A chitosan-Chlamydomonas reinhardtii protein composite carrier, its preparation method and application

Abstract

This invention discloses a chitosan-Chlamydomonas reinhardtii protein composite carrier, its preparation method, and its application. The composite carrier has a triple network structure, comprising: a three-dimensional network backbone formed by cross-linking chitosan and sodium tripolyphosphate, constituting the first network; a functional enhancing phase, including Chlamydomonas reinhardtii protein, which is embedded and anchored in the three-dimensional network backbone through a first physical interaction, constituting the second network, thereby forming a chitosan-Chlamydomonas reinhardtii protein complex with an interpenetrating network structure; and a structure-directing agent, including a hydrophobic active substance, which self-assembles and embeds into the hydrophobic microregions of the chitosan-Chlamydomonas reinhardtii protein complex through a second physical interaction, constituting the third network. The composite carrier provided by this invention is prepared using electrospinning technology, achieving high loading capacity and high encapsulation efficiency, constructing a multi-layered antioxidant defense network, and can serve as a highly efficient and stable multifunctional carrier for the preparation of functional food delivery materials.

Description

Technical Field

[0001] This invention belongs to the field of functional food delivery materials technology, specifically relating to a chitosan-Chlamydomonas reinhardtii protein composite carrier and its preparation and application. Background Technology

[0002] With the increasing global demand for functional foods, developing carrier materials capable of efficiently delivering and protecting poorly water-soluble and unstable bioactive substances is a challenge facing the field of food science. Currently, polysaccharide / protein (especially plant and microalgal proteins)-based delivery systems have attracted considerable attention due to their excellent biocompatibility and functional tunability; however, single polysaccharide / protein carriers still have limitations in terms of mechanical strength, environmental responsiveness, and multifunctional synergy. Therefore, constructing novel delivery systems with superior environmental stability, synergistic activity effects, and controllable release characteristics through material composites and structural design has become a research direction for overcoming current technological bottlenecks and meeting the development needs of next-generation functional foods.

[0003] Microalgal proteins, as an emerging sustainable protein resource, not only possess excellent nutritional and functional properties, but their abundant active groups (such as amino and carboxyl groups) also provide an ideal platform for constructing complex carriers. However, delivery systems relying on single microalgal proteins often suffer from inherent limitations in structural stability and function: these carriers are sensitive to changes in environmental pH and ionic strength, and are prone to conformational changes or dissociation under gastrointestinal or food processing conditions; simultaneously, the size and properties of their hydrophobic chambers are relatively fixed, resulting in significant bottlenecks in encapsulation efficiency and loading capacity for active substances of different polarities (especially strongly hydrophobic molecules), and making it difficult to achieve long-term, controllable release. Although existing technologies have disclosed the use of Chlamydomonas reinhardtii proteins combined with polysaccharides (such as xanthan gum) to prepare delivery systems, these methods are mostly based on physical blending to form complexes, lacking fine-tuning of protein conformation and stable chemical / physical cross-linking networks, resulting in a need to improve their structural stability and activity protection capabilities under extreme environments (such as high ionic strength and prolonged light exposure). Therefore, in order to fully realize the potential of microalgal proteins as delivery platforms and address their limitations in stability and single function, it is essential to develop composite strategies based on multiple intermolecular interactions and network structure engineering.

[0004] Melatonin, a typical hydrophobic bioactive substance, is an endogenous indoleamine with important physiological regulatory functions. It has demonstrated clear efficacy in regulating sleep rhythms, providing antioxidant defense, and neuroprotection, and is considered a highly promising functional food ingredient. However, its extremely low water solubility, high sensitivity to photothermal conditions, and rapid metabolic degradation in the gastrointestinal environment severely limit its oral bioavailability and practical application. Currently, studies have employed strategies such as cyclodextrin encapsulation or liposome encapsulation to improve its solubility and stability, but these delivery systems still generally have limitations. For example, existing technologies disclose various chitosan-encapsulated melatonin microsphere structures, but these mainly rely on single electrostatic adsorption or simple core-shell structure encapsulation, focusing on physical "encapsulation" rather than "functional synergy." They fail to fully utilize the potential biofunctional synergy between the carrier material and melatonin, and still lack structural integrity and release precision in complex physiological environments. Therefore, developing a novel delivery system that can simultaneously achieve high-efficiency loading, long-term stability, functional synergy, and precise and controllable release is crucial for fully leveraging the health benefits of melatonin and promoting its application in high-value-added functional foods. Summary of the Invention

[0005] The main objective of this invention is to provide a chitosan-Chlamydomonas reinhardtii protein composite carrier, its preparation method, and its application, so as to overcome the shortcomings of the prior art.

[0006] To achieve the aforementioned objectives, the technical solution adopted by this invention includes:

[0007] The first aspect of the present invention provides a chitosan-Chlamydomonas reinhardtii protein composite carrier, the composite carrier having a triple network structure and comprising:

[0008] The three-dimensional network framework is formed by cross-linking chitosan and sodium tripolyphosphate, and constitutes the first layer of the network;

[0009] The functional enhancement phase includes Chlamydomonas reinhardtii protein, which is embedded and anchored in the three-dimensional network backbone through a first physical action and constitutes a second network, thereby forming a chitosan-Chlamydomonas reinhardtii protein complex with an interpenetrating network structure.

[0010] The structure-directing agent includes a hydrophobic active substance, which self-assembles and embeds into the hydrophobic microdomains of the chitosan-Chlamydomonas reinhardtii protein complex through a second physical action, and forms a third network.

[0011] The first physical interaction includes electrostatic interaction and hydrogen bonding, the second physical interaction includes hydrophobic interaction and π-π stacking interaction, and the hydrophobic active substance includes melatonin.

[0012] A second aspect of the present invention provides a method for preparing the chitosan-Chlamydomonas reinhardtii protein complex carrier, comprising:

[0013] Chlamydomonas reinhardtii protein was obtained by treating Chlamydomonas reinhardtii with an alkaline dissolution and acid precipitation method.

[0014] Chitosan-acetic acid aqueous solution and sodium tripolyphosphate solution are uniformly mixed to electrostatically crosslink chitosan and sodium tripolyphosphate to form a three-dimensional network framework;

[0015] The Chlamydomonas reinhardtii protein is embedded and anchored in the three-dimensional network backbone by utilizing a first physical action to form a chitosan-Chlamydomonas reinhardtii protein complex with an interpenetrating network structure.

[0016] The chitosan-Chlamydomonas reinhardtii protein complex is uniformly mixed with a spinning aid and a hydrophobic active substance in an aqueous system. The hydrophobic active substance is then self-assembled into the hydrophobic microregions of the chitosan-Chlamydomonas reinhardtii protein complex using a second physical action to obtain a spinning solution. The spinning solution is then used to prepare a chitosan-Chlamydomonas reinhardtii protein complex carrier, wherein the hydrophobic active substance includes melatonin.

[0017] A third aspect of the invention provides the use of the chitosan-Chlamydomonas reinhardtii protein composite carrier in the preparation of functional food delivery materials or drug delivery materials.

[0018] A fourth aspect of the present invention provides a functional food delivery material comprising the chitosan-Chlamydomonas reinhardtii protein composite carrier.

[0019] Compared with the prior art, the present invention has at least the following beneficial effects:

[0020] (1) Unique “Triple Network” Structure Design: This invention is the first to construct a triple composite network structure with “chitosan-TPP crosslinked network as the backbone, Chlamydomonas reinforcing protein as the functional enhancing phase, and hydrophobic active substance as the structure directing agent”. This structure differs from the simple physical blending or single core-shell embedding in the prior art. Through a step-by-step assembly and multiple crosslinking strategy, an interpenetrating and dense network system is formed, which significantly improves the structural stability and environmental tolerance of the carrier.

[0021] (2) Excellent loading performance and biosafety: The obtained chitosan-Chlamydomonas reinhardtii protein composite carrier benefits from its dense three-dimensional network structure, which exhibits excellent loading capacity and encapsulation efficiency for hydrophobic active ingredients. At the same time, it has good hydrophobic properties and biosafety, providing an ideal platform for the efficient delivery of functional ingredients.

[0022] (3) Excellent antioxidant synergistic effect: This invention achieves a synergistic effect of three antioxidant mechanisms by combining Chlamydomonas reinhardtii protein, chitosan, and hydrophobic active substances: the antioxidant amino acid residues of Chlamydomonas reinhardtii protein directly scavenge free radicals, the chelating effect of chitosan reduces ion-catalyzed oxidation, and the hydrophobic active substances provide electron donors as highly efficient free radical scavengers. The three are not simply added together, but form a stable functional complex at the molecular level through multiple non-covalent interactions. Under extreme environmental conditions, it exhibits antioxidant stability far exceeding that of a single carrier or simple combination, achieving an unexpected technical effect of "1+1+1>3".

[0023] (4) Precise pH-responsive intestinal-targeted release: The chitosan-Chlamydomonas reinhardtii protein composite carrier microspheres of the present invention achieve precise intestinal-targeted release of hydrophobic active substances through a dual mechanism of the pH-responsive swelling properties of chitosan and the conformational unfolding of Chlamydomonas reinhardtii protein under alkaline conditions. This carrier can effectively protect the active ingredients from the damage of the gastric acid environment and trigger rapid release in the intestinal tract, significantly improving the oral bioavailability and actual efficacy of the functional active ingredients. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 This is a dispersion diagram of chitosan and chitosan-Chlamydomonas reinhardtii protein complex in Example 1 of the present invention;

[0026] Figure 2 This is a hydrophobic diagram of the Chlamydomonas reinhardtii protein and chitosan-Chlamydomonas reinhardtii protein complex in Example 1 of the present invention;

[0027] Figure 3 This is the X-ray photoelectron spectrum of the chitosan and chitosan-Chlamydomonas reinhardtii protein complex of Example 1 of the present invention;

[0028] Figure 4 This is a two-dimensional atomic force microscope image of the chitosan morphology of Example 1 of the present invention;

[0029] Figure 5 This is an atomic force microscope three-dimensional morphology image of chitosan in Example 1 of the present invention;

[0030] Figure 6 This is a scanning electron microscope image of chitosan from Example 1 of the present invention;

[0031] Figure 7 This is a two-dimensional atomic force microscope image of the chitosan-Chlamydomonas reinhardtii protein complex of Example 1 of the present invention;

[0032] Figure 8 This is an atomic force microscope three-dimensional morphology image of the chitosan-Chlamydomonas reinhardtii protein complex of Example 1 of the present invention;

[0033] Figure 9 This is a scanning electron microscope image of the chitosan-Chlamydomonas reinhardtii protein complex of Example 1 of the present invention;

[0034] Figure 10 This is a two-dimensional atomic force microscope image of the chitosan-Chlamydomonas reinhardtii protein composite carrier of Example 1 of the present invention;

[0035] Figure 11 This is an atomic force microscope three-dimensional morphology image of the chitosan-Chlamydomonas reinhardtii protein composite carrier of Example 1 of the present invention;

[0036] Figure 12 This is a scanning electron microscope image of the chitosan-Chlamydomonas reinhardtii protein composite carrier of Example 1 of the present invention;

[0037] Figure 13 These are Fourier transform infrared spectra of the chitosan-Chlamydomonas reinhardtii protein composite carriers of Examples 1-5 of the present invention and the chitosan-Chlamydomonas reinhardtii protein complex of Comparative Example 1.

[0038] Figure 14 These are thermal stability diagrams of the chitosan-Chlamydomonas reinhardtii protein composite carriers of Examples 1-5 of the present invention and the chitosan-Chlamydomonas reinhardtii protein complex of Comparative Example 1.

[0039] Figure 15 This is a UV stability diagram of the chitosan-Chlamydomonas reinhardtii protein composite carrier of Examples 1-5 of the present invention and the chitosan-Chlamydomonas reinhardtii protein complex of Comparative Example 1;

[0040] Figure 16 This is a diagram showing the loading amount of the chitosan-Chlamydomonas reinhardtii protein composite carrier in Examples 1-5 of the present invention;

[0041] Figure 17 These are encapsulation efficiency diagrams of the chitosan-Chlamydomonas reinhardtii protein composite carriers in Examples 1-5 of this invention;

[0042] Figure 18 The graphs show the antioxidant properties of the chitosan-Chlamydomonas reinhardtii protein composite carriers of Examples 1-5 and the chitosan-Chlamydomonas reinhardtii protein complex of Comparative Example 1 against 1,1-diphenyl-2-trinitrophenylhydrazine.

[0043] Figure 19This is a graph showing the antioxidant properties of the chitosan-Chlamydomonas reinhardtii protein composite carriers of Examples 1-5 and the chitosan-Chlamydomonas reinhardtii protein complex of Comparative Example 1 against 2,2-azido-bis(3-ethyl-benzothiazole-6-sulfonic acid).

[0044] Figure 20 This is a graph showing the scavenging rate of 1,1-diphenyl-2-trinitrophenylhydrazine by different pH values ​​for the chitosan-Chlamydomonas reinhardtii protein composite carrier of Example 1, the chitosan-Chlamydomonas reinhardtii protein complex of Comparative Example 1, and the chitosan-melatonin composite microspheres of Comparative Example 2.

[0045] Figure 21 This is a graph showing the scavenging rates of 2,2-azino-bis(3-ethyl-benzothiazole-6-sulfonic acid) at different pH values ​​for the chitosan-Chlamydomonas reinhardtii protein composite carrier of Example 1, the chitosan-Chlamydomonas reinhardtii protein complex of Comparative Example 1, and the chitosan-melatonin composite microspheres of Comparative Example 2.

[0046] Figure 22 This is a graph showing the scavenging rate of 1,1-diphenyl-2-trinitrophenylhydrazine by different SDS concentrations for the chitosan-Chlamydomonas reinhardtii protein composite carrier of Example 1, the chitosan-Chlamydomonas reinhardtii protein complex of Comparative Example 1, and the chitosan-melatonin composite microspheres of Comparative Example 2.

[0047] Figure 23 This is a graph showing the scavenging rates of 2,2-azino-bis(3-ethyl-benzothiazole-6-sulfonic acid) by the chitosan-Chlamydomonas reinhardtii protein composite carrier of Example 1, the chitosan-Chlamydomonas reinhardtii protein complex of Comparative Example 1, and the chitosan-melatonin composite microspheres of Comparative Example 2 at different SDS concentrations.

[0048] Figure 24 The graph shows the scavenging rates of 1,1-diphenyl-2-trinitrophenylhydrazine by the chitosan-Chlamydomonas reinhardtii protein composite carrier of Example 1, the chitosan-Chlamydomonas reinhardtii protein complex of Comparative Example 1, and the chitosan-melatonin composite microspheres of Comparative Example 2 under different UV irradiation times.

[0049] Figure 25 This is a graph showing the scavenging rate of 2,2-azino-bis(3-ethyl-benzothiazole-6-sulfonic acid) by the chitosan-Chlamydomonas reinhardtii protein composite carrier of Example 1, the chitosan-Chlamydomonas reinhardtii protein complex of Comparative Example 1, and the chitosan-melatonin composite microspheres of Comparative Example 2 under different UV irradiation times.

[0050] Figure 26 This is a graph showing the cumulative release rate of the chitosan-Chlamydomonas reinhardtii protein composite carrier of Example 1, the chitosan-melatonin composite microspheres of Comparative Example 2, and melatonin during an in vitro simulated digestion process, in response to pH.

[0051] Figure 27 This is a cytotoxicity diagram of the chitosan-Chlamydomonas reinhardtii protein composite carrier of Example 1 of the present invention;

[0052] Figure 28 This is a graph showing the relative hemolysis rate of the chitosan-Chlamydomonas reinhardtii protein composite carrier in Example 1 of the present invention. Detailed Implementation

[0053] In view of the problems existing in the above-mentioned prior art, after in-depth research, a chitosan-Chlamydomonas reinhardtii protein composite carrier, its preparation method, and its application are provided. The following will further explain the technical solution, its implementation process, and its principle.

[0054] The first aspect of the present invention provides a chitosan-Chlamydomonas reinhardtii protein composite carrier, the composite carrier having a triple network structure and comprising:

[0055] The three-dimensional network framework is formed by cross-linking chitosan and sodium tripolyphosphate, and constitutes the first layer of the network;

[0056] The functional enhancement phase includes Chlamydomonas reinhardtii protein, which is embedded and anchored in the three-dimensional network backbone through a first physical action and constitutes a second network, thereby forming a chitosan-Chlamydomonas reinhardtii protein complex with an interpenetrating network structure.

[0057] The structure-directing agent includes a hydrophobic active substance, which self-assembles and embeds into the hydrophobic microdomains of the chitosan-Chlamydomonas reinhardtii protein complex through a second physical action, and forms a third network.

[0058] The first physical interaction includes electrostatic interaction and hydrogen bonding, the second physical interaction includes hydrophobic interaction and π-π stacking interaction, and the hydrophobic active substance includes melatonin.

[0059] In some embodiments, the chitosan-Chlamydomonas reinhardtii protein composite carrier is spherical with a particle diameter of 0.45~0.75μm.

[0060] In some embodiments, the raw materials of the chitosan-Chlamydomonas reinhardtii protein composite carrier include 45-50 wt% chitosan, 2.0-3.0 wt% sodium tripolyphosphate, 25-30 wt% Chlamydomonas reinhardtii protein, 0.2-2.0 wt% melatonin, and 20-25 wt% spinning aid.

[0061] In some embodiments, the chitosan-Chlamydomonas reinhardtii protein composite carrier has a pH-responsive, smooth, and dense structure.

[0062] A second aspect of the present invention provides a method for preparing the chitosan-Chlamydomonas reinhardtii protein complex carrier, comprising:

[0063] Chlamydomonas reinhardtii protein was obtained by treating Chlamydomonas reinhardtii with an alkaline dissolution and acid precipitation method.

[0064] Chitosan-acetic acid aqueous solution and sodium tripolyphosphate solution are uniformly mixed to electrostatically crosslink chitosan and sodium tripolyphosphate to form a three-dimensional network framework;

[0065] The Chlamydomonas reinhardtii protein is embedded and anchored in the three-dimensional network backbone by utilizing a first physical action to form a chitosan-Chlamydomonas reinhardtii protein complex with an interpenetrating network structure.

[0066] The chitosan-Chlamydomonas reinhardtii protein complex is uniformly mixed with a spinning aid and a hydrophobic active substance in an aqueous system. The hydrophobic active substance is then self-assembled into the hydrophobic microregions of the chitosan-Chlamydomonas reinhardtii protein complex using a second physical action to obtain a spinning solution. The spinning solution is then used to prepare a chitosan-Chlamydomonas reinhardtii protein complex carrier, wherein the hydrophobic active substance includes melatonin.

[0067] In some implementation methods, Chlamydomonas reinhardtii protein powder is prepared by an alkali-soluble acid-precipitated method, specifically including: mixing Chlamydomonas reinhardtii powder with water at room temperature at a ratio of 1g:15mL to 1g:30mL. The mixture was homogeneously mixed at a mass-to-volume ratio of mL, and the pH was adjusted to 12.0-12.5 with an alkaline substance. The mixture was then stirred continuously at 400-600 rpm for 2-3 hours. After stirring, the mixture was centrifuged at 10000-12000 rpm for 0.5-1 hours. The supernatant was then collected, and the pH was adjusted to 3.0-3.5 with an acidic substance. The mixture was then stirred continuously at 300-500 rpm for 1-2 hours. After centrifugation at 10000-12000 rpm for 0.5-1 hours, the precipitate was collected, and the precipitate was dispersed in water. The pH was adjusted to 7.0-7.5 with an alkaline substance. The mixture was then freeze-dried at -80 to -60°C for 24-36 hours to obtain Chlamydomonas reinhardtii protein.

[0068] Furthermore, the alkaline substance includes a sodium hydroxide solution with a concentration of 0.5~1 mol / L.

[0069] Furthermore, the acidic substance includes a hydrochloric acid solution with a concentration of 0.5~1 mol / L.

[0070] Furthermore, the mass-to-volume ratio of the precipitate to water is 1:10 to 1:20 g / mL.

[0071] In some embodiments, the preparation method specifically includes: adding equal volumes of sodium tripolyphosphate solution to a chitosan-acetic acid aqueous solution in batches at room temperature, and stirring continuously at a stirring speed of 600-800 rpm for 2-3 hours to form a mixed solution containing the three-dimensional network framework.

[0072] Furthermore, the mass-to-volume ratio of chitosan to acetic acid in the chitosan-acetic acid aqueous solution is 1:50~1:200 g / mL.

[0073] Furthermore, the concentration of the acetic acid aqueous solution is 1~2% (v / v).

[0074] Furthermore, the mass-to-volume ratio of sodium tripolyphosphate to water in the sodium tripolyphosphate solution is 1:500~1:1500 g / mL.

[0075] In some embodiments, the preparation method specifically includes: mixing the Chlamydomonas reinhardtii protein powder with a mixed solution containing a three-dimensional network framework at a mass-volume ratio of 1g:100mL to 1g:750mL at room temperature, stirring continuously at a stirring speed of 300-500rpm for 6-8h, then centrifuging at a centrifugation speed of 10000-12000rpm for 0.5-1h, collecting the precipitate, and freeze-drying at -80 to -60℃ for 24-36h to obtain the chitosan-Chlamydomonas reinhardtii protein complex.

[0076] In some embodiments, the preparation method specifically includes: mixing the chitosan-Chlamydomonas reinhardtii protein complex, a spinning aid, and water uniformly at room temperature, then adding melatonin-ethanol solution and mixing uniformly under light-protected conditions to obtain the spinning solution; injecting the spinning solution into calcium chloride solution using an electrospinning device, and preparing the chitosan-Chlamydomonas reinhardtii protein composite carrier by electrospinning process.

[0077] Furthermore, the spinning aid comprises sodium alginate and polyethylene oxide, wherein the mass ratio of sodium alginate to polyethylene oxide is 1:1 to 1:1.5.

[0078] Furthermore, the mass-to-volume ratio of the chitosan-Chlamydomonas reinhardtii protein complex to water is 1:100~1:200 g / mL.

[0079] Furthermore, the mass-to-volume ratio of the spinning aid to water is 1:150 to 1:300 g / mL.

[0080] Furthermore, the mass-to-volume ratio of melatonin to ethanol in the melatonin-ethanol solution is 1:15 to 1:20 g / mL.

[0081] Furthermore, the mass ratio of chitosan-Chlamydomonas reinhardtii protein to melatonin is 10:1 to 30:1.

[0082] Furthermore, the process parameters of the electrospinning technology are as follows: the receiving liquid is a 1-2 wt% calcium chloride solution, the needle type is 21-23 G, the syringe capacity is 10-15 ml, the distance between the needle and the liquid surface is 10-15 cm, the electric field voltage is 10-15 kV, the feeding rate is 0.5-1 ml / h, the temperature is 20-25°C, the humidity is 30-50%, and the time is 10-30 h.

[0083] In some more specific implementations, the manufacturing method specifically includes the following steps:

[0084] (1) Dissolve Chlamydomonas reinhardtii powder in distilled water, adjust the pH to 12.0-12.5 with NaOH solution, and stir continuously at 400-600 rpm for 2-3 hours at room temperature. Then, centrifuge at 10000-12000 rpm for 0.5-1 hour, collect the supernatant, adjust the pH to 3.0-3.5 with HCl solution, and stir continuously at 300-500 rpm for 1-2 hours to ensure complete dissolution. Then, centrifuge again at 10000-12000 rpm for 0.5-1 hour, collect the precipitate, and redisperse it with distilled water. Finally, adjust the pH of the resulting solution to 7.0-7.5 with NaOH solution, and freeze-dry at -80 to -60℃ for 24-36 hours to obtain Chlamydomonas reinhardtii protein powder.

[0085] (2) Chitosan powder was dissolved in an aqueous acetic acid solution, and sodium tripolyphosphate was dissolved in distilled water. The mixture was stirred at 600-800 rpm for 2-3 h. An equal volume of sodium tripolyphosphate solution was added dropwise to the chitosan solution to form a primary chitosan network through electrostatic cross-linking. Then, Chlamydomonas reinhardtii protein powder was added to the above mixed solution. At room temperature, Chlamydomonas reinhardtii protein was embedded and anchored in the chitosan network through electrostatic interaction and hydrogen bonding to form a chitosan-Chlamydomonas reinhardtii protein complex with an interpenetrating network structure. The mixture was stirred at 300-500 rpm for 6-8 h. After thorough stirring, the mixture was centrifuged at 10000-12000 rpm for 0.5-1 h. The precipitate was collected and then freeze-dried at -80 to -60℃ for 24-36 h to obtain the chitosan-Chlamydomonas reinhardtii protein (CS-CRP) complex sample.

[0086] (3) Chitosan-Chlamydomonas reinhardtii protein powder was dissolved in distilled water. Sodium alginate and polyethylene oxide were added as spinning aids at a stirring speed of 400-600 rpm for 2-3 h. Then, melatonin was pre-dissolved in ethanol solution and slowly added dropwise to the above mixed solution. Melatonin self-assembled and embedded itself into the hydrophobic microdomains of the chitosan-Chlamydomonas reinhardtii protein complex through hydrophobic interactions and π-π stacking, inducing rearrangement and densification of the carrier network. After stirring at 300-500 rpm in the dark at room temperature for 10-12 h, a chitosan-Chlamydomonas reinhardtii protein-melatonin electrospinning solution was obtained. The electrospinning process parameters are as follows: the receiving liquid is a 1-2 wt% calcium chloride solution, the needle type is 21-23 G, the syringe capacity is 10-15 ml, the distance between the needle and the liquid surface is 10-15 cm, the electric field voltage is 10-15 kV, the feeding rate is 0.5-1 ml / h, the temperature is 20-25°C, the humidity is 30-50%, and the time is 10-30 h. After electric field-induced directional assembly and secondary cross-linking of calcium ions, chitosan-Chlamydomonas reinhardtii protein composite carrier microspheres are obtained.

[0087] Compared to other proteins or peptide chains, Chlamydomonas reinhardtii proteins, due to their advantages in molecular chain flexibility, molecular weight distribution, and active group arrangement, can be uniformly anchored within the chitosan backbone through physical interactions and form a self-standing network, creating a stable interpenetrating network structure. This significantly enhances the structural stability and functional synergy of the complex. Other proteins or short-chain peptides typically struggle to achieve such a homogeneous and continuous bicontinuous network structure with a rigid backbone while maintaining their own conformation.

[0088] Moreover, this invention is the first to construct a triple network structure with a chitosan-TPP crosslinked network as the backbone, Chlamydomonas reinforcing protein as the functional enhancing phase, and hydrophobic active substances as structure-directing agents. This unique structure not only provides excellent physical barrier protection for the hydrophobic active substances but also achieves a triple antioxidant synergistic mechanism at the molecular level: the antioxidant amino acid residues of Chlamydomonas reinforcing protein directly scavenge free radicals, the chelating effect of chitosan reduces metal ion-catalyzed oxidation, and the hydrophobic active substances act as highly efficient free radical scavengers and provide electron donors. The three synergistically produce antioxidant efficacy that significantly surpasses that of a single component or simple combination. This invention not only provides an innovative and scalable preparation strategy for developing high-performance active substance delivery systems based on sustainable microalgal protein resources but also elucidates, from the perspective of multiple network structures and multiple molecular interactions, the multiple mechanisms by which protein-polysaccharide composite carriers achieve enhanced stability, synergistic activity, and controlled release.

[0089] A third aspect of the invention provides the use of the chitosan-Chlamydomonas reinhardtii protein composite carrier in the preparation of functional food delivery materials or drug delivery materials.

[0090] A fourth aspect of the present invention provides a functional food delivery material comprising the chitosan-Chlamydomonas reinhardtii protein composite carrier.

[0091] For experiments not specifically described in the examples, the procedures or conditions can be performed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available. Other unmentioned raw materials and instruments are all conventionally chosen and do not involve the core technical means of this invention.

[0092] For example, the specific models or sources of the raw materials and instruments used in the embodiments and comparative examples of the present invention are as follows:

[0093] Chitosan: Deacetylation degree of 93.40%, viscosity <200 mpa.s, purchased from Shanghai Yuanye Biotechnology Co., Ltd. (China);

[0094] Melatonin: Purchased from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China);

[0095] Sodium tripolyphosphate: Purchased from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China);

[0096] Sodium alginate: purchased from Aladdin Chemical Reagent Co., Ltd.;

[0097] Polyethylene oxide: purchased from Aladdin Chemical Reagent Co., Ltd.;

[0098] Electrospinning instrument: Electrospinning machine (HZ-12, China).

[0099] Example 1

[0100] This embodiment provides a method for preparing a chitosan-Chlamydomonas reinhardtii protein composite carrier, specifically including the following steps:

[0101] (1) Dissolve 5g of Chlamydomonas reinhardtii powder in 100mL of distilled water, adjust the pH to 12.0 with 1M NaOH solution, and stir thoroughly at room temperature (500rpm, 25℃, 2h). Then, centrifuge at 12000rpm, 25℃ for 0.5h, collect the supernatant, adjust the pH to 3.5 with 1M HCl solution, and continue stirring the supernatant (300rpm, 25℃, 1h) to ensure complete dissolution. Then, centrifuge at 12000rpm, 25℃ for 0.5h, collect the precipitate, and redisperse it with distilled water (precipitate to distilled water mass-to-volume ratio of 1:20). Finally, adjust the pH of the resulting solution to 7.0 with 1M NaOH solution, and freeze-dry at -60℃ for 24h to obtain Chlamydomonas reinhardtii protein powder.

[0102] (2) Dissolve 2g of chitosan powder in 100mL of 1% (v / v) acetic acid aqueous solution, and dissolve 0.1g of sodium tripolyphosphate in 100mL of distilled water. Under continuous stirring (600rpm, 25℃), an equal volume of sodium tripolyphosphate solution was added dropwise to the chitosan solution, and stirring was continued for 2h to allow it to fully crosslink and form a chitosan primary network. Then, 1.2g of Chlamydomonas reinhardtii protein powder was added to the above 200mL mixed solution, and stirring was continued at room temperature (300rpm, 25℃, 6h) to allow Chlamydomonas reinhardtii protein to embed and anchor in the chitosan network through electrostatic interactions and hydrogen bonds, forming a chitosan-Chlamydomonas reinhardtii protein complex with an interpenetrating network structure. After thorough stirring, the mixture was centrifuged at 12000rpm, 25℃ for 0.5h, the precipitate was collected, and then freeze-dried at -60℃ for 24h to obtain the chitosan-Chlamydomonas reinhardtii protein (CS-CRP) complex sample.

[0103] (3) Dissolve 0.60 g of chitosan-Chlamydomonas reinhardtii protein powder in 100 mL of distilled water. Add 0.5 g of sodium alginate and polyethylene oxide while stirring (500 rpm, 25 °C, 3 h). Then, pre-dissolve melatonin in ethanol solution (50 mg / mL). Then, while stirring (800 rpm, 25 °C), slowly add 0.04 g of melatonin (i.e., the mass ratio of chitosan-Chlamydomonas reinhardtii protein to melatonin is 15:1) to the above mixed solution. This allows melatonin to self-assemble and embed into the hydrophobic microdomains of the chitosan-Chlamydomonas reinhardtii protein complex through hydrophobic interactions and π-π stacking, inducing rearrangement and densification of the carrier network. After stirring in the dark at room temperature (300 rpm, 25 °C, 12 h), a chitosan-Chlamydomonas reinhardtii protein-melatonin electrospinning solution is obtained. At room temperature, the above solution was dripped into a 2% calcium chloride solution using an electrospinning apparatus. Following electric field-induced directional assembly and secondary cross-linking by calcium ions, chitosan-Chlamydomonas reinhardtii protein-melatonin electrospun microspheres as described in Example 1 were obtained. The specific electrospinning conditions were as follows: a 20 ml syringe with a 22 G needle was used, the distance between the needle and the liquid surface was maintained at 10 cm, an electric field of 12 kV was applied to all solutions, the feed rate was 1 ml / h, and the temperature was maintained at 25°C and the humidity at 30% throughout the process.

[0104] The chitosan-Chlamydomonas reinhardtii protein composite carrier prepared in this embodiment has a particle diameter of approximately 0.46 μm, a loading of approximately 51.97 μg / mg, and an encapsulation efficiency of approximately 80.68%.

[0105] Example 2

[0106] The difference between this embodiment and Embodiment 1 is that:

[0107] (3) The mass ratio of chitosan-Chlamydomonas reinhardtii protein to melatonin is 30:1, that is, 0.60g of chitosan-Chlamydomonas reinhardtii protein powder and 0.02g of melatonin. The remaining steps are the same as in Example 1.

[0108] The chitosan-Chlamydomonas reinhardtii protein composite carrier prepared in this embodiment has a particle diameter of approximately 0.56 μm, a loading of approximately 18.88 μg / mg, and an encapsulation efficiency of approximately 90.52%.

[0109] Example 3

[0110] The difference between this embodiment and Embodiment 1 is that:

[0111] (3) The mass ratio of chitosan-Chlamydomonas reinhardtii protein to melatonin is 25:1, that is, 0.60g of chitosan-Chlamydomonas reinhardtii protein powder and 0.024g of melatonin. The remaining steps are the same as in Example 1.

[0112] The chitosan-Chlamydomonas reinhardtii protein composite carrier prepared in this embodiment has a particle diameter of approximately 0.53 μm, a loading of approximately 27.43 μg / mg, and an encapsulation efficiency of approximately 88.35%.

[0113] Example 4

[0114] The difference between this embodiment and Embodiment 1 is that:

[0115] (3) The mass ratio of chitosan-Chlamydomonas reinhardtii protein to melatonin is 20:1, that is, 0.60g of chitosan-Chlamydomonas reinhardtii protein powder and 0.03g of melatonin. The remaining steps are the same as in Example 1.

[0116] The chitosan-Chlamydomonas reinhardtii protein composite carrier prepared in this embodiment has a particle diameter of approximately 0.49 μm, a loading of approximately 42.59 μg / mg, and an encapsulation efficiency of approximately 85.05%.

[0117] Example 5

[0118] The difference between this embodiment and Embodiment 1 is that:

[0119] (3) The mass ratio of chitosan-Chlamydomonas reinhardtii protein to melatonin is 10:1, that is, 0.60g of chitosan-Chlamydomonas reinhardtii protein powder and 0.06g of melatonin. The remaining steps are the same as in Example 1.

[0120] The chitosan-Chlamydomonas reinhardtii protein composite carrier prepared in this embodiment has a particle diameter of approximately 0.45 μm, a loading of approximately 55.61 μg / mg, and an encapsulation efficiency of approximately 68.58%.

[0121] Example 6

[0122] The difference between this embodiment and Embodiment 1 is that:

[0123] (2) The mass-to-volume ratio of Chlamydomonas reinhardtii protein powder to the mixed solution containing the three-dimensional network framework is 1:100 g / mL, that is, 1.2 g of Chlamydomonas reinhardtii protein powder is added to 120 mL of the mixed solution, and the remaining steps are the same as in Example 1.

[0124] The chitosan-Chlamydomonas reinhardtii protein composite carrier prepared in this embodiment has a particle diameter of approximately 0.51 μm, a loading of approximately 47.21 μg / mg, and an encapsulation efficiency of approximately 74.35%.

[0125] Example 7

[0126] The difference between this embodiment and Embodiment 1 is that:

[0127] (2) The mass-to-volume ratio of Chlamydomonas reinhardtii protein powder to the mixed solution containing the three-dimensional network framework is 1:500 g / mL, that is, 1.2 g of Chlamydomonas reinhardtii protein powder is added to 600 mL of the mixed solution, and the remaining steps are the same as in Example 1.

[0128] The chitosan-Chlamydomonas reinhardtii protein composite carrier prepared in this embodiment has a particle diameter of approximately 0.65 μm, a loading of approximately 33.52 μg / mg, and an encapsulation efficiency of approximately 68.31%.

[0129] Example 8

[0130] The difference between this embodiment and Embodiment 1 is that:

[0131] (2) The mass-to-volume ratio of Chlamydomonas reinhardtii protein powder to the mixed solution containing the three-dimensional network framework is 1:750 g / mL, that is, 1.2 g of Chlamydomonas reinhardtii protein powder is added to 900 mL of the mixed solution, and the remaining steps are the same as in Example 1.

[0132] The chitosan-Chlamydomonas reinhardtii protein composite carrier prepared in this embodiment has a particle diameter of approximately 0.71 μm, a loading of approximately 23.65 μg / mg, and an encapsulation efficiency of approximately 62.15%.

[0133] Example 9

[0134] (1) Dissolve 5g of Chlamydomonas reinhardtii powder in 100mL of distilled water, adjust the pH to 12.5 with 0.5M NaOH solution, and stir thoroughly at room temperature (400rpm, 20℃, 3h). Then, centrifuge at 10000rpm, 20℃ for 1h, collect the supernatant, adjust the pH to 3 with 0.5M HCl solution, and continue stirring the supernatant (500rpm, 25℃, 2h) to ensure complete dissolution. Then, centrifuge at 10000rpm, 20℃ for 1h, collect the precipitate, and redisperse it with distilled water (precipitate to distilled water mass-to-volume ratio of 1:10). Finally, adjust the pH of the resulting solution to 7.5 with 0.5M NaOH solution, and freeze-dry at -80℃ for 36h to obtain Chlamydomonas reinhardtii protein powder.

[0135] (2) Dissolve 2g of chitosan powder in 100mL of 1% (v / v) acetic acid aqueous solution, and dissolve 0.1g of sodium tripolyphosphate in 50mL of distilled water. Under continuous stirring (800rpm, 20℃), an equal volume of sodium tripolyphosphate solution was added dropwise to the chitosan solution, and stirring was continued for 3h to allow it to fully crosslink and form a chitosan primary network. Then, 1.2g of Chlamydomonas reinhardtii protein powder was added to the above 200mL mixed solution, and stirring was continued at room temperature (500rpm, 20℃, 8h) to allow Chlamydomonas reinhardtii protein to embed and anchor in the chitosan network through electrostatic interactions and hydrogen bonds, forming a chitosan-Chlamydomonas reinhardtii protein complex with an interpenetrating network structure. After thorough stirring, the mixture was centrifuged at 10000rpm, 20℃ for 1h, the precipitate was collected, and then freeze-dried at -80℃ for 36h to obtain the chitosan-Chlamydomonas reinhardtii protein (CS-CRP) complex sample.

[0136] (3) Dissolve 0.60 g of chitosan-Chlamydomonas reinhardtii protein powder in 100 mL of distilled water. Add 0.5 g of sodium alginate and polyethylene oxide while stirring (400 rpm, 20 °C, 2 h). Then, pre-dissolve melatonin in ethanol solution (50 mg / mL). Then, while stirring (600 rpm, 20 °C), slowly add 0.04 g of melatonin (i.e., the mass ratio of chitosan-Chlamydomonas reinhardtii protein to melatonin is 15:1) to the above mixed solution. This allows melatonin to self-assemble and embed into the hydrophobic microdomains of the chitosan-Chlamydomonas reinhardtii protein complex through hydrophobic interactions and π-π stacking, inducing rearrangement and densification of the carrier network. After stirring in the dark at room temperature (500 rpm, 20 °C, 10 h), a chitosan-Chlamydomonas reinhardtii protein-melatonin electrospinning solution is obtained. At room temperature, the above solution was dripped into a 2% calcium chloride solution using an electrospinning apparatus. Following electric field-induced directional assembly and secondary cross-linking with calcium ions, chitosan-Chlamydomonas reinhardtii protein-melatonin electrospun microspheres were obtained. The specific electrospinning conditions were as follows: a 20 ml syringe with a 22 G needle was used, the needle distance from the liquid surface was maintained at 15 cm, an electric field of 15 kV was applied to all solutions, the feed rate was 0.5 ml / h, and the temperature was maintained at 20°C and the humidity at 50% throughout the process.

[0137] The chitosan-Chlamydomonas reinhardtii protein composite carrier prepared in this embodiment has a particle diameter of approximately 0.61 μm, a loading of approximately 41.16 μg / mg, and an encapsulation efficiency of approximately 68.63%.

[0138] Example 10

[0139] The difference between this embodiment and Embodiment 1 is that:

[0140] (1) The mass-to-volume ratio of the Chlamydomonas reinhardtii powder to water is 1:15 g / mL;

[0141] (2) The mass-to-volume ratio of chitosan to acetic acid in the chitosan-acetic acid aqueous solution is 1:50 g / mL; the concentration of the acetic acid aqueous solution is 2% (v / v); the mass-to-volume ratio of sodium tripolyphosphate to water in the sodium tripolyphosphate solution is 1:500 g / mL;

[0142] (3) The mass-to-volume ratio of the chitosan-Chlamydomonas reinhardtii protein complex to water is 1:100 g / mL; the mass ratio of sodium alginate to polyethylene oxide is 1:1;

[0143] The mass-to-volume ratio of the spinning aid to water is 1:150 g / mL;

[0144] The mass-to-volume ratio of melatonin to ethanol in the melatonin-ethanol solution is 1:15 g / mL.

[0145] The remaining steps are the same as in Example 1.

[0146] The chitosan-Chlamydomonas reinhardtii protein composite carrier prepared in this embodiment has a particle diameter of approximately 0.52 μm, a loading of approximately 45.23 μg / mg, and an encapsulation efficiency of approximately 72.45%.

[0147] Example 11

[0148] The difference between this embodiment and Embodiment 1 is that:

[0149] (1) The mass-to-volume ratio of the Chlamydomonas reinhardtii powder to water is 1:30 g / mL;

[0150] (2) The mass-to-volume ratio of chitosan to acetic acid in the chitosan-acetic acid aqueous solution is 1:200 g / mL; the concentration of the acetic acid aqueous solution is 2% (v / v); the mass-to-volume ratio of sodium tripolyphosphate to water in the sodium tripolyphosphate solution is 1:1500 g / mL;

[0151] (3) The mass-to-volume ratio of the chitosan-Chlamydomonas reinhardtii protein complex to water is 1:100 g / mL; the mass ratio of sodium alginate to polyethylene oxide is 1:1.5;

[0152] The mass-to-volume ratio of the spinning aid to water is 1:300 g / mL;

[0153] The melatonin-ethanol solution has a mass-to-volume ratio of melatonin to ethanol of 1:20 g / mL.

[0154] The remaining steps are the same as in Example 1.

[0155] The chitosan-Chlamydomonas reinhardtii protein composite carrier prepared in this embodiment has a particle diameter of approximately 0.63 μm, a loading of approximately 37.38 μg / mg, and an encapsulation efficiency of approximately 67.53%.

[0156] Comparative Example 1

[0157] (1) Dissolve 5g of Chlamydomonas reinhardtii powder in 100mL of distilled water, adjust the pH to 12.0 with 1M NaOH solution, and stir thoroughly at room temperature (500rpm, 25℃, 2h). Then, centrifuge at 12000rpm, 25℃ for 0.5h, collect the supernatant, adjust the pH to 3.5 with 1M HCl solution, and continue stirring the supernatant (300rpm, 25℃, 1h) to ensure complete dissolution. Then, centrifuge at 12000rpm, 25℃ for 0.5h, collect the precipitate, and redisperse it with distilled water (precipitate to distilled water mass-to-volume ratio of 1:20). Finally, adjust the pH of the resulting solution to 7.0 with 1M NaOH solution, and freeze-dry at -60℃ for 24h to obtain Chlamydomonas reinhardtii protein powder.

[0158] (2) Dissolve 2g of chitosan powder in 100mL of 1% (v / v) acetic acid aqueous solution, and dissolve 0.1g of sodium tripolyphosphate in 100mL of distilled water. Under continuous stirring (600rpm, 25℃), add an equal volume of sodium tripolyphosphate solution dropwise to the chitosan solution, and continue stirring for 2h to allow it to fully crosslink and form a chitosan primary network. Then add 1.2g of Chlamydomonas reinhardtii protein powder to the above 200mL mixed solution, and stir continuously at room temperature (300rpm, 25℃, 6h) to allow Chlamydomonas reinhardtii protein to embed and anchor in the chitosan network through electrostatic interactions and hydrogen bonds, forming a chitosan-Chlamydomonas reinhardtii protein complex with an interpenetrating network structure. After thorough stirring, the mixture was centrifuged at 12,000 rpm and 25°C for 0.5 h, and the precipitate was collected. Then, it was freeze-dried at -60°C for 24 h to obtain the chitosan-Chlamydomonas reinhardtii protein (CS-CRP) complex sample in Comparative Example 1.

[0159] Comparative Example 2

[0160] (1) Dissolve 0.60 g of chitosan powder in 100 mL of distilled water. Add 0.5 g of sodium alginate and polyethylene oxide while stirring (500 rpm, 25 °C, 3 h). Then, pre-dissolve melatonin in ethanol solution (50 mg / mL). Add 0.04 g of melatonin (i.e., the mass ratio of chitosan to melatonin is 15:1) slowly dropwise to the above mixed solution while stirring (800 rpm, 25 °C). After stirring in the dark at room temperature (300 rpm, 25 °C, 12 h), obtain a chitosan-melatonin electrospinning solution. At room temperature, dropwise inject the above solution into a 2% calcium chloride solution using an electrospinning instrument to obtain the chitosan-melatonin electrospinned microspheres in Comparative Example 2. The specific conditions for electrospinning are as follows: a 20 ml syringe is used, the needle is a 22 G type, the distance between the needle and the liquid surface is kept at 10 cm, an electric field of 12 kV is applied to all solutions, the feed rate is 1 ml / h, and the temperature is kept at 25°C and the humidity is 30% throughout the process.

[0161] Comparative Example 3

[0162] (1) Dissolve 5g of Chlamydomonas reinhardtii powder in 100mL of distilled water, adjust the pH to 12.0 with 1M NaOH solution, and stir thoroughly at room temperature (500rpm, 25℃, 2h). Then, centrifuge at 12000rpm, 25℃ for 0.5h, collect the supernatant, adjust the pH to 3.5 with 1M HCl solution, and continue stirring the supernatant (300rpm, 25℃, 1h) to ensure complete dissolution. Then, centrifuge at 12000rpm, 25℃ for 0.5h, collect the precipitate, and redisperse it with distilled water (precipitate to distilled water mass-to-volume ratio of 1:20). Finally, adjust the pH of the resulting solution to 7.0 with 1M NaOH solution, and freeze-dry at -60℃ for 24h to obtain Chlamydomonas reinhardtii protein powder.

[0163] (2) Dissolve 0.60 g of Chlamydomonas reinhardtii protein powder in 100 mL of distilled water. Add 0.5 g of sodium alginate and polyethylene oxide while stirring (500 rpm, 25 °C, 3 h). Then, pre-dissolve melatonin in ethanol solution (50 mg / mL). Then, while stirring (800 rpm, 25 °C), slowly add 0.04 g of melatonin (i.e., the mass ratio of Chlamydomonas reinhardtii protein to melatonin is 15:1) to the above mixed solution. This allows melatonin to self-assemble and embed into the hydrophobic microregions of Chlamydomonas reinhardtii protein through hydrophobic interactions and π-π stacking, inducing rearrangement and densification of the carrier network. After stirring in the dark at room temperature (300 rpm, 25 °C, 12 h), a Chlamydomonas reinhardtii protein-melatonin electrospinning solution is obtained. At room temperature, the above solution was dripped into a 2% calcium chloride solution using an electrospinning apparatus. Following electric field-induced directional assembly and secondary cross-linking by calcium ions, electrospun microspheres of Chlamydomonas reinhardtii protein and melatonin, as shown in Comparative Example 3, were obtained. The specific electrospinning conditions were as follows: a 20 ml syringe with a 22G needle was used, the distance between the needle and the liquid surface was maintained at 10 cm, an electric field of 12 kV was applied to all solutions, the feed rate was 1 ml / h, and the temperature was maintained at 25°C and the humidity at 30% throughout the process.

[0164] The Chlamydomonas reinhardtii protein-melatonin complex carrier prepared in this comparative example has a particle diameter of approximately 0.97 μm, a loading capacity of approximately 11.32 μg / mg, and an encapsulation efficiency of approximately 23.51%.

[0165] Comparative Example 4

[0166] (1) Dissolve 5g of Chlamydomonas reinhardtii powder in 100mL of distilled water, adjust the pH to 12.0 with 1M NaOH solution, and stir thoroughly at room temperature (500rpm, 25℃, 2h). Then, centrifuge at 12000rpm, 25℃ for 0.5h, collect the supernatant, adjust the pH to 3.5 with 1M HCl solution, and continue stirring the supernatant (300rpm, 25℃, 1h) to ensure complete dissolution. Then, centrifuge at 12000rpm, 25℃ for 0.5h, collect the precipitate, and redisperse it with distilled water (precipitate to distilled water mass-to-volume ratio of 1:20). Finally, adjust the pH of the resulting solution to 7.0 with 1M NaOH solution, and freeze-dry at -60℃ for 24h to obtain Chlamydomonas reinhardtii protein powder.

[0167] (2) Dissolve 2g of chitosan powder in 100mL of 1% (v / v) acetic acid aqueous solution. While stirring continuously (600rpm, 25℃), add 1.2g of Chlamydomonas reinhardtii protein powder to the above solution. Stir continuously at room temperature (300rpm, 25℃, 6h) to allow Chlamydomonas reinhardtii protein to embed and anchor in the chitosan network through electrostatic interactions and hydrogen bonds, forming a chitosan-Chlamydomonas reinhardtii protein complex with an interpenetrating network structure. After thorough stirring, centrifuge at 12000rpm, 25℃ for 0.5h, collect the precipitate, and then freeze-dry at -60℃ for 24h to obtain the chitosan-Chlamydomonas reinhardtii protein (CS-CRP) complex sample.

[0168] (3) Dissolve 0.60 g of chitosan-Chlamydomonas reinhardtii protein powder in 100 mL of distilled water. Add 0.5 g of sodium alginate and polyethylene oxide while stirring (500 rpm, 25 °C, 3 h). Then, pre-dissolve melatonin in ethanol solution (50 mg / mL). Then, while stirring (800 rpm, 25 °C), slowly add 0.04 g of melatonin (i.e., the mass ratio of chitosan-Chlamydomonas reinhardtii protein to melatonin is 15:1) to the above mixed solution. This allows melatonin to self-assemble and embed into the hydrophobic microdomains of the chitosan-Chlamydomonas reinhardtii protein complex through hydrophobic interactions and π-π stacking, inducing rearrangement and densification of the carrier network. After stirring in the dark at room temperature (300 rpm, 25 °C, 12 h), a chitosan-Chlamydomonas reinhardtii protein-melatonin electrospinning solution is obtained. At room temperature, the above solution was dripped into a 2% calcium chloride solution using an electrospinning apparatus. Following electric field-induced directional assembly and secondary cross-linking by calcium ions, chitosan-Chlamydomonas reinhardtii protein-melatonin electrospun microspheres, as shown in Comparative Example 4, were obtained. The specific electrospinning conditions were as follows: a 20 ml syringe with a 22 G needle was used, the needle distance from the liquid surface was maintained at 10 cm, an electric field of 12 kV was applied to all solutions, the feed rate was 1 ml / h, and the temperature was maintained at 25°C and the humidity at 30% throughout the process.

[0169] The chitosan-Chlamydomonas reinhardtii protein composite carrier prepared in this comparative example has a particle diameter of approximately 0.81 μm, a loading of approximately 15.12 μg / mg, and an encapsulation efficiency of approximately 36.25%.

[0170] Comparative Example 5

[0171] (1) Dissolve 5g of spirulina powder in 100mL of distilled water, adjust the pH to 12.0 with 1M NaOH solution, and stir thoroughly at room temperature (500rpm, 25℃, 2h). Then, centrifuge at 12000rpm, 25℃ for 0.5h, collect the supernatant, adjust the pH to 3.5 with 1M HCl solution, and continue stirring the supernatant (300rpm, 25℃, 1h) to ensure complete dissolution. Then, centrifuge at 12000rpm, 25℃ for 0.5h, collect the precipitate, and redisperse it with distilled water (precipitate to distilled water mass-to-volume ratio of 1:20). Finally, adjust the pH of the resulting solution to 7.0 with 1M NaOH solution, and freeze-dry at -60℃ for 24h to obtain spirulina protein powder.

[0172] (2) Dissolve 2g of chitosan powder in 100mL of 1% (v / v) acetic acid aqueous solution, and dissolve 0.1g of sodium tripolyphosphate in 100mL of distilled water. Under continuous stirring (600rpm, 25℃), add an equal volume of sodium tripolyphosphate solution dropwise to the chitosan solution, and continue stirring for 2h to allow it to fully crosslink and form a chitosan primary network. Then add 1.2g of spirulina protein powder to the above 200mL mixed solution, and stir continuously at room temperature (300rpm, 25℃, 6h) to allow the spirulina protein to embed and anchor in the chitosan network through electrostatic interactions and hydrogen bonds, forming a chitosan-spirulina protein complex with an interpenetrating network structure. After thorough stirring, centrifuge at 12000rpm, 25℃ for 0.5h, collect the precipitate, and then freeze-dry at -60℃ for 24h to obtain the chitosan-spirulina protein complex sample.

[0173] (3) Dissolve 0.60 g of chitosan-spirulina protein powder in 100 mL of distilled water. Add 0.5 g of sodium alginate and polyethylene oxide while stirring (500 rpm, 25 °C, 3 h). Then, pre-dissolve melatonin in an ethanol solution (50 mg / mL). Then, while stirring (800 rpm, 25 °C), slowly add 0.04 g of melatonin (i.e., the mass ratio of chitosan-spirulina protein to melatonin is 15:1) to the above mixed solution to allow melatonin to embed into the hydrophobic microregions of the chitosan-spirulina protein complex. After stirring in the dark at room temperature (300 rpm, 25 °C, 12 h), obtain a chitosan-spirulina protein-melatonin electrospinning solution. At room temperature, the above solution was dripped into a 2% calcium chloride solution using an electrospinning apparatus. Following electric field-induced directional assembly and secondary cross-linking by calcium ions, chitosan-spirulina protein-melatonin electrospun microspheres as described in Example 1 were obtained. The specific electrospinning conditions were as follows: a 20 ml syringe with a 22 G needle was used, the distance between the needle and the liquid surface was maintained at 10 cm, an electric field of 12 kV was applied to all solutions, the feed rate was 1 ml / h, and the temperature was maintained at 25°C and the humidity at 30% throughout the process.

[0174] The chitosan-spirulina protein-melatonin composite carrier prepared in this comparative example has a particle diameter of approximately 0.73 μm, a loading of approximately 22.35 μg / mg, and an encapsulation efficiency of approximately 41.62%.

[0175] Performance testing

[0176] Figure 1 This is a dispersion diagram of chitosan and chitosan-Chlamydomonas reinhardtii protein complex in Example 1 of the present invention; Figure 2 This is a hydrophobic diagram of the Chlamydomonas reinhardtii protein and chitosan-Chlamydomonas reinhardtii protein complex in Example 1 of the present invention; Figure 3 This is the X-ray photoelectron spectrum of the chitosan and chitosan-Chlamydomonas reinhardtii protein complex from Example 1 of this invention. Figure 1-3As shown, in terms of dispersibility, compared with chitosan (polydispersity index of 0.37), the polydispersity index of the chitosan-Chlamydomonas reinhardtii protein complex decreased to 0.26, a decrease of 29.7%. This indicates that at this ratio, Chlamydomonas reinhardtii protein is not simply mixed with chitosan, but rather embedded in the chitosan-TPP crosslinking network through electrostatic interactions and hydrogen bonds, acting as a "crosslinking point enhancer". This effectively inhibits the aggregation of chitosan chains, resulting in a more uniform particle size distribution and forming a more homogeneous and stable dispersion system. Regarding hydrophobicity, the chitosan-Chlamydomonas reinhardtii complex exhibits significantly enhanced hydrophobicity compared to Chlamydomonas reinhardtii protein. This may be attributed to the fact that, at this ratio, chitosan and Chlamydomonas reinhardtii protein form a dense and stable composite conformation through multiple non-covalent interactions. This allows hydrophobic groups within Chlamydomonas reinhardtii protein (such as aromatic or aliphatic amino acid side chains) to be more fully exposed on the surface or rearranged, creating a richer hydrophobic microenvironment than the single component. This provides ideal binding sites for the subsequent loading of the hydrophobic active substance melatonin. X-ray photoelectron spectroscopy analysis shows that, compared to chitosan, the relative intensities of C, N, and O elements in the chitosan-Chlamydomonas reinhardtii protein complex change significantly, and the characteristic S 2p signal of Chlamydomonas reinhardtii protein appears. This signal originates from sulfur-containing amino acids (such as methionine and cysteine) in Chlamydomonas reinhardtii protein. This elemental analysis confirms the successful introduction of Chlamydomonas reinhardtii protein and its effective complexation with chitosan, forming a true molecular-level complex rather than a simple physical blend. In summary, the results of dispersibility, hydrophobicity, and X-ray photoelectron spectroscopy analysis confirm that, at this ratio, chitosan and Chlamydomonas reinhardtii protein successfully constructed a composite functional carrier with uniform size distribution, good dispersibility, and significantly enhanced surface hydrophobicity, laying a structural foundation for the efficient loading of melatonin and the synergistic antioxidant effect.

[0177] Figure 4-6 These are, respectively, two-dimensional atomic force microscope (AFM) image, three-dimensional atomic force microscope (AFM) image, and scanning electron microscope (SEM) image of chitosan in Example 1 of the present invention; Figure 7-9 These are, respectively, atomic force microscopy (AFM) two-dimensional morphology image, atomic force microscopy (AFM) three-dimensional morphology image, and scanning electron microscopy (SEM) image of the chitosan-Chlamydomonas reinhardtii protein complex of Example 1 of the present invention; Figure 10-12 These are, respectively, atomic force microscopy (AFM) two-dimensional morphology images, atomic force microscopy (AFM) three-dimensional morphology images, and scanning electron microscopy (SEM) images of the chitosan-Chlamydomonas reinhardtii protein composite carrier microspheres of Example 1 of the present invention. Figure 4-12As shown, in terms of three-dimensional surface morphology, chitosan exhibits a sparse, irregular, and relatively flat granular morphology. In contrast, the chitosan-Chlamydomonas reinhardtii protein complex forms a relatively dense, uniform, and nearly spherical structure, with significantly improved dispersibility. This confirms that the introduction of Chlamydomonas reinhardtii protein may effectively regulate the aggregation behavior of chitosan, promoting the formation of more regular nanostructures and initially demonstrating the modifying effect of the "protein-enhancing phase" on the chitosan backbone. After loading melatonin, the spherical structure of the chitosan-Chlamydomonas reinhardtii protein complex carrier was maintained and its distribution became more uniform, while the overall particle size showed a decreasing trend. This indicates that melatonin is not only a passively loaded active ingredient but also acts as a "structure-directing agent," participating in and synergistically regulating the assembly process of microspheres through hydrophobic interactions and π-π stacking, inducing the formation of more dense and uniformly sized spherical particles. This reflects the active role of melatonin in the construction of the "triple network." Although a few large aggregates exist, the majority of the structure consists of well-dispersed small spherical particles, which is beneficial for increasing the specific surface area and loading efficiency of the carrier. Under scanning electron microscopy, chitosan exhibits an irregular, loose, and porous aggregated network structure. The chitosan-Chlamydomonas reinhardtii protein complex, however, displays a more irregular morphology with a rougher surface and a denser structure. This may be attributed to the enhanced intermolecular interactions and increased structural rigidity of the complex due to the introduction of Chlamydomonas reinhardtii protein, thus better resisting pore collapse during freeze-drying. After loading with melatonin, the aggregated network structure of the chitosan-Chlamydomonas reinhardtii protein complex carrier becomes more compact, with a smoother and denser surface and no obvious pore defects. This significant morphological transformation visually demonstrates the hierarchical construction process of the "triple network": chitosan-TPP provides the primary framework (first layer), Chlamydomonas reinhardtii protein insertion enhances (second layer), and melatonin-induced rearrangement densification (third layer). These three layers work synergistically and progressively to ultimately form structurally complete composite microspheres. The hydrophobicity of melatonin may have facilitated phase separation in the aqueous phase of the complex or acted as a "cross-linking point," guiding the chitosan-Chlamydomonas reinhardtii protein carrier to form a denser coating structure during assembly and subsequent drying, resulting in a macroscopically compacted structure and a smoother surface. In summary, the characterization results corroborate each other, and this systematic change in morphology directly confirms the successful introduction and complexation of Chlamydomonas reinhardtii protein and melatonin, fully demonstrating the formation process of the "triple network" structure.

[0178] Figure 13 These are the Fourier transform infrared spectra of the chitosan-Chlamydomonas reinhardtii protein composite carriers of Examples 1-5 of this invention and the chitosan-Chlamydomonas reinhardtii protein complex of Comparative Example 1. Figure 13As shown, in the infrared spectrum, compared with Comparative Example 1, the chitosan-Chlamydomonas reinhardtii protein composite carriers in Examples 1-5 exhibit significant changes in the amide bond region: the characteristic absorption peaks of the amide I band (C=O stretching vibration) and the amide II band (NH bending vibration), which were originally distinguishable around 1615 cm⁻¹, merged into a single broad peak and shifted significantly to a lower wavenumber to 1606 cm⁻¹. This phenomenon strongly suggests that melatonin molecules may not simply be physically adsorbed onto the carrier surface or pores, but rather form new, specific intermolecular interactions (such as hydrogen bonds or π-π stacking) with the C=O or NH groups in the chitosan-Chlamydomonas reinhardtii protein carrier through NH or aromatic ring groups on their indole rings. This molecular-level interaction is direct evidence of the formation of the third network (melatonin-directing network) in the "triple network". This interaction alters the local chemical environment and electron distribution of the amide bond, leading to a decrease in its characteristic vibrational frequency. Meanwhile, compared to Comparative Example 1, the broad peaks (OH stretching vibrations) in the 3200–3400 cm⁻¹ range in Examples 1-5 showed significant peak broadening after melatonin loading. This may further indicate that the introduction of melatonin reconstructed and strengthened the hydrogen bond network of the entire complex system, resulting in tighter interfacial bonding between the triple network. In summary, infrared spectroscopy analysis confirms at the molecular level that melatonin and the chitosan-Chlamydomonas reinhardtii protein carrier may have formed a hydrogen-bonded molecular interaction, which is the molecular basis for the formation of the "function-directed network" in the triple network structure. This structural evidence provides a key structural basis for the high loading efficiency, enhanced stability, and controllable release behavior subsequently exhibited by the chitosan-Chlamydomonas reinhardtii protein complex carrier.

[0179] Figure 14 These are thermal stability diagrams of the chitosan-Chlamydomonas reinhardtii protein composite carriers of Examples 1-5 of the present invention and the chitosan-Chlamydomonas reinhardtii protein complex of Comparative Example 1. Figure 15 These are UV stability diagrams of the chitosan-Chlamydomonas reinhardtii protein composite carriers of Examples 1-5 of this invention and the chitosan-Chlamydomonas reinhardtii protein complex of Comparative Example 1. Figure 14As shown, regarding thermal stability, compared to Comparative Example 1 (endothermic peak at approximately 130 °C), the endothermic peaks of the composite microspheres in Examples 1-5 shifted significantly towards higher temperatures, indicating a substantial improvement in thermal stability, reaching a peak at Example 1 (154 °C). This trend suggests that within the optimized ratio range of Example 1, stable intermolecular interactions (such as hydrogen bonds or hydrophobic interactions) formed between the melatonin molecules and the chitosan-Chlamydomonas reinhardtii protein carrier. These forces, acting as additional "crosslinking points" or "physical crosslinks," effectively limited the mobility of polymer chain segments under heat, making the bonds between the triple network more robust, thereby increasing the energy required for the thermal transition of the entire composite system, macroscopically manifested as a systemic enhancement in thermal stability. Figure 15 As shown, regarding UV stability, melatonin is highly sensitive to ultraviolet light due to the presence of a photosensitive indole ring in its molecular structure. After 10 hours of irradiation, the retention rate of free melatonin dropped sharply to 74.97%, and further decreased to 56.43% after 40 hours. In contrast, the chitosan-Chlamydomonas reinhardtii protein composite carriers in Examples 1-5 exhibited significant protective effects. With increasing melatonin addition ratio, its retention rate showed a trend of first increasing and then slightly decreasing, reaching a peak at Example 1. At this ratio, the chitosan-Chlamydomonas reinhardtii protein composite carrier exhibited significantly superior photoprotective performance, with a melatonin retention rate as high as 88.87% after 40 hours of irradiation. This superior protective effect can be attributed to the synergistic mechanism of the triple network: on the one hand, the first and second networks (chitosan-TPP backbone and Chlamydomonas reinhardtii protein reinforcing phase) embed melatonin into the hydrophobic cavity of Chlamydomonas reinhardtii protein or bind it to its hydrophobic region through hydrophobic interactions, thereby physically reducing its direct contact with ultraviolet light. On the other hand, Chlamydomonas reinhardtii protein itself, as a microalgal protein, may contain natural pigments such as phycobiliproteins, giving it excellent light absorption capabilities. It can act as an internal UV shield, actively dissipating or filtering out some of the incident UV light energy, providing dual protection for the encapsulated melatonin. The dense structure of the third network (melatonin-directing network) further enhances this protective effect. In summary, this conclusion, corroborated by the thermal stability analysis results, jointly demonstrates that the chitosan-Chlamydomonas reinhardtii protein composite carrier in Example 1 provides comprehensive and excellent environmental stability. This is due to the multiple protective mechanisms provided by the triple network structure, which is crucial for ensuring its efficacy in processing, storage, and final functional food applications.

[0180] Figure 16 This is a diagram showing the loading amount of the chitosan-Chlamydomonas reinhardtii protein composite carrier in Examples 1-5 of the present invention; Figure 17 These are encapsulation efficiency diagrams of the chitosan-Chlamydomonas reinhardtii protein composite carriers in Examples 1-5 of this invention; Figure 18The graphs show the antioxidant properties of the chitosan-Chlamydomonas reinhardtii protein composite carriers of Examples 1-5 and the chitosan-Chlamydomonas reinhardtii protein complex of Comparative Example 1 against 1,1-diphenyl-2-trinitrophenylhydrazine. Figure 19 This is a graph showing the antioxidant properties of the chitosan-Chlamydomonas reinhardtii protein composite carriers of Examples 1-5 and the chitosan-Chlamydomonas reinhardtii protein complex of Comparative Example 1 against 2,2-azido-bis(3-ethyl-benzothiazole-6-sulfonic acid). Figure 16 As shown, regarding the loading capacity, the loading capacity in Examples 1-5 gradually increased with the increase of the melatonin addition ratio. Specifically, the loading capacity gradually climbed from a lower level, reaching a near-maximum value (51.98 μg / mg) in Example 1. This significant increase in loading capacity is attributed to the abundant binding sites in the triple network structure: the porous structure of the first network provides physical storage space, the hydrophobic microregions of the second network provide hydrophobic binding sites, and the intermolecular interactions of the third network provide chemical anchoring sites; the three work synergistically to achieve efficient loading of melatonin. Figure 17 As shown, regarding encapsulation efficiency, although the encapsulation efficiency gradually decreased in Examples 1-5 as the proportion of melatonin added increased, it still maintained a high level (80.68%) in Example 1, indicating that within this range, the synergistic encapsulation ability of the triple network remained excellent, and most melatonin molecules could be effectively captured. Figure 18 and Figure 19 As shown, in terms of antioxidant performance, compared with Comparative Example 1, the antioxidant performance of Examples 1-5 showed a gradual upward trend with the increase of melatonin addition ratio. Their DPPH and ABTS free radical scavenging rates continued to climb, reaching near their highest values ​​in Example 1 (88.52% and 85.89%, respectively). This significant performance improvement reflects a triple antioxidant synergistic mechanism: melatonin, as a highly efficient free radical scavenger, provides additional electron donors through its indole structure; chitosan in the first network reduces oxidation catalysis by chelating metal ions; the antioxidant amino acid residues of Chlamydomonas reinhardtii in the second network directly scavenge free radicals; and the introduction of melatonin in the third network places the antioxidant groups in the carrier in a more favorable exposed conformation. The combined effect of these three elements achieves a synergistic effect of "1+1+1>3". Therefore, the results indicate that the intermolecular interactions among the three elements in Example 1 reached an optimal balance, achieving not only efficient loading but also a significant synergistic enhancement effect at the functional level, providing an important basis for constructing high-performance antioxidant carriers with both delivery function and bioactivity.

[0181] Figure 20This is a graph showing the scavenging rate of 1,1-diphenyl-2-trinitrophenylhydrazine by different pH values ​​for the chitosan-Chlamydomonas reinhardtii protein composite carrier of Example 1, the chitosan-Chlamydomonas reinhardtii protein complex of Comparative Example 1, and the chitosan-melatonin composite microspheres of Comparative Example 2. Figure 21 This is a graph showing the scavenging rates of 2,2-azino-bis(3-ethyl-benzothiazole-6-sulfonic acid) at different pH values ​​for the chitosan-Chlamydomonas reinhardtii protein composite carrier of Example 1, the chitosan-Chlamydomonas reinhardtii protein complex of Comparative Example 1, and the chitosan-melatonin composite microspheres of Comparative Example 2. Figure 22 This is a graph showing the scavenging rate of 1,1-diphenyl-2-trinitrophenylhydrazine by different SDS concentrations for the chitosan-Chlamydomonas reinhardtii protein composite carrier of Example 1, the chitosan-Chlamydomonas reinhardtii protein complex of Comparative Example 1, and the chitosan-melatonin composite microspheres of Comparative Example 2. Figure 23 This is a graph showing the scavenging rates of 2,2-azino-bis(3-ethyl-benzothiazole-6-sulfonic acid) by the chitosan-Chlamydomonas reinhardtii protein composite carrier of Example 1, the chitosan-Chlamydomonas reinhardtii protein complex of Comparative Example 1, and the chitosan-melatonin composite microspheres of Comparative Example 2 at different SDS concentrations. Figure 24 The graph shows the scavenging rates of 1,1-diphenyl-2-trinitrophenylhydrazine by the chitosan-Chlamydomonas reinhardtii protein composite carrier of Example 1, the chitosan-Chlamydomonas reinhardtii protein complex of Comparative Example 1, and the chitosan-melatonin composite microspheres of Comparative Example 2 under different UV irradiation times. Figure 25 This is a graph showing the scavenging rate of 2,2-azino-bis(3-ethyl-benzothiazole-6-sulfonic acid) by the chitosan-Chlamydomonas reinhardtii protein composite carrier of Example 1, the chitosan-Chlamydomonas reinhardtii protein complex of Comparative Example 1, and the chitosan-melatonin composite microspheres of Comparative Example 2 under different UV irradiation times.

[0182] like Figure 20As shown, under different pH conditions, for Comparative Example 1, its antioxidant activity showed a trend of first stabilizing and then gradually increasing across the entire pH range, especially significantly enhanced under alkaline conditions. This phenomenon may be attributed to alkaline pH inducing partial unfolding of the protein structure, leading to the exposure of antioxidant amino acid residues buried inside the molecule to the surface, thereby increasing the effective active sites that can participate in free radical scavenging. Conversely, Comparative Example 2 showed a gradual decreasing trend, with a sharp decrease in activity under alkaline conditions. This indicates that the single chitosan carrier has limited protective effect on melatonin. In an alkaline environment, melatonin undergoes structural changes or is released and degraded from the carrier due to the lack of effective protection, resulting in impaired inherent antioxidant capacity. In contrast, Example 1 exhibited more stable antioxidant performance, with only a slight increase under acidic conditions and a slight decrease under alkaline conditions, with overall fluctuations far smaller than the former two. This excellent stability reflects the multiple protective effects of the triple network structure on melatonin: even if one network is disturbed by the environment, the other networks can still maintain structural integrity and continue to protect melatonin. Figures 21-25As shown, at different SDS concentrations, the free radical scavenging activity of Comparative Example 1 initially increased and then decreased with increasing SDS concentration. When the SDS concentration increased from 0 wt% to 0.05 wt%, its scavenging activity increased slowly, indicating that low concentrations of SDS induced partial dissociation and conformational loosening of the protein, exposing more buried antioxidant active sites. However, when the SDS concentration continued to increase to 0.1-5 wt%, its scavenging activity decreased slowly, possibly because excessively high concentrations of SDS began to disrupt the secondary structure of the protein, leading to denaturation and inactivation of some active sites. For the melatonin-loaded complex, the trend was similar to that of Comparative Example 1. In the low SDS concentration range (0-0.05 wt%), the scavenging activities of Comparative Example 2 and Example 1 also showed a slow increasing trend, possibly because low concentrations of SDS slightly perturbed the carrier structure, promoting partial release of melatonin from the carrier or limited exposure of active sites. In the high SDS concentration range (0.1-5 wt%), its scavenging activity decreased. However, it is worth noting that compared to the sharp decline in Comparative Example 2 throughout the high concentration range, the decline in Example 1 was significantly smaller, and its antioxidant performance remained at a high level throughout. This stark contrast fully demonstrates that the triple network structure has a stronger structural protection capability for melatonin. Even under strong dissociation conditions, its dense multi-crosslinked network can still effectively retain and protect most of the melatonin, demonstrating the "structural redundancy" characteristic of the triple network compared to a single network. Under different UV irradiation times, due to UV-induced photodegradation, the free radical scavenging activity of free melatonin decreases rapidly over time. The scavenging activity of Comparative Example 1 decreased significantly in the first 4 hours and then tended to stabilize. This may be due to the rapid destruction of its easily accessible active groups on the surface in the initial stage, while the internally embedded active sites were maintained and continued to function due to the protective effect of the carrier structure. Comparative Example 2 showed a continuous downward trend, further indicating that a single chitosan carrier is difficult to provide long-term photoprotection. In contrast, Example 1 only decreased slightly in the first 4 hours and then tended to stabilize. However, after 12 hours of irradiation, the DPPH and ABTS free radical scavenging rates decreased by only 11.53% and 12.60%, respectively, remaining at a high level of 75.87% and 72.56%. The results indicate that the triple network structure provides excellent photoprotection for melatonin through a dual mechanism of stable encapsulation and its own light absorption properties (derived from the pigment cofactors of Chlamydomonas reinhardtii proteins), an unexpected technical effect that cannot be achieved with a single network structure. In summary, the composite microspheres in Example 1 exhibited significantly better antioxidant stability than a single chitosan carrier under different conditions, including pH changes, SDS dissociation, and UV irradiation, fully demonstrating the synergistic protective advantages of the triple network structure in extreme environments.

[0183] Figure 26This is a graph showing the cumulative release rate of chitosan-Chlamydomonas reinhardtii protein composite carrier (Example 1), chitosan-melatonin composite microspheres (Comparative Example 2), and melatonin during simulated digestion in vitro, exhibiting pH-responsiveness. Figure 26 As shown, free melatonin exhibits a slow release trend during the first 30 minutes of digestion. Subsequently, due to its low solubility in water, the release curve gradually flattens, with a final cumulative release rate of approximately 9.81%. This result indicates that the release of unencapsulated free melatonin in a simulated digestive environment is extremely limited, and its inherent low water solubility severely restricts its bioavailability in the gastrointestinal tract. In contrast, the release in Comparative Example 2 and Example 1 is significantly accelerated, exhibiting a distinct "two-stage" release pattern. In the simulated gastric juice stage (0-60 min), melatonin is released steadily and slowly with increasing digestion time; however, upon entering the simulated intestinal juice stage (60-120 min), an explosive release occurs. This release behavior can be attributed to the structural responsiveness of the carrier material under different pH conditions. In the acidic gastric environment, chitosan has a high degree of protonation, and the carrier structure is relatively compact, resulting in a slow release of melatonin mainly via diffusion. However, upon entering the neutral to weakly alkaline intestinal environment, the deprotonation of chitosan causes the carrier network to swell or even partially disintegrate, thereby triggering a rapid release of melatonin. Further analysis revealed that after 60 and 120 minutes of digestion, the cumulative release rates of Comparative Example 2 were 17.89% and 39.12%, respectively, while the cumulative release rates of Example 1 reached 24.13% and 54.23%, respectively. Moreover, the release rate of Example 1 at all time points was significantly higher than that of Comparative Example 2. This phenomenon can be attributed to the dual pH response mechanism in the triple network structure: on the one hand, the pH-responsive swelling characteristics of the first network (chitosan-TPP), and on the other hand, the conformational unfolding of the second network (Chlamydomonas reinhardtii protein) under alkaline conditions. The superposition of these two factors leads to further relaxation of the carrier network structure, thereby accelerating the diffusion and release of melatonin, demonstrating the synergistic effect of the triple network in functional response. It is noteworthy that the release of Example 1 was relatively limited in the simulated gastric juice stage, while the main release occurred in the simulated intestinal juice stage. This result indicates that the chitosan-Chlamydomonas reinhardtii protein composite carrier can effectively protect melatonin from the damage of the gastric acid environment and accurately deliver melatonin to the intestinal site for targeted release, exhibiting excellent sustained-release performance and intestinal targeting characteristics. In summary, encapsulating melatonin in a chitosan-Chlamydomonas reinhardtii protein complex not only significantly improved its release behavior in aqueous media, but also achieved programmed release with gastric protection and intestinal targeting. This precise pH-responsive behavior is precisely the expected function of the triple network structure design, which is of great value for improving the oral bioavailability of melatonin.

[0184] Figure 27 This is a cytotoxicity diagram of the chitosan-Chlamydomonas reinhardtii protein composite carrier of Example 1 of the present invention; Figure 28This is a graph showing the relative hemolysis rate of the chitosan-Chlamydomonas reinhardtii protein composite carrier from Example 1 of the present invention. Figure 27 As shown, the cytotoxicity test results confirmed that Example 1 had good biocompatibility. All treatment groups maintained high cell viability, with the lowest value still reaching 92.06%. According to ISO 10993-5-2009 standard, this value is far above the cytotoxicity threshold and falls within the safe range. Figure 28 As shown, the hemolysis experiment results indicated that the supernatant in all experimental groups remained clear and transparent, with a maximum hemolysis rate of only 1.12%, a stark contrast to the severe hemolysis induced by the deionized water positive control group, and far below the internationally recognized safety threshold of 5%. This excellent blood compatibility may be attributed to the fact that each component in the triple network structure is a naturally derived biomaterial (chitosan, Chlamydomonas reinhardtii protein, and melatonin), and that no toxic cross-linking agents or organic solvents were introduced during the preparation process. The suitable physicochemical properties of the carrier surface enable it to maintain good interfacial compatibility with the cell membrane without causing damage. In summary, the above systematic safety assessment results collectively demonstrate that the chitosan-Chlamydomonas reinhardtii protein composite carrier in Example 1 exhibits excellent performance in both cell compatibility and blood compatibility. These results fully confirm that the composite material meets internationally accepted biosafety standards, providing a crucial and solid biosafety basis for its further development and application in the field of functional foods.

[0185] In addition, the present invention has also conducted experiments with other raw materials, process operations and process conditions described in this specification, with reference to the foregoing embodiments, and has obtained relatively ideal results.

[0186] Although the invention has been described with reference to illustrative embodiments, those skilled in the art will understand that various other changes, omissions, and / or additions can be made without departing from the spirit and scope of the invention, and that elements of the embodiments can be substituted with substantially equivalents. Furthermore, many modifications can be made without departing from the scope of the invention to adapt particular situations or materials to the teachings of the invention. Therefore, this invention is not intended to be limited to the specific embodiments disclosed for carrying out the invention, but rather is intended to encompass all embodiments falling within the scope of the appended claims. Moreover, unless specifically stated otherwise, any use of the terms first, second, etc., does not indicate any order or importance, but is used to distinguish one element from another.

Claims

1. A method for preparing a chitosan-Chlamydomonas reinhardtii protein composite carrier, characterized in that, include: Chlamydomonas reinhardtii protein was obtained by treating Chlamydomonas reinhardtii with an alkaline dissolution and acid precipitation method. Chitosan-acetic acid aqueous solution and sodium tripolyphosphate solution are uniformly mixed to electrostatically crosslink chitosan and sodium tripolyphosphate, forming a mixed solution with a three-dimensional network framework. At room temperature, the Chlamydomonas reinhardtii protein powder was mixed with a mixed solution containing a three-dimensional network framework at a mass-volume ratio of 1g:100mL to 1g:750mL, and stirred continuously at a stirring speed of 300-500rpm for 6-8h. Then, the mixture was centrifuged at a centrifugation speed of 10000-12000rpm, the precipitate was collected, and the mixture was freeze-dried at -80 to -60℃ for 24-36h to obtain the chitosan-Chlamydomonas reinhardtii protein complex. The chitosan-Chlamydomonas reinhardtii protein complex is uniformly mixed with a spinning aid and a hydrophobic active substance in an aqueous system to prepare a spinning solution. The spinning solution is then used to prepare a chitosan-Chlamydomonas reinhardtii protein complex carrier by electrospinning, wherein the hydrophobic active substance includes melatonin.

2. The manufacturing method according to claim 1, characterized in that, The alkaline dissolution and acid precipitation method specifically includes: At room temperature, Chlamydomonas reinhardtii powder and water were uniformly mixed at a mass-volume ratio of 1g:15mL to 1g:30mL. The pH was adjusted to 12.0-12.5 with an alkaline substance, and the mixture was stirred continuously at 400-600 rpm for 2-3 hours. Then, the mixture was centrifuged at 10000-12000 rpm. The supernatant was collected, and the pH was adjusted to 3.0-3.5 with an acidic substance. The mixture was stirred continuously at 300-500 rpm for 1-2 hours, and then centrifuged at 10000-12000 rpm. The precipitate was collected, dispersed in water, and the pH was adjusted to 7.0-7.5 with an alkaline substance. Finally, the mixture was freeze-dried at -80 to -60℃ for 24-36 hours to obtain Chlamydomonas reinhardtii protein.

3. The manufacturing method according to claim 1, characterized in that, Specifically, it includes: At room temperature, equal volumes of sodium tripolyphosphate solution were added in batches to a chitosan-acetic acid aqueous solution, and stirred continuously at a stirring speed of 600-800 rpm for 2-3 hours to form a mixed solution containing the three-dimensional network framework. In the chitosan-acetic acid aqueous solution, the mass-volume ratio of chitosan to acetic acid aqueous solution is 1g:50mL~1g:200mL, the volume fraction of acetic acid aqueous solution is 1~2%, and the mass-volume ratio of sodium tripolyphosphate to water in the sodium tripolyphosphate solution is 1:500~1:1500 g / mL.

4. The manufacturing method according to claim 1, characterized in that, Specifically, it includes: Under room temperature conditions, the chitosan-Chlamydomonas reinhardtii protein complex, spinning aid and water are mixed evenly, and then melatonin-ethanol solution is added under light-protected conditions and mixed evenly to obtain the spinning solution; The chitosan-Chlamydomonas reinhardtii protein composite carrier is prepared by injecting the spinning solution into a calcium chloride solution using an electrospinning device and employing an electrospinning process.

5. The manufacturing method according to claim 4, characterized in that: The spinning aid comprises sodium alginate and polyethylene oxide, wherein the mass ratio of sodium alginate to polyethylene oxide is 1:1 to 1:1.

5.

6. The manufacturing method according to claim 4, characterized in that: The mass-to-volume ratio of the chitosan-Chlamydomonas reinhardtii protein complex to water is 1:100~1:200 g / mL.

7. The manufacturing method according to claim 4, characterized in that: The mass-to-volume ratio of the spinning aid to water is 1:150 to 1:300 g / mL.

8. The manufacturing method according to claim 4, characterized in that: The melatonin-ethanol solution has a mass-to-volume ratio of melatonin to ethanol of 1:15 to 1:20 g / mL.

9. The manufacturing method according to claim 4, characterized in that: The mass ratio of chitosan-Chlamydomonas reinhardtii protein to melatonin is 10:1 to 30:

1.

10. The manufacturing method according to claim 4, characterized in that: The electrospinning process parameters are as follows: the receiving liquid is a 1-2 wt% calcium chloride solution, the needle type is 21-23 G, the syringe capacity is 10-15 ml, the distance between the needle and the liquid surface is 10-15 cm, the electric field voltage is 10-15 kV, the feeding rate is 0.5-1 ml / h, the temperature is 20-25°C, the humidity is 30-50%, and the time is 10-30 h.

11. The chitosan-Chlamydomonas reinhardtii protein composite carrier prepared by any one of claims 1-10, characterized in that, The chitosan-Chlamydomonas reinhardtii protein composite carrier is spherical with a particle diameter of 0.45~0.75μm.

12. The use of the chitosan-Chlamydomonas reinhardtii protein composite carrier according to claim 11 in the preparation of functional food or drug delivery materials.

13. A functional food delivery material, characterized in that, Includes the chitosan-Chlamydomonas reinhardtii protein complex carrier as described in claim 11.

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