Beverage additive containing N-acetylglucosamine and application of beverage additive in beverage

Abstract

The invention belongs to the technical field of food additives and functional beverages, and particularly relates to a beverage additive containing N-acetylglucosamine and application of the beverage additive in beverages, the additive adopts a four-layer embedding structure and comprises a beta-cyclodextrin-NAG complex, a sodium alginate-chitosan layer, a pectin-whey protein layer and an eudragit FS30D targeting layer, the stability and bioavailability of the NAG are improved, and the multi-target regulation effect is achieved through the composite formula design. In addition, components such as sodium hyaluronate, fructo-oligosaccharide, probiotic spores and the like are also combined, so that joint health, intestinal regulation and anti-inflammatory effects are synergistically enhanced. The preparation method comprises the steps of enzymatic deacetylation, electrodialysis purification, microcapsule embedding and the like, and has the characteristic of green production. The beverage prepared based on the additive also has the advantages of good taste, long shelf life and the like, and an innovative solution is provided for the functional beverage market.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of food additives and functional beverages, and specifically relates to a beverage additive containing N-acetyl glucosamine and application thereof in beverages. BACKGROUND

[0002] With the rapid development of the functional beverage market, N-acetyl glucosamine (NAG) has gradually become a research hotspot due to its potential efficacy in joint health, skin moisturizing, and intestinal regulation. However, in the prior art, traditional NAG production relies on chemical methods (such as hydrochloric acid hydrolysis) of crustacean raw materials, which has problems of high pollution, low purity, and high cost. Although the whole biological method improves the yield through strain modification, the cost of the immobilized carrier is high, and the downstream purification needs to rely on special resins (such as HPD600 resin) and electric field flow field coupling membrane separation equipment, which has a high industrialization threshold.

[0003] Secondly, the stability and bioavailability of N-acetyl glucosamine in the prior art are insufficient; due to the influence of pH and temperature on NAG in a liquid environment, the active ingredient is lost. The existing microcapsule technology (such as single-layer sodium alginate embedding) has poor resistance to acidic environment and low release efficiency, which cannot meet the application requirements of acidic beverages such as fruit juice.

[0004] In addition, the function is single and the synergistic effect is insufficient; NAG in the prior art is mainly used for single function, the addition amount is high, and there is a lack of synergistic design with other active ingredients (such as hyaluronic acid and prebiotics), which leads to increased cost and insufficient market competitiveness. SUMMARY

[0005] In order to solve the problems in the prior art, the purpose of the present application is to provide a beverage additive containing N-acetyl glucosamine and its application in beverages, which solves the problems of low bioavailability, insufficient stability, and single function of NAG in liquid beverages in the prior art through composite formula design, microcapsule embedding technology, and green production process.

[0006] The technical scheme of the present application is: a beverage additive containing N-acetyl glucosamine, characterized in that it is composed of the following weight components: 0.05-0.15 parts of N-acetyl glucosamine microcapsules, 0.05-0.15 parts of sodium hyaluronate, 0.3-0.8 parts of fructooligosaccharide, 0.1-0.3 parts of buffer, 0.01-0.05 parts of preservative, 0.01-0.03 parts of probiotic spores, 0.02-0.05 parts of curcumin liposomes, and the balance is water. The N-acetyl glucosamine microcapsules have a four-layer embedding structure, and from the inside to the outside, they are: a β-cyclodextrin-NAG complex, a sodium alginate-chitosan layer, and a pectin-whey protein layer. The preparation method of the N-acetyl glucosamine microcapsule comprises the following steps: S1, preparing N-acetyl glucosamine raw material: through an enzymatic process, cooperating with magnetic immobilized enzyme, and coupling with electrodialysis purification process, N-acetyl glucosamine raw material is obtained; S2, preparing inner layer β-cyclodextrin-NAG complex by using β-cyclodextrin and N-acetyl glucosamine prepared in step S1; S3, using the β-cyclodextrin-NAG complex prepared in step S2 to perform middle layer sodium alginate-chitosan embedding to obtain middle layer embedded microspheres; S4, performing outer layer pectin-whey protein coating on the middle layer embedded microspheres obtained in step S3 to obtain three-layer embedded NAG microcapsules.

[0007] As preferred, the step S1 of preparing N-acetyl glucosamine raw material comprises the following steps: S11, enzymatic deacetylation: chitin is first crushed, and then mixed with gene engineering bacteria fermentation liquor at a mass concentration of 1:20; then magnetic immobilized enzyme is added, and reaction is performed at 55-65°C for 18-24 hours; S12, magnetic field recovery: after enzymatic deacetylation, the reaction liquid is used to separate the magnetic immobilized enzyme by using a permanent magnet, and the recovery rate is above 95%; S13, electrodialysis purification: after magnetic field recovery, the reaction liquid is removed by an anion exchange membrane electrodialysis system to remove impurity ions, and in this process, the system voltage is 18-24 V, and the reaction liquid flow rate is 0.8-1.2 L / h; S14, the reaction liquid after electrodialysis purification is vacuum concentrated at 50°C to NAG concentration≥25 g / L, and after spray drying, N-acetyl glucosamine raw material powder is obtained, and the purity is≥98%.

[0008] As preferred, the step S2 of preparing inner layer β-cyclodextrin-NAG complex comprises the following steps: S21, solution preparation: β-cyclodextrin is mixed with deionized water at a mass concentration of 1:20, and heated to 55-65°C for stirring and dissolution; S22, complexation reaction: N-acetyl glucosamine raw material powder is added to the solution prepared in step S21 at a molar ratio of 1:2 with β-cyclodextrin, kept at 55-65°C constant temperature for 1.5-2.5 hours for stirring to form inclusion complex; S23, centrifugal purification: the solution in step S22 is subjected to centrifugal treatment at 7000-8000 rpm for 10-20 minutes to remove unembedded N-acetyl glucosamine raw material, and the precipitate is collected; S24, freeze-drying: the precipitate in step S23 is pre-frozen at a temperature of -50±5℃ for 18-24 hours, and then vacuum freeze-dried for 48 hours, and the dried product is a powder.

[0009] As preferred, S31, sodium alginate dispersion preparation: sodium alginate is dissolved in deionized water at a mass concentration of 1:50, and stirred until completely dissolved; S32, complex dispersion: the β-cyclodextrin-NAG complex in step S2 is added to the sodium alginate solution at a mass concentration of 1:10, and dispersed by ultrasonic for 15-25 minutes; S33, dropwise ball formation: the mixed solution obtained in step S32 is dropped into a crosslinking solution containing 1% chitosan and 0.1M CaCl2 using a syringe pump, the pH is maintained at 5-6, and the droplet diameter is controlled to be 1-2 mm; S34, crosslinking and solidification: the crosslinking solution in step S33 is allowed to stand at 20-30℃ for 20-40 minutes, so that the mixed solution forms gel microspheres, and after standing, it is filtered and washed with deionized water for more than 3 times to obtain solidified middle-layer embedding microspheres.

[0010] As preferred, the outer-layer pectin-whey protein coating process in step S4 comprises the following steps: S41, coating solution preparation: pectin and whey protein are mixed at a mass concentration of 1:1, and dissolved in a pH 7.0 phosphate buffer, and the mass concentration of the phosphate buffer is 2%; S42, coating treatment: the middle-layer microspheres are immersed in the coating solution obtained in step S41, and stirred at a constant temperature of 55-65℃ for 15-30 minutes to form an outer-layer film; S43, heat treatment and solidification: heat treatment at a temperature of 75-85℃ for 8-12 minutes to promote the Maillard reaction between pectin and whey protein and enhance the film strength; S44, drying and storage: dried under vacuum at 35-45℃ until the moisture content is less than 5%, and the three-layer embedded NAG microcapsules are obtained, and stored in a sealed and light-proof manner.

[0011] As preferred, it further comprises step S5, a colon-targeting layer process is performed on the three-layer embedded NAG microcapsules obtained in step S4; specifically comprising the following steps: S51, coating solution preparation: Eutec FS30D and pectin are dispersed in deionized water at a mass concentration of 1:1, and the total concentration is within 5%; S52, fluidized bed coating: place the three-layer embedded NAG microcapsules in the fluidized bed, spray the coating prepared in step S51, control the temperature environment to be 35-45 DEG C, and the spraying rate is 0.2-0.4 mL / min, so that a 10-15 mu m target layer is formed on the outside of the three-layer embedded NAG microcapsules.

[0012] As preferred, the magnetic immobilized enzyme in step S1 is chitosan-coated Fe3O4, and the preparation method comprises the following steps: First, Fe3O4 nanoparticles with a particle size of 40-60 nm are synthesized by a coprecipitation method, then chitosan with a degree of deacetylation greater than 90% is dissolved in a 1% acetic acid solution, and then mixed with Fe3O4 nanoparticles at a mass concentration ratio of 1:5, and finally cross-linked and dried to obtain.

[0013] The core of the present application is to protect the stability of active ingredients such as N-acetylglucosamine, curcumin and probiotic spores through multi-component synergistic effect and multi-layer delivery technology, and to improve the bioavailability through targeted delivery and sustained release. In addition, it also has the effect of multi-target regulation, such as the regulation of joints, intestinal tract and inflammatory response.

[0014] The detailed analysis is as follows: The role of the inner β-cyclodextrin-NAG complex is that β-cyclodextrin forms an inclusion compound with N-acetylglucosamine through a hydrophobic cavity, thereby improving its water solubility and antioxidant stability and avoiding oxidative degradation during processing.

[0015] The role of the middle sodium alginate-chitosan layer is that anionic polymer sodium alginate and cationic polymer chitosan form a semi-permeable membrane through electrostatic interaction, resist gastric acid erosion, and protect NAG from being destroyed by gastric acid. In addition, the adhesion of chitosan can prolong the intestinal retention time and promote subsequent absorption.

[0016] The role of the outer pectin-whey protein layer is that pectin forms a gel barrier under acidic conditions to further resist gastric acid. Whey protein undergoes Maillard reaction with pectin through heat treatment to form a dense cross-linked network, thereby enhancing the mechanical strength and preventing the microcapsules from being broken during processing (such as homogenization and sterilization).

[0017] The role of the outermost Eutectic FS30D target layer is that based on the pH-sensitive polymer, it dissolves in the colon environment to realize colon-targeted release and synergize with intestinal probiotics.

[0018] The four-layer embedding of the present application increases the retention rate of N-acetylglucosamine in a high-temperature, gastric acid environment to more than 90%, which is much higher than the 60% or less of the traditional process; and in terms of targeted release, N-acetylglucosamine is released at the target site in the intestinal tract / colon, and the bioavailability is increased by 30-50%.

[0019] The mechanism of sodium hyaluronate and N-acetylglucosamine in synergistic effect on the repair of articular cartilage and the relief of joint pain is as follows: Both sodium hyaluronate and N-acetylglucosamine are components of articular cartilage matrix, wherein sodium hyaluronate (HA) is a glycosaminoglycan skeleton, and N-acetylglucosamine (NAG) is a precursor of synthetic glycosaminoglycan. HA promotes chondrocyte proliferation by binding to CD44 receptors, and NAG provides raw materials for the synthesis of glycosaminoglycans, both of which synergistically stimulate cartilage repair, increase the efficiency of articular cartilage repair, and shorten the time for the relief of joint pain.

[0020] The mechanism of the synergistic effect of fructooligosaccharides (such as prebiotics) and spores of probiotics is as follows: As prebiotics, fructooligosaccharides (FOS) selectively promote the proliferation of intestinal bifidobacteria and lactobacilli; Probiotic spores (such as Bacillus coagulans) are activated after colonizing the intestine, secrete antibacterial peptides to inhibit pathogenic bacteria (such as Escherichia coli), and metabolize FOS to produce short-chain fatty acids (SCFAs, such as butyric acid), which reduce the intestinal pH and further inhibit the growth of pathogenic bacteria; SCFA feedback enhancement: butyric acid activates GPR41 / 43 receptors, promotes intestinal barrier function, and reduces endotoxin into the blood, indirectly relieving joint inflammation.

[0021] The intestinal flora diversity index (Shannon index) is increased by 20-30%, and the serum inflammatory factor (such as IL-6, TNF-α) level is reduced by 40-60%.

[0022] In the present application, the technical integration of the multi-layer delivery system is as follows: Gastric acid-intestinal fluid responsive release mechanism: the outer pectin-whey protein layer forms a dense gel in gastric acid (pH 1-3) to block H⁺ permeation; the middle sodium alginate-chitosan layer gradually swells in intestinal fluid (pH 6-7) to release the inner complex; the targeting layer (Eudragit FS30D) only dissolves in the colon (pH ≥7) to achieve precise delivery.

[0023] Component release kinetics matching: probiotic spores start to proliferate after being activated in the intestine (6-8 hours are required), which matches the fermentation period of fructooligosaccharides (4-6 hours), ensuring continuous supply of SCFAs; NAG microcapsules are released slowly in the intestine (half-life period of 8-12 hours), which is synchronized with the metabolic period of articular cartilage.

[0024] Therefore, compared with the prior art, the beverage additive containing N-acetylglucosamine provided by the application has the following advantages: The application breaks through the limitations of traditional formula, such as easy degradation of active ingredients, poor targeting, single target action, etc., through multi-level embedding technology and cross-system collaborative design (joint-intestine-immune). Four-layer embedding system: combining beta-cyclodextrin complexation, polyelectrolyte complex membrane, and Maillard reaction membrane to achieve multi-environmental adaptability protection; Intestine-joint axis regulation: through the prebiotic-probiotic-SCFAs pathway, the intestinal health is associated with joint inflammation relief to form a systematic intervention; Liposome and microcapsule dual carrier: simultaneously solving the stability and bioavailability problems of hydrophilic (NAG) and liposoluble (curcumin) components.

[0025] The application also provides a beverage containing the beverage additive.

[0026] The beverage includes 0.5-1.5 parts of the additive, 5-8 parts of natural fruit juice, 0.1-0.3 parts of sweetener, 0.01-0.03 parts of citric acid, 0.02-0.05 parts of vitamin C, and the balance is water. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 The process flow diagram of the beverage additive containing N-acetylglucosamine of the application is shown. DETAILED DESCRIPTION

[0028] The application is further described through the description of specific embodiments, but this is not a limitation of the application. Those skilled in the art can make various modifications or improvements according to the basic idea of the application, as long as they do not deviate from the basic idea of the application, and they are within the protection scope of the application.

[0029] In the following examples and comparative examples, the reagents not specifically mentioned are conventional reagents, which can be purchased from conventional reagent production and sales companies. The methods used are existing technologies, unless otherwise specified. Example 1

[0030] A beverage additive containing N-acetyl glucosamine is a basic three-layer embedded N-acetyl glucosamine microcapsule beverage additive, which comprises the following components by weight: 0.1 parts of N-acetyl glucosamine microcapsules, 0.1 parts of sodium hyaluronate, 0.5 parts of fructooligosaccharide, 0.2 parts of sodium citrate, 0.03 parts of potassium sorbate, 0.02 parts of Bacillus coagulans spores, 0.03 parts of curcumin liposomes, and water to 100 parts.

[0031] The preparation method of the N-acetyl glucosamine microcapsule is as shown in Figure 1 The preparation method of the N-acetyl glucosamine microcapsule is as shown in Step S1: Enzymatic preparation of NAG raw material; S11: Chitin is crushed to 80 mesh, mixed with genetically engineered bacteria (recombinant Bacillus subtilis) fermentation broth at a ratio of 1:20, and chitosan-coated Fe3O4 magnetic immobilized enzyme (enzyme loading 15 U / g) is added, and reacted at 60°C for 20 h.

[0032] S12: Recover the magnetic enzyme with a permanent magnet (0.5 T), with a recovery rate of 98.2%.

[0033] S13: Electrodialysis purification (anion membrane, voltage 20 V, flow rate 1.0 L / h), desalination rate 92.5%.

[0034] S14: Concentrate to NAG concentration 28 g / L, spray dry to powder, purity 96.3%.

[0035] Step S2: β-cyclodextrin complexation; S21: β-cyclodextrin is mixed with deionized water at a ratio of 1:20 and dissolved at 60°C.

[0036] S22: Add NAG (molar ratio 1:2), stir at 60°C for 2 h, and the inclusion rate is 88.7%.

[0037] S23: Centrifugation (8000 rpm, 15 min), collect the precipitate.

[0038] S24: Pre-freeze at -50°C and freeze-dry to get loose powder.

[0039] Step S3: Middle layer embedding; S31: Dissolve sodium alginate 1:50, ultrasonic dispersion of β-cyclodextrin-NAG complex (1:10).

[0040] S32: Drop 2% CaCl2-1% chitosan crosslinking solution, droplet diameter 1.5 mm.

[0041] S33: Stand at 25°C for 30 min, get microspheres with particle size 1.8±0.2 mm, encapsulation efficiency 91.5%.

[0042] Step S4: outer layer coating; S41: Pectin-whey protein (1:1) dissolved in 2% pH 7.0 phosphate buffer.

[0043] S42: 60℃ stirring coating for 20 min, heat treatment at 80℃ for 10 min.

[0044] S43: vacuum drying to moisture content of 4.2%, obtaining three-layer microcapsules with an embedding rate of 89.3%. Example 2

[0045] On the basis of Example 1, step S5 is added to perform a colon targeting layer process on the three-layer NAG microcapsules obtained in step S4; specifically comprising the following steps: S51: Eutectic FS30D and pectin are mixed at a ratio of 1:1 to form a 5% dispersion.

[0046] S52: fluidized bed coating (40℃, 0.3 mL / min), forming a 12±2 μm targeting layer, with a coating efficiency of 94.6%.

[0047] In vitro release test (simulated gastrointestinal fluid): Gastric juice (pH 1.2) 2 h release rate: 7.8% vs. 15.3% of Example 1; Intestinal juice (pH 6.8) 4 h release rate: 68.9% vs. 82.4% of Example 1; Colonic juice (pH 7.4) 12 h cumulative release: 92.1% vs. 85.7% of Example 1. Example 3

[0048] In this example, the preparation of magnetic immobilized enzyme was optimized, so that the yield of N-acetylglucosamine microcapsules reached 38.7 g / L, while the yield of N-acetylglucosamine microcapsules by traditional process was about 28.5 g / L; Among them, the particle size of Fe3O4 is 50 nm, the mass ratio of chitosan (degree of deacetylation 95%) to Fe3O4 is 1:5, the crosslinking agent is glutaraldehyde (0.5%), the enzyme activity retention rate is 98.5%, and the activity retention rate after 10 times of reuse is 89.7%, while the enzyme activity retention rate of traditional carrier is 82.3%, and the activity retention rate after 10 times of reuse is 63.2%. Example 4

[0049] In this example, the components of NAG microcapsules are adjusted to 0.05 parts, sodium hyaluronate 0.05 parts, fructooligosaccharide 0.3 parts, buffer 0.1 parts, preservative 0.01 parts, probiotic spores 0.01 parts, curcumin liposomes 0.02 parts, and the balance is water, which are mixed to obtain; The preparation process is adjusted as follows: The preparation method of the N-acetyl glucosamine microcapsule comprises the following steps: S1, preparing N-acetyl glucosamine raw material: obtaining N-acetyl glucosamine raw material through an enzymatic process, cooperating with magnetic immobilized enzyme, and coupling with electrodialysis purification process; the step S1 of preparing N-acetyl glucosamine raw material comprises the following steps: S11, enzymatic deacetylation: first, crushing chitin, then mixing it with the fermentation broth of genetically engineered bacteria at a mass concentration of 1:20; then adding magnetic immobilized enzyme, and reacting at 55°C for 24 hours; S12, magnetic field recovery: using a permanent magnet to separate the magnetic immobilized enzyme after enzymatic deacetylation, and the recovery rate is more than 95%; S13, electrodialysis purification: removing impurity ions from the reaction liquid after magnetic field recovery through an anion exchange membrane electrodialysis system, and in this process, the system voltage is 18 V and the reaction liquid flow rate is 0.8 L / h; S14, vacuum concentrating the reaction liquid after electrodialysis purification to NAG concentration ≥25 g / L at 50°C, and then spray drying to obtain N-acetyl glucosamine raw material powder with purity ≥98%.

[0050] S2, preparing inner layer β-cyclodextrin-NAG complex using β-cyclodextrin and the prepared N-acetyl glucosamine in step S1; the step S2 of preparing inner layer β-cyclodextrin-NAG complex comprises the following steps: S21, solution preparation: mixing β-cyclodextrin with deionized water at a mass concentration ratio of 1:20, heating to 55°C and stirring to dissolve; S22, complexation reaction: adding N-acetyl glucosamine raw material powder to the solution prepared in step S21 at a molar ratio of 1:2 with β-cyclodextrin, keeping constant temperature at 55°C for 2.5 hours, and forming inclusion compound; S23, centrifugal purification: centrifuging the solution in step S22 at 7000 rpm for 20 minutes to remove unembedded N-acetyl glucosamine raw material, and collecting the precipitate; S24, freeze-drying: pre-freezing the precipitate in step S23 in a temperature environment of-55°C for 18 hours, and then vacuum freeze-drying for 48 hours, and obtaining powder-like complex after drying.

[0051] S3, using the prepared β-cyclodextrin-NAG complex in step S2 to perform middle layer sodium alginate-chitosan embedding to obtain middle layer embedded microspheres, and the specific process is as follows: S31, sodium alginate dispersion liquid preparation: dissolving sodium alginate in deionized water at a mass concentration ratio of 1:50, and stirring until completely dissolved; S32, complex dispersion: the β-cyclodextrin-NAG complex in step S2 is added into the sodium alginate solution with a mass concentration of 1:10, and dispersed by ultrasonic for 15-25 minutes; S33, drop forming: the mixed solution obtained in step S32 is dropped into the crosslinking solution containing 1% chitosan and 0.1M CaCl2 using a syringe pump, the pH is maintained between 5-6, and the drop diameter is controlled to be 1-2 mm; S34, crosslinking and solidification: the crosslinking solution in step S33 is placed at 20-30°C for 20-40 minutes to make the mixed solution form gel microspheres, and then filtered and washed with deionized water for more than 3 times to obtain the solidified middle-layer embedding microspheres.

[0052] S4, the middle-layer embedding microspheres obtained in step S3 are coated with an outer layer of pectin-whey protein to obtain three-layer embedding NAG microcapsules, As preferred, the outer layer pectin-whey protein coating process in step S4 comprises the following steps: S41, coating solution preparation: pectin and whey protein are mixed with a mass concentration of 1:1 and dissolved in a pH 7.0 phosphate buffer, and the mass concentration of the phosphate buffer is 2%; S42, coating treatment: the middle-layer microspheres are immersed in the coating solution obtained in step S41 and stirred at a constant temperature of 55-65°C for 15-30 minutes to form an outer layer film; S43, heat treatment and solidification: heat treatment is performed at a temperature of 75-85°C for 8-12 minutes to promote the Maillard reaction of pectin and whey protein and enhance the film strength; S44, drying and preservation: dried under vacuum at 35-45°C until the moisture content is less than 5%, and the three-layer embedding NAG microcapsules are obtained, which are sealed and stored in the dark.

[0053] Performance test: In vitro release (simulated gastric juice 2h / enteric juice 4h): Gastric juice release rate: 18.6% ±1.2%; Intestinal juice release rate: 89.7% ±1.8%; Anti-inflammatory effect (LPS-induced RAW264.7 cells): TNF-α inhibition rate: 73.5% ±2.9%; IL-6 inhibition rate: 68.4% ±3.3%.

[0054] Stability risk: the microcapsule breakage rate is 8.3% ±0.7% after 30 days of storage, and that of Example 1 is 4.1%. Example 5

[0055] In this embodiment, the components of the NAG microcapsule are adjusted to 0.15 parts, sodium hyaluronate 0.15 parts, fructooligosaccharide 0.8 parts, buffer 0.3 parts, preservative 0.05 parts, probiotic spores 0.03 parts, curcumin liposome 0.05 parts, and the balance is water. Mixing is obtained; the preparation process is adjusted as follows: The preparation method of the N-acetyl glucosamine microcapsule comprises the following steps: S1, preparation of N-acetyl glucosamine raw material: through enzyme process, synergistic magnetic immobilized enzyme, and coupling electrodialysis purification process, N-acetyl glucosamine raw material is obtained; the step S1 of preparing N-acetyl glucosamine raw material comprises the following steps: S11, enzyme deacetylation: first, chitin is crushed, then mixed with gene engineering bacteria fermentation broth with a mass concentration of 1:20; then add magnetic immobilized enzyme, react at 65℃ for 18 hours; S12, magnetic field recovery: the reaction solution after enzyme deacetylation is used to separate magnetic immobilized enzyme by permanent magnet, and the recovery rate is more than 95%; S13, electrodialysis purification: the reaction solution after magnetic field recovery is removed by anion exchange membrane electrodialysis system, and in this process, the system voltage is 24 V, and the reaction solution flow rate is 1.2 L / h; S14, the reaction solution after electrodialysis purification is vacuum concentrated at 50℃ to NAG concentration≥25 g / L, and after spray drying, N-acetyl glucosamine raw material powder with purity≥98% is obtained.

[0056] S2, using β-cyclodextrin and N-acetyl glucosamine prepared in step S1 to prepare inner layer β-cyclodextrin-NAG complex; the step S2 of preparing inner layer β-cyclodextrin-NAG complex comprises the following steps: S21, solution preparation: mix β-cyclodextrin with deionized water in a mass concentration ratio of 1:20, heat to 65℃ and stir to dissolve; S22, complexation reaction: add N-acetyl glucosamine raw material powder to the solution prepared in step S21 with β-cyclodextrin in a molar ratio of 1:2, keep constant temperature at 65℃ for 1.5 hours, and form inclusion compound; S23, centrifugal purification: centrifuge the solution in step S22 at 8000 rpm for 10 minutes to remove unembedded N-acetyl glucosamine raw material, and collect the precipitate; S24, freeze-drying: the precipitate in step S23 is pre-frozen at a temperature of-45℃ for 24 hours, and then vacuum freeze-dried for 48 hours, and the dried powder is obtained.

[0057] S3, using the prepared β-cyclodextrin-NAG complex in step S2 to perform middle-layer sodium alginate-chitosan embedding to obtain middle-layer embedded microspheres, and the specific process is as follows: S31, sodium alginate dispersion preparation: sodium alginate is dissolved in deionized water at a mass concentration ratio of 1:50, and stirred until completely dissolved; S32, complex dispersion: the β-cyclodextrin-NAG complex in step S2 is added to the sodium alginate solution at a mass concentration ratio of 1:10, and ultrasonic dispersion is performed for 15-25 minutes; S33, dropwise sphere formation: the mixed solution obtained in step S32 is dropped into a crosslinking solution containing 1% chitosan and 0.1M CaCl2 using a syringe pump, the pH is maintained at 5-6, and the droplet diameter is controlled to be 1-2 mm; S34, crosslinking and solidification: the crosslinking solution in step S33 is placed at 20-30°C for 20-40 minutes to allow the mixed solution to form gel microspheres, and after standing, it is filtered and washed with deionized water for more than 3 times to obtain solidified middle-layer embedded microspheres.

[0058] S4, outer-layer pectin-whey protein coating is performed on the middle-layer embedded microspheres obtained in step S3 to obtain three-layer embedded NAG microcapsules, As preferred, the outer-layer pectin-whey protein coating process in step S4 includes the following steps: S41, coating liquid preparation: pectin and whey protein are mixed at a mass concentration ratio of 1:1 and dissolved in a pH 7.0 phosphate buffer, and the mass concentration of the phosphate buffer is 2%; S42, coating treatment: the middle-layer microspheres are immersed in the coating liquid obtained in step S41, and stirred at a constant temperature of 55-65°C for 15-30 minutes to form an outer-layer film; S43, heat treatment and solidification: heat treatment is performed at a temperature of 75-85°C for 8-12 minutes to promote the Maillard reaction of pectin and whey protein and enhance the film strength; S44, drying and storage: dried under vacuum at 35-45°C until the moisture content is less than 5%, and the three-layer embedded NAG microcapsules are obtained, and stored in a sealed and light-proof manner.

[0059] Performance test: In vitro release (simulated gastric juice for 2h and intestinal juice for 4h): Gastric juice release rate: 9.2% ±0.8%; Intestinal juice release rate: 71.5% ±2.1%; Anti-inflammatory effect (LPS-induced RAW264.7 cells): TNF-α inhibition rate: 52.3% ±3.1%; IL-6 inhibition rate: 47.8% ± 2.7%.

[0060] The chemical method NAG combined with single-layer embedding was used, specifically, NAG was prepared by hydrochloric acid hydrolysis of chitin with a purity of 88.5%, and single-layer sodium alginate embedding was used, and a CaCl2 cross-linking solution with a mass concentration of 2% was used, and the gastric juice 2 h release rate was 42.6%, and the intestinal juice 4 h release rate was 93.2%. Without electrodialysis purification, step S13 was omitted, and HPD600 resin purification was used instead, obtaining NAG with a purity of 89.7% and a desalination rate of 78.3%, and the production cost increased by 35%. Without fructooligosaccharide formula, the fructooligosaccharide was removed from the formula, and the in vitro probiotic proliferation rate decreased by 42.3% compared with example 1. The NAG microcapsule used an ultra-range low dose, specifically 0.03 parts (the rest was the same as example 1), and the anti-inflammatory effect was represented by a TNF-α inhibition rate of only 38.2% ± 2.5%, which was significantly lower than 52.3% of example 4. The NAG microcapsule used an ultra-range high dose, specifically 0.2 parts (the rest was the same as example 1), and stability problems occurred, with a breakage rate of 15.6% ± 1.1% after 30 days of storage, and flocculation phenomenon occurred. The microcapsule acid resistance test was performed, specifically, the microcapsules of example 1 and control group 1 were placed in pH 2.0 HCl at 37°C and oscillated, and after 2 h, the NAG retention rate in example 1 was 92.4%, and that in comparative example 1 was 67.8%. After 4 h, the NAG retention rate in example 1 was 85.3%, and that in comparative example 1 was 53.2%. The synergistic anti-inflammatory effect test was performed, using a LPS-induced RAW264.7 cell inflammation model, the TNF-α inhibition rate in the formula group of example 1 was 68.9%, and the IL-6 inhibition rate was 63.2%; the single NAG group was 42.7%, and the IL-6 inhibition rate was 38.5%. The intestinal flora regulation test was performed, in vitro simulation of colon fermentation (72 h), the abundance of bifidobacterium in example 1 increased by 2.8 times, and the proportion of butyrate-producing bacteria was 35.7%; the abundance of bifidobacterium in comparative example 3 increased by 1.2 times, and the proportion of butyrate-producing bacteria in comparative example 1 was 22.4%.

[0061] The above examples only exemplarily illustrate the principles and effects of the present application, and are not used to limit the present application. Any person skilled in the art can modify or change the above examples without departing from the spirit and scope of the present application. Therefore, all equivalent modifications or changes completed by those skilled in the art without departing from the spirit and technical thought disclosed by the present application should be covered by the claims of the present application.

Claims

1. A beverage additive containing N-acetylglucosamine, characterized in that, It is composed of the following components by weight: The following ingredients are present: 0.05-0.15 parts N-acetylglucosamine microcapsules, 0.05-0.15 parts sodium hyaluronate, 0.3-0.8 parts fructooligosaccharides, 0.1-0.3 parts buffer, 0.01-0.05 parts preservative, 0.01-0.03 parts probiotic spores, 0.02-0.05 parts curcumin liposomes, and the balance being water. Mix these ingredients to obtain the final product. The N-acetylglucosamine microcapsules have a four-layer encapsulation structure, from the inside to the outside: β-cyclodextrin-NAG complex, sodium alginate-chitosan layer, and pectin-whey protein layer; The method for preparing the N-acetylglucosamine microcapsules includes the following steps: S1. Preparation of N-acetylglucosamine raw material: N-acetylglucosamine raw material is obtained by enzymatic process, synergistic magnetic immobilization of enzyme, and coupled with electrodialysis purification process. S2. Prepare an inner β-cyclodextrin-NAG complex using β-cyclodextrin and N-acetylglucosamine prepared in step S1. S3. Using the β-cyclodextrin-NAG complex prepared in step S2, sodium alginate-chitosan is used for middle-layer embedding to obtain middle-layer embedded microspheres. S4. Coat the middle-layer embedded microspheres obtained in step S3 with an outer layer of pectin-whey protein to obtain three-layer embedded NAG microcapsules.

2. The beverage additive containing N-acetylglucosamine according to claim 1, characterized in that, The preparation of N-acetylglucosamine raw material in step S1 includes the following steps: S11, Enzymatic deacetylation: First, pulverize the chitin, then mix it with the fermentation broth of genetically engineered bacteria at a mass concentration of 1:20; then add the magnetically immobilized enzyme and react at 55-65℃ for 18-24 hours. S12, Magnetic field recovery: The reaction solution after enzymatic deacetylation is separated and magnetically immobilized enzymes using a permanent magnet; S13. Electrodialysis purification: The reaction solution after magnetic field recovery is passed through an anion exchange membrane electrodialysis system to remove impurity ions. During this process, the system voltage is 18-24 V and the reaction solution flow rate is 0.8-1.2 L / h. S14. The reaction solution purified by electrodialysis is concentrated under vacuum at 50°C to a NAG concentration ≥25 g / L, and then spray-dried to obtain N-acetylglucosamine raw material powder.

3. The beverage additive containing N-acetylglucosamine according to claim 2, characterized in that, The preparation of the inner β-cyclodextrin-NAG complex in step S2 includes the following steps: S21. Solution preparation: Mix β-cyclodextrin and deionized water at a mass concentration ratio of 1:20, heat to 55-65℃ and stir to dissolve. S22. Complexation reaction: N-acetylglucosamine raw material powder is added to the solution prepared in step S21 at a molar ratio of 1:2 with β-cyclodextrin, and stirred at a constant temperature of 55-65℃ for 1.5-2.5 hours to form an inclusion complex; S23. Centrifugal purification: Centrifuge the solution from step S22 to remove the unencapsulated N-acetylglucosamine raw material and collect the precipitate. S24. Freeze-drying: The precipitate from step S23 is pre-frozen at a temperature of -50±5℃, and then freeze-dried under vacuum to obtain a powdered complex.

4. The beverage additive containing N-acetylglucosamine according to claim 3, characterized in that, The intermediate layer sodium alginate-chitosan encapsulation process in step S3 includes the following steps: S31. Preparation of sodium alginate dispersion: Dissolve sodium alginate in deionized water at a mass concentration ratio of 1:50 and stir until completely dissolved. S32. Complex dispersion: Add the β-cyclodextrin-NAG complex from step S2 to sodium alginate solution at a mass concentration of 1:10, and disperse using ultrasound for 15-25 minutes. S33, Droplet addition to form spheres: Use an injection pump to drop the mixture obtained in step S32 into the cross-linking liquid containing chitosan, and control the droplet diameter to 1-2 mm; S34. Crosslinking and curing: The crosslinking solution in step S33 is allowed to stand at 20-30℃ for 20-40 minutes to form gel microspheres. After standing, the mixture is filtered to obtain cured middle-layer embedded microspheres.

5. The beverage additive containing N-acetylglucosamine according to claim 4, characterized in that, The outer pectin-whey protein coating process in step S4 includes the following steps: S41. Preparation of coating solution: Mix pectin and whey protein at a mass ratio of 1:1 and dissolve them in pH 7.0 phosphate buffer solution, wherein the mass concentration of the phosphate buffer solution is 2%. S42. Coating treatment: Immerse the middle layer microspheres in the coating liquid obtained in step S41, and stir for 15-30 minutes under constant temperature of 55-65℃ to form an outer layer film. S43. Heat treatment curing: Heat treatment at 75-85℃ for 8-12 minutes to promote the Maillard reaction between pectin and whey protein and enhance film strength. S44. Dry storage: Dry under vacuum at 35-45℃ until the moisture content is below 5% to obtain three-layer embedded NAG microcapsules, which should be sealed and stored away from light.

6. The beverage additive containing N-acetylglucosamine according to claim 1, characterized in that, The process also includes step S5, which involves further processing the three-layer NAG microcapsules obtained in step S4 with a colon-targeting layer; specifically, it includes the following steps: S51. Preparation of coating solution: Disperse Eute FS30D and pectin in deionized water at a mass ratio of 1:1, with a total concentration of no more than 5%. S52, Fluidized Bed Coating: Place the three-layer embedded NAG microcapsules in a fluidized bed, and spray the coating using the coating liquid prepared in step S51. Control the temperature environment at 35-45℃ and the spray rate at 0.2-0.4 mL / min to form a 10-15 μm targeting layer on the outside of the three-layer embedded NAG microcapsules.

7. The beverage additive containing N-acetylglucosamine according to claim 1, characterized in that, The magnetically immobilized enzyme in step S1 is chitosan-coated Fe3O4, and its preparation method includes the following steps: First, Fe3O4 nanoparticles with a particle size of 40-60 nm are synthesized by co-precipitation. Then, chitosan with a degree of deacetylation greater than 90% is dissolved in 1% acetic acid solution and mixed with Fe3O4 nanoparticles at a mass concentration ratio of 1:

5. Finally, the mixture is cross-linked and dried to obtain the final product.

8. The use of a beverage additive containing N-acetylglucosamine according to any one of claims 1 to 7 in the preparation of beverages with joint health care, anti-inflammatory and intestinal flora regulation effects.

9. The application according to claim 8, characterized in that, The beverage includes natural fruit juice, sweeteners, citric acid, vitamins, and water.

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