A method for preparing DHA from Schizochytrium based on pH-responsive microcapsules and its spray freeze-drying preparation system

Abstract

This invention relates to the field of microalgae functional component preparation, and particularly to a method for preparing DHA from Schizochytrium based on pH-responsive microcapsules and its spray freeze-drying preparation system, by constructing sodium alginate / Ca 2+ The cross-linked inner layer and the chitosan outer layer grafted with pH-sensitive groups are double-encapsulated, and combined with synergistic processes such as nano-atomization, liquid nitrogen quenching and vacuum sublimation drying, the problems of easy oxidation and inactivation of Schizochytrium DHA microcapsules, non-targeted release from the gastrointestinal tract and long cycle and low efficiency of traditional freeze-drying processes are solved.

Description

Technical Field

[0001] This invention relates to the field of microalgae functional component preparation, specifically a method for preparing DHA from Schizochytrium based on pH-responsive microcapsules and its spray freeze-drying preparation system. Background Technology

[0002] DHA (docosahexaenoic acid) is an important omega-3 polyunsaturated fatty acid with various biological functions, including promoting nerve and retinal development, anti-inflammation, and cardiovascular protection. Among algae, *Schizochytrium* sp. is widely studied and applied due to its high DHA content. However, the DHA molecule contains multiple double bonds, making it susceptible to oxidation and inactivation. Oxidative losses during preparation and storage remain a major bottleneck limiting its functional efficacy.

[0003] Currently, DHA stabilization mainly relies on microencapsulation technology, with common wall materials including gelatin, sodium alginate, maltodextrin, and chitosan. While single-layer encapsulation wall materials can isolate oxygen to some extent, they still have the following limitations: Severe oxidation loss: During conventional spray drying or freeze drying, ice crystals formed inside the microcapsules or localized high temperatures from heat treatment can puncture the wall material, exposing DHA to oxygen and triggering a peroxidation reaction, resulting in an increase in peroxide value and a significant decrease in activity.

[0004] Non-targeted release: Commercially available microcapsules often release most of their DHA in gastric juice (pH 2–3). Early release in the gastrointestinal tract cannot achieve targeted absorption in the intestine, reducing the bioavailability of DHA.

[0005] Furthermore, conventional freeze-drying processes suffer from lengthy procedures (drying time often exceeds 24 hours), high energy consumption, and low production efficiency, severely hindering the industrial application of DHA microcapsules. Therefore, there is an urgent need for a novel microcapsule preparation technology and system that can improve the antioxidant stability of DHA, achieve targeted release into the intestines, and maintain high preparation efficiency. Summary of the Invention

[0006] This invention addresses the problems of easy oxidation and inactivation of *Schizochytrium* DHA microcapsules, non-targeted gastrointestinal release, and the long cycle and low efficiency of traditional freeze-drying processes in existing technologies. It proposes a method for preparing *Schizochytrium* DHA based on pH-responsive bilayer microcapsules and its spray freeze-drying preparation system. This method uses sodium alginate / Ca... 2+ The inner layer of the microcapsule is constructed by a cross-linked network, and the outer layer is constructed by electrostatic coating of chitosan with grafted pH-sensitive groups. It remains stable in acidic environments and rapidly dissociates in alkaline environments. Combined with processes such as two-fluid atomization, liquid nitrogen quenching and vacuum sublimation drying, it effectively inhibits ice crystal puncture, shortens the freeze-drying cycle, and significantly improves the antioxidant stability and intestinal-targeted release performance of DHA.

[0007] The technical solution of the present invention is as follows: A method for preparing DHA from Schizochytrium based on pH-responsive microcapsules, characterized by comprising: a) Double-layer encapsulation: The DHA oil from *Schizochytrium* was mixed with a sodium alginate solution, and Ca was added. 2+ Cross-linking forms an inner layer embedding solution; then it is mixed with a chitosan solution grafted with pH-sensitive groups, and electrostatic adsorption forms an outer layer; b) Freeze-drying: The double-layer embedding solution is atomized and then frozen, and then sublimated and dried under vacuum until the remaining moisture content is ≤3%.

[0008] Preferably, the sodium alginate concentration is 2%–3%, and the degree of deacetylation of the chitosan is ≥90%.

[0009] Preferably, rosmarinic acid and vitamin E are added to the embedding solution in step a at a mass ratio of 1:2 as antioxidants.

[0010] Preferably, the atomization uses a dual-fluid nozzle with a nozzle orifice diameter of 0.5 mm and a pressure of 0.3 MPa, resulting in an average droplet size of ≤100 nm.

[0011] Preferably, the freezing step includes instantaneous liquid nitrogen spray cooling and programmed cooling to -50°C, with a cooling rate ≥550°C / s.

[0012] Preferably, the sublimation drying is carried out under vacuum of 10 Pa, with the plate temperature rising from -50°C to 25°C, and the total drying time ≤ 6 hours.

[0013] Preferably, the inner layer DHA loading is 5%–15% (mass fraction).

[0014] A type of DHA microcapsule from Schizochytrium, the microcapsule being composed of sodium alginate / Ca 2+ It consists of an inner layer and an outer layer of chitosan grafted with pH-sensitive groups.

[0015] A spray freeze-drying preparation system based on the method of claim 1, characterized in that it comprises: Atomizing unit for atomizing double-layer embedding fluid using a dual-fluid nozzle; Freezing unit, used for rapid cooling and programmed temperature reduction via liquid nitrogen spray; The freeze-drying unit is used to sublimate and dry the water content to ≤3% under vacuum conditions.

[0016] Preferably, the atomization unit, freezing unit, and freeze-drying unit are controlled collaboratively by a multi-objective optimization algorithm to balance the microcapsule particle size, ice crystal size, and drying efficiency.

[0017] Through the above technical solution, the present invention has the following significant beneficial effects: Significantly improves oxidation stability: Utilizing sodium alginate / Ca 2+ The cross-linked inner layer works synergistically with antioxidants to extend the oxidation induction period of DHA from 4h to ≥48h, significantly reducing the incidence of peroxidation.

[0018] Achieve targeted release into the gut The outer chitosan is grafted with pH-sensitive groups, and the release rate is <10% in simulated gastric juice (pH 2–3) and ≥90% in simulated intestinal juice (pH 7.4) within 30 minutes, effectively avoiding premature release in the stomach and improving intestinal absorption efficiency.

[0019] Shorten freeze-drying cycle and improve production efficiency By combining two-fluid nano-atomization (droplets ≤100nm), liquid nitrogen quenching (cooling rate ≥550 ℃ / s), and vacuum sublimation (total drying time ≤6h), the freeze-drying cycle is shortened by ≥70% compared to traditional methods, significantly reducing energy consumption and equipment downtime.

[0020] Nanoscale particle size and structural integrity After atomization, the microcapsules have an average particle size of ≤100nm, uniform inner and outer layers, strong resistance to ice crystal penetration, and intact morphology and good dispersibility after drying.

[0021] Industrialization is highly feasible and controllable The preparation system uses a multi-objective optimization algorithm to collaboratively control the parameters of atomization, quenching and sublimation, which can be adjusted online according to the production scale, making it easy to scale up production and ensure product consistency.

[0022] In addition to the aforementioned expected technical effects, the bilayer pH-responsive microcapsule system of the present invention also exhibits the following unexpected beneficial effects: Enhances intestinal adhesion and prolongs retention time in the body After the outer layer of chitosan dissociates in an alkaline environment, the remaining aminopolysaccharide fragments have a certain degree of adhesion to the intestinal mucosa, which allows the microcapsules to stay in the intestine for a longer time, further improving the absorption and utilization rate of DHA.

[0023] Synergistic antibacterial and preservative effects Rosmarinic acid itself has natural antibacterial activity. When embedded in the inner layer of microcapsules, it not only protects DHA but also inhibits the growth of microorganisms on the capsule surface, significantly extending the product's shelf life and reducing the need for preservatives.

[0024] High humidity and heat stability Thanks to the dual protection of cross-linked sodium alginate and antioxidant network, the microcapsules can still maintain a DHA content of >90% under high temperature and high humidity (40℃ / 75%RH) conditions, without the need for strict low temperature storage, thus reducing subsequent storage and transportation costs.

[0025] Multi-functional carrying capacity This system can simultaneously encapsulate other hydrophobic active substances (such as vitamins A and E) without significantly increasing particle size, achieving the preparation of compound microcapsules with "one dose, multiple effects", thus broadening the application prospects of this invention in the field of functional foods and nutritional supplements.

[0026] In summary, this invention constructs sodium alginate / Ca 2+ The system features a dual encapsulation of a cross-linked inner layer and a chitosan outer layer grafted with pH-sensitive groups. Combined with synergistic processes such as nano-atomization, liquid nitrogen quenching, and vacuum sublimation drying, it not only significantly extends the oxidation induction period of DHA and achieves targeted release into the intestine, but also greatly shortens the freeze-drying cycle and improves production efficiency. In addition, the system unexpectedly exhibits excellent intestinal adhesion, antibacterial preservation, and high hydrothermal stability, and has the potential to co-load various active substances, fully meeting the industrialization needs of functional foods and nutritional supplements. Detailed Implementation

[0027] To facilitate understanding by those skilled in the art, the present invention will provide a more detailed description of the technical solutions in conjunction with the embodiments, but this is not intended to limit the scope of protection of the present invention.

[0028] Example 1: Preparation of bilayer microcapsules Raw material preparation DHA oil from Schizochytrium (purity ≥95%); Sodium alginate (food grade, molecular weight 100–200 kDa). CaCl2·2H2O; Chitosan (degree of deacetylation ≥90%, molecular weight 50–100 kDa); Succinic anhydride; Rosmarinic acid and vitamin E (mass ratio 1:2); Deionized water.

[0029] Inner layer embedding solution preparation At room temperature, 2.5 g of sodium alginate was dissolved in 97.5 g of deionized water and stirred until completely dissolved to obtain a 2.5% (w / w) sodium alginate solution. Add 10g of Schizochytrium DHA oil to the solution and emulsify using a high-speed homogenizer (10,000 rpm) for 2 min; Slowly add 20 mL of 0.2 mol / L CaCl2 solution, and continue stirring for 30 min to achieve a sodium alginate / CaCl2 ratio.2+ Cross-linking is performed to obtain a uniform inner layer embedding solution.

[0030] Preparation of outer coating solution Dissolve 1.5g of chitosan in 100mL of 1% (v / v) acetic acid aqueous solution and stir until clear; Add 0.5g of succinic anhydride to the chitosan solution and react for 1 hour to graft some of the amino groups with pH-sensitive groups. Add 0.1g of rosmarinic acid and 0.2g of vitamin E to the solution in sequence, stir well, and let stand for 20 minutes to obtain the outer coating solution.

[0031] Construction of a double-layer embedding system The inner layer embedding solution is slowly added to the outer layer coating solution at a ratio of 1:1 (v / v), and electrostatic adsorption is carried out for 10 minutes to obtain a double-layer embedding suspension.

[0032] Example 2: Spray freeze-drying process atomization Equipment: Dual-fluid nozzle, orifice diameter 0.5mm; Conditions: Liquid phase pressure 0.3 MPa, gas phase pressure 0.2 MPa, room temperature; Result: The average droplet size was approximately 80 nm.

[0033] Liquid nitrogen quenching The atomizing spray is placed directly into the liquid nitrogen atomization chamber, with a liquid nitrogen flow rate of 5 L / min; The programmed cooling instantly drops the room temperature to -50 ℃, with a cooling rate of approximately 600 ℃ / s.

[0034] Sublimation drying Place the quenched sample in a freeze dryer; Pre-freezing stage: –50 ℃, maintain for 30 min; Main drying stage: vacuum 10 Pa, plate temperature increased from -50 ℃ to 25 ℃ at a rate of 5 ℃ / h, total drying time 6h; Final moisture content ≤3%.

[0035] Example 3: Performance Evaluation Release performance test Simulated gastric fluid: 0.1 mol / L HCl–pepsin solution (pH 2.0), 37 ℃, 30 min, release rate determined; Simulated intestinal fluid: phosphate buffer (pH 7.4), 37 ℃, 30 min, release rate determined; Results: Gastric juice release rate was 8.2%, and intestinal juice release rate was 92.5%.

[0036] Oxidative stability test Acceleration conditions: 60 ℃, avoidance of light, sealed container, 7 days; Peroxide value determination: The international standard ISO 3960 method was adopted; Results: The peroxide value of the experimental group was 2.1 meq / kg, and that of the control group (without antioxidant) was 28.3 meq / kg.

[0037] High humidity and heat stability Conditions: 40 ℃ / 75% RH, stored for 30 days; DHA residue determination: GC method; Result: Residual DHA content >90%.

[0038] Microscopic morphology observation The surface and cross-section of the dried microcapsules were observed using a scanning electron microscope (SEM), which showed that the particle size was about 80–120 nm, and the surface was smooth and without cracks.

[0039] Example 4: Multifunctional Co-loading Application In Example 1, an additional 5% vitamin A oil solution was added to the double-layer encapsulation solution, and simultaneous spray freeze-drying was performed to evaluate the co-loading performance of the system. The results showed that the obtained compound microcapsules had a particle size of approximately 95 nm, and the loading rates of both DHA and vitamin A were >85%. The release curves were similar to those of the single DHA formulation, indicating that the system possesses excellent multifunctional co-loading capabilities.

Claims

1. A method for preparing DHA from Schizochytrium based on pH-responsive microcapsules, characterized in that, include: a) Double-layer encapsulation: The DHA oil from *Schizochytrium* was mixed with a sodium alginate solution, and Ca was added. 2+ Cross-linking forms the inner layer embedding fluid; Then, it is mixed with a chitosan solution grafted with pH-sensitive groups, and an outer layer is formed by electrostatic adsorption. b) Freeze-drying: The double-layer embedding solution is atomized and then frozen, and then sublimated and dried under vacuum until the remaining moisture content is ≤3%.

2. The method according to claim 1, characterized in that, The sodium alginate concentration is 2%–3%, and the degree of deacetylation of the chitosan is ≥90%.

3. The method according to claim 1, characterized in that, Rosmarinic acid and vitamin E were added to the embedding solution in step a at a mass ratio of 1:2 as antioxidants.

4. The method according to claim 1, characterized in that, The atomization uses a dual-fluid nozzle with a nozzle orifice diameter of 0.5 mm and a pressure of 0.3 MPa, resulting in droplets with an average particle size of ≤100 nm.

5. The method according to claim 1, characterized in that, The freezing step includes instantaneous liquid nitrogen spray cooling and programmed cooling to -50°C, with a cooling rate ≥550°C / s.

6. The method according to claim 1, characterized in that, The sublimation drying was carried out under a vacuum of 10 Pa, with the plate temperature rising from -50°C to 25°C, and the total drying time ≤ 6 hours.

7. The method according to claim 1, characterized in that, The inner layer DHA loading is 5%–15% (mass fraction).

8. A type of Schizochytrium DHA microcapsule, characterized in that, The microcapsules are made of sodium alginate / Ca 2+ It consists of an inner layer and an outer layer of chitosan grafted with pH-sensitive groups.

9. A spray freeze-drying preparation system based on the method of claim 1, characterized in that, include: Atomizing unit for atomizing double-layer embedding fluid using a dual-fluid nozzle; Freezing unit, used for rapid cooling and programmed temperature reduction via liquid nitrogen spray; The freeze-drying unit is used to sublimate and dry the water content to ≤3% under vacuum conditions.

10. The system according to claim 9, characterized in that, The atomization unit, freezing unit, and freeze-drying unit are controlled collaboratively by a multi-objective optimization algorithm to balance microcapsule particle size, ice crystal size, and drying efficiency.