-neuraminic acid composite powder, vitamin C, vitamin E, sodium hyaluronate, microcrystalline cellulose, lactose, magnesium stearate, silicon dioxide, natural flavor and inulin into a three-dimensional mixer in sequence according to the proportion, set the mixing speed to 17 rpm, the mixing time to 17 min, and obtain a uniformly mixed dry powder.

[0053] S2. Transfer the mixed dry powder to the granulation equipment, slowly add the mixed solution of sorbitol and deionized water as a binder, control the stirring rate at 115 rpm, and stir for 7 min until uniform wet granules are formed.

[0054] S3. The wet granules were transferred to a hot air circulation drying oven, and the drying temperature was set to 53 °C and the drying time was set to 3 h, until the moisture content of the granules was less than 3%. After drying, the dry granules were sieved through a 17-mesh screen to obtain smooth and uniform granules.

[0055] S4. Transfer the evenly lubricated granules to the tablet press, set the tableting pressure to 17 kN, the tableting speed to 36 tablets / min, the tablet weight to 0.4g, and the tablet hardness to 66N. After the finished tablets pass the quality inspection, put them into aluminum foil bags or plastic bottles, and seal them for storage.

[0056] Example 3 An N-acetylneuraminic acid tablet comprises the following components, measured by weight: 16 parts of chitosan-neuraminic acid composite powder, 4.0 parts of vitamin C, 2.0 parts of vitamin E, 8.0 parts of sodium hyaluronate, 42 parts of microcrystalline cellulose, 16 parts of lactose, 4.0 parts of sorbitol, 0.8 parts of magnesium stearate, 0.8 parts of silicon dioxide, 8 parts of inulin and 0.3 parts of natural flavor.

[0057] The preparation method of the chitosan-neuraminic acid composite powder of the invention comprises the following steps: placing N-acetylneuraminic acid gel in a low-temperature freezing environment at -56°C for pre-freezing, the pre-freezing time being 156 minutes, then placing the frozen hydrogel in a freeze dryer, setting the vacuum degree to 16 Pa, controlling the temperature at -48°C, performing main drying, the main drying time being 31 hours, gradually increasing the freeze dryer plate temperature to 28°C after the main drying, controlling the vacuum degree at 13 Pa, performing analytical drying to remove residual bound water, the analytical drying time being 276 minutes, and forming a loose porous block after freeze drying; then mechanically crushing the loose porous block into fine powder, sieving with a 92-mesh sieve, and finally obtaining the chitosan-neuraminic acid composite powder.

[0058] The preparation method of the N-acetylneuraminic acid gel of the present invention comprises the following steps: first, by weight, 5.0 parts of chitosan are weighed and dissolved in 130 parts of a 1.5 vol.% glacial acetic acid aqueous solution, and the mixture is stirred at a stirring rate of 460 rpm for 39 minutes to obtain a chitosan solution, then 14 parts of N-acetylneuraminic acid, 0.3 parts of transglutaminase and 2.0 parts of soy protein are added to the chitosan solution, the mixed solution is heated to 63°C, and the mixture is stirred at a stirring rate of 460 rpm for 39 minutes to obtain a uniform solution, and then the uniform solution is subjected to ultrasonic treatment, the ultrasonic treatment power is set to 112 W, and the time is 8 minutes. After the ultrasonic treatment is completed, the obtained uniform solution is divided into a mold, and a freeze-thaw treatment is performed, the freeze-thaw treatment temperature is set to -23°C, the freezing time is 17 hours, and then the mixture is thawed at room temperature for 10 hours, and the freeze-thaw-thaw process is repeated 3 times in total to achieve preliminary physical crosslinking of chitosan and N-acetylneuraminic acid, thereby forming the N-acetylneuraminic acid gel.

[0059] The preparation method of N-acetylneuraminic acid of the present invention comprises the following steps: A1. Egg white pretreatment: fresh egg white is diluted, filtered through a 200-mesh sieve, allowed to stand, and pH adjusted to obtain an egg white solution suitable for enzymatic hydrolysis; A2. Enzymatic hydrolysis reaction: the pretreated egg white solution is added with papain, and enzymatic hydrolysis is performed under suitable temperature and stirring conditions, and the enzyme activity is inactivated by heating to obtain an enzymatic solution; A3. Centrifugation and preliminary purification: The enzymatic hydrolysate is subjected to high-speed centrifugation, ethanol precipitation and filtration to obtain a preliminary purified N-acetylneuraminic acid solution; A4. Dialysis and lyophilization: The preliminarily purified solution was dialyzed through a dialysis bag and freeze-dried to obtain a crude N-acetylneuraminic acid powder; A5. HPLC purification: The crude powder is dissolved and purified by HPLC, the target component is collected and the solvent is removed by rotary evaporation to obtain N-acetylneuraminic acid powder.

[0060] The detailed process of step A1 of the present invention is: using egg white as a raw material, mixing fresh egg white and deionized water in a volume ratio of 1:1.8, filtering through a 200-mesh sieve to remove impurities and unevenly distributed protein particles; placing the filtered egg white solution at 5 ° C for 78 minutes, and adjusting the pH of the solution to 6.3 with 1 mol / L hydrochloric acid or 1 mol / L sodium hydroxide to obtain a pretreated egg white solution.

[0061] The detailed process of step A2 of the present invention is as follows: placing the pretreated egg white solution in a reaction container, adding 1.0% volume of α-(2-3, 6, 8, 9) neuraminidase to the pretreated egg white solution, setting the stirring rate to 204 rpm, adjusting the reaction temperature to 48 ° C, and continuously stirring for 2.1 h. After the enzymatic hydrolysis reaction is completed, the reaction solution is quickly heated to 93 ° C and maintained for 10 min, and then cooled to obtain an enzymatic hydrolyzate.

[0062] The detailed process of step A3 of the present invention is as follows: transferring the cooled enzymatic hydrolyzate to a high-speed centrifuge, centrifuging at 5 ° C and 26000 rpm for 23 min, collecting the supernatant obtained by centrifugation as a crude extract of N-acetylneuraminic acid; slowly adding ethanol to the crude extract, the volume ratio of the crude extract to ethanol is 1:4.2, the addition rate is controlled at 2.6 mL / min, and the stirring rate is maintained at 230 rpm until the solution is fully mixed; placing the mixed solution in an ice bath and standing for 32 min to precipitate protein and other high molecular impurities; then placing the solution in a high-speed centrifuge again, centrifuging at 5 ° C and 26000 rpm for 23 min, collecting the obtained supernatant, and filtering through a filter membrane with a pore size of 0.35 μm to remove suspended impurities to obtain a preliminarily purified N-acetylneuraminic acid solution.

[0063] The detailed process of step A4 of the present invention is as follows: the obtained preliminary purified solution is transferred to a dialysis bag with a molecular weight cutoff range of 3300Da, and is placed in deionized water at 5 ° C for dialysis. The dialysis time is 22 h, during which the dialysate is replaced every 4 h, and the volume of the dialysate replaced each time is 3 times the volume of the dialysis solution to ensure that small molecule impurities and salt ions are fully removed; after the dialysis is completed, the dialysate is transferred to a freeze dryer and freeze-dried at -48 ° C for 27 h until the liquid is completely evaporated to obtain a crude N-acetylneuraminic acid powder.

[0064] The detailed process of step A5 of the present invention is as follows: dissolving the obtained crude N-acetylneuraminic acid powder in deionized water, wherein the mass ratio of the N-acetylneuraminic acid powder to the deionized water is 1:13, the stirring rate is controlled at 230 rpm, and the stirring time is 18 min, so as to ensure that the powder is completely dissolved; the dissolved solution is filtered through a filter membrane with a pore size of 0.22 μm and then placed in a high performance liquid chromatography device for further purification; an Aminex HPX-87H chromatographic column is used, and the chromatographic conditions are set to a mobile phase concentration of 5.2 mM / L sulfuric acid, a flow rate of 0.6 mL / min, a detection wavelength of 211 nm, and a column temperature of 36 ° C; the components corresponding to the target peak are collected, and the solvent is removed by a rotary evaporator at a water bath temperature of 46 ° C, and the rotary evaporation time is 48 min, and finally the N-acetylneuraminic acid powder is obtained.

[0065] A method for preparing an N-acetylneuraminic acid tablet of the present invention comprises the following steps: S1. Put chitosan-neuraminic acid composite powder, vitamin C, vitamin E, sodium hyaluronate, microcrystalline cellulose, lactose, magnesium stearate, silicon dioxide, natural flavor and inulin into a three-dimensional mixer in sequence according to the proportion, set the mixing speed to 18 rpm, and the mixing time to 18 min to obtain a uniformly mixed dry powder.

[0066] S2. Transfer the mixed dry powder to the granulation equipment, slowly add the mixed solution of sorbitol and deionized water as a binder, control the stirring rate at 130 rpm, and stir for 8 min until uniform wet granules are formed.

[0067] S3. The wet granules were transferred to a hot air circulation drying oven, and the drying temperature was set to 56 °C and the drying time was set to 3 h, until the moisture content of the granules was less than 3%. After drying, the dry granules were sieved through an 18-mesh screen to obtain smooth and uniform granules.

[0068] S4. Transfer the evenly lubricated granules to the tablet press, set the tableting pressure to 18 kN, the tableting speed to 42 tablets / min, the tablet weight to 0.42g, and the tablet hardness to 72N. After the finished tablets pass the quality inspection, put them into aluminum foil bags or plastic bottles, and seal them for storage.

[0069] Example 4 An N-acetylneuraminic acid tablet comprises the following components, measured by weight: 20 parts of chitosan-neuraminic acid composite powder, 5.0 parts of vitamin C, 3.0 parts of vitamin E, 10.0 parts of sodium hyaluronate, 50 parts of microcrystalline cellulose, 20 parts of lactose, 5.0 parts of sorbitol, 1.0 part of magnesium stearate, 1.0 part of silicon dioxide, 10 parts of inulin, and 0.5 part of natural flavor.

[0070] The preparation method of the chitosan-neuraminic acid composite powder of the present invention comprises the following steps: placing N-acetylneuraminic acid gel in a low-temperature freezing environment of −60° C. for pre-freezing, the pre-freezing time being 180 minutes, then placing the frozen hydrogel in a freeze dryer, setting the vacuum degree to 20 Pa, controlling the temperature at −50° C., performing main drying, the main drying time being 36 hours, gradually increasing the freeze dryer plate temperature to 30° C., controlling the vacuum degree at 15 Pa, performing analytical drying to remove residual bound water, the analytical drying time being 300 minutes, and forming a loose porous block after freeze drying, then mechanically crushing the loose porous block into fine powder, sieving with a 100-mesh sieve, and finally obtaining the chitosan-neuraminic acid composite powder.

[0071] The preparation method of N-acetylneuraminic acid gel of the present invention is as follows: first, by weight, 6.0 parts of chitosan are weighed and dissolved in 150 parts of 1.5 vol.% glacial acetic acid aqueous solution, stirred at a stirring rate of 500 rpm for 45 minutes to obtain a chitosan solution, then 16 parts of N-acetylneuraminic acid, 0.5 parts of transglutaminase and 3.0 parts of soy protein are added to the chitosan solution, the mixed solution is heated to 65°C, stirred at a stirring rate of 500 rpm for 45 minutes to obtain a uniform solution, then the uniform solution is subjected to ultrasonic treatment, the ultrasonic treatment power is set to 120 W, the time is 10 minutes, after the completion of the ultrasonic treatment, the obtained uniform solution is divided into a mold, and a freeze-thaw treatment is performed, the freeze-thaw treatment temperature is set to -25°C, the freezing time is 20 hours, and then thawed at room temperature for 12 hours, and the freeze-thaw-thaw process is repeated 3 times in total to achieve preliminary physical crosslinking of chitosan and N-acetylneuraminic acid, thereby forming N-acetylneuraminic acid gel.

[0072] The preparation method of N-acetylneuraminic acid of the present invention comprises the following steps: A1. Egg white pretreatment: fresh egg white is diluted, filtered through a 200-mesh sieve, allowed to stand, and pH adjusted to obtain an egg white solution suitable for enzymatic hydrolysis; A2. Enzymatic hydrolysis reaction: the pretreated egg white solution is added with papain, and enzymatic hydrolysis is performed under suitable temperature and stirring conditions, and the enzyme activity is inactivated by heating to obtain an enzymatic solution; A3. Centrifugation and preliminary purification: The enzymatic hydrolysate is subjected to high-speed centrifugation, trichloroacetic acid precipitation and filtration to obtain a preliminary purified N-acetylneuraminic acid solution; A4. Dialysis and lyophilization: The preliminarily purified solution was dialyzed through a dialysis bag and freeze-dried to obtain a crude N-acetylneuraminic acid powder; A5. HPLC purification: The crude powder is dissolved and purified by HPLC, the target component is collected and the solvent is removed by rotary evaporation to obtain N-acetylneuraminic acid powder.

[0073] The detailed process of step A1 of the present invention is: using egg white as a raw material, mixing fresh egg white and deionized water in a volume ratio of 1:2, filtering through a 200-mesh sieve to remove impurities and unevenly distributed protein particles; placing the filtered egg white solution at 6 ° C for 90 minutes, and adjusting the pH of the solution to 6.5 with 1 mol / L hydrochloric acid or 1 mol / L sodium hydroxide to obtain a pretreated egg white solution.

[0074] The detailed process of step A2 of the present invention is as follows: placing the pretreated egg white solution in a reaction container, adding 1.2% volume of α-(2-3,6,8,9) neuraminidase of the pretreated egg white solution, setting the stirring rate to 220 rpm, adjusting the reaction temperature to 50 ° C, and continuously stirring for 2.5 h. After the enzymatic hydrolysis reaction is completed, the reaction solution is quickly heated to 95 ° C and maintained for 12 min, and then cooled to obtain an enzymatic hydrolyzate.

[0075] The detailed process of step A3 of the present invention is as follows: transferring the cooled enzymatic hydrolyzate to a high-speed centrifuge, centrifuging at 6 ° C and 30000 rpm for 25 min, collecting the supernatant obtained by centrifugation as a crude extract of N-acetylneuraminic acid; slowly adding ethanol to the crude extract, the volume ratio of the crude extract to ethanol is 1:5.0, the addition rate is controlled at 3 mL / min, and the stirring rate is maintained at 250 rpm until the solution is fully mixed; placing the mixed solution in an ice bath and standing for 35 min to precipitate protein and other high molecular impurities; then placing the solution in a high-speed centrifuge again, centrifuging at 6 ° C and 30000 rpm for 25 min, collecting the obtained supernatant, filtering through a filter membrane with a pore size of 0.45 μm to remove suspended impurities, and obtaining a preliminarily purified N-acetylneuraminic acid solution.

[0076] The detailed process of step A4 of the present invention is as follows: the obtained preliminary purified solution is transferred to a dialysis bag with a molecular weight cutoff range of 3500Da, and is placed in deionized water at 6 ° C for dialysis. The dialysis time is 24 h, during which the dialysate is replaced every 5 h, and the volume of the dialysate replaced each time is 3 times the volume of the dialysis solution to ensure that small molecule impurities and salt ions are fully removed; after the dialysis is completed, the dialysate is transferred to a freeze dryer and freeze-dried at -50 ° C for 30 h until the liquid is completely evaporated to obtain a crude N-acetylneuraminic acid powder.

[0077] The detailed process of step A5 of the present invention is as follows: dissolving the obtained crude N-acetylneuraminic acid powder in deionized water, wherein the mass ratio of the N-acetylneuraminic acid powder to the deionized water is 1:15, the stirring rate is controlled at 250 rpm, and the stirring time is 20 min, so as to ensure that the powder is completely dissolved; the dissolved solution is filtered through a filter membrane with a pore size of 0.22 μm and then placed in a high performance liquid chromatography device for further purification; an Aminex HPX-87H chromatographic column is used, and the chromatographic conditions are set to a mobile phase concentration of 6 mM / L sulfuric acid, a flow rate of 0.7 mL / min, a detection wavelength of 215 nm, and a column temperature of 40 ° C; the components corresponding to the target peak are collected, and the solvent is removed by a rotary evaporator at a water bath temperature of 50 ° C, and the rotary evaporation time is 60 min, and finally the N-acetylneuraminic acid powder is obtained.

[0078] A method for preparing an N-acetylneuraminic acid tablet of the present invention comprises the following steps: S1. Put chitosan-neuraminic acid composite powder, vitamin C, vitamin E, sodium hyaluronate, microcrystalline cellulose, lactose, magnesium stearate, silicon dioxide, natural flavor and inulin into a three-dimensional mixer in sequence according to the proportion, set the mixing speed to 20 rpm, and the mixing time to 20 min to obtain a uniformly mixed dry powder.

[0079] S2. Transfer the mixed dry powder to the granulation equipment, slowly add the mixed solution of sorbitol and deionized water as a binder, control the stirring rate at 150 rpm, and stir for 10 min until uniform wet granules are formed.

[0080] S3. The wet granules were transferred to a hot air circulation drying oven, and the drying temperature was set to 60 °C and the drying time was set to 4 h, until the moisture content of the granules was less than 3%. After drying, the dry granules were sieved through a 20-mesh screen to obtain smooth and uniform granules.

[0081] S4. Transfer the evenly lubricated granules to the tablet press, set the tableting pressure to 20 kN, the tableting speed to 50 tablets / min, the tablet weight to 0.5g, and the tablet hardness to 80N. After the finished tablets pass the quality inspection, put them into aluminum foil bags or plastic bottles, and seal them for storage.

[0082] Comparative Example 1 The method is basically the same as Example 1, except that the chitosan-neuraminic acid composite powder is not added to the tablets.

[0083] Comparative Example 2 The same as Example 1, except that vitamin C is not added to the tablets.

[0084] Comparative Example 3 The method is substantially the same as Example 1, except that sodium hyaluronate is not added to the tablet.

[0085] Comparative Example 4 The method is substantially the same as Example 1, except that sorbitol is not added to the tablets.

[0086] Comparative Example 5 The same as Example 1, except that magnesium stearate is not added to the tablet.

[0087] Comparative Example 6 The method is basically the same as Example 1, except that the pH of the egg white solution is not adjusted and the original pH is kept at 7.0. The results show that the enzymolysis efficiency is significantly reduced, the reaction time is extended to more than 4 hours, and the purity of the final product is less than 80%.

[0088] Comparative Example 7 The method is basically the same as Example 1, except that the molecular weight cutoff range of the dialysis bag is adjusted to 5000 Da. The results show that the small molecule impurities cannot be completely removed, resulting in the purity of the final N-acetylneuraminic acid powder being only 85%.

[0089] Comparative Example 8 The method is basically the same as Example 1, except that the drying temperature in S3 is increased to 70° C. The results show that the active ingredients inside the granules are partially degraded and the active content of the tablets is reduced.

[0090] Performance Test: This experiment solves the problem of easy degradation of N-acetylneuraminic acid in a complex system by detecting the stability of N-acetylneuraminic acid in the tablet of the present invention. The experiment selected the N-acetylneuraminic acid tablet of the present invention and the N-acetylneuraminic acid solution without chitosan coating as the control group, and carried out stability tests under the conditions of simulated gastric fluid (pH 1.2) and simulated intestinal fluid (pH 6.8), respectively, with the temperature controlled at 37°C to simulate the internal environment of the human body, and set the time points of 0 h and 4 h. The content of N-acetylneuraminic acid in the sample was detected by high performance liquid chromatography (HPLC), and its degradation rate (the percentage of initial concentration to residual concentration) was calculated.

[0091] This experiment evaluated the dissolution efficiency and bioavailability of N-acetylneuraminic acid in the tablets of the present invention, and verified its efficient utilization in complex systems. The tablets of the present invention and pure N-acetylneuraminic acid powder were used as controls in the experiment, and dissolution experiments were carried out in simulated gastric fluid (pH 1.2) and simulated intestinal fluid (pH 6.8). The dissolution process was monitored by a dissolution instrument (speed 100 rpm, temperature 37°C), sampling was performed every 10 minutes, and the dissolution within 1 hour was recorded. HPLC was used to detect the concentration of N-acetylneuraminic acid in the sample, and the dissolution curve was drawn and the cumulative release percentage was calculated. The results showed that the dissolution rate of the tablets of the present invention was more uniform and the cumulative release ratio was higher, which fully demonstrated its efficient utilization in complex systems.

[0092] This experiment tests the antioxidant properties of the tablets and evaluates their synergistic effects. The experiment selected the tablets of the present invention, the comparative tablets without vitamin C and vitamin E (non-antioxidant group), and the pure N-acetylneuraminic acid powder as controls, and tested the antioxidant capacity by DPPH free radical scavenging experiment (1,1-diphenyl-2-picrylhydrazyl). After the tablet sample was dissolved, the DPPH solution was added, and the absorbance was measured at a wavelength of 517 nm after 30 minutes of reaction, and the free radical scavenging rate (%) was calculated.

[0093] This experiment mainly tests the stability of tablets and evaluates their long-term storage performance under high temperature and high humidity conditions through accelerated conditions. The tablets were placed at 40°C and 75% relative humidity, and the test period was set to 0, 1, 2, and 3 months. The content of N-acetylneuraminic acid in the tablets was tested (HPLC) and the changes in the appearance hardness of the tablets were observed.

[0094] The properties of the tablets of Examples 1 to 4 and Comparative Examples 1 to 10 are summarized in Table 1.

[0095] Table 1 Performance summary of the tablets of Examples 1 to 4 and Comparative Examples 1 to 10

[0096] Chitosan plays a key role in tablet packaging and protection, which can significantly reduce the degradation rate of tablets and improve dissolution rate and stability. Lack of chitosan will lead to loose internal structure of tablets, reduced dissolution rate and significantly decreased stability, indicating that chitosan is essential for the protection and release performance of tablets. Sorbitol, as a binder, can enhance the formability and mechanical stability of tablet particles. Its absence will lead to a decrease in tablet hardness and a negative impact on the dissolution rate, indicating the important role of binders in improving the mechanical properties and release properties of tablets. Vitamin C, as an antioxidant component, can significantly improve the free radical scavenging rate of tablets. Its absence will lead to a decrease in antioxidant capacity, indicating its key role in the antioxidant properties of tablets. In addition, drying time and tableting pressure also have an important influence on tablet performance. Insufficient drying will lead to residual moisture, thereby reducing the stability of tablets, while insufficient tableting pressure will affect the hardness and mechanical stability of tablets, further illustrating the necessity of tablet formulation and process optimization for performance improvement.

[0097] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, ordinary technicians in the relevant field should understand that all equivalent structural changes made under the concept of the present invention and using the contents of the present invention specification and drawings should be covered within the scope of protection of the claims of the present invention.

Claims

1. An N-acetylneuraminic acid tablet, characterized in that: The invention comprises the following components in parts by weight: 10-20 parts of chitosan-neuraminic acid composite powder, 2.0-5.0 parts of vitamin C, 1.0-3.0 parts of vitamin E, 5.0-10.0 parts of sodium hyaluronate, 30-50 parts of microcrystalline cellulose, 10-20 parts of lactose, 2.0-5.0 parts of sorbitol, 0.5-1.0 parts of magnesium stearate, 0.5-1.0 parts of silicon dioxide, 5-10 parts of inulin and 0.1-0.5 parts of natural flavor.

2. An N-acetylneuraminic acid tablet according to claim 1, characterized in that: The preparation method of the chitosan-neuraminic acid composite powder is as follows: by weight, N-acetylneuraminic acid gel is placed in a low-temperature freezing environment of −50~−60 °C for pre-freezing, the pre-freezing time is 120~180min, then the frozen hydrogel is placed in a freeze dryer, the vacuum degree is set to 10~20 Pa, the temperature is controlled at −50~−45 °C, main drying is performed, the main drying time is 24~36 h, after the main drying is completed, the freeze dryer plate temperature is gradually increased to 25~30 °C, the vacuum degree is controlled at 10~15 Pa, analytical drying is performed to remove residual bound water, the analytical drying time is 240~300min, and the loose porous block after freeze drying is then mechanically crushed into fine powder, sieved with an 80~100 mesh screen, and finally the chitosan-neuraminic acid composite powder is obtained.

3. An N-acetylneuraminic acid tablet according to claim 2, characterized in that: The preparation method of the N-acetylneuraminic acid gel is as follows: first, weigh 3.0-6.0 parts of chitosan and dissolve them in 100-150 parts of 1.5 vol.% glacial acetic acid aqueous solution, stir at a stirring rate of 400-500 rpm for 30-45 min to obtain a chitosan solution, then add 10-16 parts of N-acetylneuraminic acid, 0.1-0.5 parts of transglutaminase and 1.0-3.0 parts of soy protein to the chitosan solution, heat the mixed solution to 60-65°C, stir at a stirring rate of 400-500 rpm for 30-45 min to obtain a uniform solution, then subject the uniform solution to ultrasonic treatment, the ultrasonic treatment power is set to 100-120 W, the time is 5-10 min, after the completion of the ultrasonic treatment, the obtained uniform solution is divided into a mold, and subjected to freeze-thaw treatment, the freeze-thaw treatment temperature is set to -20--25°C, and the freezing time is 12-20 h, and then thawed at room temperature for 8-12 h, and the freeze-thaw-thaw process was repeated 3 times to achieve the initial physical cross-linking of chitosan and N-acetylneuraminic acid, thereby forming N-acetylneuraminic acid gel.

4. An N-acetylneuraminic acid tablet according to claim 3, characterized in that: The preparation method of described N-acetylneuraminic acid comprises the following steps: A1. Egg white pretreatment: fresh egg white is diluted, filtered through a 200-mesh sieve, allowed to stand, and pH adjusted to obtain an egg white solution suitable for enzymatic hydrolysis; A2. Enzymatic hydrolysis reaction: the pretreated egg white solution is added with papain, and enzymatic hydrolysis is performed under suitable temperature and stirring conditions, and the enzyme activity is inactivated by heating to obtain an enzymatic solution; A3. Centrifugation and preliminary purification: The enzymatic hydrolysate is subjected to high-speed centrifugation, ethanol precipitation and filtration to obtain a preliminary purified N-acetylneuraminic acid solution; A4. Dialysis and lyophilization: The preliminarily purified solution was dialyzed through a dialysis bag and freeze-dried to obtain a crude N-acetylneuraminic acid powder; A5. HPLC purification: The crude powder is dissolved and purified by HPLC, the target component is collected and the solvent is removed by rotary evaporation to obtain N-acetylneuraminic acid powder.

5. An N-acetylneuraminic acid tablet according to claim 4, characterized in that: The detailed process of step A1 is as follows: using egg white as raw material, mixing fresh egg white and deionized water in a volume ratio of 1: (1.5-2), filtering through a 200-mesh sieve to remove impurities and unevenly distributed protein particles; placing the filtered egg white solution at 4-6 ° C for 60-90 minutes, and adjusting the pH of the solution to 6.0-6.5 with 1 mol / L hydrochloric acid or 1 mol / L sodium hydroxide to obtain a pretreated egg white solution.

6. An N-acetylneuraminic acid tablet according to claim 4, characterized in that: The detailed process of step A2 is as follows: placing the pretreated egg white solution in a reaction container, adding 0.8-1.2% volume of α-(2-3, 6, 8, 9) neuraminidase to the pretreated egg white solution, setting the stirring rate to 180-220 rpm, adjusting the reaction temperature to 45-50 ° C, and continuously stirring for 1.5-2.5 h. After the enzymatic hydrolysis reaction is completed, the reaction solution is quickly heated to 90-95 ° C and maintained for 8-12 min, and then cooled to obtain an enzymatic hydrolyzate.

7. An N-acetylneuraminic acid tablet according to claim 4, characterized in that: The detailed process of step A3 is as follows: transferring the cooled enzymatic hydrolysate to a high-speed centrifuge, centrifuging at 4-6 °C and 20000-30000 rpm for 20-25 min, collecting the supernatant obtained by centrifugation as a crude extract of N-acetylneuraminic acid; slowly adding ethanol to the crude extract, the volume ratio of the crude extract to ethanol is 1:(3.0-5.0), the addition rate is controlled at 2-3 mL / min, and the stirring rate is maintained at 200-250 rpm until the solution is fully mixed; placing the mixed solution in an ice bath and standing for 25-35 min to precipitate proteins and other high molecular impurities; then placing the solution in a high-speed centrifuge again, centrifuging at 4-6 °C and 20000-30000 rpm for 20-25 min, collecting the obtained supernatant, and filtering through a filter membrane with a pore size of 0.22-0.45 μm to remove suspended impurities to obtain a preliminarily purified N-acetylneuraminic acid solution.

8. An N-acetylneuraminic acid tablet according to claim 4, characterized in that: The detailed process of step A4 is as follows: the obtained preliminary purified solution is transferred to a dialysis bag with a molecular weight cutoff range of 3000~3500 Da, and placed in deionized water at 4~6 ° C for dialysis, the dialysis time is 20~24 h, during which the dialysate is replaced every 3~5 h, and the volume of the dialysate replaced each time is 3 times the volume of the dialysis solution to ensure sufficient removal of small molecule impurities and salt ions; after the dialysis is completed, the dialysate is transferred to a freeze dryer and freeze-dried at -45~-50 ° C for 24~30 h until the liquid is completely evaporated to obtain a crude N-acetylneuraminic acid powder.

9. An N-acetylneuraminic acid tablet according to claim 4, characterized in that: The detailed process of step A5 is as follows: dissolving the obtained crude N-acetylneuraminic acid powder in deionized water, wherein the mass ratio of N-acetylneuraminic acid powder to deionized water is 1:(10-15), the stirring rate is controlled at 200-250 rpm, and the stirring time is 15-20 min to ensure that the powder is completely dissolved; the dissolved solution is filtered through a filter membrane with a pore size of 0.22 μm and placed in a high performance liquid chromatography device for further purification; an Aminex HPX-87H chromatographic column is used, and the chromatographic conditions are set to a mobile phase concentration of 4-6 mM / L sulfuric acid, a flow rate of 0.5-0.7 mL / min, a detection wavelength of 205-215 nm, and a column temperature of 30-40 °C; the components corresponding to the target peak are collected, and the solvent is removed by a rotary evaporator at a water bath temperature of 40-50 °C, and the rotary evaporation time is 30-60 min, and finally N-acetylneuraminic acid powder is obtained.

10. A method for preparing an N-acetylneuraminic acid tablet as claimed in any one of claims 1 to 9, characterized in that The following steps are involved: S1. Chitosan-neuraminic acid composite powder, vitamin C, vitamin E, sodium hyaluronate, microcrystalline cellulose, lactose, magnesium stearate, silicon dioxide, natural flavor and inulin are sequentially put into a three-dimensional mixer according to the proportion, and the mixing speed is set to 15-20 rpm and the mixing time is 15-20 min to obtain a uniformly mixed dry powder; S2. The mixed dry powder is transferred to a granulation device, and a mixed solution of sorbitol and deionized water is slowly added as a binder. The stirring rate is controlled at 100-150 rpm and the stirring time is 5-10 min until uniform wet granules are formed; S3. The wet granules are transferred to a hot air circulation drying oven, the drying temperature is set to 50-60 ° C, the drying time is 2-4 hours, until the moisture content of the granules is less than 3%, after the drying is completed, the dry granules are sieved through a 16-20 mesh screen to obtain smooth and uniform granules; S4. Transfer the evenly lubricated granules to the tablet press, set the tableting pressure to 15~20 kN, the tableting speed to 30~50 tablets / min, the tablet weight to 0.3~0.5g, and the tablet hardness to 60~80N. After the finished tablets pass the quality inspection, put them into aluminum foil bags or plastic bottles, and seal them for storage.

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