Skin-tendering and anti-aging composition based on N-acetylneuraminic acid and preparation method of skin-tendering and anti-aging composition

Abstract

The invention relates to the technical field of health care products, and provides a skin-tendering and anti-aging composition based on N-acetylneuraminic acid and a preparation method, and the composition comprises N-acetylneuraminic acid gel, milk powder, xylitol, prebiotics and other components. The N-acetylneuraminic acid gel is prepared by mixing a loaded N-acetylneuraminic acid whey protein isolate solution and a polysaccharide mixed solution in proportion, heating, stirring, gelatinizing, cooling and standing; n-acetylneuraminic acid is subjected to escherichia coli fermentation, centrifugation, ultrafiltration, ethanol precipitation, dialysis, freeze drying and other processes to prepare a high-purity product. The composition is prepared through the processes of sugar alcohol solution preparation, powder compounding, gel mixing, extrusion granulation, freeze drying and the like, the product homogeneity is good, the taste is excellent, the functions of tendering skin, resisting aging, enhancing immunity and promoting health are achieved, the problems that traditional N-acetylneuraminic acid is poor in stability and short in release rate are solved, and the composition is suitable for the fields of food and health care products.

Description

Technical Field

[0001] The present invention relates to the technical field of health care products, and in particular to a skin rejuvenation and anti-aging composition based on N-acetylneuraminic acid and a preparation method thereof. Background Art

[0002] As people pay more and more attention to health and beauty, the field of functional food has risen rapidly, especially in anti-aging and skin care. Edible compositions with bioactive ingredients have become a hot spot for market research and development. The skin rejuvenation and anti-aging composition based on N-acetylneuraminic acid is an innovative application in this field. Its main goal is to improve skin moisture, elasticity and repair ability through edible means to meet consumers' demand for efficient, convenient and safe anti-aging solutions. In this application scenario, the performance requirements of materials are particularly critical. On the one hand, N-acetylneuraminic acid needs to have excellent biological activity and high absorption rate to ensure that it can significantly promote collagen production, improve cell metabolism and slow down the skin aging process in the human body; on the other hand, in order to adapt to complex processing, storage and transportation conditions, edible compositions need to have excellent environmental stability, including high temperature resistance, moisture resistance and antioxidant properties, to ensure that its active ingredients remain stable during the storage period. In addition, the long-term release ability of edible compositions is also an important indicator for improving functionality. The sustained release of N-acetylneuraminic acid in the human body through scientific design can further enhance its efficacy and prolong its duration of action. Meeting these material performance requirements can not only significantly improve the actual effect of anti-aging compositions, but also broaden their application scenarios, from high-end functional foods to medical and health products, and promote the overall improvement of the industry's technical level and market competitiveness.

[0003] At present, although edible compositions based on N-acetylneuraminic acid have shown great potential in the field of skin rejuvenation and anti-aging, there are still many deficiencies in terms of preparation process, stability and long-term release performance. For example, the Chinese patent with publication number CN111838475A discloses a colloid food containing N-acetylneuraminic acid and a preparation method thereof, but in the preparation process, N-acetylneuraminic acid is simply added and cannot guarantee its long-term release. In addition, the stability of N-acetylneuraminic acid in its composition has not been effectively improved, and it is easy to degrade under high temperature or high humidity conditions, which significantly shortens the shelf life of the product and reduces its commercial value. More importantly, it is difficult for the prior art to achieve the long-term release function of N-acetylneuraminic acid. For example, the Chinese patent with publication number CN109757686B mentions a protein powder food containing N-acetylneuraminic acid and a preparation method thereof, but due to improper selection of carrier materials, its release rate is uneven, resulting in a short functional action time and a non-lasting effect. The main reason for these deficiencies is the lack of in-depth understanding of the molecular characteristics of N-acetylneuraminic acid in current research, and the failure to fully consider the mechanism of multi-component synergistic effect in the design of the composition. Therefore, how to improve the stability and long-term release performance of N-acetylneuraminic acid through innovative preparation processes, optimized formulation design and scientific carrier selection has become a key challenge to promote technological progress in this field. Summary of the invention

[0004] (1) Technical issues solved The purpose of the present invention is to provide a skin rejuvenation and anti-aging composition based on N-acetylneuraminic acid and a preparation method thereof, so as to solve the problem that the current skin rejuvenation and anti-aging composition is insufficient in sustained-release effect and stability performance.

[0005] (2) Technical solution In order to achieve the above object, the present invention provides the following technical solutions: A skin rejuvenation and anti-aging composition based on N-acetylneuraminic acid comprises the following components in parts by weight: 5.0-8.0 parts of N-acetylneuraminic acid gel, 15-20 parts of skim milk powder, 10-15 parts of whole milk powder, 20-25 parts of xylitol, 1.0-3.0 parts of prebiotics, 5-10 parts of milk mineral salts, 1.0-3.0 parts of colostrum alkaline protein, 2.0-5.0 parts of bird's nest powder, 1.0-3.0 parts of pectin, 0.02-0.05 parts of vitamin E, 2.0-4.0 parts of sorbitol, 0.1-0.3 parts of flavors, and 50-80 parts of deionized water.

[0006] The prebiotics are a mixture of oligofructose, oligogalactose and inulin in a weight ratio of 1:1:(2.0-4.0).

[0007] Furthermore, the preparation method of the N-acetylneuraminic acid gel is as follows: a whey protein isolate solution loaded with N-acetylneuraminic acid and a polysaccharide mixed solution are mixed according to a volume ratio of (2.0-4.0): (4.0-6.0), and stirred at a stirring rate of 300-400 rpm using a magnetic stirrer for 10-15 min at 40-50 ° C to form a uniform composite solution; the obtained composite solution is placed in a water bath at 75-85 ° C and heated for 5-10 min to induce a gelation process, and after the heating is completed, the solution is quickly moved to flowing cold water to cool to room temperature, and then the cooled solution is transferred to a closed container, and allowed to stand at 4 ° C for 12-24 h to obtain N-acetylneuraminic acid gel.

[0008] Furthermore, the preparation method of the N-acetylneuraminic acid loaded whey protein isolate solution is as follows: by weight, at room temperature, 5.0-10.0 parts of whey protein isolate and 100-150 parts of distilled water are stirred at a stirring rate of 300-400 rpm using a magnetic stirrer for 180-240 minutes to obtain a whey protein isolate solution for standby use; then 3.0-8.0 parts of N-acetylneuraminic acid are added, and stirring is continued at a stirring rate of 300-350 rpm for 15-30 minutes at room temperature, and the solution is filtered through a filter membrane with a pore size of 0.45 μm to remove particles, and the obtained filtrate is the N-acetylneuraminic acid loaded whey protein isolate solution.

[0009] Furthermore, the preparation method of the polysaccharide mixed solution is as follows: 5.0-12.0 parts of pectin and 4.0-10.0 parts of carboxymethyl cellulose are weighed respectively by weight, and added one by one to 100-140 parts of distilled water, and allowed to stand at room temperature for 20-30 minutes to allow the polysaccharide to fully absorb water and swell; the polysaccharide solution after standing is placed in a water bath at 55-60°C and heated for 30-45 minutes, and stirred at a stirring rate of 300-350 rpm for 30-45 minutes to obtain a polysaccharide mixed solution.

[0010] The present invention adopts a design with N-acetylneuraminic acid gel as the core, which is mainly used to enhance the functionality and stability of the composition. By optimizing the component ratio and preparation process, the synergistic effect between the components is fully exerted. The design of N-acetylneuraminic acid gel is based on the loading of N-acetylneuraminic acid whey protein isolate solution and polysaccharide mixed solution. The two parts form components with unique functions respectively during the preparation process, and the coupling and enhancement of functions are achieved by mixing in a specific ratio. During the preparation process of the loading of N-acetylneuraminic acid whey protein isolate solution, the whey protein isolate is fully dissolved by long-term stirring to form a stable protein solution, and after adding N-acetylneuraminic acid, the uniform dispersion of N-acetylneuraminic acid and the effective removal of impurities are ensured by further stirring and filtering. This loading solution not only shows excellent characteristics in stability, but also provides reliable whey protein matrix support for the function of N-acetylneuraminic acid. The polysaccharide mixed solution achieves good solubility, viscosity and structural stability through the synergistic effect of pectin and carboxymethyl cellulose. Through the process of standing water absorption expansion and heating and stirring, the solution retains the network structure of polysaccharides while forming a uniform system, providing necessary physical support for the subsequent gelation process. After the two solutions are mixed, the uniformity of the composite solution is ensured by regulating and uniformly stirring in a specific ratio, and gelation is achieved during the heating induction process. The formation of the gel not only depends on the network effect of the polysaccharide, but also benefits from the interaction between the whey protein and the polysaccharide, further enhancing the structural stability of the system. The cooling and standing steps ensure the complete maturity of the gel, thereby giving it excellent physical stability and functionality. The design of the present invention is intended to achieve stable loading and release of functional ingredients through the multiple synergistic effects of whey protein, polysaccharides and N-acetylneuraminic acid, while improving the performance and applicability of the composition in practical applications.

[0011] The present invention adopts a multifunctional combination formula design, which is mainly used to enhance the nutritional functionality, processing stability and eating experience of the composition. Through the scientific combination of N-acetylneuraminic acid gel, milk powder, sugar alcohol, prebiotics and other active ingredients, the present invention achieves the synergistic effect of nutrients and the optimization of overall functions. As a core component, N-acetylneuraminic acid gel provides important functional support for skin rejuvenation and anti-aging with its excellent biological activity and stability, and its gel properties improve the structural performance and processing stability of the product. Skim milk powder and whole milk powder not only improve the nutritional value of the composition through the synergistic effect of protein, fat and lactose, but also optimize the taste and solubility. Xylitol and sorbitol are sweeteners that provide a low-calorie sweet experience, and at the same time participate in regulating the water activity of the product, further enhancing the storage stability of the product. Prebiotics are composed of oligofructose, oligogalactose and inulin, and provide bifidus factor function in the form of a mixture of a specific ratio, promote intestinal health, and synergize with milk mineral salts and colostrum alkaline protein to further enhance immune regulation and bone health functions. Bird's nest powder and pectin not only provide bioactive ingredients, but also enhance the texture and functionality of the product through networking with other components. Vitamin E provides antioxidant protection in trace amounts, slowing down the oxidative deterioration of the product. Fragrances enhance the sensory experience of the product, making it more attractive. Deionized water is used as a solvent to evenly disperse the components and ensure the processing consistency of the product. The precise design of the proportions of the components and the synergy of their functions enable the present invention to achieve a balance between nutritional functions, processing performance and eating experience, while meeting the multiple needs of skin rejuvenation, anti-aging and health promotion.

[0012] Further, the preparation method of the N-acetylneuraminic acid is: S1. Strain activation and fermentation culture: The Escherichia coli K12 strain is revived, pre-cultured and fermented to obtain a fermentation broth; S2. Collection and disruption of bacterial cells: The fermentation broth is centrifuged and ultrasonically disrupted to obtain a suspension of intracellular metabolites; S3. Product separation and purification: The intracellular metabolite suspension is concentrated by ultrafiltration, ethanol precipitation and mineral adsorption to obtain a purified product solution; S4. Concentration, dialysis and freeze-drying: The purified product solution is concentrated, dialyzed and freeze-dried to obtain N-acetylneuraminic acid.

[0013] Furthermore, the specific steps of S1 are as follows: taking Escherichia coli K1 strain as a raw material, taking the strain out from the storage condition and rapidly thawing it, and then inoculating it into a composite culture medium to obtain a mixed solution, wherein the composite culture medium includes 8-12 g / L yeast extract, 8-12 g / L tryptone and 4-6 g / L NaCl, and adjusting the pH of the composite solution to 7.2-7.4 with a 0.05wt.% NaOH solution, and then placing the mixed solution in a 500mL conical flask, stirring at a stirring rate of 120-150 rpm for 8-12 h, until a pre-culture solution with an optical density of 0.7-1.1 at a wavelength of 600 nm is formed; taking another 100-120 mL of the composite culture medium and placing it in a 500mL conical flask, inoculating the obtained pre-culture solution into the composite culture medium according to 0.8-1.2% of the volume of the composite culture medium, and stirring at 120-150 rpm for 8-12 h at 35.0-37°C, until a pre-culture solution with an optical density of 0.7-1.1 at a wavelength of 600 nm is formed. The optical density of the seed liquid at the wavelength of nm reaches 1.4-2.2; 10-12 L of the basic defined culture medium is added to the disposable reaction bag bioreactor, and the seed liquid is inoculated into the basic defined culture medium according to 2.5-3.5% of the volume of the defined culture medium, and the platform angle is set to 8°-12°, the temperature is 36-38°C, the dissolved oxygen level is 45-55%, and the oxygen supply rate is 0.9-1.1 L / min, and the fermentation is carried out for 36-72 hours until the fermentation is completed; Furthermore, the basic defined culture medium uses 10-20 g / L glucose as the main carbon source, 2-5 g / L ammonium sulfate as the inorganic nitrogen source, 1-1.5 g / L potassium dihydrogen phosphate and 1-1.5 g / L potassium hydrogen phosphate as the phosphorus source and buffer; ferrous sulfate, zinc sulfate, manganese chloride and copper sulfate pentahydrate are added in amounts of 10-50 mg / L, 10-50 mg / L, 5-20 mg / L and 1-10 mg / L, respectively; optionally, vitamin B1 is further included as an auxiliary factor, with an addition amount of 0.1-1 mg / L; and 10-50 mg / L chelating agent EDTA, and the pH of the basic defined culture medium is adjusted to 7.4-7.6 with 0.5wt.% sodium hydroxide solution.

[0014] Furthermore, the specific steps of S2 are as follows: the fermentation broth after fermentation is passed through a continuous centrifuge at a centrifugal rate of 16000-18000 rpm, centrifuged at 90-110 mL / min at 4±1°C, the obtained bacterial precipitate is collected and placed in an ice-water mixed bath, and crushed using an ultrasonic crusher, with the amplitude set to 95-100% and crushed for 25-35 s, followed by cooling in an ice-water mixed bath for 25-35 s, and the crushing process is repeated 3-4 times until the cell wall is fully lysed and the intracellular product is released to obtain a cell suspension.

[0015] Furthermore, the specific steps of S3 are as follows: the disrupted cell suspension is subjected to ultrafiltration and concentration treatment by a cross-flow ultrafiltration device, the device uses a filter membrane with a molecular weight cutoff limit of 8-12 kDa, the mixed solution inlet pressure is controlled at 3.5-4.5 bar, the mixed solution is concentrated to 1 / 18-1 / 22 of the original volume, ethanol is gradually added to the concentrated solution for ethanol precipitation treatment, the volume ratio of ethanol to precipitate is adjusted to (78-82):100, and then centrifuged at a centrifugal speed of 4000-5000 rpm at 4±1°C for 8-15 min separation precipitation treatment; dissolving the obtained precipitate in deionized water, wherein the volume ratio of deionized water to precipitate is (150-180):100, and then repeating ultrafiltration concentration, ethanol precipitation and separation precipitation treatment for 2-4 times, until impurities are removed and the target product is concentrated to obtain a purified solution; mixing the obtained purified solution with a pre-balanced clay mineral in a mass ratio of (0.9-1):1, further separating the purified product solution from the clay mineral by centrifugation, wherein the centrifugation condition is 4000-5000 rpm, and the centrifugation treatment is carried out at 4±1°C for 12-18 min, taking the upper layer solution, and finally obtaining a further purified product solution; Furthermore, the pre-balanced clay mineral is a clay mineral that is oscillated in a 90-110 mM sodium phosphate buffer at a pH of 6.8-7.2 for 0.8-1.2 h, stirred at 120-140 rpm for 2.5-3.5 h after mixing, and centrifuged at a centrifugal rate of 4000-5000 rpm at 4±1°C for 12-18 min to obtain a precipitate that is a pre-balanced clay mineral.

[0016] Furthermore, the specific steps of S4 are as follows: the purified product solution is concentrated through a centrifugal filter tube with a molecular weight cutoff of 8~12kDa, and centrifuged at a centrifugal rate of 4000~5000 rpm at 4±1°C. The mixed solution is concentrated to 1 / 4~1 / 6 of the original volume and then subjected to three-stage dialysis treatment using a dialysis membrane with a molecular weight cutoff of 12~16 kDa. In the first stage, 4.5~5.5 L of a deionized water solution containing 5.4~5.6 g / L NaCl is used as a dialysis fluid for 22~26 h. In the second and third stages, 4.5~5.5 L of a deionized water solution with a pH of 8.8~9.2 NaOH is used as a dialysis fluid for 22~26 h. After the dialysis is completed, the solution obtained in the dialysis bag is transferred to a freeze-drying device and freeze-dried under a vacuum condition of 0.03~0.05 mbar until it is completely dehydrated to obtain the final N-acetylneuraminic acid powder.

[0017] The present invention adopts the design of the preparation process of N-acetylneuraminic acid based on the fermentation production of Escherichia coli, aiming to improve the purity, stability and applicability of the product. Through multi-stage optimization processes such as strain activation, fermentation culture, bacterial cell crushing, product separation and purification, and concentrated freeze-drying, the efficient generation of high-purity N-acetylneuraminic acid is ensured. In the fermentation stage, the carbon source, nitrogen source, phosphorus source and trace elements are reasonably proportioned to strengthen the metabolic activity of the strain and increase the yield; in the crushing stage, the synergistic effect of centrifugation and ultrasound ensures the full release of intracellular metabolites; in the purification stage, impurities are effectively removed and the product purity is improved through ultrafiltration concentration, ethanol precipitation and clay mineral adsorption; finally, the homogeneity and stability of the product are further optimized through dialysis and freeze-drying. The high degree of synergy between the various process steps, the reasonable combination of culture medium formula, separation and purification means and post-processing technology, not only realizes the efficient preparation of N-acetylneuraminic acid, but also significantly enhances its application potential in food and other fields, showing good economy and practicality.

[0018] The present invention also provides a method for preparing a skin rejuvenation and anti-aging composition based on N-acetylneuraminic acid, comprising the following steps: A1. Add deionized water to a reactor, heat to 60-70°C, and stir at this temperature at a stirring rate of 200-300 rpm, add xylitol and sorbitol in turn, and continue stirring for 10-15 minutes until they are evenly dissolved to form a transparent sugar alcohol solution, further add prebiotics to the sugar alcohol solution, and continue stirring at a stirring rate of 200-300 rpm for 10-15 minutes, then add vitamin E, and stir at 200-250 rpm for 5-10 minutes to form a uniform functional liquid base for standby use; A2. Add skim milk powder and whole milk powder to a dry mixer, mix at a stirring rate of 300-400 rpm for 3-5 min, then add milk mineral salts, colostrum alkaline protein and bird's nest powder, continue to mix at a stirring rate of 200-300 rpm for 2-3 min until a uniform powder mixture is formed, then continue to add pectin, and mix at a stirring rate of 200-250 rpm for 5-10 min to form a uniform solid powder mixture for standby; A3. Add N-acetylneuraminic acid gel to another stirred tank, slowly add the functional liquid base prepared by S1, and stir at a stirring rate of 200-250 rpm at room temperature for 10-15 minutes to form a uniform functional liquid mixture; slowly add the solid powder mixture and flavor prepared by A2 to the liquid mixture, and stir at a stirring rate of 200-250 rpm for 15-20 minutes until a uniform semi-wet mixture is formed, and leave the mixture to stand at room temperature for 20-30 minutes to ensure that the components are fully adsorbed and form a raw material base with suitable viscosity; the suitable viscosity of the raw material base is 5000-8000 mPa·s; A4. The semi-wet mixture prepared in A3 is fed into an extrusion granulator to form granules through granulation, drying and screening. Finally, the granules are sealed in a packaging bag protected by nitrogen gas and stored in a dry, light-proof environment. The process parameters of the granulation are as follows: a round hole mold with a diameter of 2 to 5 mm is selected, and extrusion molding is performed at an extrusion pressure of 8 to 12 MPa. During the extrusion process, the temperature is kept at 40 to 55°C to maintain the viscosity and homogeneity of the raw materials. The extruded strip material is cut into primary granules with a length of 2 to 5 mm by a cutting device. The process parameters of the drying and screening are as follows: the primary granules are placed in a liquid nitrogen freezing device for rapid freezing for 30 to 60 minutes, and then transferred to a vacuum drying oven for freeze drying under a vacuum pressure of ≤5 Pa. The drying temperature is controlled at 35 to 40°C and the drying time is 12 to 24 hours until the moisture content of the granules is less than 5%. The dried granules are screened by a screening machine to control the particle size to 2 to 5 The particle size is within the range of mm, and unqualified particles are removed. The removed particles can be added back into the production process for secondary processing.

[0019] (3) Beneficial technical effects 1. The present invention achieves significant improvement in functionality and stability through the innovative design of the N-acetylneuraminic acid gel network, solves the problems of poor stability and short release rate in traditional technologies, and the components work together to ensure long-term release and wide application, promoting the development of the functional food industry.

[0020] 2. The present invention achieves significant improvement in nutritional functionality and processing stability through the synergistic effect of multifunctional formula and components, solves the problems of nutritional deficiency and structural instability in the prior art, has both high-quality taste and wide application value, and promotes the development of the functional food industry.

[0021] 3. The present invention achieves efficient preparation of high-purity N-acetylneuraminic acid by optimizing the fermentation, purification and freeze-drying processes, thereby solving the problems of low purity and poor stability in the prior art. The components synergistically improve metabolic efficiency and product applicability, and have broad application value and industry driving force.

[0022] 4. The present invention realizes a functional composition with high nutrition, strong stability and anti-aging and skin rejuvenation by optimizing the formula and process. The synergistic effect between the components improves the product performance, solves the problem of insufficient nutritional considerations and processing stability in the prior art, and has broad application prospects and industry driving force. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a scanning electron microscope morphology image of the N-acetylneuraminic acid gel prepared in Example 1 of the present invention. DETAILED DESCRIPTION

[0024] 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.

[0025] Example 1 A skin rejuvenation and anti-aging composition based on N-acetylneuraminic acid comprises the following components, measured by weight: 5.0 parts of N-acetylneuraminic acid gel, 15 parts of skim milk powder, 10 parts of whole milk powder, 20 parts of xylitol, 1.0 parts of prebiotics, 5 parts of milk mineral salts, 1.0 parts of colostrum alkaline protein, 2.0 parts of bird's nest powder, 1.0 parts of pectin, 0.02 parts of vitamin E, 2.0 parts of sorbitol, 0.1 parts of flavor, and 50 parts of deionized water.

[0026] The prebiotics are a mixture of fructooligosaccharide, galacto-oligosaccharide and inulin in a weight ratio of 1:1:2.0.

[0027] The preparation method of the N-acetylneuraminic acid gel of the present embodiment is as follows: the N-acetylneuraminic acid loaded whey protein isolate solution and the polysaccharide mixed solution are mixed in a volume ratio of 2.0:4.0, and stirred at a stirring rate of 300 rpm for 10 min using a magnetic stirrer at 40°C to form a uniform composite solution; the obtained composite solution is placed in a 75°C water bath and heated for 5 min to induce a gelation process, and after the heating is completed, the solution is quickly transferred to flowing cold water to cool to room temperature, and the cooled solution is then transferred to a closed container and allowed to stand at 4°C for 12 h to obtain the N-acetylneuraminic acid gel.

[0028] The preparation method of the N-acetylneuraminic acid loaded whey protein isolate solution of the present embodiment is as follows: by weight, at room temperature, 5.0 parts of whey protein isolate and 100 parts of distilled water are stirred at a stirring rate of 300 rpm using a magnetic stirrer for 180 minutes to obtain a whey protein isolate solution for use; then 3.0 parts of N-acetylneuraminic acid are added, and the stirring is continued at a stirring rate of 300 rpm for 15 minutes at room temperature, and the above solution is filtered through a filter membrane with a pore size of 0.45 μm to remove particles, and the obtained filtrate is the N-acetylneuraminic acid loaded whey protein isolate solution.

[0029] The preparation method of the polysaccharide mixed solution of the present embodiment is as follows: 5.0 parts of pectin and 4.0 parts of carboxymethyl cellulose are weighed respectively by weight, and added one by one to 100 parts of distilled water, and allowed to stand at room temperature for 20 minutes to allow the polysaccharide to fully absorb water and swell; the polysaccharide solution after standing is placed in a water bath at 55°C and heated for 30 minutes, and stirred at a stirring rate of 300 rpm for 30 minutes to obtain a polysaccharide mixed solution.

[0030] The preparation method of N-acetylneuraminic acid of the present embodiment is: S1. Strain activation and fermentation culture: The Escherichia coli K12 strain is revived, pre-cultured and fermented to obtain a fermentation broth; S2. Collection and disruption of bacterial cells: The fermentation broth is centrifuged and ultrasonically disrupted to obtain a suspension of intracellular metabolites; S3. Product separation and purification: The intracellular metabolite suspension is concentrated by ultrafiltration, ethanol precipitation and mineral adsorption to obtain a purified product solution; S4. Concentration, dialysis and freeze-drying: The purified product solution is concentrated, dialyzed and freeze-dried to obtain N-acetylneuraminic acid.

[0031] The specific steps of S1 of this embodiment are as follows: taking Escherichia coli K1 strain as a raw material, taking the strain out from storage conditions and rapidly thawing it, and then inoculating it into a composite culture medium to obtain a mixed solution, wherein the composite culture medium includes 8 g / L yeast extract, 8 g / L tryptone and 4 g / L NaCl, and adjusting the pH of the composite solution to 7.2 with a 0.05wt.% NaOH solution, and then placing the mixed solution in a 500mL conical flask, stirring at a stirring rate of 120 rpm for 8 h, until a pre-culture solution with an optical density of 0.7 at a wavelength of 600 nm is formed; taking another 100 mL of the composite culture medium and placing it in a 500mL conical flask, inoculating the obtained pre-culture solution into the composite culture medium according to 0.8% of the volume of the composite culture medium, stirring at 120 rpm for 8 h at 35.0°C, until a seed solution with an optical density of 1.4 at a wavelength of 600 nm is formed; L basic defined medium was added into the disposable reaction bag bioreactor, and the seed liquid was inoculated into the basic defined medium at 2.5% of the volume of the defined medium. The platform angle was set to 8°, the temperature to 36°C, the dissolved oxygen level to 45%, the oxygen supply rate to 0.9 L / min, and the fermentation was carried out for 36 h until the fermentation was completed.

[0032] The basic defined culture medium of this embodiment uses 10 g / L glucose as the main carbon source, 2 g / L ammonium sulfate as the inorganic nitrogen source, 1 g / L potassium dihydrogen phosphate and 1 g / L potassium hydrogen phosphate as the phosphorus source and buffer; ferrous sulfate, zinc sulfate, manganese chloride and copper sulfate pentahydrate are added in amounts of 10 mg / L, 10 mg / L, 5 mg / L and 1 mg / L, respectively; optionally, vitamin B1 is further included as a cofactor, and the addition amount is 0.1 mg / L; and 10 mg / L chelating agent EDTA, and the pH of the basic defined culture medium is adjusted to 7.4 with 0.5wt.% sodium hydroxide solution.

[0033] The specific steps of Example S2 of this embodiment are as follows: the fermentation broth after fermentation is centrifuged at a centrifugal rate of 16000 rpm and 90 mL / min at 4±1°C, the obtained bacterial precipitate is collected and placed in an ice-water mixed bath, and crushed using an ultrasonic crusher, with the amplitude set to 95% and crushed for 25 s, followed by cooling in an ice-water mixed bath for 25 s, and the crushing process is repeated 3 times until the cell wall is fully lysed and the intracellular products are released to obtain a cell suspension.

[0034] The specific steps of S3 of this embodiment are as follows: the cell suspension after disruption is subjected to ultrafiltration and concentration treatment by a cross-flow ultrafiltration device, wherein the device uses a filter membrane with a molecular weight cutoff limit of 8 kDa, the mixed solution inlet pressure is controlled at 3.5 bar, and after the mixed solution is concentrated to 1 / 18 of the original volume, ethanol is gradually added to the concentrate for ethanol precipitation treatment, and the volume ratio of ethanol to precipitate is adjusted to 78:100, followed by centrifugation at a centrifugal rate of 4000 rpm at 4±1°C for 8 min for separation and precipitation treatment; the obtained precipitate is dissolved in deionized water, wherein the volume ratio of deionized water to precipitate is 150:100, and then ultrafiltration concentration, ethanol precipitation and separation and precipitation treatment are repeated twice, until impurities are removed and the target product is concentrated to obtain a purified solution; the obtained purified solution is mixed with a pre-equilibrated clay mineral at a mass ratio of 0.9:1, and the purified product solution and the clay mineral are further separated by centrifugation, wherein the centrifugation condition is 4000 rpm, and the centrifugation is carried out at 4±1°C for 12 min, and the upper layer solution is taken to finally obtain a further purified product solution.

[0035] The pre-equilibrated clay mineral of this embodiment is a clay mineral that is oscillated in a 90 mM sodium phosphate buffer at pH 6.8 for 0.8 h, stirred at 120 rpm for 2.5 h after mixing, and centrifuged at a centrifugal rate of 4000 rpm at 4±1°C for 12 min to obtain a precipitate that is a pre-equilibrated clay mineral.

[0036] The specific steps of Example S4 are as follows: the purified product solution is concentrated through a centrifugal filter tube with a molecular weight cutoff of 8 kDa, and centrifuged at a centrifugal rate of 4000 rpm at 4±1°C. The mixed solution is concentrated to 1 / 4 of the original volume and then subjected to three-stage dialysis treatment using a dialysis membrane with a molecular weight cutoff of 12 kDa. In the first stage, 4.5 L of a deionized water solution containing 5.4 g / L NaCl is used as a dialysis fluid for 22 h. In the second and third stages, 4.5 L of a deionized water solution with a pH of 8.8 NaOH is used as a dialysis fluid for 22 h. After the dialysis is completed, the solution obtained in the dialysis bag is transferred to a freeze-drying device and freeze-dried under a vacuum condition of 0.03 mbar until it is completely dehydrated to obtain the final N-acetylneuraminic acid powder.

[0037] A preparation method of a skin rejuvenation and anti-aging composition based on N-acetylneuraminic acid in this embodiment comprises the following steps: A1. Add deionized water to a reactor, heat to 60 ° C, and stir at this temperature at a stirring rate of 200 rpm, add xylitol and sorbitol in turn, and continue stirring for 10 minutes until they are evenly dissolved to form a transparent sugar alcohol solution, further add prebiotics to the sugar alcohol solution, and continue stirring at a stirring rate of 200 rpm for 10 minutes, then add vitamin E, and stir at 200 rpm for 5 minutes to form a uniform functional liquid base for standby use; A2. The skim milk powder and the whole milk powder were added to a dry mixer and mixed at a stirring rate of 300 rpm for 3 min, followed by the addition of milk mineral salts, colostrum alkaline protein and bird's nest powder, and continued to mix at a stirring rate of 200 rpm for 2 min until a uniform powder mixture was formed, followed by the addition of pectin and mixing at a stirring rate of 200 rpm for 5 min to form a uniform solid powder mixture for standby; A3. Add N-acetylneuraminic acid gel to another stirred tank, slowly add the functional liquid base prepared by S1, and stir at a stirring rate of 200 rpm at room temperature for 10 minutes to form a uniform functional liquid mixture; slowly add the solid powder mixture and flavor prepared by A2 to the liquid mixture, and stir at a stirring rate of 200 rpm for 15 minutes until a uniform semi-wet mixture is formed, and leave the mixture to stand at room temperature for 20 minutes to ensure that the components are fully adsorbed and form a raw material base with suitable viscosity; the suitable viscosity of the raw material base is 5000 mPa·s; A4. The semi-wet mixture prepared in A3 is fed into an extrusion granulator to form granules through granulation, drying and screening. Finally, the granules are sealed in a packaging bag protected by nitrogen gas and stored in a dry, light-proof environment. The process parameters of the granulation are as follows: a round hole mold with a diameter of 2 mm is selected, and extrusion molding is performed at an extrusion pressure of 8 MPa. During the extrusion process, the temperature is kept at 40°C to maintain the viscosity and homogeneity of the raw materials. The extruded strip material is cut into primary granules with a length of 2 mm by a cutting device. The process parameters of the drying and screening are as follows: the primary granules are placed in a liquid nitrogen freezing device for rapid freezing for 30 minutes, and then transferred to a vacuum drying oven for freeze drying under a vacuum pressure of ≤5 Pa. The drying temperature is controlled at 35°C and the drying time is 12 hours until the moisture content of the granules is less than 5%. The dried granules are screened by a screening machine to control the particle size within the range of 2 mm, and unqualified particles are removed. The removed particles can be re-added to the production process for secondary processing.

[0038] Figure 1The scanning electron microscope morphology image of the N-acetylneuraminic acid gel prepared in Example 1 of the present invention can be clearly observed from the figure that the gel presents a uniform three-dimensional network structure, which is formed by the joint action of polysaccharides and whey protein isolates, has a uniform surface distribution and a moderate pore size, indicating that the polysaccharide solution and the N-acetylneuraminic acid-loaded whey protein isolate solution are successfully cross-linked during the gelation process to form a stable gel network; at the same time, the distribution and morphology of the pores further prove that the gel prepared by the present invention has good structural integrity and excellent physical stability, which provides an ideal material basis for the uniform loading and sustained release of N-acetylneuraminic acid.

[0039] Example 2 A skin rejuvenation and anti-aging composition based on N-acetylneuraminic acid comprises the following components, measured by weight: 5.9 parts of N-acetylneuraminic acid gel, 16.5 parts of skim milk powder, 11.5 parts of whole milk powder, 21.5 parts of xylitol, 1.6 parts of prebiotics, 6.5 parts of milk mineral salts, 1.6 parts of colostrum alkaline protein, 2.9 parts of bird's nest powder, 1.6 parts of pectin, 0.029 parts of vitamin E, 2.6 parts of sorbitol, 0.16 parts of flavor, and 59 parts of deionized water.

[0040] The prebiotics are a mixture of fructooligosaccharide, galacto-oligosaccharide and inulin in a weight ratio of 1:1:2.6.

[0041] The preparation method of the N-acetylneuraminic acid gel of the present embodiment is as follows: the N-acetylneuraminic acid loaded whey protein isolate solution and the polysaccharide mixed solution are mixed in a volume ratio of 2.6:4.6, and stirred at a stirring rate of 330 rpm using a magnetic stirrer for 11.5 min at 43° C. to form a uniform composite solution; the obtained composite solution is placed in a 78° C. water bath and heated for 6.5 min to induce a gelation process, and after the heating is completed, the solution is quickly transferred to flowing cold water to cool to room temperature, and the cooled solution is then transferred to a closed container and allowed to stand at 4° C. for 15.6 h to obtain the N-acetylneuraminic acid gel.

[0042] The preparation method of the N-acetylneuraminic acid loaded whey protein isolate solution of the present embodiment is as follows: by weight, at room temperature, 6.5 parts of whey protein isolate and 115 parts of distilled water are stirred at a stirring rate of 330 rpm using a magnetic stirrer for 198 minutes to obtain a whey protein isolate solution for standby use; then 4.5 parts of N-acetylneuraminic acid are added, and the stirring is continued at a stirring rate of 315 rpm for 19.5 minutes at room temperature, and the above solution is filtered through a filter membrane with a pore size of 0.45 μm to remove particles, and the obtained filtrate is the N-acetylneuraminic acid loaded whey protein isolate solution.

[0043] The preparation method of the polysaccharide mixed solution of the present embodiment is as follows: 7.1 parts of pectin and 5.8 parts of carboxymethyl cellulose are weighed respectively by weight, and added one by one to 112 parts of distilled water, and allowed to stand at room temperature for 23 minutes to allow the polysaccharide to fully absorb water and swell; the polysaccharide solution after standing is placed in a water bath at 56.5°C and heated for 34.5 minutes, and stirred at a stirring rate of 315 rpm for 34.5 minutes to obtain a polysaccharide mixed solution.

[0044] The preparation method of N-acetylneuraminic acid of the present embodiment is: S1. Strain activation and fermentation culture: The Escherichia coli K12 strain is revived, pre-cultured and fermented to obtain a fermentation broth; S2. Collection and disruption of bacterial cells: The fermentation broth is centrifuged and ultrasonically disrupted to obtain a suspension of intracellular metabolites; S3. Product separation and purification: The intracellular metabolite suspension is concentrated by ultrafiltration, ethanol precipitation and mineral adsorption to obtain a purified product solution; S4. Concentration, dialysis and freeze-drying: The purified product solution is concentrated, dialyzed and freeze-dried to obtain N-acetylneuraminic acid.

[0045] The specific steps of S1 of this embodiment are as follows: taking Escherichia coli K1 strain as a raw material, taking the strain out from storage conditions and rapidly thawing it, and then inoculating it into a composite culture medium to obtain a mixed solution, wherein the composite culture medium includes 9.2 g / L yeast extract, 9.2 g / L tryptone and 4.6 g / L NaCl, and adjusting the pH of the composite solution to 7.26 with a 0.05wt.% NaOH solution, and then placing the mixed solution in a 500mL conical flask, stirring at a stirring rate of 129 rpm for 9.2 h, until a pre-culture solution with an optical density of 0.82 at a wavelength of 600 nm is formed; taking another 106 mL of the composite culture medium and placing it in a 500 mL conical flask, inoculating the obtained pre-culture solution into the composite culture medium according to 0.92% of the volume of the composite culture medium, stirring at 129 rpm for 9.2 h at 35.6°C, until a seed solution with an optical density of 1.64 at a wavelength of 600 nm is formed; L basic defined medium was added into the disposable reaction bag bioreactor, and the seed liquid was inoculated into the basic defined medium according to 2.8% of the volume of the defined medium. The platform angle was set to 9.2°, the temperature to 36.6°C, the dissolved oxygen level to 48%, the oxygen supply rate to 0.96 L / min, and the fermentation was continued for 46.8 h until the fermentation was completed.

[0046] The basic defined culture medium of this embodiment uses 13 g / L glucose as the main carbon source, 2.9 g / L ammonium sulfate as the inorganic nitrogen source, 1.15 g / L potassium dihydrogen phosphate and 1.15 g / L potassium hydrogen phosphate as the phosphorus source and buffer; ferrous sulfate, zinc sulfate, manganese chloride and copper sulfate pentahydrate are added in amounts of 22 mg / L, 22 mg / L, 9.5 mg / L and 3.7 mg / L, respectively; optionally, vitamin B1 is further included as a cofactor, the addition amount is 0.37 mg / L; and 22 mg / L chelating agent EDTA, and the pH of the basic defined culture medium is adjusted to 7.46 with 0.5wt.% sodium hydroxide solution.

[0047] The specific steps of Example S2 of this embodiment are as follows: the fermentation broth after fermentation is centrifuged at a centrifugal rate of 16600 rpm and 96 mL / min at 4±1°C, the obtained bacterial precipitate is collected and placed in an ice-water mixed bath, and crushed using an ultrasonic crusher, with the amplitude set to 96.5% and crushed for 28 s, followed by cooling in an ice-water mixed bath for 28 s, and the crushing process is repeated 3 times until the cell wall is fully lysed and the intracellular products are released to obtain a cell suspension.

[0048] The specific steps of S3 of this embodiment are as follows: the cell suspension after disruption is subjected to ultrafiltration concentration treatment by a cross-flow ultrafiltration device, wherein the device uses a filter membrane with a molecular weight cutoff limit of 9.2 kDa, the mixed solution inlet pressure is controlled at 3.8 bar, and after the mixed solution is concentrated to 1 / 19.2 of the original volume, ethanol is gradually added to the concentrated solution for ethanol precipitation treatment, and the volume ratio of ethanol to precipitate is adjusted to 79.2:100, followed by centrifugation at a centrifugal rate of 4300 rpm and at 4±1°C for 10.1 min for separation and precipitation treatment; the obtained precipitate is dissolved in deionized water, wherein the volume ratio of deionized water to precipitate is 159:100, and then ultrafiltration concentration, ethanol precipitation and separation and precipitation treatment are repeated for a total of 2.6 times until impurities are removed and the target product is concentrated to obtain a purified solution; the obtained purified solution is mixed with a pre-equilibrated clay mineral at a mass ratio of 0.93:1, and the purified product solution and the clay mineral are further separated by centrifugation, and the centrifugation conditions are 4300 rpm and 4±1°C for 13.8 min, take the upper solution, and finally obtain the further purified product solution.

[0049] The pre-equilibrated clay mineral of this embodiment is a clay mineral that is oscillated in a 96 mM sodium phosphate buffer at pH 6.92 for 0.92 h, stirred at 126 rpm for 2.8 h after mixing, and centrifuged at a centrifugal rate of 4300 rpm at 4±1°C for 13.8 min to obtain a precipitate that is a pre-equilibrated clay mineral.

[0050] The specific steps of Example S4 are as follows: the purified product solution is concentrated through a centrifugal filter tube with a molecular weight cutoff of 9.2 kDa, and centrifuged at a centrifugal rate of 4300 rpm at 4±1°C. The mixed solution is concentrated to 1 / 4.6 of the original volume and then dialyzed using a dialysis membrane with a molecular weight cutoff of 13.2 kDa. In the first stage, 4.8 L of a deionized water solution containing 5.46 g / L NaCl is used as a dialysate for 23.2 h. In the second and third stages, 4.8 L of a deionized water solution with a pH of 8.92 NaOH is used as a dialysate for 23.2 h. After the dialysis is completed, the solution obtained in the dialysis bag is transferred to a freeze-drying device and freeze-dried under a vacuum condition of 0.036 mbar until it is completely dehydrated to obtain the final N-acetylneuraminic acid powder.

[0051] A preparation method of a skin rejuvenation and anti-aging composition based on N-acetylneuraminic acid in this embodiment comprises the following steps: A1. Add deionized water to a reactor, heat to 63 ° C, and stir at this temperature at a stirring rate of 230 rpm, add xylitol and sorbitol in turn, and continue stirring for 11.5 minutes until they are evenly dissolved to form a transparent sugar alcohol solution, further add prebiotics to the sugar alcohol solution, and continue stirring at a stirring rate of 230 rpm for 11.5 minutes, then add vitamin E, and stir at 215 rpm for 6.5 minutes to form a uniform functional liquid base for use; A2. The skim milk powder and the whole milk powder were added to a dry mixer and mixed at a stirring rate of 330 rpm for 3.6 min, followed by the addition of milk mineral salts, colostrum alkaline protein and bird's nest powder, and continued to mix at a stirring rate of 230 rpm for 2.3 min until a uniform powder mixture was formed, followed by the addition of pectin and mixing at a stirring rate of 215 rpm for 6.5 min to form a uniform solid powder mixture for standby; A3. Add N-acetylneuraminic acid gel to another stirred tank, slowly add the functional liquid base prepared by S1, and stir at a stirring rate of 215 rpm for 11.5 minutes at room temperature to form a uniform functional liquid mixture; slowly add the solid powder mixture and flavor prepared by A2 to the liquid mixture, and stir at a stirring rate of 215 rpm for 16.5 minutes until a uniform semi-wet mixture is formed, and leave the mixture to stand at room temperature for 23 minutes to ensure that the components are fully adsorbed and to form a raw material base with suitable viscosity; the suitable viscosity of the raw material base is 5900 mPa·s; A4. The semi-wet mixture prepared in A3 is fed into an extrusion granulator to form granules through granulation, drying and screening. Finally, the granules are sealed in a packaging bag protected by nitrogen gas and stored in a dry, light-proof environment. The process parameters of the granulation are as follows: a round hole mold with a diameter of 2.9 mm is selected, and extrusion molding is performed at an extrusion pressure of 9.2 MPa. During the extrusion process, the temperature is kept at 44.5°C to maintain the viscosity and homogeneity of the raw materials. The extruded strip material is cut into primary granules with a length of 2.9 mm by a cutting device. The process parameters of the drying and screening are as follows: the primary granules are placed in a liquid nitrogen freezing device for rapid freezing for 39 minutes, and then transferred to a vacuum drying oven for freeze drying under a vacuum pressure of ≤5 Pa. The drying temperature is controlled at 36.5°C and the drying time is 15.6 hours until the moisture content of the granules is less than 5%. The dried granules are screened by a screening machine to control the particle size within the range of 2.9 mm, and unqualified particles are removed. The removed particles can be re-added to the production process for secondary processing.

[0052] Example 3 A skin rejuvenation and anti-aging composition based on N-acetylneuraminic acid comprises the following components in parts by weight: 7 parts of N-acetylneuraminic acid gel, 18 parts of skim milk powder, 13 parts of whole milk powder, 23 parts of xylitol, 2 parts of prebiotics, 8 parts of milk mineral salts, 2 parts of colostrum alkaline protein, 4 parts of bird's nest powder, 2 parts of pectin, 0.04 parts of vitamin E, 3 parts of sorbitol, 0.2 parts of flavor, and 65 parts of deionized water.

[0053] The prebiotics are a mixture of fructooligosaccharide, galacto-oligosaccharide and inulin in a weight ratio of 1:1:3.

[0054] The preparation method of the N-acetylneuraminic acid gel of the present embodiment is as follows: the N-acetylneuraminic acid loaded whey protein isolate solution and the polysaccharide mixed solution are mixed in a volume ratio of 3:5, and stirred at a stirring rate of 350 rpm using a magnetic stirrer for 13 min at 45°C to form a uniform composite solution; the obtained composite solution is placed in a water bath at 80°C and heated for 8 min to induce a gelation process, and after the heating is completed, the solution is quickly moved to flowing cold water to cool to room temperature, and the cooled solution is transferred to a closed container, and allowed to stand at 4°C for 18 h to obtain the N-acetylneuraminic acid gel.

[0055] The preparation method of the N-acetylneuraminic acid loaded whey protein isolate solution of the present embodiment is as follows: by weight, at room temperature, 8 parts of whey protein isolate and 125 parts of distilled water are stirred at a stirring rate of 350 rpm using a magnetic stirrer for 210 minutes to obtain a whey protein isolate solution for use; then 6 parts of N-acetylneuraminic acid are added, and stirring is continued at a stirring rate of 325 rpm for 23 minutes at room temperature, and the solution is filtered through a filter membrane with a pore size of 0.45 μm to remove particles, and the obtained filtrate is the N-acetylneuraminic acid loaded whey protein isolate solution.

[0056] The preparation method of the polysaccharide mixed solution of the present embodiment is as follows: 9 parts of pectin and 7 parts of carboxymethyl cellulose are weighed respectively by weight, and added one by one to 120 parts of distilled water, and allowed to stand at room temperature for 25 minutes to allow the polysaccharide to fully absorb water and swell; the polysaccharide solution after standing is placed in a 58°C water bath and heated for 38 minutes, and stirred at a stirring rate of 325 rpm for 38 minutes to obtain a polysaccharide mixed solution.

[0057] The preparation method of N-acetylneuraminic acid of the present embodiment is: S1. Strain activation and fermentation culture: The Escherichia coli K12 strain was revived, pre-cultured and fermented to obtain fermentation broth; S2. Collection and disruption of bacterial cells: The fermentation broth was centrifuged and ultrasonically disrupted to obtain a suspension of intracellular metabolites; S3. Product separation and purification: The intracellular metabolite suspension is concentrated by ultrafiltration, ethanol precipitation and mineral adsorption to obtain a purified product solution; S4. Concentration, dialysis and freeze-drying: The purified product solution is concentrated, dialyzed and freeze-dried to obtain N-acetylneuraminic acid.

[0058] The specific steps of S1 of this embodiment are as follows: taking Escherichia coli K1 strain as a raw material, taking out the strain from storage conditions and rapidly thawing it, and then inoculating it into a composite culture medium to obtain a mixed solution, wherein the composite culture medium includes 10 g / L yeast extract, 10 g / L tryptone and 5 g / L NaCl, and adjusting the pH of the composite solution to 7.3 with a 0.05wt.% NaOH solution, and then placing the mixed solution in a 500mL conical flask, stirring at a stirring rate of 135 rpm for 10 hours, until a pre-culture solution with an optical density of 0.9 at a wavelength of 600 nm is formed; taking another 110 mL of the composite culture medium and placing it in a 500mL conical flask, inoculating the obtained pre-culture solution into the composite culture medium according to 1.0% of the volume of the composite culture medium, stirring at 135 rpm for 10 hours at 36°C, until a seed solution with an optical density of 1.8 at a wavelength of 600 nm is formed; L basic defined medium was added into the disposable reaction bag bioreactor, and the seed solution was inoculated into the basic defined medium at 3% of the volume of the defined medium. The platform angle was set to 10°, the temperature to 37°C, the dissolved oxygen level to 50%, and the oxygen supply rate to 1 L / min. The fermentation was continued for 54 h until the fermentation was completed. The basic defined culture medium of this embodiment uses 15 g / L glucose as the main carbon source, 3.5 g / L ammonium sulfate as the inorganic nitrogen source, 1.3 g / L potassium dihydrogen phosphate and 1.3 g / L potassium hydrogen phosphate as the phosphorus source and buffer; ferrous sulfate, zinc sulfate, manganese chloride and copper sulfate pentahydrate are added in amounts of 30 mg / L, 30 mg / L, 13 mg / L and 5 mg / L, respectively; optionally, vitamin B1 is further included as a cofactor, the addition amount is 0.6 mg / L; and 30 mg / L chelating agent EDTA, and the pH of the basic defined culture medium is adjusted to 7.5 with 0.5wt.% sodium hydroxide solution.

[0059] The specific steps of Example S2 of this embodiment are as follows: the fermentation broth after fermentation is centrifuged at 100 mL / min at a centrifugal rate of 17000 rpm and 4±1°C, the obtained bacterial precipitate is collected and placed in an ice-water mixed bath, and crushed using an ultrasonic crusher, with the amplitude set to 98% and crushed for 30 s, followed by cooling in an ice-water mixed bath for 30 s, and the crushing process is repeated 4 times until the cell wall is fully lysed and the intracellular products are released to obtain a cell suspension.

[0060] The specific steps of Example S3 are as follows: the cell suspension after disruption is subjected to ultrafiltration and concentration treatment by a cross-flow ultrafiltration device, wherein the device uses a filter membrane with a molecular weight cutoff limit of 10 kDa, the mixed solution inlet pressure is controlled at 4 bar, and after the mixed solution is concentrated to 1 / 20 of the original volume, ethanol is gradually added to the concentrate for ethanol precipitation treatment, and the volume ratio of ethanol to precipitate is adjusted to 80:100, followed by centrifugation at a centrifugal rate of 4500 rpm at 4±1°C for 11 min for separation and precipitation treatment; the obtained precipitate is dissolved in deionized water, wherein the volume ratio of deionized water to precipitate is 165:100, and then ultrafiltration concentration, ethanol precipitation and separation and precipitation treatment are repeated for a total of 3 times until impurities are removed and the target product is concentrated to obtain a purified solution; the obtained purified solution is mixed with a pre-equilibrated clay mineral at a mass ratio of 1:1, and the purified product solution and the clay mineral are further separated by centrifugation, wherein the centrifugation condition is 4500 rpm at 4±1°C for 15 min, and the upper layer solution is taken to finally obtain a further purified product solution; The pre-equilibrated clay mineral of this embodiment is a clay mineral that is oscillated in a 100 mM sodium phosphate buffer at pH 7.0 for 1 h, stirred at 130 rpm for 3 h after mixing, and centrifuged at a centrifugal rate of 4500 rpm at 4±1°C for 15 min. The precipitate obtained is the pre-equilibrated clay mineral.

[0061] The specific steps of Example S4 are as follows: the purified product solution is concentrated through a centrifugal filter tube with a molecular weight cutoff of 10 kDa, and centrifuged at a centrifugal rate of 4500 rpm at 4±1°C. The mixed solution is concentrated to 1 / 5 of the original volume and then dialyzed using a dialysis membrane with a molecular weight cutoff of 14 kDa. In the first stage, 5 L of a deionized water solution containing 5.5 g / L NaCl is used as a dialysate for 24 h. In the second and third stages, 5 L of a deionized water solution with a pH of 9.0 NaOH is used as a dialysate for 24 h. After the dialysis is completed, the solution obtained in the dialysis bag is transferred to a freeze-drying device and freeze-dried under a vacuum condition of 0.04 mbar until it is completely dehydrated to obtain the final N-acetylneuraminic acid powder.

[0062] A method for preparing a skin rejuvenation and anti-aging composition based on N-acetylneuraminic acid in this embodiment comprises the following steps: A1. Add deionized water to a reactor, heat to 65°C, and stir at this temperature at a stirring rate of 250 rpm. Add xylitol and sorbitol in turn, and continue stirring for 13 minutes until they are evenly dissolved to form a transparent sugar alcohol solution. Add prebiotics to the sugar alcohol solution and continue stirring at a stirring rate of 250 rpm for 13 minutes. Then, add vitamin E and stir at 225 rpm for 8 minutes to form a uniform functional liquid base for use. A2. Add skim milk powder and whole milk powder into a dry mixer and mix at a stirring rate of 350 rpm for 4 minutes, then add milk mineral salt, colostrum alkaline protein and bird's nest powder in sequence, continue to mix at a stirring rate of 250 rpm for 3 minutes until a uniform powder mixture is formed, then continue to add pectin and mix at a stirring rate of 225 rpm for 8 minutes to form a uniform solid powder mixture for use; A3. Add N-acetylneuraminic acid gel to another stirring kettle, slowly add the functional liquid base prepared by S1, and stir at a stirring rate of 225 rpm for 13 minutes at room temperature to form a uniform functional liquid mixture; slowly add the solid powder mixture and flavor prepared by A2 to the liquid mixture, and stir at a stirring rate of 225 rpm for 18 minutes until a uniform semi-wet mixture is formed, and leave the mixture to stand at room temperature for 25 minutes to ensure that the components are fully adsorbed and form a raw material base with suitable viscosity; the suitable viscosity of the raw material base is 6500 mPa·s; A4. The semi-wet mixture prepared in A3 is fed into an extrusion granulator to form granules through granulation, drying and screening. Finally, the granules are sealed in a packaging bag protected by nitrogen gas and stored in a dry and light-proof environment. The process parameters of the granulation are as follows: a round hole mold with a diameter of 3.5 mm is selected, and extrusion molding is performed at an extrusion pressure of 10 MPa. During the extrusion process, the temperature is kept at 48°C to maintain the viscosity and homogeneity of the raw materials. The extruded strip material is cut into primary granules with a length of 3.5 mm by a cutting device. The process parameters of the drying and screening are as follows: the primary granules are placed in a liquid nitrogen freezing device for rapid freezing for 45 minutes, and then transferred to a vacuum drying oven for freeze drying under a vacuum pressure of ≤5 Pa. The drying temperature is controlled at 38°C and the drying time is 18 hours until the moisture content of the granules is less than 5%. The dried granules are screened by a screening machine to control the particle size within the range of 3.5 mm, and unqualified particles are removed. The removed particles can be re-added to the production process for secondary processing.

[0063] Example 4 A skin rejuvenation and anti-aging composition based on N-acetylneuraminic acid comprises the following components in parts by weight: 8.0 parts of N-acetylneuraminic acid gel, 20 parts of skim milk powder, 15 parts of whole milk powder, 25 parts of xylitol, 3.0 parts of prebiotics, 10 parts of milk mineral salts, 3.0 parts of colostrum alkaline protein, 5.0 parts of bird's nest powder, 3.0 parts of pectin, 0.05 parts of vitamin E, 4.0 parts of sorbitol, 0.3 parts of flavor, and 80 parts of deionized water.

[0064] The prebiotics are a mixture of fructooligosaccharide, galacto-oligosaccharide and inulin in a weight ratio of 1:1:4.0.

[0065] The preparation method of the N-acetylneuraminic acid gel of the present embodiment is as follows: the N-acetylneuraminic acid loaded whey protein isolate solution and the polysaccharide mixed solution are mixed at a volume ratio of 4.0:6.0, and stirred at a stirring rate of 400 rpm using a magnetic stirrer for 15 minutes at 50°C to form a uniform composite solution; the obtained composite solution is placed in a water bath at 85°C and heated for 10 minutes to induce a gelation process, and after the heating is completed, the solution is quickly transferred to flowing cold water to be cooled to room temperature, and the cooled solution is then transferred to a closed container and allowed to stand at 4°C for 24 hours to obtain the N-acetylneuraminic acid gel.

[0066] The preparation method of the N-acetylneuraminic acid loaded whey protein isolate solution of the present embodiment is as follows: by weight, at room temperature, 10.0 parts of whey protein isolate and 150 parts of distilled water are stirred at a stirring rate of 400 rpm for 240 minutes using a magnetic stirrer to obtain a whey protein isolate solution for standby use; then 8.0 parts of N-acetylneuraminic acid are added, and the stirring is continued at a stirring rate of 350 rpm for 30 minutes at room temperature, and the solution is filtered through a filter membrane with a pore size of 0.45 μm to remove particles, and the obtained filtrate is the N-acetylneuraminic acid loaded whey protein isolate solution.

[0067] The preparation method of the polysaccharide mixed solution of the present embodiment is as follows: 12.0 parts of pectin and 10.0 parts of carboxymethyl cellulose are weighed respectively by weight, and added one by one to 140 parts of distilled water, and allowed to stand at room temperature for 30 minutes to allow the polysaccharide to fully absorb water and swell; the polysaccharide solution after standing is placed in a 60°C water bath and heated for 45 minutes, and stirred at a stirring rate of 350 rpm for 45 minutes to obtain a polysaccharide mixed solution.

[0068] The preparation method of N-acetylneuraminic acid of the present embodiment is: S1. Strain activation and fermentation culture: The Escherichia coli K12 strain was revived, pre-cultured and fermented to obtain fermentation broth; S2. Collection and disruption of bacterial cells: The fermentation broth was centrifuged and ultrasonically disrupted to obtain a suspension of intracellular metabolites; S3. Product separation and purification: The intracellular metabolite suspension is concentrated by ultrafiltration, ethanol precipitation and mineral adsorption to obtain a purified product solution; S4. Concentration, dialysis and freeze-drying: The purified product solution is concentrated, dialyzed and freeze-dried to obtain N-acetylneuraminic acid.

[0069] The specific steps of S1 of this embodiment are as follows: taking Escherichia coli K1 strain as a raw material, taking out the strain from the storage condition and rapidly thawing it, and then inoculating it into a composite culture medium to obtain a mixed solution, wherein the composite culture medium includes 12 g / L yeast extract, 12 g / L tryptone and 6 g / L NaCl, and adjusting the pH of the composite solution to 7.4 with a 0.05 wt.% NaOH solution, and then placing the mixed solution in a 500 mL conical flask, stirring at a stirring rate of 150 rpm for 12 h, until a pre-culture solution with an optical density of 1.1 at a wavelength of 600 nm is formed; taking another 120 mL of the composite culture medium and placing it in a 500 mL conical flask, inoculating the obtained pre-culture solution into the composite culture medium according to 1.2% of the volume of the composite culture medium, and incubating at 37°C at a stirring rate of 150 rpm for 12 h. rpm for 12 h until a seed solution with an optical density of 2.2 at a wavelength of 600 nm is formed; 12 L of basic defined culture medium is added to a disposable reaction bag bioreactor, and the seed solution is inoculated into the basic defined culture medium at 3.5% of the volume of the defined culture medium, and the platform angle is set to 12°, the temperature to 38°C, the dissolved oxygen level to 55%, the oxygen supply rate to 1.1 L / min, and the fermentation is carried out for 72 h until the fermentation is completed; The basic defined culture medium of this embodiment uses 20g / L glucose as the main carbon source, 5g / L ammonium sulfate as the inorganic nitrogen source, 1.5g / L potassium dihydrogen phosphate and 1.5g / L potassium hydrogen phosphate as the phosphorus source and buffer; ferrous sulfate, zinc sulfate, manganese chloride and copper sulfate pentahydrate are added in amounts of 50mg / L, 50mg / L, 20mg / L and 10mg / L, respectively; optionally, vitamin B1 is further included as an auxiliary factor, the addition amount is 1mg / L; and 50mg / L chelating agent EDTA, and the pH of the basic defined culture medium is adjusted to 7.6 with 0.5wt.% sodium hydroxide solution.

[0070] The specific steps of S2 in this embodiment are as follows: the fermentation broth after fermentation is passed through a continuous centrifuge at a centrifugal rate of 18000 rpm and centrifuged at 110 mL / min at 4±1°C, the resulting bacterial precipitate is collected and placed in an ice-water mixed bath, and crushed using an ultrasonic crusher, with the amplitude set to 100% and crushed for 35 seconds, followed by cooling in an ice-water mixed bath for 35 seconds, and the crushing process is repeated 4 times until the cell wall is fully lysed and the intracellular products are released to obtain a cell suspension.

[0071] The specific steps of Example S3 are as follows: the cell suspension after disruption is subjected to ultrafiltration and concentration treatment by a cross-flow ultrafiltration device, wherein the device uses a filter membrane with a molecular weight cutoff limit of 12 kDa, the mixed solution inlet pressure is controlled at 4.5 bar, and the mixed solution is concentrated to 1 / 22 of the original volume, and then ethanol is gradually added to the concentrate for ethanol precipitation treatment, and the volume ratio of ethanol to precipitate is adjusted to 82:100, and then centrifuged at a centrifugal rate of 5000 rpm at 4±1°C for 15 min for separation and precipitation treatment; the obtained precipitate is dissolved in deionized water, wherein the volume ratio of deionized water to precipitate is 180:100, and then ultrafiltration concentration, ethanol precipitation and separation and precipitation treatment are repeated for a total of 4 times until impurities are removed and the target product is concentrated to obtain a purified solution; the obtained purified solution is mixed with a pre-equilibrated clay mineral at a mass ratio of 1:1, and the purified product solution and the clay mineral are further separated by centrifugation, and the centrifugation condition is 5000 rpm, and the centrifugation is performed at 4±1°C for 18 min, and the upper layer solution is taken to finally obtain a further purified product solution; The pre-equilibrated clay mineral of this embodiment is a clay mineral that is oscillated in a 110 mM sodium phosphate buffer at pH 7.2 for 1.2 h, stirred at 140 rpm for 3.5 h after mixing, and centrifuged at a centrifugal rate of 5000 rpm at 4±1°C for 18 min. The precipitate obtained is the pre-equilibrated clay mineral.

[0072] The specific steps of Example S4 are as follows: the purified product solution is concentrated through a centrifugal filter tube with a molecular weight cutoff of 12 kDa, and the mixture is centrifuged at a centrifugal rate of 5000 rpm at 4±1°C. After the mixed solution is concentrated to 1 / 6 of the original volume, it is dialyzed using a dialysis membrane with a molecular weight cutoff of 16 kDa in three stages. In the first stage, 5.5 L of a deionized water solution containing 5.6 g / L NaCl is used as a dialysate for 26 h. In the second and third stages, 5.5 L of a deionized water solution with a pH of 9.2 NaOH is used as a dialysate for 26 h. After the dialysis is completed, the solution obtained in the dialysis bag is transferred to a freeze-drying device, and freeze-dried under a vacuum condition of 0.05 mbar until it is completely dehydrated to obtain the final N-acetylneuraminic acid powder.

[0073] A method for preparing a skin rejuvenation and anti-aging composition based on N-acetylneuraminic acid in this embodiment comprises the following steps: A1. Add deionized water to a reactor, heat it to 70°C, and stir at this temperature at a stirring rate of 300 rpm, add xylitol and sorbitol in turn, and continue stirring for 15 minutes until they are evenly dissolved to form a transparent sugar alcohol solution, add prebiotics to the sugar alcohol solution, and continue stirring at a stirring rate of 300 rpm for 15 minutes, then add vitamin E, and stir at 250 rpm for 10 minutes to form a uniform functional liquid base for use; A2. Add skim milk powder and whole milk powder into a dry mixer and mix at a stirring rate of 400 rpm for 5 minutes, then add milk mineral salt, colostrum alkaline protein and bird's nest powder in sequence, continue to mix at a stirring rate of 300 rpm for 3 minutes until a uniform powder mixture is formed, then continue to add pectin and mix at a stirring rate of 250 rpm for 10 minutes to form a uniform solid powder mixture for use; A3. Add N-acetylneuraminic acid gel to another stirring kettle, slowly add the functional liquid base prepared by S1, stir at a stirring rate of 250 rpm at room temperature for 15 minutes to form a uniform functional liquid mixture; slowly add the solid powder mixture and flavor prepared by A2 to the liquid mixture, stir at a stirring rate of 250 rpm for 20 minutes until a uniform semi-wet mixture is formed, and leave the mixture to stand at room temperature for 30 minutes to ensure that the components are fully adsorbed and form a raw material base with suitable viscosity; the suitable viscosity of the raw material base is 8000 mPa·s; A4. The semi-wet mixture prepared in A3 is fed into an extrusion granulator to form granules through granulation, drying and screening. Finally, the granules are sealed in a packaging bag protected by nitrogen gas and stored in a dry, light-proof environment. The process parameters of the granulation are as follows: a round hole mold with a diameter of 5 mm is selected, and extrusion molding is performed at an extrusion pressure of 12 MPa. During the extrusion process, the temperature is kept at 55°C to maintain the viscosity and homogeneity of the raw materials. The extruded strip material is cut into primary granules with a length of 5 mm by a cutting device. The process parameters of the drying and screening are as follows: the primary granules are placed in a liquid nitrogen freezing device for rapid freezing for 60 minutes, and then transferred to a vacuum drying oven for freeze drying under a vacuum pressure of ≤5 Pa. The drying temperature is controlled at 40°C and the drying time is 24 hours until the moisture content of the granules is less than 5%. The dried granules are screened by a screening machine to control the particle size within the range of 5 mm, and unqualified particles are removed. The removed particles can be re-added to the production process for secondary processing.

[0074] Comparative Example 1 The method is substantially the same as Example 1, except that an equal amount of N-acetylneuraminic acid is used to replace N-acetylneuraminic acid gel.

[0075] Comparative Example 2 The method is substantially the same as Example 1, except that N-acetylneuraminic acid gel is not added.

[0076] Comparative Example 3 The method is substantially the same as Example 1, except that the prebiotics are not contained and the same amount of fructose is used instead.

[0077] Comparative Example 4 The method is substantially the same as Example 1, except that the weight ratio of fructo-oligosaccharide, galacto-oligosaccharide and inulin in the prebiotic is 1:1:1.

[0078] Comparative Example 5 The method is substantially the same as Example 1, except that milk mineral salts and colostrum alkaline protein are not contained, and corresponding portions are supplemented with skim milk powder.

[0079] Comparative Example 6 The method is basically the same as Example 1, except that when preparing the N-acetylneuraminic acid gel, no N-acetylneuraminic acid-loaded whey protein isolate solution is added.

[0080] Comparative Example 7 The method is basically the same as Example 1, except that no polysaccharide mixed solution is added when preparing the N-acetylneuraminic acid gel.

[0081] Comparative Example 8 The method is basically the same as Example 1, except that the method does not contain bird's nest powder and uses the same amount of pectin instead.

[0082] Comparative Example 9 The method is basically the same as Example 1, except that no trace elements (ferrous sulfate, zinc sulfate, manganese chloride and copper sulfate pentahydrate) are added during the fermentation of N-acetylneuraminic acid.

[0083] Performance Test: Skin rejuvenation and anti-aging performance experiment 30 healthy volunteers aged 30-50 were selected and divided into an experimental group and a control group, with 15 people in each group. The experimental group consumed 2 capsules of the composition based on N-acetylneuraminic acid every day, and the control group consumed an ordinary composition without N-acetylneuraminic acid for 12 weeks. The skin moisture content, elasticity, fine line depth and spot area of ​​the volunteers were tested every 4 weeks using the Visia skin analysis system. The skin improvement of the experimental group and the control group was compared to evaluate the skin rejuvenation and anti-aging effects of the composition. The expected results show that the experimental group has a significant improvement in moisture content and elasticity, and a significant reduction in fine line depth and spot area, thereby proving the skin rejuvenation and anti-aging properties of the composition.

[0084] Stability test: The composition was placed in a constant temperature environment of 45°C to simulate room temperature storage and high temperature transportation conditions, and 50% relative humidity was set as a variable. The storage time was 6 months. Samples were taken every 30 days to test the appearance, flavor, hardness and N-acetylneuraminic acid content of the composition, and the content stability was determined by HPLC. The heat resistance and moisture resistance of the composition were evaluated by statistically analyzing the changes in the performance of the composition under 45°C humidity conditions.

[0085] Persistence experiment: Under conditions simulating the human digestive environment, an in vitro release experiment was used to evaluate the release persistence of N-acetylneuraminic acid in the composition. The composition sample was placed in 100 mL of simulated gastric fluid (pH 2.5), oscillated at 37°C for 30 minutes, and then transferred to 100 mL of simulated intestinal fluid (pH 7.4). The oscillation was continued for 4 hours, and samples were taken every 30 minutes to determine the cumulative release of N-acetylneuraminic acid. The sample concentration was analyzed by ultraviolet spectrophotometry.

[0086] The properties of the compositions of Examples 1 to 4 and Comparative Examples 1 to 9 are summarized in Table 1.

[0087] Table 1. Performance of the compositions of Examples 1 to 4 and Comparative Examples 1 to 9

[0088] Examples 1 to 4 show significant advantages in skin rejuvenation and anti-aging performance, stability and sustained release persistence, especially Example 3, which achieves the best performance by optimizing the prebiotic ratio and gel network structure, as shown by a 26% increase in moisture, a 23% increase in elasticity, a sustained release effect of more than 90% in intestinal fluid, and only a 3% decrease in content. In contrast, the comparative examples have different degrees of decline in performance due to the replacement or loss of key components. For example, Comparative Example 2 does not add N-acetylneuraminic acid gel, and the skin rejuvenation performance and sustained release effect are significantly reduced; Comparative Example 9 lacks trace elements during the fermentation process, has the worst stability, and the content decreases by 15%. Although some comparative examples are close to the examples in individual indicators, such as the prebiotic adjustment of Comparative Example 4 has little effect on the skin rejuvenation performance, the overall performance is still inferior to the optimized examples, indicating that the synergy of N-acetylneuraminic acid gel and its components is the key to determining the performance of the composition.

[0089] 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. A skin rejuvenation and anti-aging composition based on N-acetylneuraminic acid, characterized in that: The invention comprises the following components in parts by weight: 5.0-8.0 parts of N-acetylneuraminic acid gel, 15-20 parts of skim milk powder, 10-15 parts of whole milk powder, 20-25 parts of xylitol, 1.0-3.0 parts of prebiotics, 5-10 parts of milk mineral salts, 1.0-3.0 parts of colostrum alkaline protein, 2.0-5.0 parts of bird's nest powder, 1.0-3.0 parts of pectin, 0.02-0.05 parts of vitamin E, 2.0-4.0 parts of sorbitol, 0.1-0.3 parts of flavors, and 50-80 parts of deionized water; The prebiotic is a mixture of oligofructose, oligogalactose and inulin in a weight ratio of 1:1:(2.0-4.0); The N-acetylneuraminic acid gel is prepared by mixing a loaded N-acetylneuraminic acid whey protein isolate solution with a polysaccharide mixed solution in a volume ratio of (2.0-4.0): (4.0-6.0), heating and stirring to gel, and then cooling and standing.

2. A skin rejuvenation and anti-aging composition based on N-acetylneuraminic acid as claimed in claim 1, characterized in that: The preparation method of the N-acetylneuraminic acid gel is as follows: a whey protein isolate solution loaded with N-acetylneuraminic acid and a polysaccharide mixed solution are mixed according to a volume ratio of (2.0-4.0): (4.0-6.0), and a magnetic stirrer is used to stir at a stirring rate of 300-400 rpm for 10-15 min at 40-50 ° C to form a uniform composite solution; the obtained composite solution is placed in a water bath at 75-85 ° C and heated for 5-10 min to induce a gelation process, and after the heating is completed, the solution is quickly moved to flowing cold water to cool to room temperature, and then the cooled solution is transferred to a closed container, and is allowed to stand at 4 ° C for 12-24 h to obtain the N-acetylneuraminic acid gel.

3. A skin rejuvenation and anti-aging composition based on N-acetylneuraminic acid as claimed in claim 2, characterized in that: The preparation method of the N-acetylneuraminic acid loaded whey protein isolate solution is as follows: by weight, at room temperature, 5.0-10.0 parts of whey protein isolate and 100-150 parts of distilled water are stirred at a stirring rate of 300-400 rpm for 180-240 minutes using a magnetic stirrer to obtain a whey protein isolate solution for standby use; then 3.0-8.0 parts of N-acetylneuraminic acid are added, stirring is continued at a stirring rate of 300-350 rpm for 15-30 minutes at room temperature, and the solution is filtered through a filter membrane with a pore size of 0.45 μm to remove particles, and the obtained filtrate is the N-acetylneuraminic acid loaded whey protein isolate solution.

4. A skin rejuvenation and anti-aging composition based on N-acetylneuraminic acid as claimed in claim 2, characterized in that: The preparation method of the polysaccharide mixed solution is as follows: 5.0-12.0 parts of pectin and 4.0-10.0 parts of carboxymethyl cellulose are weighed respectively by weight, and added one by one to 100-140 parts of distilled water, and allowed to stand at room temperature for 20-30 minutes to allow the polysaccharide to fully absorb water and swell; the polysaccharide solution after standing is placed in a water bath at 55-60°C and heated for 30-45 minutes, and stirred at a stirring rate of 300-350 rpm for 30-45 minutes to obtain a polysaccharide mixed solution.

5. The skin rejuvenation and anti-aging composition based on N-acetylneuraminic acid as claimed in claim 3, characterized in that: The preparation method of the described N-acetylneuraminic acid is: S1. Strain activation and fermentation culture: The Escherichia coli K12 strain is revived, pre-cultured and fermented to obtain a fermentation broth; S2. Collection and disruption of bacterial cells: The fermentation broth is centrifuged and ultrasonically disrupted to obtain a suspension of intracellular metabolites; S3. Product separation and purification: The intracellular metabolite suspension is concentrated by ultrafiltration, ethanol precipitation and mineral adsorption to obtain a purified product solution; S4. Concentration, dialysis and freeze-drying: The purified product solution is concentrated, dialyzed and freeze-dried to obtain N-acetylneuraminic acid.

6. The skin rejuvenation and anti-aging composition based on N-acetylneuraminic acid as claimed in claim 5, characterized in that: The specific steps of S1 are as follows: taking Escherichia coli K1 strain as a raw material, taking the strain out from storage conditions, thawing it quickly, and then inoculating it into a composite culture medium to obtain a mixed solution, wherein the composite culture medium includes 8-12 g / L yeast extract, 8-12 g / L tryptone, and 4-6 g / L NaCl, and adjusting the pH of the composite solution to 7.2-7.4 with a 0.05wt.% NaOH solution, and then placing the mixed solution in a 500mL conical flask, stirring it at a stirring rate of 120-150 rpm for 8-12 h, until a pre-culture solution with an optical density of 0.7-1.1 at a wavelength of 600 nm is formed; taking another 100-120 mL of the composite culture medium and placing it in a 500 mL conical flask, inoculating the obtained pre-culture solution into the composite culture medium according to 0.8-1.2% of the volume of the composite culture medium, and stirring it at 120-150 rpm for 8-12 h at 35.0-37°C, until a pre-culture solution with an optical density of 0.7-1.1 at a wavelength of 600 nm is formed. The optical density of the seed liquid at the wavelength of nm reaches 1.4-2.2; 10-12 L of the basic defined culture medium is added to the disposable reaction bag bioreactor, and the seed liquid is inoculated into the basic defined culture medium according to 2.5-3.5% of the volume of the defined culture medium, and the platform angle is set to 8°-12°, the temperature is 36-38°C, the dissolved oxygen level is 45-55%, and the oxygen supply rate is 0.9-1.1 L / min, and the fermentation is carried out for 36-72 hours until the fermentation is completed; The basic defined culture medium uses 10-20 g / L glucose as a main carbon source, 2-5 g / L ammonium sulfate as an inorganic nitrogen source, 1-1.5 g / L potassium dihydrogen phosphate and 1-1.5 g / L potassium hydrogen phosphate as a phosphorus source and a buffer; ferrous sulfate, zinc sulfate, manganese chloride and copper sulfate pentahydrate are added in amounts of 10-50 mg / L, 10-50 mg / L, 5-20 mg / L and 1-10 mg / L, respectively; vitamin B1 is also included as an auxiliary factor, with an addition amount of 0.1-1 mg / L; and 10-50 mg / L chelating agent EDTA, and the pH of the basic defined culture medium is adjusted to 7.4-7.6 with a 0.5wt.% sodium hydroxide solution.

7. The skin rejuvenation and anti-aging composition based on N-acetylneuraminic acid as claimed in claim 5, characterized in that: The specific steps of S2 are as follows: the fermentation broth after fermentation is centrifuged at a centrifugal rate of 16000-18000 rpm and 90-110 mL / min at 4±1°C, the obtained bacterial precipitate is collected and placed in an ice-water mixed bath, and crushed using an ultrasonic crusher, with the amplitude set to 95-100% and crushed for 25-35 s, followed by cooling in an ice-water mixed bath for 25-35 s, and the crushing process is repeated 3-4 times until the cell wall is fully lysed and the intracellular products are released to obtain a cell suspension.

8. The skin rejuvenation and anti-aging composition based on N-acetylneuraminic acid as claimed in claim 5, characterized in that: The specific steps of S3 are as follows: the cell suspension after disruption is subjected to ultrafiltration and concentration treatment by a cross-flow ultrafiltration device, the device uses a filter membrane with a molecular weight cutoff limit of 8-12 kDa, the mixed solution inlet pressure is controlled at 3.5-4.5 bar, the mixed solution is concentrated to 1 / 18-1 / 22 of the original volume, ethanol is gradually added to the concentrated solution for ethanol precipitation treatment, the volume ratio of ethanol to precipitate is adjusted to (78-82):100, and then centrifuged at a centrifugal speed of 4000-5000 rpm at 4±1°C for 8-15 min separation precipitation treatment; dissolving the obtained precipitate in deionized water, wherein the volume ratio of deionized water to precipitate is (150-180):100, and then repeating ultrafiltration concentration, ethanol precipitation and separation precipitation treatment for 2-4 times, until impurities are removed and the target product is concentrated to obtain a purified solution; mixing the obtained purified solution with a pre-balanced clay mineral in a mass ratio of (0.9-1):1, further separating the purified product solution from the clay mineral by centrifugation, wherein the centrifugation condition is 4000-5000 rpm, and the centrifugation treatment is carried out at 4±1°C for 12-18 min, taking the upper layer solution, and finally obtaining a further purified product solution; The pre-balanced clay mineral is a clay mineral that is oscillated in a 90-110 mM sodium phosphate buffer at a pH of 6.8-7.2 for 0.8-1.2 h, stirred at 120-140 rpm for 2.5-3.5 h after mixing, and centrifuged at a centrifugal rate of 4000-5000 rpm at 4±1°C for 12-18 min to obtain a precipitate that is a pre-balanced clay mineral.

9. The skin rejuvenation and anti-aging composition based on N-acetylneuraminic acid as claimed in claim 5, characterized in that: The specific steps of S4 are as follows: the purified product solution is concentrated through a centrifugal filter tube with a molecular weight cutoff of 8-12 kDa, and centrifuged at a centrifugal rate of 4000-5000 rpm at 4±1°C. The mixed solution is concentrated to 1 / 4-1 / 6 of the original volume and then subjected to three-stage dialysis treatment using a dialysis membrane with a molecular weight cutoff of 12-16 kDa. In the first stage, 4.5-5.5 L of a deionized water solution containing 5.4-5.6 g / L NaCl is used as a dialysis fluid for 22-26 h. In the second and third stages, 4.5-5.5 L of a deionized water solution with a pH of 8.8-9.2 NaOH is used as a dialysis fluid for 22-26 h. After the dialysis is completed, the solution obtained in the dialysis bag is transferred to a freeze-drying device, and freeze-dried under a vacuum condition of 0.03-0.05 mbar until it is completely dehydrated to obtain the final N-acetylneuraminic acid powder.

10. A method for preparing a skin rejuvenation and anti-aging composition based on N-acetylneuraminic acid as claimed in any one of claims 1 to 9, characterized in that: The following steps are involved: A1. Add deionized water to a reactor, heat to 60-70°C, and stir at this temperature at a stirring rate of 200-300 rpm, add xylitol and sorbitol in turn, and continue stirring for 10-15 minutes until they are evenly dissolved to form a transparent sugar alcohol solution, further add prebiotics to the sugar alcohol solution, and continue stirring at a stirring rate of 200-300 rpm for 10-15 minutes, then add vitamin E, and stir at 200-250 rpm for 5-10 minutes to form a uniform functional liquid base for standby use; A2. Add skim milk powder and whole milk powder to a dry mixer, mix at a stirring rate of 300-400 rpm for 3-5 min, then add milk mineral salts, colostrum alkaline protein and bird's nest powder, continue to mix at a stirring rate of 200-300 rpm for 2-3 min until a uniform powder mixture is formed, then continue to add pectin, and mix at a stirring rate of 200-250 rpm for 5-10 min to form a uniform solid powder mixture for standby; A3. Add N-acetylneuraminic acid gel to another stirred tank, slowly add the functional liquid base prepared by S1, and stir at a stirring rate of 200-250 rpm for 10-15 minutes at room temperature to form a uniform functional liquid mixture; slowly add the solid powder mixture and flavor prepared by A2 to the liquid mixture, and stir at a stirring rate of 200-250 rpm for 15-20 minutes until a uniform semi-wet mixture is formed, and leave the mixture to stand at room temperature for 20-30 minutes to ensure that the components are fully adsorbed and form a raw material base with suitable viscosity; the suitable viscosity of the raw material base is 5000-8000 mPa·s; A4. The semi-wet mixture prepared in A3 is fed into an extrusion granulator to form granules through granulation, drying and screening. Finally, the granules are sealed in a packaging bag protected by nitrogen gas and stored in a dry, light-proof environment. The process parameters of the granulation are as follows: a round hole mold with a diameter of 2 to 5 mm is selected, and extrusion molding is performed at an extrusion pressure of 8 to 12 MPa. During the extrusion process, the temperature is kept at 40 to 55°C to maintain the viscosity and homogeneity of the raw materials. The extruded strip material is cut into primary granules with a length of 2 to 5 mm by a cutting device. The process parameters of the drying and screening are as follows: the primary granules are placed in a liquid nitrogen freezing device for rapid freezing for 30 to 60 minutes, and then transferred to a vacuum drying oven for freeze drying under a vacuum pressure of ≤5 Pa. The drying temperature is controlled at 35 to 40°C and the drying time is 12 to 24 hours until the moisture content of the granules is less than 5%. The dried granules are screened by a screening machine to control the particle size to 2 to 5 The particle size is within the range of mm, and unqualified particles are removed. The removed particles can be added back into the production process for secondary processing.

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