A myofibrillar protein-k-carrageenan oligosaccharide complex, and a preparation method and application thereof

Abstract

The application belongs to the technical field of new biological materials, and discloses a myofibrillar protein-kappa-carrageenan oligosaccharide complex as well as a preparation method and application thereof. The complex TMP / κCOS is prepared by using tilapia myofibrillar protein (TMP) and kappa-carrageenan oligosaccharide (κCOS) as raw materials through a pH driving method (the optimal process is that the volume ratio of TMP to κCOS is 1:1, the concentration of TMP is 1.2%, and the concentration of κCOS is 0.4%). The complex can load curcumin (CUR) to form a TMP / κCOS / CUR complex. The particle size of the complex is 280-295 nm, the CUR loading rate is greater than or equal to 76%, and the complex has good ion stability and thermal stability. In in-vitro digestion, the CUR gastric juice release is less than or equal to 15%, and the intestinal juice release is greater than or equal to 74%, so that the intestinal targeted delivery is expected to be realized, and the high value of tilapia processing by-products is promoted.

Description

Technical Field

[0001] This invention belongs to the field of novel biomaterials technology. It relates to a myofibrillar protein-κ-carrageenan oligosaccharide complex, its preparation method and application. Background Technology

[0002] Curcumin, a natural hydrophobic flavonoid, exists primarily in three forms: curcumin, dimethoxycurcumin, and di-desmethylcurcumin. Among these, curcumin exhibits the most prominent activity and antioxidant capacity, possessing various physiological activities such as anti-inflammatory, antibacterial, antitumor, and cardiovascular and neurological function regulation. Its safety has been widely verified, making it highly promising for applications in health foods and pharmaceuticals. However, the practical application of curcumin is limited by its inherent drawbacks: poor water solubility, low oral absorption efficiency, rapid metabolic rate, and its molecular configuration is easily degraded by light, temperature, pH, and solution polarity, resulting in significantly low bioavailability and hindering the full realization of its functional value.

[0003] To address the challenge of curcumin delivery, current technologies primarily employ nanocarriers for encapsulation and protection. Common carrier materials include polysaccharides, proteins, and protein-polysaccharide complexes. For instance, soluble pea protein isolate can form nanoparticles to encapsulate curcumin, exhibiting good anti-tumor potential and low cytotoxicity to normal cells. Soybean soluble polysaccharides, as carriers, effectively enhance the water solubility and stability of curcumin, providing a new direction for colorectal cancer treatment. Casein, with its hydrophobic-hydrophilic amino acid distribution characteristics, is widely used for encapsulating hydrophobic compounds to improve their bioavailability. Nanocomposites formed from heat-denatured lactoferrin and pectin can achieve an encapsulation efficiency of up to 85% for curcumin, enabling controlled release. While these carriers improve the application performance of curcumin to some extent, most carrier materials rely on plant proteins or specific polysaccharides, resulting in limitations in source or insufficient compatibility with fish-derived proteins. Furthermore, there is still room for improvement in the efficiency of intestinal-targeted delivery of lipid-soluble active ingredients.

[0004] Myofibrillar protein (TMP), a core component of meat protein, accounts for 55-60% of total muscle protein. It is not only highly nutritious, easily digestible, and low in allergens, but also possesses functional properties such as emulsification, gel formation, and water retention, making it an excellent protein substrate for constructing nanocarriers. my country has abundant tilapia farming resources, but the tilapia industry faces challenges such as low processing levels, product homogeneity, and low added value, leading to inventory backlog and price declines. There is an urgent need to expand the industrial chain through the high-value utilization of TMP. Currently, research on myofibrillar protein mainly focuses on animal sources such as chicken and pork, with limited research on fish-derived TMP such as tilapia. While existing modifications of fish-derived TMP involve coupling with glucosamine and dextran to improve their solubility and flowability through Maillard reactions or non-covalent interactions, the possibility of combining it with specific oligosaccharides to construct curcumin delivery carriers has not yet been explored, failing to fully utilize the resource advantages and functional potential of tilapia TMP.

[0005] κ-Carrageenan oligosaccharides (κCOS), as degradation products of carrageenan, possess characteristics such as low molecular weight, good water solubility, and high absorption efficiency. They also exhibit immunomodulatory, antioxidant, anti-inflammatory, and protein oxidation-inhibiting biological activities. κCOS can significantly inhibit the formation of carbonyl and dihydrotyrosine groups in TMP, maintain total sulfhydryl content, enhance the emulsifying activity and stability of TMP, extend shelf life, and simultaneously delay the formation of calcium ions. 2+ - Decreased ATPase activity inhibits the increase of TMP surface hydrophobicity. However, existing research on κCOS mainly focuses on its oxidative protection of TMP, without addressing its application in forming a complex to carry lipid-soluble active ingredients (such as curcumin). It has failed to combine the bioactivity of κCOS with the carrier function of TMP to form a system with both protective and delivery efficacy.

[0006] In terms of preparation technology, pH-driven methods, as a solvent-free and low-energy nanocarrier preparation technique, induce protein "dissociation-refolding" by adjusting the solution pH value, utilizing the hydrophobic regions of the protein to spontaneously encapsulate active ingredients. This method has been used for curcumin encapsulation in systems such as casein and whey protein isolates. However, current research has not optimized the pH-driven method for the specific tilapia TMP / κCOS system. Key process parameters such as the concentration ratio of TMP to κCOS remain unclear, making it difficult to obtain composite carriers with suitable particle size, good stability, and high encapsulation capacity, thus failing to meet the requirements for efficient curcumin delivery.

[0007] In summary, current curcumin delivery vectors suffer from the following technical shortcomings: First, the sources of materials for curcumin delivery vectors are limited, and research on composite vectors of fish-derived TMP and κCOS is lacking; second, the high-value utilization of tilapia TMP is insufficient, and its potential for constructing functional vectors by combining it with κCOS has not been explored; third, green preparation processes for the tilapia TMP / κCOS system are missing. Therefore, developing a tilapia TMP / κCOS-based complex to solve the curcumin delivery problem and meet the needs of tilapia industry upgrading has become an urgent technical challenge. Summary of the Invention

[0008] To address the aforementioned technical problems, this invention proposes a pH-driven composite method to form a stable complex by combining tilapia-derived myofibrillar protein (TMP) with κ-carrageenan oligosaccharide (κCOS). This complex can be further loaded with fat-soluble active ingredients such as curcumin. This strategy not only improves the solubility and stability of TMP under low ionic strength, promoting the high-value conversion of tilapia processing byproducts, but also protects the loaded fat-soluble active ingredients from degradation by light, heat, and digestion, improving their loading rate and in vitro targeted release effect. Simultaneously, it avoids the particle size defects of existing carrageenan-based TMP composites, thus constructing a composite carrier system that combines high-value raw material utilization, active ingredient protection, and efficient delivery, providing a solution for the utilization of fish-derived proteins and the delivery of fat-soluble active ingredients in health foods and pharmaceuticals. To achieve this technical objective, this invention provides the following technical solution:

[0009] In a first aspect, the present invention provides a myofibrillar protein-κ-carrageenan oligosaccharide complex, wherein the components of the myofibrillar protein-κ-carrageenan oligosaccharide complex are: a 1:1 ratio of tilapia myofibrillar protein solution with a concentration of 1.2% w / v to a κ-carrageenan oligosaccharide solution with a concentration of 0.4% w / v.

[0010] Furthermore, in the above-mentioned myofibrillar protein-κ-carrageenan oligosaccharide complex, the tilapia myofibrillar protein is extracted by the following method:

[0011] Take the white flesh of tilapia, add 3-5 times the volume of pre-cooled first PBS extract, homogenize and centrifuge, discard the supernatant and scrape off the white flocculent material on the surface of the precipitate, repeat homogenization and centrifugation once;

[0012] Collect the precipitate, add 3-5 times the volume of the second PBS extract, homogenize, and centrifuge.

[0013] Collect the precipitate, add 0.1-0.3 mol / L NaCl solution at a volume ratio of 1:3-5 to homogenize, filter through 4 layers of gauze and centrifuge. The resulting precipitate is TMP.

[0014] The first PBS extract consisted of: EDTA 0.5 mmol / L, NaCl 137 mmol / L, Na2HPO4 10 mmol / L, KH2PO4 1.8 mmol / L, and water as the solvent.

[0015] The second PBS extract consisted of: NaCl 137 mmol / L, Na2HPO4 10 mmol / L, KH2PO4 1.8 mmol / L, and water as the solvent.

[0016] Furthermore, in the above myofibrillar protein-κ-carrageenan oligosaccharide complex, the first homogenization was performed for 90 seconds, and the second homogenization was performed for 60 seconds; the centrifugation conditions were: 6000~8000 r / min, 4±1℃ for 10~15 min.

[0017] Secondly, the present invention provides a method for preparing the above-mentioned tilapia myofibrillar protein-κ-carrageenan oligosaccharide complex, comprising the following steps:

[0018] (1) Preparation of TMP solution: Prepare a 1.2% TMP solution with deionized water, adjust the pH to 12, and hydrate it under cold.

[0019] (2) Preparation of κCOS solution: Prepare a κCOS solution with a concentration of 0.4% using deionized water, adjust the pH to 12, and hydrate it under cold conditions;

[0020] (3) Mixing reaction: Mix the TMP solution from step (1) with the κCOS solution from step (2) at a volume ratio of 1:1 and stir.

[0021] (4) Neutralization and stabilization: Neutralize the mixture from step (3) to pH=7 with HCl solution and continue stirring;

[0022] (5) Purification: Centrifuge the mixture from step (4) to remove the precipitate, and take the supernatant as the complex.

[0023] Furthermore, in step (1) of the above method, the hydration temperature of the TMP solution is 4±1℃, the hydration time is 12h±2h, and a sealed container is used during the hydration process to avoid concentration deviation caused by solution evaporation.

[0024] Furthermore, in steps (3) to (4) of the above method, the stirring rate is 400±50 rpm, and the ambient temperature is maintained at 25±2℃ during the preparation process to avoid temperature fluctuations causing aggregation of tilapia myofibril protein or κ-carrageenan oligosaccharide molecules.

[0025] Furthermore, in step (4) of the above method, when neutralizing with 1 mol / L HCl, HCl is added dropwise while stirring to ensure that the pH of the reaction system changes uniformly.

[0026] Thirdly, the present invention provides the application of the above-mentioned myofibrillar protein-κ-carrageenan oligosaccharide complex in the preparation of a lipid-soluble active ingredient delivery system.

[0027] Furthermore, in the above applications, the fat-soluble active ingredient is curcumin.

[0028] Furthermore, in the above applications, the delivery system is used to prepare health foods or medicines.

[0029] The present invention, "a myofibrillar protein-κ-carrageenan oligosaccharide complex, its preparation method and application", has at least the following beneficial effects:

[0030] 1. Achieving high-value utilization of tilapia myofibrillar protein (TMP)

[0031] Tilapia is an important aquaculture species globally, but its processing level is low and the high-value utilization of by-products is insufficient. This solution uses tilapia TMP as the core protein substrate, employing a specific extraction process (multiple homogenizations and centrifugations with 4 volumes of PBS buffer to obtain salt-soluble TMP precipitate) to ensure TMP purity. Simultaneously, it is compounded with κ-carrageenan oligosaccharide (κCOS) to improve the defects of TMP, such as easy polymerization and poor solubility under low ionic strength. Experiments show that the solubility stability of TMP is significantly improved after compounding, and the application limitations of terrestrial animal or plant proteins are avoided, promoting the transformation of tilapia processing by-products into high-value-added carrier materials.

[0032] 2. Excellent composite physical and chemical properties, meeting the core requirements of nanocarriers.

[0033] Compared with other carrageenan-based substrates (κ-carrageenan polysaccharide, Iota-carrageenan polysaccharide, and oligosaccharides), the κCOS / TMP composite selected in this scheme shows significantly better performance: κ-carrageenan polysaccharide (κCG) has a lower potential and lower binding rate after being composited with TMP; Iota-carrageenan polysaccharide (ICG) has a higher binding rate after being composited with TMP, but the product particle size is too large; Iota-carrageenan oligosaccharide (ICOS) has a high binding rate after being composited with TMP, but the particle size is also too large. κCG, ICG, and ICOS cannot meet the requirements of nanocarriers; while the TMP / κCOS complex has an average particle size of 280~295nm, a zeta potential of -25.0 to -29.8mV, less protein precipitation after centrifugation, a high binding rate, and the particle size remains stable within the above range after being diluted 25 times with ultrapure water, possessing the dispersibility and structural stability required for nanocarriers.

[0034] 3. Highly efficient loading and targeted delivery of lipid-soluble active ingredients, enhancing bioavailability.

[0035] To address the issues of poor water solubility, easy degradation, and low bioavailability of fat-soluble components such as curcumin, the TMP / κCOS complex in this formulation can efficiently load curcumin with a loading rate of ≥76%. Furthermore, in vitro digestion experiments show that after loading curcumin, the release rate in gastric juice (pH=2.8) is only 14.2% (to avoid premature degradation in the stomach), while the final release rate in intestinal juice (pH=7.0) reaches 74.7%, which is significantly better than free curcumin (in vitro bioavailability of 59.8%). This achieves targeted and sustained release of the active ingredient into the intestine, thereby improving its bioavailability.

[0036] 4. The complex exhibits strong stability and is adaptable to various application scenarios.

[0037] Experiments have shown that the TMP / κCOS complex exhibits no significant abnormal changes in particle size and potential within the NaCl concentration range of 0–400 mmol / L, demonstrating good ionic stability. After heat treatment at 40–80 °C for 30 min, the particle size initially increases and then decreases, while the absolute value of the potential remains stable (≥20 mV at some temperatures). The curcumin release rate is controllable, and the thermal stability is excellent. It can adapt to various environments such as health food processing, storage, and human digestion, thus expanding its application scope. Attached Figure Description

[0038] Figure 1 This is a diagram showing the appearance of the solution after centrifugation of the myofibrillar protein (TMP)-κ-carrageenan (κCG) nanocomposite (i.e., TMP / κCG). Figure 1 In the table, A represents the TMP blank group, B represents the TMP / 0.1% κCG group, C represents the TMP / 0.2% κCG group, D represents the TMP / 0.3% κCG group, E represents the TMP / 0.4% κCG group, F represents the TMP / 0.5% κCG group, and G represents the 0.5% κCG blank group.

[0039] Figure 2 The graph shows the particle size and potential measurement results of the TMP / κCG complex. Figure 2 In the graph, the line graph represents the zeta potential, and the bar graph represents the particle size.

[0040] Figure 3 This is a diagram showing the appearance of the solution after centrifugation of the myofibrillar protein (TMP)-Iota-carrageenan (ICG) (i.e., TMP / ICG) nanocomposite. Figure 3 In the table, A represents the TMP blank group, B represents the TMP / 0.1% ICG group, C represents the TMP / 0.2% ICG group, D represents the TMP / 0.3% ICG group, E represents the TMP / 0.4% ICG group, F represents the TMP / 0.5% ICG group, and G represents the 0.5% ICG blank group.

[0041] Figure 4The graph shows the particle size and potential measurement results of the TMP / ICG complex. Figure 4 In the graph, the line graph represents the zeta potential, and the bar graph represents the particle size.

[0042] Figure 5 This is a diagram of the solution appearance of the myofibrillar protein (TMP)-Iota-carrageenan oligosaccharide (ICOS) nanocomposite (i.e., TMP / ICOS) before centrifugation. Figure 5 In the diagram, A represents the TMP blank group, B represents the TMP / 0.1% ICOS group, C represents the TMP / 0.2% ICOS group, D represents the TMP / 0.3% ICOS group, E represents the TMP / 0.4% ICOS group, F represents the TMP / 0.5% ICOS group, and G represents the 0.5% ICOS blank group.

[0043] Figure 6 This is a diagram showing the appearance of the TMP / ICOS complex solution after centrifugation. Figure 6 In Chinese, A~G have the same meaning. Figure 5 .

[0044] Figure 7 The graph shows the particle size and potential measurement results of the TMP / ICOS complex. Figure 7 In the graph, the line graph represents the zeta potential, and the bar graph represents the particle size.

[0045] Figure 8 This is a diagram of the solution appearance of the myofibrillar protein (TMP)-κ-carrageenan oligosaccharide (κCOS) nanocomplex (i.e., TMP / κCOS) before centrifugation. Figure 8 In the table, A represents the 0.4% κCOS blank group, B represents the 0.4% TMP / κCOS group, C represents the 0.8% TMP / κCOS group, D represents the 1.2% TMP / κCOS group, E represents the 1.6% TMP / κCOS group, F represents the 2.0% TMP / κCOS group, and G represents the TMP blank group.

[0046] Figure 9 This is a diagram showing the appearance of the TMP / κCOS complex solution after centrifugation. Figure 9 In Chinese, A~G have the same meaning. Figure 8 .

[0047] Figure 10 The figure shows the particle size and potential measurement results of the TMP / κCOS complex. Figure 10 In the graph, the line graph represents the zeta potential, and the bar graph represents the particle size.

[0048] Figure 11The effects of different concentrations of κCOS and TMP on the particle size and potential of the TMP / κCOS complex, and the comparison of particle size and potential of TMP, TMP / κCOS, and TMP / κCOS / CUR complex (i.e., TMP / κCOS loaded with CUR). Figure 11 In the figures, (A) shows the effect of different κCOS concentrations (0~0.5% w / v) on particle size and potential; (B) shows the effect of different TMP concentrations (0~2.0% w / v) on particle size and potential; and (C) shows the particle size and potential of TMP, TMP / κCOS, and TMP / κCOS / CUR complex (i.e., TMP / κCOS loaded with CUR).

[0049] Figure 12 SDS-PAGE gel images of TMP, TMP / κCOS, and TMP / κCOS / CUR. Figure 12 In this context, "Marker" represents the 16-270 kDa protein molecular weight standard, "T" represents the TMP group, "T / κ" represents the TMP / κCOS group, and "T / κ / C" represents the TMP / κCOS / CUR group.

[0050] Figure 13 The intrinsic fluorescence spectrum (A), far-ultraviolet CD spectrum (B), and secondary structure content (C) of TMP, TMP / κCOS, and TMP / κCOS / CUR are shown. Figure 13 In the diagram, (D) represents the infrared spectra of κCOS (a), TMP (b), TMP / κCOS (c), TMP / κCOS / CUR (d), and CUR (e).

[0051] Figure 14 Scanning electron microscope (5000×) images of TMP (A), κCOS (B), TMP / κCOS (C), and TMP / κCOS / CUR (D).

[0052] Figure 15 The graph shows the results of surface hydrophobicity measurements for TMP, TMP / κCOS, and TMP / κCOS / CUR.

[0053] Figure 16 For different NaCl concentrations ( Figure 16 (A) in the middle), different temperatures ( Figure 16 Figure (B) shows the particle size and potential results of the TMP / κCOS complex. Figure 16 In the graph, the line graph represents the zeta potential, and the bar graph represents the particle size.

[0054] Figure 17 The CUR release rate of the TMP / κCOS / CUR complex at different temperatures (A) and under simulated in vitro digestion conditions (B) is shown. Detailed Implementation

[0055] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0056] Unless otherwise specified in the examples, the methods or conditions described in the literature in this field or in accordance with the product instructions shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0057] The main experimental materials and instruments used in this embodiment are as follows:

[0058] I. Experimental Materials

[0059] Tilapia: All were purchased from the local seafood market in Zhanjiang, Guangdong, China; Processing method: Fresh tilapia were slaughtered, gutted, and the white meat was used to extract tilapia myofibrillar protein (TMP).

[0060] κ-Carrageenan (κCG), Iota-Carrageenan (ICG), Iota-Carrageenan Oligosaccharide (ICOS), κ-Carrageenan Oligosaccharide (κCOS): Qingdao Bozhi Huili Biotechnology Co., Ltd.;

[0061] Curcumin (CUR): Shanghai Aladdin Biochemical Technology Co., Ltd.;

[0062] Pepsin, pancreatic enzymes, and porcine bile salts: Beijing Solarbio Co., Ltd.

[0063] II. Test Instruments

[0064] ATASIZER NANO ZSE Nanoparticle Potentiometer: Malvern Instruments, UK;

[0065] High-speed refrigerated centrifuge, Hitachi, Japan;

[0066] Grinding machine, Joyoung Company, China;

[0067] F-7000 fluorescence spectrometer: Hitachi, Japan;

[0068] TEN SOR 27 Fourier Transform Infrared Spectrometer (FTIR): BRUKER GmbH, Germany;

[0069] Chirascan V100 Circular Dichroism Spectrometer: Applied Photophysics Ltd, UK;

[0070] UV-2600 Spectrophotometer: Shimadzu Corporation, Japan;

[0071] S-4800 Scanning Electron Microscope (SEM): Hitachi, Japan;

[0072] Fully automated microplate reader: Thermo Fisher Scientific, USA;

[0073] Vertical electrophoresis apparatus: Beyotime.

[0074] Example 1

[0075] This embodiment describes the feasibility verification of combining different carrageenan-based substrates with tilapia myofibrillar protein (TMP).

[0076] I. Experimental Objective

[0077] Composite systems of tilapia myofibrillar protein (TMP) with different types of carrageenan substrates (κ-carrageenan polysaccharide, Iota-carrageenan polysaccharide, Iota-carrageenan oligosaccharide, and κ-carrageenan oligosaccharide) were prepared by pH-driven method. The composite effect of each system was evaluated from the perspectives of appearance, particle size, and zeta potential, and the core substrates that can form stable nanoscale composites were screened.

[0078] II. Test Materials

[0079] Tilapia myofibrillar protein (TMP): Preparation method: Take tilapia white meat purchased from Zhanjiang aquatic product market, add 4 times the volume of pre-cooled first PBS extraction buffer (containing EDTA 0.5 mmol / L, NaCl 137 mmol / L, Na2HPO4 10 mmol / L, KH2PO4 1.8 mmol / L), homogenize for 90 s, centrifuge at 8000 r / min, 4℃ for 15 min, discard the supernatant and scrape off the white flocculent material on the surface of the precipitate, repeat homogenization and centrifugation once; collect the precipitate, add 4 times the volume of second PBS buffer (containing NaCl 137 mmol / L, Na2HPO4 10 mmol / L, KH2PO4 1.8 mmol / L), homogenize for 60 s, centrifuge under the same conditions; then add 4 times the volume of 0.1 mol / L NaCl solution, homogenize for 60 s, filter through 4 layers of gauze, centrifuge the filtrate at 8000 r / min, 4℃ for 15 min, the obtained precipitate is TMP, store at 4℃ and use within 48 h.

[0080] Carrageenan-based substrates: κ-carrageenan polysaccharide (κCG), Iota-carrageenan polysaccharide (ICG), Iota-carrageenan oligosaccharide (ICOS), and κ-carrageenan oligosaccharide (κCOS), all of analytical grade.

[0081] III. Test Methods

[0082] (I) Preparation of composite system (pH-driven method)

[0083] Four types of TMP-carrageenan composite systems were constructed, and the specific steps are as follows:

[0084] 1. Preparation of basic solution

[0085] Preparation of TMP solutions: Dissolve TMP in deionized water, adjust the pH to 12.0, and prepare TMP solutions of 0.4%, 0.8%, 1.0%, 1.2%, 1.6%, and 2.0% (w / v) respectively. Hydrate at 4°C for 12 hours under sealed conditions to ensure complete protein hydration.

[0086] Preparation of carrageenan-based substrate solutions: κCG, ICG, ICOS, and κCOS solutions with concentration gradients of 0.1%, 0.2%, 0.3%, 0.4%, and 0.5% (w / v) were prepared respectively. The pH of each solution was adjusted to 12.0 with 2 mol / L NaOH and hydrated at 4°C for 12 h under sealed conditions.

[0087] 2. Construction of composite systems

[0088] At room temperature (25±2℃), TMP solution was mixed with κCG, ICG, ICOS and κCOS solutions at a volume ratio of 1:1 and magnetically stirred at 400 rpm for 1 h to ensure complete reaction.

[0089] Neutralize the mixture to pH 7.0 with 1 mol / L HCl, stirring continuously at 400 rpm for 2 hours to stabilize the system;

[0090] Centrifuge each mixture at 2000g and 4℃ for 10min to remove precipitates and retain the supernatant as the composite system to be tested.

[0091] Meanwhile, TMP blank group and blank groups of each carrageenan substrate (treated under the same conditions) were set up as controls.

[0092] (II) Detection Methods

[0093] 1. Visual observation: Take pictures of the solution state of each composite system before and after centrifugation, record the clarity of the solution, color change and amount of precipitation, and compare the differences between the groups.

[0094] 2. Particle size and zeta potential determination: The supernatant after centrifugation was diluted 25 times with ultrapure water, and the particle size and zeta potential were determined using a ZATASIZERNANO ZSE nanoparticle size potentiometer at 25±0.1℃. Each sample was measured 3 times, and the average value was taken.

[0095] IV. Experimental Results and Analysis

[0096] (I) The combined effect of TMP and κ-carrageenan (κCG)

[0097] In this experiment, 1.0% TMP solution was mixed with 0.1%~0.5% κCG solution, and 1.0% TMP solution and 0.5% κCG solution were set as blank controls.

[0098] 1. Appearance Results

[0099] The appearance of the TMP / κCG complex solution after centrifugation is as follows: Figure 1 As shown. A is the TMP blank group, B-F are the TMP combined with 0.1-0.5% κCG groups, and G is the 0.5% κCG blank group. Figure 1 It is evident that a large amount of protein precipitate was generated in both the TMP / κCG solutions after centrifugation, indicating that the binding rate of κCG to TMP was low and TMP was not fully utilized.

[0100] 2. Particle size and potential results

[0101] The results of TMP / κCG complex particle size and zeta potential measurements are as follows: Figure 2 As shown, the line graph represents the zeta potential, and the bar graph represents the particle size. Figure 2 It can be seen that there was no significant difference in zeta potential between groups B-F and the TMP blank group. However, with the increase of κCG concentration, the particle size showed a trend of first increasing and then decreasing, indicating that the two bind, but excessive κCG content leads to precipitation. After TMP binds to κCG, the particle size is less than 200 nm, and the absolute value of the zeta potential is between 15 and 18 mV, which is relatively small. Figures 1-2 It can be seen that the potential of κCG after complexing with TMP is small and the binding rate is low.

[0102] (II) The combined effect of TMP and Iota-carrageenan (ICG)

[0103] In this experiment, 1.0% TMP solution was mixed with 0.1%~0.5% ICG solution, and 1.0% TMP solution and 0.5% ICG solution were set as blank controls.

[0104] 1. Appearance Results

[0105] The appearance of the TMP / ICG complex solution after centrifugation is as follows: Figure 3 As shown in the figure. A is the TMP blank group, B~F are the TMP and 0.1~0.5% ICG complex groups, and G is the 0.5% ICG blank group. After centrifugation, the TMP / ICG complex was gel-like and coagulated at the bottom of the centrifuge, failing to form a homogeneous supernatant, indicating that the two combined to form a complex and aggregated.

[0106] 2. Particle size and potential results

[0107] The results of TMP / ICG complex particle size and zeta potential measurements are as follows: Figure 4 As shown, the line graph represents the zeta potential, and the bar graph represents the particle size. Figure 4 It can be seen that after TMP is combined with 0.1~0.5% ICG, the particle size is greater than 2000nm, and although the absolute value of the potential reaches about 50mV, the product is gel-like, which does not meet the requirement of uniform dispersion of nanocarriers. In summary... Figures 3-4 It can be seen that ICG has a high binding rate with TMP, but the product particle size is too large.

[0108] (III) The combined effect of TMP and Iota-carrageenan oligosaccharides (ICOS)

[0109] In this experiment, 1.0% TMP solution was mixed with 0.1%~0.5% ICOS solution, and 1.0% TMP solution and 0.5% ICOS solution were set as blank controls.

[0110] 1. Appearance Results

[0111] The appearance of the TMP / ICOS complex solution before centrifugation is as follows: Figure 5 As shown, the appearance after centrifugation is as follows Figure 6 As shown in the figure, A represents the TMP blank group, B-F represent the TMP / ICOS complex groups (0.1-0.5% ICOS), and G represents the 0.5% ICOS blank group. After centrifugation, the protein precipitation in the TMP / ICOS complex solution gradually decreased, with the 0.5% ICOS concentration group (group F) showing the least protein precipitation, indicating a high binding rate between TMP and ICOS.

[0112] 2. Particle size and potential results

[0113] The results of TMP / ICOS complex particle size and zeta potential measurements are as follows: Figure 7 As shown, the line graph represents the zeta potential, and the bar graph represents the particle size. Figure 7 It can be seen that the particle size and potential of the 0.1%~0.5% concentration groups (groups B~F) show a trend of first increasing and then decreasing, with the particle size of group F, which has the highest binding rate, being too large (close to 400nm). In summary... Figures 5-7 It can be seen that ICOS and TMP have a high binding rate after being combined, but the particle size is also too large.

[0114] (iv) The combined effect of TMP and κ-carrageenan oligosaccharide (κCOS)

[0115] In this experiment, 0.4–2.0% TMP solutions were mixed with 0.4% κCOS solutions, and 1.0% TMP solutions and 0.4% κCOS solutions were set as blank controls.

[0116] 1. Appearance Results

[0117] The appearance of the TMP / κCOS complex solution before centrifugation is as follows: Figure 8 As shown, the appearance after centrifugation is as follows Figure 9 As shown. A is the 0.4% κCOS blank group, B~F are the κCOS and 0.4~2.0% TMP complex groups, and G is the TMP blank group. Before centrifugation, the color of the complex solution gradually darkened with increasing TMP concentration. Figure 8 After centrifugation, a small amount of protein precipitate was observed in all groups, but significantly less than in the TMP control group. Figure 9 This indicates that TMP has a high binding rate to κCOS.

[0118] 2. Particle size and potential results

[0119] The results of TMP / κCOS complex particle size and zeta potential determination are as follows: Figure 10 As shown, the line graph represents the zeta potential, and the bar graph represents the particle size. Figure 10 It can be seen that with the increase of TMP concentration, the particle size of the TMP / κCOS complex first decreases and then increases, with significant differences among the groups. When the TMP concentration is 1.2%, the nanocomposite particle size is the smallest, at 289.9 nm, and the absolute value of the zeta potential is -29.1 mV. Figures 8-10 It can be seen that κCOS has a high binding rate with TMP and the product has a small particle size, making it suitable for the preparation of complexes.

[0120] V. Conclusion

[0121] Through the verification of the feasibility of combining four types of carrageenan-based substrates with TMP, only κ-carrageenan oligosaccharide (κCOS) and TMP can form a stable complex through the pH-driven method (high binding rate, particle size up to 289.9 nm, zeta potential -29.1 mV). Therefore, κCOS is used as the core carrageenan substrate for constructing TMP-based complexes.

[0122] Example 2

[0123] This embodiment describes the optimization of process parameters for the preparation of the TMP / κCOS complex.

[0124] I. Experimental Objective

[0125] Based on Example 1, which has determined that κ-carrageenan oligosaccharide (κCOS) is a suitable substrate for tilapia myofibrillar protein (TMP), this example optimizes the key process parameters of κCOS concentration and TMP concentration using a single-factor variable method, and screens out the best preparation process that meets the requirements of nanocarriers in terms of particle size and zeta potential, providing a stable composite substrate system for subsequent loading of lipophilic active ingredients and performance verification.

[0126] II. Test Methods

[0127] (I) Single-factor variable optimization design

[0128] Using the particle size (core indicator) and zeta potential (stability indicator) of the complex as evaluation criteria, single-factor experiments were conducted to optimize the κCOS concentration and TMP concentration, respectively. The following experiments were set up:

[0129] 1. κCOS concentration optimization: With the TMP solution concentration fixed at 1.0% (w / v), the κCOS concentration gradient was set to 0.1%, 0.2%, 0.3%, 0.4%, and 0.5% (w / v).

[0130] 2. TMP Concentration Optimization: Based on the optimal κCOS concentration screened in the first step, the TMP concentration gradient was set to 0.4%, 0.8%, 1.2%, 1.6%, and 2.0% (w / v). The volume ratio of TMP to κCOS solution was fixed at 1:1.

[0131] (II) Preparation of TMP / κCOS complex (pH-driven method)

[0132] 1. Preparation of basic solution

[0133] TMP solution: Dissolve TMP in deionized water according to the above concentration gradient, stir magnetically until completely dispersed, adjust the pH to 12.0 with 2 mol / L NaOH, and hydrate at 4℃ for 12 h to ensure complete protein hydration.

[0134] κCOS solution: Dissolve κCOS in deionized water according to the above concentration gradient. Stir magnetically until completely transparent, then adjust the pH to 12.0 with 2 mol / L NaOH and hydrate at 4℃ for 12 h to ensure that κCOS is fully dissolved.

[0135] 2. Construction of composite systems

[0136] Mix TMP and κCOS solutions at a volume ratio of 1:1 at the same temperature (room temperature, to avoid temperature differences affecting mixing), and magnetically stir at 400 rpm for 1 h (to ensure sufficient contact and reaction). Slowly neutralize the mixture with 1 mol / L HCl to pH 7.0, and continue stirring at 400 rpm for 2 h to stabilize the structure. Centrifuge the mixture at 2000g and 4℃ for 10 min to remove unreacted large particles, and collect the supernatant as the TMP / κCOS complex sample to be tested. At the same time, set up a TMP blank group (1.0% (w / v) TMP solution, treated under the same conditions) and a κCOS blank group (only the optimal concentration of κCOS solution, treated under the same conditions) as controls.

[0137] (III) Detection Methods

[0138] The sample to be tested was diluted 25 times with ultrapure water. The particle size and zeta potential were measured using a ZATASIZER NANO ZSE nanoparticle size potentiometer at 25±0.1℃. Each sample was measured three times and the average value was taken.

[0139] III. Experimental Results and Analysis

[0140] (I) Results of κCOS Concentration Optimization

[0141] 1. Particle size variation trend

[0142] like Figure 11 As shown in (A): the particle size of the TMP blank group (1.0% w / v) was 62.82±3.91 nm. After being combined with κCOS, the particle size showed a trend of first decreasing and then increasing: when the κCOS concentration was 0.1%, due to the low concentration of κCOS, only a small amount adhered to the TMP surface and no stable composite structure was formed, resulting in particle aggregation and a larger particle size; when the κCOS concentration increased to 0.4%, κCOS and TMP molecules were fully integrated and formed dense nanoparticles through hydrogen bonding and electrostatic interaction, at which point the particle size reached the minimum value of 289.97±4.51 nm; when the κCOS concentration exceeded 0.4%, the particle size increased to 399.67±1.25 nm. Due to the excessive accumulation of κCOS molecules on the TMP surface, particle aggregation was triggered, and the dispersibility decreased.

[0143] 2. Trend of zeta potential changes

[0144] like Figure 11 As shown in (A): the potential of the TMP blank group was -3.96±0.23mV. After recombination with κCOS, the absolute value of the potential gradually increased with the increase of κCOS concentration. When the κCOS concentration was 0.4%, the potential reached -29.16±0.61mV (absolute value ≥25mV). This is because the sulfate group carried by κCOS forms an electrostatic interaction with the acidic amino acid residues on the TMP surface, increasing the negative charge density on the particle surface, enhancing the electrostatic repulsion, and reducing aggregation. When the κCOS concentration was 0.5%, the potential was -30.27±2.67mV, but due to the large particle size, the stability was still inferior to the 0.4% concentration group.

[0145] Conclusion: The optimal concentration of κCOS was determined to be 0.4% (w / v), at which the particle size of the complex was 289.97±4.51 nm, PDI=0.33±0.05, and potential was -29.16±0.61 mV.

[0146] (II) Results of TMP Concentration Optimization

[0147] 1. Particle size variation trend

[0148] like Figure 11As shown in (B): with a fixed κCOS concentration of 0.4% (w / v), the particle size decreased significantly with increasing TMP concentration, reaching a minimum at a TMP concentration of 1.2% (w / v) with a particle size of 291.17 ± 5.37 nm. This is because under these conditions, the ratio of TMP molecules to κCOS molecules is well-matched, forming a dense three-dimensional "protein-oligosaccharide" structure, resulting in uniform particle dispersion.

[0149] 2. Trend of zeta potential changes

[0150] like Figure 11 As shown in (B): with a fixed κCOS concentration of 0.4% (w / v), the absolute value of the potential increases with the increase of TMP concentration. When the TMP concentration is 1.2% (w / v), the potential value reaches -29.8±1.67mV. At this time, the electrostatic interaction between TMP and κCOS is strong, and the surface charge density of the particles is stable. When the TMP concentration is 1.6% and 2.0%, although the potential is still ≥25mV, the overall stability decreases due to the increase in particle size.

[0151] Conclusion: Comprehensive Figure 11 Based on (A) and (B), the optimal TMP concentration was determined to be 1.2% (w / v). At this concentration, the composite particle size was 291.17±5.37 nm and the potential was -29.8±1.67 mV, which met the requirements for nanocarriers.

[0152] IV. Final Optimization Process Determination

[0153] Based on the above optimization results, the optimal preparation process parameters for the TMP / κCOS complex are: TMP concentration 1.2% (w / v), κCOS concentration 0.4% (w / v), TMP to κCOS solution volume ratio 1:1, and preparation using a pH-driven method.

[0154] Example 3

[0155] This embodiment describes the structural characterization of the TMP / κCOS complex and the TMP / κCOS / CUR complex.

[0156] I. Experimental Objective

[0157] The complexation mechanism and structural characteristics of tilapia myofibrillar protein (TMP) and κ-carrageenan oligosaccharide (κCOS) were analyzed from multiple dimensions, including molecular polymerization state, protein secondary / tertiary structure, intermolecular interactions, microstructure and surface properties. This study verified the structural stability and adaptability of tilapia myofibrillar protein (TMP) and κ-carrageenan oligosaccharide (κCOS) as carriers of lipid-soluble active ingredients.

[0158] II. Test Samples

[0159] TMP / κCOS complex: prepared according to the optimal process determined in Example 2 (TMP concentration 1.2% w / v, κCOS concentration 0.4% w / v, volume ratio 1:1, pH driven method).

[0160] TMP / κCOS / CUR complex: 0.2 mg / mL curcumin (CUR) was added when TMP and κCOS were mixed, and the reaction was stirred in the dark.

[0161] Control samples: pure TMP (TMP blank group in Example 2), pure κCOS (Qingdao Bozhi Huili Biotechnology Co., Ltd.), and pure CUR (Shanghai Aladdin Biochemical Technology Co., Ltd.).

[0162] III. Test Methods

[0163] (a) SDS-PAGE electrophoresis analysis (protein polymerization status)

[0164] 1. Sample preparation: Take pure TMP, TMP / κCOS, and TMP / κCOS / CUR samples, and adjust the protein concentration to 4 mg / mL with deionized water; mix the protein solution and loading buffer at a volume ratio of 4:1, heat in a boiling water bath for 5 min to denature the protein, centrifuge at 13000 r / min for 3 min, and take the supernatant for later use.

[0165] 2. Electrophoresis procedure: Install a 12% precast gel, add electrophoresis buffer, and add 7.5 μL of sample to each well (add 5 μL of 16~270 kDa protein molecular weight standard to the marker well); set the voltage to 120V and perform constant voltage electrophoresis for 2 hours.

[0166] 3. Staining and destaining: After electrophoresis, remove the gel and place it in Coomassie Brilliant Blue R-250 staining solution and shake for 20 minutes. After rinsing the gel with distilled water, place it in pure water and shake to destain until the background is clear. Take a picture to record the staining.

[0167] (ii) Endogenous fluorescence spectroscopy analysis (protein tertiary structure)

[0168] 1. Sample dilution: Dilute pure TMP, TMP / κCOS, and TMP / κCOS / CUR samples 500 times with deionized water (ensure the concentration is within the linear range of the instrument's detection).

[0169] 2. Parameter settings: excitation wavelength 390nm, emission wavelength 470nm, slit width 5nm, photomultiplier tube voltage 600V, scanning speed 100nm / min.

[0170] 3. Measurement and Recording: Using deionized water as a blank control, the fluorescence intensity of the three groups of samples was measured, the endogenous fluorescence spectrum was plotted, and the maximum fluorescence wavelength and relative fluorescence intensity were recorded.

[0171] (III) Circular dichroism (CD) analysis (protein secondary structure)

[0172] 1. Sample dilution: Dilute pure TMP, TMP / κCOS, and TMP / κCOS / CUR samples 10 times with deionized water (to ensure the light path is clear and free from scattering interference).

[0173] 2. Parameter settings: scanning wavelength 190~250nm (far ultraviolet region, reflecting peptide bond conformation), optical path 1cm, bandwidth 1.0nm, scanning speed 60nm / min, response time 0.25s, detection temperature 25℃.

[0174] 3. Data processing: Using deionized water as a blank to subtract the background, CDNN software was used to calculate the relative contents of α-helices, β-sheets, β-turns and random coils in the samples to analyze the changes in secondary structure.

[0175] (iv) Fourier transform infrared spectroscopy (FTIR) analysis (intermolecular interactions)

[0176] 1. Sample preparation: Pure TMP, pure κCOS, TMP / κCOS, and TMP / κCOS / CUR samples were freeze-dried to constant weight, and then mixed with potassium bromide at a mass ratio of 1:20. The mixtures were then ground into powder in an agate mortar (particle size <2μm to avoid scattering).

[0177] 2. Tableting and Scanning: The mixed powder is placed into a tableting mold, and a pressure of 10 MPa is applied and held for 1 minute to form transparent tablets; an FTIR spectrometer is used to scan the tablets at 400~4000 cm⁻¹. -1 Wavenumber range scanning, resolution 4cm -1 , scanned 32 times.

[0178] 3. Background subtraction: Using pure potassium bromide thin film as the background, after subtracting background interference, the shifts and intensity changes of characteristic absorption peaks (-OH, amide I band, amide II band, amide III band, and sulfate group) were analyzed.

[0179] (v) Scanning electron microscopy (SEM) analysis (microscopic morphology)

[0180] 1. Sample pretreatment: Freeze-dry pure TMP, pure κCOS, TMP / κCOS, and TMP / κCOS / CUR samples until completely dry to avoid moisture affecting observation.

[0181] 2. Sample fixation and gold sputtering: Fix the freeze-dried sample on the sample stage with conductive adhesive and place it in the gold sputtering machine for gold sputtering (gold layer thickness 5~10nm, to ensure conductivity).

[0182] 3. Observation and photography: The microstructure of the sample was observed using SEM at an accelerating voltage of 10kV and a magnification of 5000×. Representative areas were selected for photography and recording, and structural features (such as whether a three-dimensional network structure is formed and the size of the pores) were analyzed.

[0183] (vi) Surface hydrophobicity analysis (surface properties)

[0184] 1. Sample dilution: Dilute pure TMP, TMP / κCOS, and TMP / κCOS / CUR samples to 0.01, 0.02, 0.04, 0.06, and 0.08 mg / mL, respectively, with 20 mmol / L PBS buffer (pH 7.0).

[0185] 2. Fluorescence reaction: Take one portion (290 μL) of diluted sample and mix it with 10 μL of ANS solution with a concentration of 8 mmol / L. Let it stand in the dark for 5 min (to ensure that ANS is fully bound to the hydrophobic groups).

[0186] 3. Fluorescence assay: The fluorescence intensity of the mixture was measured using a fully automated microplate reader with an excitation wavelength of 390 nm and an emission wavelength of 470 nm. The slope of the linear regression equation plotted against the protein concentration is the surface hydrophobicity index (H0). The larger the H0, the stronger the surface hydrophobicity.

[0187] IV. Experimental Results and Analysis

[0188] (a) SDS-PAGE electrophoresis results (protein polymerization status)

[0189] SDS-PAGE gel images of TMP, TMP / κCOS, and TMP / κCOS / CUR are shown below. Figure 12 As shown: The pure TMP group (lane T) exhibits clear bands of myosin heavy chain (MHC, 220 kDa), actin (44 kDa), and tropomyosin (37 kDa), consistent with the typical protein composition of tilapia TMP. The MHC bands in the TMP / κCOS group (lane T / κ) and the TMP / κCOS / CUR group (lane T / κ / C) almost completely disappear, and dark high-molecular-weight bands appear at the top of the stacking gel.

[0190] The results showed that κCOS crosslinked with the MHC of TMP, or induced TMP molecules to form a polymer through covalent bonds, and the addition of CUR did not destroy the polymer structure, confirming that TMP and κCOS formed a stable complex system.

[0191] (II) Endogenous fluorescence spectroscopy results (protein tertiary structure)

[0192] like Figure 13 As shown in (A), the maximum fluorescence wavelength of pure TMP is 331 nm, and the relative fluorescence intensity is 523.8, indicating that the tryptophan residues in TMP are exposed to a hydrophobic microenvironment. After TMP is complexed with κCOS, the maximum fluorescence wavelength blue-shifts to 311 nm, and the relative fluorescence intensity decreases to 72.5, with a blue shift of 20 nm; while the maximum fluorescence wavelength and intensity of the TMP / κCOS / CUR group are not significantly different from those of TMP / κCOS (p>0.05).

[0193] The results showed that when κCOS, as an oligosaccharide, binds to TMP, it generates a steric hindrance effect, which promotes the migration of tryptophan residues into the molecule (reducing the polarity of the microenvironment), resulting in a decrease in fluorescence intensity and a blue shift in wavelength, confirming that the tertiary structure of TMP has been altered. CUR did not further affect the tryptophan microenvironment, indicating that it was encapsulated in the hydrophobic core of TMP / κCOS.

[0194] (III) Circular dichroism (CD) results (protein secondary structure)

[0195] like Figure 13 As shown in (B) and (C) in the diagram: CD spectrum characteristics: Pure TMP has a positive peak at 192 nm and negative peaks at 208 nm and 222 nm (typical α-helical characteristic peaks). The TMP / κCOS group shows a significant increase in the intensity of the positive peak at 191 nm, and a decrease in the intensity of the negative peaks at 207-210 nm with a slight blue shift, indicating that the secondary structure of TMP and κCOS has changed after recombination. The spectrum of the TMP / κCOS / CUR group is not significantly different from that of the TMP / κCOS group.

[0196] Secondary structure content: Pure TMP contained 39% α-helices, 18% β-sheets, 12% β-turns, and 31% random coils. After TMP was combined with κCOS, the α-helix content increased to 76% (p<0.05), the β-sheet content decreased to 3%, and the random coil content decreased to 10% (p<0.05), while the β-turn content remained largely unchanged. Compared to κCOS / CUR, the secondary structure of TMP / κCOS / CUR showed no significant change.

[0197] Figure 13 The results in (B) and (C) show that κCOS promotes the rearrangement of the secondary structure of TMP through intermolecular interactions, increasing α-helices (ordered structure) and decreasing random coils (disordered structure), which significantly improves the structural stability of the complex. CUR does not affect the secondary structure of TMP, further confirming that it is encapsulated inside rather than participating in molecular interactions.

[0198] (iv) Fourier transform infrared (FTIR) spectroscopy results (intermolecular interactions)

[0199] Figure 13 (D) shows the Fourier transform infrared (FTIR) spectra of different samples. The horizontal axis represents wavenumber and the vertical axis represents absorbance. The samples include: TMP, κCOS, TMP / κCOS, TMP / κCOS / CUR, and CUR curves.

[0200] TMP curve: 1652cm -1 (Amide I band, C=O stretching), 1541cm -1 (Amide II tape, CN stretch), 1236cm -1 (Amide III band, NH angular vibration coupled with CN stretching) is a characteristic peak of the amide group in proteins; 2964 cm⁻¹ -1 The peak for CH tensile vibration is 3435 cm⁻¹. -1 This is the peak of the OH bending vibration in protein molecules.

[0201] κCOS curve: 850cm -1 924cm -1 1259cm -1 The absorption peak at 1070 cm⁻¹ corresponds to a sulfate group (characteristic peaks of 4-sulfate-β-D-galactose, α-D-3,6-dehydrated galactose, and sulfate, respectively). -1 The peak at this location is a characteristic peak of the COC stretching vibration of the pyranose ring.

[0202] TMP / κCOS curve: 3440.7 cm compared to TMP. -1 The -OH absorption band at the site broadened significantly and the absorption intensity increased, indicating that the number of -OH groups increased after the protein molecule bound to κCOS. At the same time, the absorption peaks of amide I, amide II, and amide III bands showed a blue shift and an increase in intensity, reflecting that the interaction between TMP and κCOS led to a conformational change in the protein.

[0203] TMP / κCOS / CUR curve: Characteristic peaks of TMP / κCOS / CUR (e.g., 3509 cm⁻¹) -1 1633cm -1 The overlap of characteristic peaks of CUR with those of TMP / κCOS, or the narrowing of characteristic peaks of TMP / κCOS, suggests that CUR is encapsulated in the hydrophobic core of the TMP / κCOS complex through hydrophobic interactions.

[0204] CUR curve: at 3509cm -1 A -OH stretching vibration peak appears at 1633 cm⁻¹. -1 The peak at 1506 cm⁻¹ represents the stretching vibration of the carbonyl group (C=O) of the benzene ring. -1 and 1276cm -1The peaks at these locations correspond to the stretching vibration peaks of the benzene ring and the CO stretching vibration peaks in the glycosidic bond, respectively, which are typical infrared characteristics of CUR.

[0205] Figure 13 (D) indicates that TMP and κCOS achieve molecular self-assembly through hydrogen bonding (change in the -OH absorption band), electrostatic interaction (sulfate group and charged residue of TMP), and hydrophobic interaction (amide band shift); CUR is encapsulated in the composite core through hydrophobic interaction and does not participate in covalent interaction.

[0206] (v) Scanning electron microscopy (SEM) results (microscopic morphology)

[0207] Scanning electron microscopy of TMP, κCOS, TMP / κCOS, and TMP / κCOS / CUR, such as... Figure 14 As shown: Pure TMP ( Figure 14 The surface structure of (A) is rough and banded, with wrinkles and cracks; pure κCOS ( Figure 14 The particles in sample (B) have smooth, dense surfaces and a layered structure; TMP / κCOS ( Figure 14 (C) forms a continuous three-dimensional network of pores with a pore size of approximately 1–5 μm, confirming that TMP undergoes protein secondary structure unfolding in the strong base of the pH-driven method, fully complexing with κCOS to construct an ordered spatial structure; TMP / κCOS / CUR ( Figure 14 The (D) in the middle still maintains a three-dimensional mesh structure. The added CUR is attached to and wrapped inside the mesh structure of TMP / κOS, and is filled to form a wrapping and delivery of CUR.

[0208] The results show that the three-dimensional network structure provides sufficient encapsulation space for CUR. The addition of CUR does not disrupt the network framework, but fills the pores only through hydrophobic interactions, further confirming the structural adaptability of the composite.

[0209] (vi) Surface hydrophobicity results (surface properties)

[0210] The surface hydrophobicity measurement results of TMP, TMP / κCOS, and TMP / κCOS / CUR are as follows: Figure 15 As shown: the surface hydrophobicity index (H0) of pure TMP is 586.67, indicating that its surface exposes a large number of hydrophobic groups; the H0 of the TMP / κCOS group is reduced to 286.55, which is significantly lower than that of pure TMP. This is because κCOS carries a large number of hydrophilic groups, which bind with TMP and reduce the hydrophobicity of TMP / κCOS; the H0 of the TMP / κCOS / CUR group is 102.84, which is further reduced compared with the TMP / κCOS group.

[0211] The results showed that the hydrophilic groups (-OH, sulfate groups) carried by κCOS covered the surface of TMP or combined with the hydrophobic groups inside TMP, which significantly reduced the surface hydrophobicity of the complex; CUR was wrapped in a hydrophobic core and did not increase the exposure of surface hydrophobic groups, confirming the improvement of surface hydrophilicity of the complex.

[0212] V. Conclusion

[0213] At the molecular level, κCOS induces tertiary folding of TMP (inward shift of tryptophan residues) and ordered secondary structure (increased α-helices), reducing surface hydrophobicity. At the microscopic level, a continuous three-dimensional network of porous structures is constructed, providing encapsulation space for CURs. At the polymerization level, TMP and κCOS crosslink to form a polymeric complex, with CURs efficiently encapsulated without disrupting the complex structure. This indicates that TMP and κCOS achieve molecular self-assembly through hydrogen bonding, electrostatic interactions, and hydrophobic interactions, forming a multi-scale stable structure. This structural feature enables the TMP / κCOS complex to meet the core requirements (structural stability, hydrophilic surface, and hydrophobic interior) as a carrier of lipophilic active ingredients, laying the foundation for subsequent CUR loading and stability verification.

[0214] Example 4

[0215] This embodiment describes the verification of the ionic and thermal stability of the TMP / κCOS complex.

[0216] I. Experimental Objective

[0217] Using the TMP / κCOS complex (TMP / κCOS / CUR) loaded with curcumin (CUR) as the research object, the particle size, zeta potential and CUR release rate of the complex were measured by simulating the ionic strength (NaCl concentration) and temperature environment during the processing, storage and human digestion of health food, and verifying its ionic stability and thermal stability, so as to provide data support for its subsequent application in the field of health food.

[0218] II. Test Materials

[0219] TMP / κCOS / CUR: Prepared according to the optimal process determined in Example 2 (TMP concentration 1.2% w / v, κCOS concentration 0.4% w / v, volume ratio 1:1, pH driven method). Add 0.2 mg / mL CUR when mixing TMP and κCOS, stir at 400 rpm for 1 h in the dark, neutralize to pH 7.0 and continue stirring for 2 h, centrifuge at 2000g for 10 min and collect the supernatant, store at 4℃ in the dark for later use.

[0220] Control sample: Free CUR solution (0.2 mg / mL, dissolved in anhydrous ethanol and then diluted with deionized water) was used to compare the differences in CUR release.

[0221] III. Test Methods

[0222] (a) Verification of ion stability

[0223] 1. Sample preparation: Take an equal amount (10 mL) of TMP / κCOS complex and mix it with 10 mL of NaCl solution of different concentrations (0, 50, 100, 200, 400, 800 mmol / L) to obtain a mixture system with a final NaCl concentration of 0, 25, 50, 100, 200, 400 mmol / L.

[0224] 2. Storage and testing: Store the mixture at 25°C in the dark for 24 hours, gently shaking it once every 6 hours during this period (to avoid particle sedimentation due to standing); after 24 hours, take samples to determine the particle size and zeta potential.

[0225] (II) Thermal stability verification

[0226] 1. Sample preparation: Take 10 mL of TMP / κCOS and TMP / κCOS / CUR complex and place them in 5 clean transparent centrifuge tubes, numbered 1 to 5 respectively.

[0227] 2. Heat treatment: Place the centrifuge tubes in constant temperature water baths at 40, 50, 60, 70 and 80°C respectively, and heat for 30 minutes in the dark; after heating, immediately place the centrifuge tubes in an ice box for rapid cooling.

[0228] 3. Detection indicators and methods: TMP / κCOS particle size and zeta potential were determined using the same ion stability determination method, and the particle size was measured using a nanoparticle size potentiometer after dilution.

[0229] An appropriate amount of CUR was dissolved in anhydrous ethanol, vortexed for 10 min, sonicated for 30 min, and placed in a refrigerator overnight to ensure complete dissolution. Different concentrations (2, 4, 6, 8, 12, 16, 20 mg / L) of CUR were prepared. The absorbance of CUR was measured at 422 nm using a UV spectrophotometer, and a standard curve was plotted.

[0230] Curcumin-anhydrous ethanol standard curve: y = 0.1323x + 0.00128 (R²) 2 =0.9996)

[0231] Determination of CUR content in the complex (TMP / κCOS / CUR): Take 1 mL of the complex and centrifuge at 10000g for 15 min. Take 0.5 mL of the supernatant, add 1 mL of anhydrous ethanol, vortex for 1 min, and centrifuge at 500g for 10 min to ensure complete dissolution of CUR from the sample group. After dilution five times, measure the absorbance of the ethanol supernatant at 422 nm. Substitute this absorbance into the curcumin-anhydrous ethanol standard curve to obtain the CUR concentration.

[0232] CUR retention rate (%) = (C1V1 / C 总 V2) × 100%;

[0233] In the formula, C1 is the concentration of CUR after heating (mg / L), and V1 is the volume of the solution after heating (L); C 总 V1 represents the total concentration of CUR loaded in the complex before heating (mg / L), and V2 represents the solution volume before heating (L).

[0234] IV. Experimental Results and Analysis

[0235] (a) Results of ion stability

[0236] 1. Particle size variation trend

[0237] like Figure 16 As shown in (A): when the NaCl concentration was 0 mmol / L, the TMP / κCOS particle size was 285.7 ± 19.7 nm; as the NaCl concentration increased from 0 to 100 mmol / L, the particle size gradually decreased to 218.1 ± 0.3 nm. This is because low concentrations of NaCl promote the solubilization of TMP (salt-soluble protein), exposing more internal polar groups and resulting in a tighter binding with κCOS; as the NaCl concentration increased from 100 to 400 mmol / L, the particle size gradually increased to 265 ± 3.8 nm. This is because the electrostatic shielding effect of high concentrations of NaCl neutralizes the negative charge on the surface of the complex, leading to a decrease in electrostatic repulsion between particles and slight aggregation. Overall, within the NaCl concentration range of 0–400 mmol / L, the particle size remained consistently between 200 and 300 nm, meeting the requirements for nanocarriers (≤500 nm), with no significant aggregation.

[0238] 2. Trend of zeta potential changes

[0239] like Figure 16 As shown in (A): when the NaCl concentration is 0 mmol / L, the potential is -18.7 ± 0.3 mV; as the NaCl concentration increases, the absolute value of the potential gradually decreases, dropping to -6.1 ± 0.9 mV at 400 mmol / L; the reason for the change is the Na+ content in NaCl. + The anionic charge on the surface of the complex (the sulfate group of κCOS and the acidic amino acid residue of TMP) was neutralized, reducing the electrostatic repulsion between particles; however, the absolute value of the potential remained ≥5mV and the particle size did not change drastically, confirming that the complex still has a certain degree of dispersion stability.

[0240] (II) Thermal stability results

[0241] 1. Particle size variation trend

[0242] like Figure 16As shown in (B): when the temperature rises from 40℃ to 50℃, the particle size increases to 348.1±21.4nm, due to slight swelling of the protein upon heating, resulting in increased particle volume; when the temperature rises from 50℃ to 80℃, the particle size gradually decreases to 272.8±1.9nm, because κCOS promotes the rearrangement of TMP molecules at high temperatures, forming a denser nanostructure and inhibiting complex aggregation; the particle size in each temperature group is within the range of 240~350nm, with little difference and no significant abnormalities.

[0243] 2. Trend of zeta potential changes

[0244] like Figure 16 As shown in (B) of the figure: the potential is -28±0.3mV at 40℃, the absolute value of the potential drops to the lowest level at 60℃, but is still ≥20mV, and the absolute value of the potential rises back to 30mV at 70~80℃. This indicates that TMP / κCOS has good high temperature resistance.

[0245] 3. Changes in CUR retention rate

[0246] like Figure 17 As shown in (A), the CUR retention rate was 87.2±3.4% at 40℃, and after heating to 80℃ for 30 minutes, the retention rate was still 54.9±1.3%. Heat treatment at neutral pH accelerates the degradation of curcumin, which greatly limits its application in processing. This nanocomposite demonstrates stable encapsulation of CUR.

[0247] V. Conclusion

[0248] Ionic stability: The TMP / κCOS complex maintains a particle size of 200-300 nm and an absolute potential of 5-20 mV in the NaCl concentration range of 0-400 mmol / L, and can adapt to the low-salt to medium-salt environment commonly found in health food processing.

[0249] Thermal stability: The TMP / κCOS complex maintains a particle size of 240~350nm and an absolute potential of 20~30mV in the temperature range of 40~80℃. After high-temperature heating, the CUR retention rate of the TMP / κCOS / CUR complex is ≥54%, and it can still maintain structural stability at high temperatures. It can withstand sterilization (such as pasteurization) and conventional storage temperatures.

[0250] In summary, the TMP / κCOS complex possesses good ionic and thermal stability, meeting the application requirements in the health food field.

[0251] Example 5

[0252] This embodiment describes the CUR loading rate and in vitro digestion and delivery performance test of the TMP / κCOS / CUR complex.

[0253] I. Experimental Objective

[0254] Based on the optimal process determined in Example 2, a TMP / κCOS complex (TMP / κCOS / CUR) loaded with curcumin (CUR) was prepared. Its physicochemical properties (particle size, zeta potential, CUR loading rate) were measured. The release pattern of CUR in gastric and intestinal fluid stages was analyzed through in vitro simulated gastrointestinal digestion experiments to verify the intestinal targeted delivery capability of the complex for CUR, providing data support for its practical application as a delivery carrier of lipid-soluble active ingredients.

[0255] II. Test Materials

[0256] Tilapia myofibrillar protein (TMP), κ-carrageenan oligosaccharide (κCOS), curcumin (CUR): Same as in the previous examples.

[0257] Simulated digestion reagent:

[0258] Simulated gastric juice (SGF): containing pepsin (3000U / mg, Beijing Solarbio Co., Ltd.) 3.2mg / mL, NaCl 2.0mg / mL, and pH adjusted to 2.8 with 1mol / L HCl.

[0259] Simulated intestinal fluid (SIF): contains 2.4 mg / mL pancreatic enzyme (4000 U / mg, Beijing Solarbio Co., Ltd.), 5.0 mg / mL porcine bile salts (Beijing Solarbio Co., Ltd.), 120 mmol / L NaCl, and 10 mmol / L CaCl2.

[0260] Control sample: Free CUR solution: Take 0.2 mg / mL CUR stock solution and dilute it with deionized water to the same concentration as the initial CUR in the complex, to compare the differences in CUR release.

[0261] III. Test Methods

[0262] (a) Determination of physicochemical properties

[0263] TMP / κCOS / CUR was prepared according to Example 4, and the particle size and zeta potential were measured.

[0264] (II) Determination of CUR loading rate

[0265] Refer to Example 4 to determine the CUR concentration in the TMP / κCOS / CUR solution.

[0266] Packing capacity calculation: The absorbance of the sample was measured at 422 nm and substituted into the standard curve (y = 0.1323x + 0.00128, R0). 2 =0.9996) Calculate the concentration of bound CUR in the supernatant (mg / L), and calculate the encapsulation loading rate EE according to the following formula:

[0267] EE (%) = CV / m;

[0268] In the formula, C is the concentration of curcumin loaded in the complex solution (mg / L), V is the volume of the complex solution (L), and m is the total amount of curcumin added (mg).

[0269] (III) In vitro simulated gastrointestinal digestion test

[0270] 1. Gastric juice stage (0~60min)

[0271] Take 10 mL of TMP / κCOS / CUR complex and mix it with 10 mL of simulated gastric juice (SGF, pH 2.8). Correct the pH to 2.8 with 2 mol / L HCl. Incubate the mixture in a constant temperature water bath at 37℃ and 120 r / min. Take 2 mL samples at 30 min and 60 min respectively (denoted as S30 and S60). Immediately after sampling, place the samples in an ice bath (to terminate pepsin activity), centrifuge at 10000g for 15 min, collect the supernatant, dilute with anhydrous ethanol, and measure the absorbance of CUR. Substitute the values ​​into the standard curve to calculate the mass of free CUR in the supernatant (m). t ).

[0272] 2. Intestinal fluid stage (60~180min)

[0273] After 60 min of incubation in the gastric juice stage, 20 mL of simulated intestinal fluid (SIF, pH 7.0) was added to the remaining digestive fluid, and the pH was corrected to 7.0 with 2 mol / L NaOH. Incubation continued at 37℃ and 120 r / min. Samples of 2 mL were taken at 90 min, 120 min, 150 min, and 180 min (denoted as S90, S120, S150, and S180, respectively). The reaction was terminated by ice bath after sampling, followed by centrifugation, dilution, and absorbance measurement as in step 1. The mass of free CUR in the supernatant (m³) was calculated. t ).

[0274] 3. Release rate calculation: The release rate of CUR at different time points is calculated using the following formula:

[0275] Release rate (%) = m t / m 总 ×100%;

[0276] In the formula, m t The mass (mg) of free CUR in the supernatant at a certain time point; m 总 This refers to the total amount of CUR added during preparation (mg).

[0277] 4. Control experiment: Using free CUR solution as a control, an in vitro digestion experiment was performed following the same steps as above to determine its release rate at each time point and compare the delivery difference between the TMP / κCOS / CUR complex and free CUR.

[0278] (iv) Data validity control

[0279] All experiments were conducted under light-protected conditions to avoid CUR degradation (CUR is photosensitive); particle size, potential, loading rate and release rate were measured three times and the average value was taken.

[0280] IV. Experimental Results and Analysis

[0281] (I) Physicochemical properties of the TMP / κCOS / CUR complex

[0282] 1. Particle size and zeta potential

[0283] like Figure 11 As shown in (C): the average particle size of the TMP / κCOS / CUR complex is 489.9±11.4 nm, which is slightly larger than that of the TMP / κCOS complex without CUR loading (291.17±0.79 nm, results of Example 2); the zeta potential is -25.44±0.68 mV, with an absolute value ≥25 mV.

[0284] The results showed that CUR was encapsulated in the hydrophobic core of TMP / κCOS through hydrophobic interactions, leading to particle aggregation. The filling of CUR molecules slightly increased the particle size, and the complex exhibited good dispersibility and stability.

[0285] 2. CUR load factor

[0286] The TMP / κCOS / CUR complex was found to have a CUR loading rate of 76.76%, which is significantly higher than the theoretical loading rate corresponding to the solubility of free CUR in water. This confirms that the TMP / κCOS complex can effectively encapsulate hydrophobic CUR and solve its poor water solubility problem.

[0287] (II) Release pattern of CUR during in vitro simulated digestion

[0288] 1. Release curve characteristics

[0289] The CUR release rate of the TMP / κCOS / CUR complex under simulated in vitro digestion conditions is as follows: Figure 17 As shown in (B), the release process of CUR is divided into two stages:

[0290] Gastric juice phase (0~60 min): CUR release rate increases slowly, reaching 14.2% at 60 min. The release rate is low in this phase because in the acidic environment of SGF (pH 2.8), the TMP / κCOS complex forms large-scale electrostatic aggregates. The dense structure hinders CUR leakage. At the same time, the steric hindrance effect of κCOS buries the sites on TMP that bind to pepsin, reducing complex degradation and preventing premature CUR release.

[0291] Intestinal fluid stage (60~180min): The CUR release rate increases rapidly, reaching 74.7% at 180min. The release rate is significantly improved in this stage. Due to the neutral environment of SIF (pH 7.0) and the presence of bile salts, the stable structure of TMP / κCOS is disrupted (hydrogen bonds and electrostatic interactions are weakened), the pore size of the complex increases, and at the same time, pancreatic enzymes degrade TMP molecules, which promotes the release of a large amount of encapsulated CUR, thus achieving targeted delivery to the intestine.

[0292] 2. Comparison with free CUR

[0293] The release rate of free CUR in the gastric juice stage (60 min) was 58.3%, which was much higher than that of the TMP / κCOS / CUR complex (14.2%). Furthermore, free CUR is easily degraded in gastric juice, resulting in a reduced amount that can be released in the subsequent intestinal juice stage. The final release rate of free CUR after in vitro digestion (180 min) was 59.8%, which was lower than that of the TMP / κCOS / CUR complex (74.7%).

[0294] The results showed that the TMP / κCOS / CUR complex can effectively protect CUR from gastric acid environment damage, reduce premature release in the stomach, and efficiently release CUR in the intestinal stage, significantly improving the targeted delivery efficiency and bioavailability of CUR.

[0295] V. Conclusion

[0296] Feasibility of complex preparation: Using a pH-driven method, a TMP / κCOS / CUR complex was successfully prepared with TMP 1.2% w / v, κCOS 0.4% w / v, and a volume ratio of 1:1. The complex had a particle size of 284.07±0.88 nm, a zeta potential of -25.44±0.68 mV, and a CUR loading of 76.76%, exhibiting excellent and stable physicochemical properties.

[0297] Excellent in vitro delivery performance: The complex exhibits a targeting characteristic of "low release in the stomach and high release in the intestine" during in vitro digestion, with a release rate of 14.2% in the gastric juice stage and a final release rate of 74.7% in the intestinal juice stage, which is significantly better than free CUR. It can effectively protect CUR from gastric juice degradation and achieve intestinal targeted delivery.

[0298] In summary, the TMP / κCOS / CUR complex possesses the core capability to serve as an intestinal-targeted delivery carrier for lipid-soluble active ingredients (such as CUR), and can be used for the delivery of functional active ingredients in the field of health foods.

[0299] The embodiments described above are only some, not all, of the embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments. All other embodiments obtained by those skilled in the art through related deductions and substitutions based on the inventive concept, without inventive effort, are within the scope of protection of the present invention.

Claims

1. A myofibrillar protein-κ-carrageenan oligosaccharide complex, characterized in that, The composition of the myofibrillar protein-κ-carrageenan oligosaccharide complex is: 1:1 (by volume ratio) of 1.2% w / v tilapia myofibrillar protein solution and 0.4% w / v κ-carrageenan oligosaccharide solution.

2. The tilapia myofibrillar protein-κ-carrageenan oligosaccharide complex according to claim 1, characterized in that, The tilapia myofibrillar protein was extracted using the following method: Take the white flesh of tilapia, add 3-5 times the volume of pre-cooled first PBS extract, homogenize and centrifuge, discard the supernatant and scrape off the white flocculent material on the surface of the precipitate, repeat homogenization and centrifugation once; Collect the precipitate, add 3-5 times the volume of the second PBS extract, homogenize, and centrifuge. Collect the precipitate, add 0.1-0.3 mol / L NaCl solution at a volume ratio of 1:3-5 to homogenize, filter through 4 layers of gauze and centrifuge. The resulting precipitate is TMP. The first PBS extract consisted of: EDTA 0.5 mmol / L, NaCl 137 mmol / L, Na2HPO4 10 mmol / L, KH2PO4 1.8 mmol / L, and water as the solvent. The second PBS extract consisted of: NaCl 137 mmol / L, Na2HPO4 10 mmol / L, KH2PO4 1.8 mmol / L, and water as the solvent.

3. The tilapia myofibrillar protein-κ-carrageenan oligosaccharide complex according to claim 2, characterized in that, The first homogenization lasted 90 seconds, and the second homogenization lasted 60 seconds. Centrifugation conditions: 6000~8000 r / min, 4±1℃ for 10~15 min.

4. A method for preparing the myofibrillar protein-κ-carrageenan oligosaccharide complex according to any one of claims 1 to 3, characterized in that, Includes the following steps: (1) Preparation of TMP solution: Prepare a 1.2% TMP solution with deionized water, adjust the pH to 12, and hydrate it under cold. (2) Preparation of κCOS solution: Prepare a κCOS solution with a concentration of 0.4% using deionized water, adjust the pH to 12, and hydrate it under cold conditions; (3) Mixing reaction: Mix the TMP solution from step (1) with the κCOS solution from step (2) at a volume ratio of 1:1 and stir. (4) Neutralization and stabilization: Neutralize the mixture from step (3) to pH=7 with HCl solution and continue stirring; (5) Purification: Centrifuge the mixture from step (4) to remove the precipitate, and take the supernatant as the complex.

5. The preparation method according to claim 4, characterized in that, In step (1), the hydration temperature of the TMP solution is 4±1℃, the hydration time is 12h±2h, and a sealed container is used during the hydration process to avoid concentration deviation caused by solution evaporation.

6. The preparation method according to claim 4, characterized in that, In steps (3) to (4), the stirring rate is 400±50 rpm, and the ambient temperature is kept at 25±2℃ during the preparation process to avoid temperature fluctuations causing the aggregation of tilapia myofibril protein or κ-carrageenan oligosaccharide molecules.

7. The preparation method according to claim 4, characterized in that, In step (4), when neutralizing with 1 mol / L HCl, HCl is added dropwise while stirring to ensure that the pH of the reaction system changes uniformly.

8. The application of the myofibrillar protein-κ-carrageenan oligosaccharide complex according to any one of claims 1 to 3 in the preparation of a lipid-soluble active ingredient delivery system, characterized in that, The fat-soluble active ingredient is curcumin.

9. The application according to claim 8, characterized in that, The delivery system is used to prepare health foods or medicines.

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